11-cis-retinal and Retinal-Degeneration

Physiological equilibrium in the retina depends on coordinated work between rod and cone photoreceptors and can be compromised by the expression of mutant proteins leading to inherited retinal degeneration (IRD). IRD is a diverse group of retinal dystrophies with multifaceted molecular mechanisms that are not fully understood. In this review, we focus on the contribution of chronically activated unfolded protein response (UPR) to inherited retinal pathogenesis, placing special emphasis on studies employing genetically modified animal models. As constitutively active UPR in degenerating retinas may activate pro-apoptotic programs associated with oxidative stress, pro-inflammatory signaling, dysfunctional autophagy, free cytosolic Ca

Topics: Animals; Autophagy; Disease Management; Endoplasmic Reticulum Stress; Humans; Retinal Degeneration; Rhodopsin; Signal Transduction; Unfolded Protein Response

The aim of this study was to understand the relationship between the findings of spectral-domain optical coherence tomography (SD-OCT) of previously reported animal models of retinitis pigmentosa (RP) associated with known genetic mutations and their background structural and functional changes.. We reviewed previous publications reporting the SD-OCT findings of animal models of RP and summarized the characteristic findings of SD-OCT in nine different animal models (. Despite the various abnormal structural changes found in these different animal models, progressive thinning of the outer nuclear layer (ONL) and hyperreflective change in the inner and outer segment (IS-OS) layers of the photoreceptors were commonly observed on SD-OCT. In the rapidly progressive severe photoreceptor degeneration seen in rd10 and

Topics: Animals; Arrestins; cis-trans-Isomerases; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Humans; Mice; Mice, Knockout; Rats; Rats, Transgenic; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

The review discusses the prospects of using rhodopsin as an optogenetic tool for prosthetics of degenerative (blind) eye retina and the principles of optogenetic techniques. Retinal-containing proteins that depolarize/hyperpolarize the plasma membrane of nerve cells and, accordingly, excite/inhibit physiological activity of neurons, are described. The problem of what cells of the degenerative retina can be treated with what particular rhodopsins is discussed in detail. Viruses and promoters required for the rhodopsin gene delivery into the degenerative retina cells are described. In conclusion, main concepts and tasks associated with the optogenetic prosthetic treatment of degenerative retina employing rhodopsins are presented.

Topics: Animals; Genetic Therapy; Humans; Optogenetics; Photoreceptor Cells; Retina; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin

Rhodopsin is the light receptor in photoreceptor cells of the retina and a prototypical G protein-coupled receptor. Two types of quaternary structures can be adopted by rhodopsin. If rhodopsin folds and attains a proper tertiary structure, it can then form oligomers and nanodomains within the photoreceptor cell membrane. In contrast, if rhodopsin misfolds, it cannot progress through the biosynthetic pathway and instead will form aggregates that can cause retinal degenerative disease. In this review, emerging views are highlighted on the supramolecular organization of rhodopsin within the membrane of photoreceptor cells and the aggregation of rhodopsin that can lead to retinal degeneration.

Topics: Animals; Cell Membrane; Humans; Photoreceptor Cells, Vertebrate; Protein Domains; Protein Folding; Retinal Degeneration; Rhodopsin

Retinal trafficking proteins are involved in molecular assemblies that govern protein transport, orchestrate cellular events involved in cilia formation, regulate signal transduction, autophagy and endocytic trafficking, all of which if not properly controlled initiate retinal degeneration. Improper function and or trafficking of these proteins and molecular networks they are involved in cause a detrimental cascade of neural retinal remodeling due to cell death, resulting as devastating blinding diseases. A universal finding in retinal degenerative diseases is the profound detection of retinal remodeling, occurring as a phased modification of neural retinal function and structure, which begins at the molecular level. Retinal remodeling instigated by aberrant trafficking of proteins encompasses many forms of retinal degenerations, such as the diverse forms of retinitis pigmentosa (RP) and disorders that resemble RP through mutations in the rhodopsin gene, retinal ciliopathies, and some forms of glaucoma and age-related macular degeneration (AMD). As a large majority of genes associated with these different retinopathies are overlapping, it is imperative to understand their underlying molecular mechanisms. This review will discuss some of the most recent discoveries in vertebrate retinal remodeling and retinal degenerations caused by protein mistrafficking.

Topics: Animals; Cell Death; Cell Movement; Humans; Mutation; Photoreceptor Cells, Vertebrate; Protein Transport; Retina; Retinal Degeneration; Rhodopsin; Signal Transduction

Because of its favorable anatomical and immunological characteristics, the eye has been at the forefront of translational gene therapy. Dozens of promising proofs of concept have been obtained in animal models of inherited retinal degenerations (IRDs), and some of them have been relayed to the clinic. The results from the first clinical trials for a congenital form of blindness have generated great interest and have demonstrated the safety and efficacy of intraocular administrations of viral vectors in humans. However, this progress has also generated new questions and posed challenges that need to be addressed to further expand the applicability of gene therapy in the eye, including safe delivery of viral vectors to the outer retina, treatment of dominant IRDs as well as of IRDs caused by mutations in large genes, and, finally, selection of the appropriate IRDs and patients to maximize the efficacy of gene transfer. This review summarizes the strategies that are currently being exploited to overcome these challenges and drive the clinical development of retinal gene therapy.

Topics: Animals; Clinical Trials as Topic; Genetic Therapy; Genetic Vectors; Humans; Retina; Retinal Degeneration; Rhodopsin; Transduction, Genetic

Rhodopsin is a seven-transmembrane G protein-coupled receptor (GPCR) and is the main component of the photoreceptor outer segment (OS), a ciliary compartment essential for vision. Because the OSs are incapable of protein synthesis, rhodopsin must first be synthesized in the inner segments (ISs) and subsequently trafficked across the connecting cilia to the OSs where it participates in the phototransduction cascade. Rapid turnover of the OS necessitates a high rate of synthesis and efficient trafficking of rhodopsin to the cilia. This cilia-targeting mechanism is shared among other ciliary-localized GPCRs. In this review, we will discuss the process of rhodopsin trafficking from the IS to the OS beginning with the trafficking signals present on the protein. Starting from the endoplasmic reticulum and the Golgi apparatus within the IS, we will cover the molecular components assisting the biogenesis and the proper sorting. We will also review the confirmed binding and interacting partners that help target rhodopsin toward the connecting cilium as well as the cilia-localized components which direct proteins into the proper compartments of the OS. While rhodopsin is the most critical and abundant component of the photoreceptor OS, mutations in the rhodopsin gene commonly lead to its mislocalization within the photoreceptors. In addition to covering the trafficking patterns of rhodopsin, we will also review some of the most common rhodopsin mutants which cause mistrafficking and subsequent death of photoreceptors. Toward the goal of understanding the pathogenesis, three major mechanisms of aberrant trafficking as well as putative mechanisms of photoreceptor degeneration will be discussed.

Topics: Animals; Arrestins; Biological Transport; Cilia; Golgi Apparatus; Humans; Light Signal Transduction; Mice; Mutation; Protein Conformation; Protein Denaturation; Protein Folding; Protein Transport; Receptors, G-Protein-Coupled; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Signal Transduction

Retinal degenerations are a group of clinically and genetically heterogeneous disorders characterised by progressive loss of vision due to neurodegeneration. The retina is a highly specialised tissue with a unique architecture and maintaining homeostasis in all the different retinal cell types is crucial for healthy vision. The retina can be exposed to a variety of environmental insults and stress, including light-induced damage, oxidative stress and inherited mutations that can lead to protein misfolding. Within retinal cells there are different mechanisms to cope with disturbances in proteostasis, such as the heat shock response, the unfolded protein response and autophagy. In this review, we discuss the multiple responses of the retina to different types of stress involved in retinal degenerations, such as retinitis pigmentosa, age-related macular degeneration and glaucoma. Understanding the mechanisms that maintain and re-establish proteostasis in the retina is important for developing new therapeutic approaches to fight blindness.

Topics: Animals; Humans; Mutation; Proteostasis Deficiencies; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinal Vessels; Rhodopsin; Stress, Physiological

Rhodopsins (Rh) are G protein-coupled receptors that function as light-sensors in photoreceptors. In humans, Rh mutations cause retinitis pigmentosa (RP), a degenerative disease that ultimately results in blindness. Studies in Drosophila have provided many insights into basic Rh biology and have identified pathways that lead to retinal degeneration. It has been shown that, because Rh is very abundant in photoreceptors, its accumulation in numerous organelles induces severe stress and results in degeneration of these cells. Moreover, genetic lesions that affect proper activation of membrane-bound Rh lead to disruption in Ca(2+) homeostasis which also causes photoreceptor degeneration. We review here the molecular signals involved in Rh homeostasis and the mechanisms underlying retinal degeneration in flies, and discuss possible links to human diseases.

Topics: Animals; Drosophila melanogaster; Homeostasis; Humans; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

In light of the elucidation of the molecular pathogenesis of some dominantly inherited retinal degenerations over the past two decades, it is timely to explore possible means of therapeutic intervention for such diseases. However, the presence of significant levels of intergenic and intragenic genetic heterogeneity in this group of dominant conditions represents a barrier to the development of therapies focused on correcting the primary genetic defect. More than 60 genes have been implicated in dominant retinopathies and indeed over 150 different mutations in the rhodopsin gene alone have been identified in patients with autosomal dominant retinitis pigmentosa. Employing next-generation sequencing to characterise populations of retinal degeneration patients genetically over the coming years will beyond doubt serve to highlight further the immense genetic heterogeneity inherent in this group of disorders. Such diversity in genetic aetiologies has promoted the search for therapeutic solutions for dominantly inherited retinopathies that are independent of disease-causing mutations. The various approaches being considered to provide mutation-independent therapies for these dominant conditions will be discussed in the review, as will the preclinical data supporting the further development of such strategies.

Topics: Animals; Dependovirus; Gene Expression; Gene Silencing; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA Interference

Topics: Animals; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Topics: Endoplasmic Reticulum; Glycoproteins; Humans; Photoreceptor Cells, Vertebrate; Proteostasis Deficiencies; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa is a retinal degeneration transmitted by varied modes of inheritance and affects approximately 1 in 4000 individuals. The photoreceptors of the outer retina, as well as the retinal pigmented epithelium which supports the outer retina metabolically and structurally, are the retinal regions most affected by the disorder. In several forms of retinitis pigmentosa, the mislocalization of the rod photoreceptor protein rhodopsin is thought to be a contributing factor underlying the pathophysiology seen in patients. The mutations causing this mislocalization often occur in genes coding proteins involved in ciliary formation, vesicular transport, rod outer segment disc formation, and stability, as well as the rhodopsin protein itself. Often, these mutations result in the most early-onset cases of both recessive and dominant retinitis pigmentosa, and the following presents a discussion of the proteins, their degenerative phenotypes, and possible treatments of the disease.

Topics: Animals; Humans; Models, Biological; Mutant Proteins; Ophthalmology; Protein Transport; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Mutations that cause rhodopsin misfolding and retention within the endoplasmic reticulum (ER) are a prominent cause of retinitis pigmentosa. Here, we discuss the hypothesis that the failure of photoreceptor neurons to adapt to the stress caused by rhodopsin accumulation in the ER leads to a global collapse of homeostasis and to retinal degeneration. We review the molecular mechanisms underlying the activity of local ER conformational sensors and stress-relaying modules and consider how ER-derived stress signals are amplified and implemented to impact on downstream processes, including rhodopsin clearance and cell fate control. The emerging view is that alterations to the systems responsible for the detection, transduction and implementation of ER stress might be used therapeutically to treat retinitis pigmentosa.

Topics: Animals; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Gene Expression Regulation; Humans; Molecular Targeted Therapy; Mutation; Proteostasis Deficiencies; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Stress, Physiological

The retina is a highly complex and specialized organ that performs preliminary analysis of visual information. Composed of highly metabolically active tissue, the retina requires a precise and well-balanced means of maintaining its functional activity during extended periods of time. Maintenance and regulation of a vast array of different structural and functional proteins is required for normal function of the retina. This process is referred to as protein homeostasis and involves a variety of activities, including protein synthesis, folding, transport, degradation, elimination, and recycling. Deregulation of any of these activities can lead to malfunctioning of the retina, from subtle subclinical signs to severe retinal degenerative diseases leading to blindness. Examples of retinal degenerative diseases caused by disruption of protein homeostasis include retinitis pigmentosa and Stargardt's disease. A detailed discussion of the role of disruption in protein homeostasis in these and other retinal diseases is presented, followed by examples of some existing and potential treatments.

Topics: Alzheimer Disease; Eye Proteins; Homeostasis; Humans; Parkinson Disease; Protein Folding; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous group of diseases that cause blindness. Mutations within the rhodopsin gene account for approximately 25% of autosomal dominantly inherited RP cases. Therefore, understanding the mechanisms causing rhodopsin-mediated RP has a significant health impact. To date, results from multiple labs indicate that rhodopsin-mediated RP pathogenesis does not share a common mechanism of degeneration. There is strong evidence that multiple mechanisms are involved, including protein misfolding, mislocalization, release of toxic products, and aberrant signaling. Development of effective treatments requires investigation of the mechanism involved in the different rhodopsin mutations. This chapter focuses on the mechanisms by which rhodopsin mutations cause retinal degeneration, as well as potential therapeutic strategies to treat the disease.

Topics: Animals; Humans; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Drosophila visual transduction is the fastest known G-protein-coupled signaling cascade and has therefore served as a genetically tractable animal model for characterizing rapid responses to sensory stimulation. Mutations in over 30 genes have been identified, which affect activation, adaptation, or termination of the photoresponse. Based on analyses of these genes, a model for phototransduction has emerged, which involves phosphoinoside signaling and culminates with opening of the TRP and TRPL cation channels. Many of the proteins that function in phototransduction are coupled to the PDZ containing scaffold protein INAD and form a supramolecular signaling complex, the signalplex. Arrestin, TRPL, and G alpha(q) undergo dynamic light-dependent trafficking, and these movements function in long-term adaptation. Other proteins play important roles either in the formation or maturation of rhodopsin, or in regeneration of phosphatidylinositol 4,5-bisphosphate (PIP2), which is required for the photoresponse. Mutation of nearly any gene that functions in the photoresponse results in retinal degeneration. The underlying bases of photoreceptor cell death are diverse and involve mechanisms such as excessive endocytosis of rhodopsin due to stable rhodopsin/arrestin complexes and abnormally low or high levels of Ca2+. Drosophila visual transduction appears to have particular relevance to the cascade in the intrinsically photosensitive retinal ganglion cells in mammals, as the photoresponse in these latter cells appears to operate through a remarkably similar mechanism.

Topics: Adaptation, Ocular; Animals; Calcium Channels; Drosophila; Drosophila Proteins; Eye Proteins; Genes, Insect; GTP-Binding Proteins; Light; Multiprotein Complexes; Phosphatidylinositol 4,5-Diphosphate; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin; Transient Receptor Potential Channels; Vision, Ocular; Vitamin A

Multipotent progenitor cells have now been isolated from the brain and retina, expanded in culture, and transplanted to the central nervous system (CNS). Work in rodent models has shown that progenitor cells derived from the CNS readily engraft in the diseased retina of mature recipients, where they develop morphologies appropriate to the local microenvironment and express mature markers, including the photoreceptor protein rhodopsin. There is also evidence for graft-associated rescue of host photoreceptors and preservation of light sensitivity in the degenerating retina. Graft survival does not necessarily require immune suppression, as CNS progenitors can behave as an immunoprivileged cell type. The use of biodegradable polymers results in an organised implant and further improves graft survival. Efforts are underway at present to extend this work to the pig, with initial results showing engraftment in both the neural retina and retinal pigment epithelium (RPE).

Topics: Absorbable Implants; Animals; Cell Differentiation; Cell Movement; Cell Survival; Central Nervous System; Models, Animal; Multipotent Stem Cells; Polymers; Retina; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Swine; Transplantation, Heterologous

Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summarises the clinical evidence for a modifying role of light exposure in retinal degeneration and experimental evidence from animal models, focusing on retinitis pigmentosa with regional degeneration, Oguchi disease, and Stargardt macular dystrophy. These cases illustrate distinct pathophysiological roles for light, and suggest that light restriction may benefit carefully defined subsets of patients.

Topics: Animals; Corneal Dystrophies, Hereditary; Disease Models, Animal; Humans; Light; Mice; Rats; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa is a group of retinal degenerative diseases, within the broad family of hereditary retinopathies, for which there is no cure at present. Mutations in different genes coding for proteins related to the metabolism of photoreceptor cells, and to the visual phototransduction cascade, are the cause of this disease. Rhodopsin, the photoreceptor protein responsible for light absorption--and key in the first stages of vision--is one of the most studied molecules of the retina. Mutations in the opsin gene account for about 25% of all cases of autosomal dominant retinitis pigmentosa. Recent crystallization of this receptor in its inactive dark state has revealed new structural details yielding further insights into the intra and intermolecular mechanismsin which the protein is involved as a result of its activation.Furthermore, the in vitro study of recombinant rhodopsins carrying mutations previously found in retinitis pigmentosa patients (by means of spectroscopic and functional techniques) has shed new light on the structural requirements for its correct function, as well as the molecular defects underlying the mechanism of photoreceptor cell death. In this study, the main findings of the recent investigations carried out in this field are presented. The relevant information obtained at the molecular level is bound to facilitate our understandingof the molecular processes that will allow suitable therapiesfor different retinal degenerative diseases, particularly retinitis pigmentosa, to be proposed.

Topics: Humans; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship

The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.

Topics: Animals; Apoptosis; Electroretinography; Fundus Oculi; Mice; Mice, Mutant Strains; Models, Animal; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Rhodopsin

Tapetoretinal degenerations are a common cause for vision problems, but have until recently not been amenable to rational treatment. With rapidly increasing insights into basic neurobiology and pathobiology this has now begun to change. From having been a relatively small group of largely unknown yet fairly prevalent disorders, they are rapidly forming a large set of well defined diseases, and it is easy to predict that our knowledge about them will continue to increase for many years to come. Vitamin A (15,000 IU daily) is currently the only rational treatment available. However, in experimental animals, therapy strategies are now actively being developed along several different lines. Apoptotic photoreceptor cell death can be delayed with different drugs, and at least one of them, diltiazem, is approved for human use in cardiovascular diseases. It remains to be seen if it has any clinically significant effect in human tapetoretinal degenerations. Other strategies aim at counteracting the production of harmful protein variants, acting either on DNA or mRNA levels. Transgenes can also be used to induce the production of important but missing metabolic components. Finally, cells or retina sheets can be transplanted, either to replace failing cells or as a source for missing trophic factors. Neither of these strategies has yet been transferred to humans, but trials are under way. With the high increase in the flow of new information on tapetoretinal disorders, much more precise diagnoses and much improved treatments are soon to be expected, augmenting considerably the possibilities for ophthalmologists to help patients with such diseases. It is not likely that there will be a single treatment for all the many varieties. Instead, we are most likely going to see pharmacological treatments for some of them, DNA transfers for some, and transplantations for others.

Topics: Amino Acid Sequence; Animals; Electrooculography; Electroretinography; Genetic Therapy; Humans; Molecular Sequence Data; Mutation; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Retina; Retinal Degeneration; Rhodopsin; Vitamin A

Many degenerative diseases involve apoptotic cell death--can they be treated with apoptosis inhibitors, while protecting the normal physiological function of the rescued cells? Reason for optimism comes from a recent study of mutant flies with an analogue of the human degenerative disease retinitis pigmentosa.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Cysteine Proteinase Inhibitors; Drosophila melanogaster; Drosophila Proteins; Eye Proteins; Humans; Inhibitor of Apoptosis Proteins; Nerve Degeneration; Phosphoprotein Phosphatases; Retinal Degeneration; Rhodopsin; Viral Proteins

Insight into the molecular basis of inherited photoreceptor cell degeneration has been rapidly evolving during the last decade. The Drosophila Rh1 rhodopsin gene was the first gene shown to cause retinal degeneration when mutated. Many more degeneration-causing mutations in genes encoding rhodopsin and other photoreceptor proteins have been isolated since then in both, Drosophila and humans. To date some 70 mutations of the Drosophila Rh1 gene have been isolated, most of them have been characterized at the molecular level, and more than 60% of them cause retinal degeneration. This review lists the known Rh1 mutations that cause retinal degeneration up to April 1998, gives an overview on the ultrastructural and biochemical correlates of photoreceptor cell degeneration, and suggests a system for the classification of degeneration-causing Rh1 mutations.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Drosophila melanogaster; Genotype; Molecular Sequence Data; Phenotype; Photoreceptor Cells, Invertebrate; Point Mutation; Retinal Degeneration; Rhodopsin

Topics: Amino Acid Sequence; DNA Mutational Analysis; Electroretinography; Eye Proteins; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Sequence Data; Nerve Tissue Proteins; Peripherins; Prognosis; Retinal Degeneration; Rhodopsin

The function of the retina is to detect light and to send appropriate signals to the brain in response. Inherited diseases that cause the retina to degenerate, leading to either partial or total blindness, affect approximately 1 in 3000 people. Rapid progress is being made in identifying the genetic causes of common, inherited retinal diseases, such as retinitis pigmentosa and macular degeneration, as well as some of the rare forms of retinal disease. Linkage studies of large families and candidate-gene screening of known retinal genes have already identified 59 independent genetic loci that can cause retinal degeneration. The astounding genetic and clinical heterogeneity that is being revealed is a 'nightmare' for those interested in molecular diagnostics but, at the same time, provides great insight into functional aspects of the normal retina.

Topics: Chromosome Mapping; Genetic Diseases, Inborn; Humans; Models, Biological; Pedigree; Retina; Retinal Degeneration; Rhodopsin

To assess the state of knowledge of photoreceptor dystrophies.. The current literature concerning photoreceptor dystrophies is reviewed, and their potential impact on concepts of pathogenesis of disease and clinical practice is assessed.. As a result of cooperative investigative work between researchers in various disciplines, major advances in the classification of retinal photoreceptor dystrophies have been made. Until recently, classification of retinal dystrophies was based on clinical observation alone, and it was evident that this method was imprecise and of limited value. Largely through the work of molecular biologists, it has been shown that diseases clinically indistinguishable from one another may be a result of mutations on a variety of genes; conversely, different mutations on a single gene may give rise to a variety of phenotypes. It is reassuring that it is possible to generate concepts as to potential pathogenetic mechanisms that exist in retinal dystrophies in light of this new knowledge. More important for the clinician is the potential impact on clinical practice. There is as yet no therapy by which the course of most of these disorders can be modified. However, there is a considerable body of work in which therapeutic intervention is being explored, and many researchers now see treatment as a justifiable objective of their work.. Knowledge of the causative mutation is of value to the clinician in that it provides a precise diagnosis and allows the distribution of the abnormal gene to be documented fully within a family. To take full advantage of the opportunities provided by current research, clinical practice will have to be modified, particularly if therapy can be justified.

Topics: Amino Acid Sequence; Cell Death; Chromosome Mapping; Humans; Molecular Sequence Data; Mutation; Photoreceptor Cells; Retinal Degeneration; Rhodopsin

Two different molecular biological approaches to the disease-causing genes of genetic eye diseases are described. In gyrate atrophy of the chroid and retina where the biochemical defect was identified as inactivation of ornithine aminotransferase, the gene was cloned by using antibody for the enzyme. In most genetic eye diseases, however, the biochemical defects are unknown. Positional cloning and/or the candidate gene approach are used to identify the disease-causing genes for these diseases. The genes of chroideremia and Norrie disease were cloned by positional cloning. Several genes expressed in the photoreceptor cells have been identified recently and may be the genes causing progressive degeneration of the retina and choroid. Rhodopsin, peripherin (RDS), rom-1, and beta subunit-cGMP phosphodiesterase are identified as the disease-causing genes for retinitis pigmentosa by the candidate gene approach.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Choroid Diseases; Cloning, Molecular; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells; Retinal Degeneration; Retinal Diseases; Rhodopsin; RNA, Messenger

Topics: Animals; Antigens; Arrestin; Drosophila melanogaster; Eye Proteins; Genes; GTP-Binding Proteins; Ion Channel Gating; Ion Channels; Photoreceptor Cells; Protein Kinase C; Retina; Retinal Degeneration; Rhodopsin; Signal Transduction; Type C Phospholipases; Vision, Ocular

Mutagenesis studies and comparisons of natural variants of rhodopsin and related visual pigments have led to new insights concerning photoreceptor function. The studies identify domains important for receptor folding, the residues that set the wavelength of absorption for the ligand 11-cis retinal, and residues, that when mutated, trigger the cell death of photoreceptors.

Topics: Amino Acid Sequence; Animals; Eye Proteins; Molecular Sequence Data; Mutation; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Rod Opsins

Topics: Animals; Cell Membrane; Chemical Phenomena; Chemistry; Eye Proteins; Fatty Acids; Glycolipids; Humans; Light; Lipid Metabolism; Lipids; Oxidation-Reduction; Phospholipids; Photoreceptor Cells; Retina; Retinal Degeneration; Rhodopsin

Retinal pigment epithelium (RPE) is a specialized pigmented monolayer dedicated to retinal support and protection. Given the fact that photoreceptor outer segments are the primary energy resource of RPE metabolism, it follows that, when photoreceptor function is compromised, RPE cells are impaired and vice versa. In retinitis pigmentosa (RP), genetic mutations lead to a massive degeneration of photoreceptors but only few studies have addressed systematically the consequences of rod and cone death on RPE cells, which, among others, undergo an abnormal organization of tight junctions (TJs) and a compromised barrier function. The biological mechanisms driving these barrier reorganizations are largely unknown. Studies aimed at addressing general and mutation-independent changes of the RPE in RP are relevant to reveal new pathogenic mechanisms of this heterogeneous family of diseases and prospectively develop effective therapeutic strategies. Here, we take advantage of a mouse model of RP in which retinal degeneration is spatially restricted to investigate a possible involvement of inflammatory responses in RPE remodeling. By immunostaining for Zona Occludens-1 (ZO-1), a structural and functional marker of TJs with pleiotropic functions, we found a partial rescue of TJs organization following local restoration of retinal organization, revealing that TJs structure can recover. Since lack of ZO-1 from TJs can alter cell density, we counted RPE cells without finding any differences between degenerated and controls animals, indicating preservation of RPE cells. However, we found an increased number of immune cells adhering to the RPE apical surface and a spatial correlation with areas of abnormal ZO-1 distribution. This suggests that inflammatory processes following photoreceptor degeneration can be responsible for TJs alterations during RP progression and deserve further investigation.

Topics: Animals; Epithelial Cells; Mice; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin

We aimed to verify whether the intravitreal injection of small molecule compounds alone can create photoreceptor cells in mouse models of retinal degeneration. Primary cultured mouse Müller cells were stimulated in vitro with combinations of candidate compounds and the rhodopsin expression was measured on day 7 using polymerase chain reaction and immunostaining. We used 6-week-old N-methyl-N-nitrosourea-treated and 4-week-old rd10 mice as representative in vivo models of retinal degeneration. The optimal combination of compounds selected via in vitro screening was injected into the vitreous and the changes in rhodopsin expression were investigated on day 7 using polymerase chain reaction and immunostaining. The origin of rhodopsin-positive cells was also analyzed via lineage tracing and the recovery of retinal function was assessed using electroretinography. The in vitro mRNA expression of rhodopsin in Müller cells increased 30-fold, and 25% of the Müller cells expressed rhodopsin protein 7 days after stimulation with a combination of 4 compounds: transforming growth factor-β inhibitor, bone morphogenetic protein inhibitor, glycogen synthase kinase 3 inhibitor, and γ-secretase inhibitor. The in vivo rhodopsin mRNA expression and the number of rhodopsin-positive cells in the outer retina were significantly increased on day 7 after the intravitreal injection of these 4 compounds in both N-methyl-N-nitrosourea-treated and rd10 mice. Lineage tracing in td-Tomato mice treated with N-methyl-N-nitrosourea suggested that the rhodopsin-positive cells originated from endogenous Müller cells, accompanied with the recovery of the rhodopsin-derived scotopic function. It was suggested that rhodopsin-positive cells generated by compound stimulation contributes to the recovery of retinal function impaired by degeneration.

Topics: Animals; Disease Models, Animal; Electroretinography; Intravitreal Injections; Methylnitrosourea; Mice; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger

Chronic endoplasmic reticulum (ER) stress is the underlying cause of many degenerative diseases, including autosomal dominant retinitis pigmentosa (adRP). In adRP, mutant rhodopsins accumulate and cause ER stress. This destabilizes wild-type rhodopsin and triggers photoreceptor cell degeneration. To reveal the mechanisms by which these mutant rhodopsins exert their dominant-negative effects, we established an in vivo fluorescence reporter system to monitor mutant and wild-type rhodopsin in Drosophila. By performing a genome-wide genetic screen, we found that PERK signaling plays a key role in maintaining rhodopsin homeostasis by attenuating IRE1 activities. Degradation of wild-type rhodopsin is mediated by selective autophagy of ER, which is induced by uncontrolled IRE1/XBP1 signaling and insufficient proteasome activities. Moreover, upregulation of PERK signaling prevents autophagy and suppresses retinal degeneration in the adRP model. These findings establish a pathological role for autophagy in this neurodegenerative condition and indicate that promoting PERK activity could be used to treat ER stress-related neuropathies, including adRP.

Topics: Animals; Autophagy; Drosophila; Drosophila Proteins; eIF-2 Kinase; Endoribonucleases; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration.. We used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H.. OCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation.. These data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration.

Topics: Aging; Animals; Endoplasmic Reticulum Stress; Endoribonucleases; Mice; Protein Serine-Threonine Kinases; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Unfolded Protein Response

The need for robust and reliable animal models is a crucial step in studying any disease. This certainly applies to inherited retinal degenerative diseases, in which mutations of retinal specific genes result in photoreceptor cell death and subsequent visual loss. Animal models of retinal gene mutations have proven valuable to our understanding of disease mechanisms and as tools to evaluate therapeutic intervention strategies. Notable among these models are mice with a mutation of the rhodopsin gene at amino acid 23 in which proline is substituted for histidine (Rho-P23H). The RHO-P23H mutation is the most common cause of autosomal dominant retinitis pigmentosa. Here, we provide a brief review of the Rho-P23H mouse models currently available for research.

Topics: Animals; Disease Models, Animal; Mice; Mutation; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Most forms of outer retinal degenerative diseases involve the ectopic accumulation of microglia/macrophages in the subretinal space, including retinitis pigmentosa. However, their role in the loss of photoreceptor function during retinal degeneration remains unknown. Here, we examined the effect of conditional microglial depletion on photoreceptor numbers and visual function in mice with the rhodopsin P23H mutation, a dominant form of retinitis pigmentosa in humans. We found that microglial depletion led to an elevated level of rhodopsin and increased photoreceptor layer thickness. However, overall electrophysiological functions of the retina were reduced with microglial depletion. Therefore, these results identify an essential role of microglia specially in preserving visual function in outer retinal degeneration.

Topics: Animals; Disease Models, Animal; Humans; Mice; Microglia; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, we performed an unbiased in vivo RNAi screen with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1

Topics: Animals; Caspases; Drosophila; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Topics: Animals; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinaldehyde; Rhodopsin; Strigiformes; Whales

Topics: Animals; Disease Models, Animal; Electroretinography; Humans; Mice; Mice, Transgenic; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Arrestins; Disease Models, Animal; Electroretinography; Glucose; Lactic Acid; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Catalytic hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase 6 (PDE6) is critical in phototransduction signalling in photoreceptors. Mutations in the genes encoding any of the three PDE6 subunits are associated with retinitis pigmentosa, the most common form of inherited retinal diseases. The RD1 mouse carries a naturally occurring nonsense mutation in the Pde6b gene. The RD1 mouse retina rapidly degenerates and fails to form rod photoreceptor outer segments due to the elevated cGMP level and subsequent excessive Ca2+ influx. In this study, we aim to test whether the PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, rescues photoreceptors in the RD1 retina.. Electroporation used the PDE5 expression plasmid to transfect neonatal RD1 mice. The mouse retina degeneration was assessed by retinal sections' stains with DAPI. The expression and localization of phototransduction proteins in photoreceptors were analysed by immunostaining. The expression of proteins in cultured cells was analysed by immunoblotting.. The exogenous PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, prevents photoreceptor degeneration in RD1 mice. Unlike endogenous photoreceptor-specific PDE6 localised in the outer segments of photoreceptors, ectopically- expressed PDE5 was distributed in inner segments and synaptic terminals. PDE5 also promoted the development of the outer segments in RD1 mice. PDE5 co-expression with rhodopsin in cultured cells showed enhanced rhodopsin expression.. Lowering the cGMP level in photoreceptors by PDE5 is sufficient to rescue photoreceptors in RD1 retinas. cGMP may also play a role in rhodopsin expression regulation in photoreceptors.

Topics: Animals; Calcium; Codon, Nonsense; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Guanosine Monophosphate; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Topics: Animals; Dietary Supplements; Mice; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Vitamin A

The P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal-dominant retinitis pigmentosa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has yet to be elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and wild-type (WT) rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant human rhodopsin allele and the WT mouse rhodopsin were reduced, but protein levels revealed selective degradation of the mutant protein. These results suggest that the mutant rods undergo an adaptative process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP. This article has an associated First Person interview with the first author of the paper.

Topics: Animals; Disease Models, Animal; Humans; Mice; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger

Photoreceptor connecting cilium (CC) is structurally analogous to the transition zone (TZ) of primary cilia and gates the molecular trafficking between the inner and the outer segment (OS). Retinal dystrophies with underlying CC defects are manifested in a broad array of syndromic conditions known as ciliopathies as well as nonsyndromic retinal degenerations. Despite extensive studies, many questions remain in the mechanism of protein trafficking across the photoreceptor CC. Here, we genetically inactivated mouse Tmem138, a gene encoding a putative transmembrane protein localized to the ciliary TZ and linked to ciliopathies. Germline deletion of Tmem138 abolished OS morphogenesis, followed by rapid photoreceptor degeneration. Tmem138 was found localized to the photoreceptor CC and was required for localization of Ahi1 to the distal subdomain of the CC. Among the examined set of OS proteins, rhodopsin was mislocalized throughout the mutant cell body prior to OS morphogenesis. Ablation of Tmem138 in mature rods recapitulated the molecular changes in the germline mutants, causing failure of disc renewal and disintegration of the OS. Furthermore, Tmem138 interacts reciprocally with rhodopsin and a related protein Tmem231, and the ciliary localization of the latter was also altered in the mutant photoreceptors. Taken together, these results suggest a crucial role of Tmem138 in the functional organization of the CC, which is essential for rhodopsin localization and OS biogenesis.

Topics: Cilia; Ciliopathies; Humans; Membrane Proteins; Photoreceptor Connecting Cilium; Retinal Degeneration; Rhodopsin

Polyglutamylation is a dynamic posttranslational modification where glutamate residues are added to substrate proteins by 8 tubulin tyrosine ligase-like (TTLL) family members (writers) and removed by the 6 member Nna1/CCP family of carboxypeptidases (erasers). Genetic disruption of polyglutamylation leading to hyperglutamylation causes neurodegenerative phenotypes in humans and animal models; the best characterized being the Purkinje cell degeneration (pcd) mouse, a mutant of the gene encoding Nna1/CCP1, the prototypic eraser. Emphasizing the functional importance of the balance between glutamate addition and elimination, loss of TTLL1 prevents Purkinje cell degeneration in pcd. However, whether Ttll1 loss protects other vulnerable neurons in pcd, or if elimination of other TTLLs provides protection is largely unknown. Here using a mouse genetic rescue strategy, we characterized the contribution of Ttll1, 4, 5, 7, or 11 to the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mutants. Ttll1 deficiency attenuates Purkinje cell loss and function and reduces olfactory bulb mitral cell death and retinal photoreceptor degeneration. Moreover, degeneration of photoreceptors in pcd is preceded by impaired rhodopsin trafficking to the rod outer segment and likely represents the causal defect leading to degeneration as this too is rescued by elimination of TTLL1. Although TTLLs have similar catalytic properties on model substrates and several are highly expressed in Purkinje cells (e.g. TTLL5 and 7), besides TTLL1 only TTLL4 deficiency attenuated degeneration of Purkinje and mitral cells in pcd. Additionally, TTLL4 loss partially rescued photoreceptor degeneration and impaired rhodopsin trafficking. Despite their common properties, the polyglutamylation profile changes promoted by TTLL1 and TTLL4 deficiencies in pcd mice are very different. We also report that loss of anabolic TTLL5 synergizes with loss of catabolic Nna1/CCP1 to promote photoreceptor degeneration. Finally, male infertility in pcd is not rescued by loss of any Ttll. These data provide insight into the complexity of polyglutamate homeostasis and function in vivo and potential routes to ameliorate disorders caused by disrupted polyglutamylation.

Topics: Animals; Glutamic Acid; GTP-Binding Proteins; Male; Phenotype; Purkinje Cells; Retinal Degeneration; Rhodopsin

The correct expression of folded, functional rhodopsin (Rho) is critical for visual perception. However, this seven-transmembrane helical G protein-coupled receptor is prone to mutations with pathological consequences of retinal degeneration in retinitis pigmentosa (RP) due to Rho misfolding. Pharmacological chaperones that stabilize the inherited Rho variants by assisting their folding and membrane targeting could slow the progression of RP. In this study, we employed virtual screening of synthetic compounds with a natural product scaffold in conjunction with in vitro and in vivo evaluations to discover a novel chromenone-containing small molecule with favorable pharmacological properties that stabilize rod opsin. This compound reversibly binds to unliganded bovine rod opsin with an EC50 value comparable to the 9-cis-retinal chromophore analog and partially rescued membrane trafficking of multiple RP-related rod opsin variants in vitro. Importantly, this novel ligand of rod opsin was effective in vivo in murine models, protecting photoreceptors from deterioration caused by either bright light or genetic insult. Together, our current study suggests potential broad therapeutic implications of the new chromenone-containing non-retinoid small molecule against retinal diseases associated with photoreceptor degeneration.

Topics: Animals; Biological Products; Cattle; Ligands; Mice; Receptors, G-Protein-Coupled; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Light detection in retinal rod photoreceptors is initiated by activation of the visual pigment rhodopsin. A critical, yet often-overlooked, step enabling efficient perception of light is rhodopsin dephosphorylation mediated by protein phosphatase 2A (PP2A). PP2A deficiency has been reported to impair rhodopsin regeneration after phosphorylation by G protein receptor kinase 1 (GRK1) and binding of arrestin (Arr1), thereby delaying rod dark adaptation. However, its effects on the viability of photoreceptors in the absence of GRK1 and Arr1 remain unclear. Here, we investigated the effects of PP2A deficiency in the absence of GRK1 or Arr1, both of which have been implicated in Oguchi disease, a form of night blindness.. Rod-specific mice lacking the predominant catalytic Cα-subunit of PP2A were crossed with the Grk1-/- or Arr1-/- strains to obtain double knockout lines. Rod photoreceptor viability was analyzed in histological cross-sections of the retina stained with hematoxylin and eosin, and rod function was evaluated by ex vivo electroretinography.. PP2A deficiency alone did not impair photoreceptor viability up to 12 months of age. Retinal degeneration was more pronounced in rods lacking GRK1 compared to rods lacking Arr1, and degeneration was accelerated in both Grk1-/- or Arr1-/- strains where PP2A was also deleted. In Arr1-/- mice, rod maximal photoresponse amplitudes were reduced by 80% at 3 months, and this diminution was enhanced further with concomitant PP2A deficiency.. These results suggest that although PP2A is not required for the survival of rods, its deletion accelerates the degeneration induced by the absence of either GRK1 or Arr1.

Topics: Animals; Arrestin; G-Protein-Coupled Receptor Kinase 1; Mice; Mice, Knockout; Protein Phosphatase 2; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Daily biological rhythms are fundamental to retinal physiology and visual function. They are generated by a local circadian clock composed of a network of cell type/layer-specific, coupled oscillators. Animal models of retinal degeneration have been instrumental in characterizing the anatomical organization of the retinal clock. However, it is still unclear, among the multiple cell-types composing the retina, which ones are essential for proper circadian function. In this study, we used a previously well-characterized mouse model for autosomal dominant retinitis pigmentosa to examine the relationship between rod degeneration and the retinal circadian clock. This model carries the P23H mutation in rhodopsin, which induces mild rod degeneration in heterozygous and rapid loss of photoreceptors in homozygous genotypes. By measuring PER2::LUC bioluminescence rhythms, we show that the retinal clock in

Topics: Animals; Circadian Clocks; Circadian Rhythm; Mice; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Rhodopsin is essential for phototransduction, and many rhodopsin mutations cause heritable retinal degenerations. The P23H rhodopsin variant generates a misfolded rhodopsin protein that photoreceptors quickly target for degradation by mechanisms that are incompletely understood. To gain insight into how P23H rhodopsin is removed from rods, we used mass spectrometry to identify protein interaction partners of P23H rhodopsin immunopurified from Rho

Topics: Animals; Biology; Disease Models, Animal; Mice; Mutation; Quality Control; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; RNA, Messenger; Ubiquitins

Retinitis pigmentosa (RP) is a heterogeneous group of inherited disorders characterized by photoreceptor degeneration. The rhodopsin gene (RHO) is the most frequent cause of autosomal dominant RP (ADRP), yet it remains unclear how RHO mutations cause heterogeneous phenotypes. Energy failure is a main cause of the secondary cone death during RP progression; however, its role in primary rod death induced by ADRP RHO mutants is unknown.. Three RHO missense mutations were chosen from different clinical classes. Wild-type (WT) RHO and its mutants, P23H (class B1), R135L (class A), and G188R (class B2), were overexpressed in 661w cells, a mouse photoreceptor cell line, and their effects on oxidative phosphorylation (OXPHOS) and aerobic glycolysis were compared separately.. Here, we report that energy failure is an early event in the cell death caused by overexpression of WT RHO and its mutants. RHO overexpression leads to OXPHOS deficiency, which might be a result of mitochondrial loss. Nonetheless, only in WT RHO and P23H groups, energy stress triggers AMP-activated protein kinase activation and metabolic reprogramming to increase glycolysis. Metabolic reprogramming impairment in R135L and G188R groups might be the reason why energy failure and cell injury are much more severe in those groups.. Our results imply that overexpression of RHO missense mutants have distinct impacts on the two energy metabolic pathways, which might be related to their heterogeneous phenotypes.

Topics: Animals; Energy Metabolism; Mice; Mutation; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.

Topics: Animals; Drosophila; Drosophila Proteins; Mutation; Phenotype; Photoreceptor Cells, Invertebrate; Retina; Retinal Degeneration; Rhodopsin; Transcriptome

Inherited retinal degenerations (IRD) are a leading cause of visual impairment and can result from mutations in any one of a multitude of genes. Mutations in the light-sensing protein rhodopsin (RHO) is a leading cause of IRD with the most common of those being a missense mutation that results in substitution of proline-23 with histidine. This variant, also known as P23H-RHO, results in rhodopsin misfolding, initiation of endoplasmic reticulum stress, the unfolded protein response, and activation of cell death pathways. In this study, we investigate the effect of α-crystallins on photoreceptor survival in a mouse model of IRD secondary to P23H-RHO. We find that knockout of either αA- or αB-crystallin results in increased intraretinal inflammation, activation of apoptosis and necroptosis, and photoreceptor death. Our data suggest an important role for the ⍺-crystallins in regulating photoreceptor survival in the P23H-RHO mouse model of IRD.

Topics: Animals; Apoptosis; Cell Death; Crystallins; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Unfolded Protein Response

Patients with diabetes often experience visual defects before any retinal pathologies are detected. The molecular mechanism for the visual defects in early diabetes has not been elucidated. Our previous study reported that in early diabetic retinopathy (DR), rhodopsin levels were reduced due to impaired 11-

Topics: Animals; Apoptosis; Blotting, Western; Diabetic Retinopathy; Eye Proteins; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Mice; Mice, Transgenic; Oxidative Stress; Retinal Degeneration; Retinol-Binding Proteins; Rhodopsin; Tomography, Optical Coherence

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in

Topics: Alleles; Animals; Drosophila; Eye Proteins; Gene Expression Regulation; Genetic Predisposition to Disease; Genotype; Mutation; Photoreceptor Cells; Retinal Degeneration; Rhodopsin; RNA Splicing; RNA, Messenger; Spliceosomes

Oxidative stress is a common cause of neurodegeneration and plays a central role in retinal degenerative diseases. Heme oxygenase-1 (HMOX1) is a redox-regulated enzyme that is induced in neurodegenerative diseases and acts against oxidative stress but can also promote cell death, a phenomenon that is still unexplained in molecular terms. Here, we test whether HMOX1 has opposing effects during retinal degeneration and investigate the molecular mechanisms behind its pro-apoptotic role.. Basal and induced levels of HMOX1 in retinas are examined during light-induced retinal degeneration in mice. Light damage-independent HMOX1 induction at two different expression levels is achieved by intraocular injection of different doses of an adeno-associated virus vector expressing HMOX1. Activation of Müller glial cells, retinal morphology and photoreceptor cell death are examined using hematoxylin-eosin staining, TUNEL assays, immunostaining and retinal function are evaluated with electroretinograms. Downstream gene expression of HMOX1 is analyzed by RNA-seq, qPCR examination and western blotting. The role of one of these genes, the pro-apoptotic DNA damage inducible transcript 3 (Ddit3), is analyzed in a line of knockout mice.. Light-induced retinal degeneration leads to photoreceptor degeneration and concomitant HMOX1 induction. HMOX1 expression at low levels before light exposure prevents photoreceptor degeneration but expression at high levels directly induces photoreceptor degeneration even without light stress. Photoreceptor degeneration following high level expression of HMOX1 is associated with a mislocalization of rhodopsin in photoreceptors and an increase in the expression of DDIT3. Genetic deletion of Ddit3 in knockout mice prevents photoreceptor cell degeneration normally resulting from high level HMOX1 expression.. The results reveal that the expression levels determine whether HMOX1 is protective or deleterious in the retina. Furthermore, in contrast to the protective low dose of HMOX1, the deleterious high dose is associated with induction of DDIT3 and endoplasmic reticulum stress as manifested, for instance, in rhodopsin mislocalization. Hence, future applications of HMOX1 or its regulated targets in gene therapy approaches should carefully consider expression levels in order to avoid potentially devastating effects.

Topics: Animals; Endoplasmic Reticulum Stress; Ependymoglial Cells; Light; Mice, Knockout; Oxidative Stress; Retina; Retinal Degeneration; Rhodopsin; Transcription Factor CHOP

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

Topics: Animals; Chemokine CCL2; Female; Gene Expression Profiling; Gene Regulatory Networks; Glial Fibrillary Acidic Protein; GTP-Binding Proteins; Male; Mice; Mice, Transgenic; Neuroglia; Neuroprotection; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Beclin-1; Cell Cycle Proteins; Disease Models, Animal; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-akt; Retinal Degeneration; Rhodopsin; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin-Protein Ligases; Ubiquitination

Environmental changes in the retina, including oxidative stress-induced cell death, influence photoreceptor degeneration in Retinitis Pigmentosa (RP). Previously, we tested and discovered that a cytoprotective chaperone protein, clusterin, produced robust preservation of rod photoreceptors of a rat autosomal dominant rhodopsin transgenic model of RP, S334ter-line3. To investigate the biochemical and molecular cytoprotective pathways of clusterin, we examined and compared a known source of cone cell death, nitric oxide (NO), observing nNOS expression using antibody against nNOS in RP retinas with intravitreal injections of saline, clusterin (10 μg/ml), or a non-isoform-selective NOS inhibitor (25 mM), L-NAME, or with an intraperitoneal injection (IP) of L-NAME (100 mg/kg). Rhodopsin-immunoreactive rod photoreceptor cells and nNOS-immunoreactive cells were quantified with immunohistochemistry in the presence or absence of L-NAME or clusterin, and the total nNOS retinal expression was determined by immunoblot analysis. In this study, the level of nNOS expression was significantly up-regulated postnatally (P) at P15 (P < 0.05), P30 (P < 0.001) and P60 (P < 0.0001) in RP retinas compared to normal controls. Clusterin treatment suppressed the up-regulated nNOS expression in RP retinas (P < 0.0001) and was enhanced in Type II amacrine cells. Additionally, IP injection of L-NAME at P15 prolonged rod survival in the later stage of RP retinas (P < 0.001). Conversely, rod survival in L-NAME-treated RP retinas was not equivalent to the rod survival number seen in clusterin-treated retinas, which suggests induction of nNOS expression in RP retinas and its reduction by clusterin is only partly responsible for the rescue observed through the reduction of nNOS expression in S334ter-line3 rat retinas.

Topics: Animals; Cell Survival; Clusterin; Disease Models, Animal; Gene Expression; Gene Expression Regulation; Male; Neurons; Nitric Oxide Synthase; Oxidative Stress; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho

Topics: Activating Transcription Factor 6; Animals; Disease Models, Animal; Female; Homeostasis; Male; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a Rho

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Knockdown Techniques; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Mice; MicroRNAs; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA; RNA Interference; RNA Splicing

Nerve growth factor (NGF) is a neuroprotective molecule performing not only on central and peripheral neurons but also on cells of the visual system. Human retinitis pigmentosa (RP) is a major cause of blindness worldwide, and a resolute therapy is still lacking. Recent studies have shown that ocular NGF administration exerts a protective action on damaged retinal cells of mammalians, including human beings, although whether NGF also protects photoreceptors is not clear.We used the Royal College of Surgeons (RCS) strain in this study. The RCS is a rodent affected by inherited retinitis pigmentosa (RP) during postnatal life. For this study, we investigated whether ocular NGF treatment reduces/stops the progression of photoreceptor degeneration of rats with RP.This study was carried out in vitro on isolated photoreceptors to further investigate the action on these cells and whether the action is direct or mediated.The results indicate that ocular NGF administration can protect photoreceptors from degeneration into a model developing inherited RP and that the NGF action is direct. In this regard, we observed that binding of NGF to its receptor modulates expression of rhodopsin, a specific biological marker for photoreceptor survival and functionality.Part of the data reported in this chapter has been published in a previous study.

Topics: Animals; Disease Models, Animal; Nerve Growth Factor; Photoreceptor Cells; Rats; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.

Topics: Animals; Apoptosis; Calcium; Calpain; eIF-2 Kinase; Endoplasmic Reticulum Stress; Enzyme Activation; Intracellular Space; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Protein Folding; Protein Kinase Inhibitors; Retinal Degeneration; Rhodopsin; Unfolded Protein Response

Death of retinal photoreceptors is the basis of prevalent blinding diseases. Since steroids might have a therapeutic role in retinal degenerations, we compared the protective effects of dexamethasone and progesterone on photoreceptor death induced by mifepristone and light exposure. Therefore, we studied the effective protection doses for each steroid in the two models. In addition, we analyzed changes in the levels of pro- and antiapoptotic molecules, glucocorticoid receptors α and β (GRα and GRβ), and rhodopsin under conditions of successful protection and photoreceptor survival. Mifepristone and light exposure selectively damaged photoreceptors. In light exposed retinas, photoreceptors mainly disappeared in the dorsotemporal region, while mifepristone produced a uniform damage. Dexamethasone and progesterone, at the same dose of 4 mg/kg/day for 2 days, preserved over 88% photoreceptor nuclei in both models. Assessment of cell death regulators showed that, in control retinas, both steroids activated BCL-X

Topics: Animals; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Caspase 3; Cell Survival; Dexamethasone; Glucocorticoids; Hormone Antagonists; Immunohistochemistry; Light; Male; Mice, Inbred BALB C; Mifepristone; Photoreceptor Cells, Vertebrate; Progesterone; Radiation Injuries, Experimental; Receptors, Glucocorticoid; Retinal Degeneration; Rhodopsin

Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Expression; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Injections, Intraocular; Mice; Mice, Knockout; Mice, Transgenic; Ophthalmoscopy; Parvovirinae; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Staining and Labeling; Tomography, Optical Coherence

The use of gene therapy may allow replacement of the defective gene. Minigenes, such as cDNAs, are often used. However, these may not express normal physiological genetic profiles due to lack of crucial endogenous regulatory elements. We constructed DNA nanoparticles (NPs) that contain either the mouse or human full-length rhodopsin genomic locus, including endogenous promoters, all introns, and flanking regulatory sequences of the 15-16 kb genomic rhodopsin DNA inserts. We transduced the NPs into primary retinal cell cultures from the rhodopsin knockout (RKO) mouse in vitro and into the RKO mouse in vivo and compared the effects on different functions to plasmid cDNA NP counterparts that were driven by ubiquitous promoters. Our results demonstrate that genomic DNA vectors resulted in long-term high levels of physiological transgene expression over a period of 5 months. In contrast, the cDNA counterparts exhibited low levels of expression with sensitivity to the endoplasmic reticulum (ER) stress mechanism using the same transgene copy number both in vitro and in vivo. This study demonstrates for the first time the transducing of the rhodopsin genomic locus using compacted DNA NPs.

Topics: Animals; Disease Models, Animal; DNA; Endoplasmic Reticulum Stress; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Humans; Mice; Mice, Knockout; Nanoparticles; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transgenes

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αβγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.

Topics: Animals; Cell Death; cis-trans-Isomerases; Cyclic GMP; Disease Models, Animal; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transducin

Rhodopsin is the G protein-coupled receptor in rod photoreceptor cells that initiates vision upon photon capture. The light receptor is normally locked in an inactive state in the dark by the covalently bound inverse agonist 11-cis retinal. Mutations can render the receptor active even in the absence of light. This constitutive activity can desensitize rod photoreceptor cells and lead to night blindness. A G90D mutation in rhodopsin causes the receptor to be constitutively active and leads to congenital stationary night blindness, which is generally thought to be devoid of retinal degeneration. The constitutively active species responsible for the night blindness phenotype is unclear. Moreover, the classification as a stationary disease devoid of retinal degeneration is also misleading. A transgenic mouse model for congenital stationary night blindness that expresses the G90D rhodopsin mutant was examined to better understand the origin of constitutive activity and the potential for retinal degeneration. Heterozygous mice for the G90D mutation did not exhibit retinal degeneration whereas homozygous mice exhibited progressive retinal degeneration. Only a modest reversal of retinal degeneration was observed when transducin signaling was eliminated genetically, indicating that some of the retinal degeneration occurred in a transducin-independent manner. Biochemical studies on purified rhodopsin from mice indicated that multiple species can potentially contribute to the constitutive activity causing night blindness.

Topics: Animals; Heterozygote; Homozygote; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutation; Night Blindness; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Transducin

Human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelium (RPE) cells are therapeutic cells that have been shown to be promising in the rescue of lost photoreceptors. In this study, we generated hiPSC from human epidermal keratinocytes and subsequently differentiated them into RPE cells to investigate their ability to influence the retinal functions of the Royal College of Surgeon (RCS) rats.. Keratinocytes were reprogrammed to hiPSC using a non-integrating Sendai reprogramming system. Established hiPSCs were differentiated into RPE cells, and complete characterization was performed. Next, the suspension of hiPSC-RPE cells was transplanted into the subretinal space of 3-week-old RCS rats (. hiPSC-derived RPE cells exhibited naïve RPE cell properties and functionality that provided trophic support and the transient rescue of photoreceptor cells.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Transplantation; Electroretinography; Epidermal Cells; Heterografts; Humans; Immunohistochemistry; Induced Pluripotent Stem Cells; Keratinocytes; Male; Microscopy, Electron, Transmission; Opsins; Phagocytosis; Photoreceptor Cells, Vertebrate; Rats, Mutant Strains; Real-Time Polymerase Chain Reaction; Retinal Degeneration; Retinal Pigment Epithelium; Rhodopsin; Tomography, Optical Coherence

The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.

Topics: Animals; Cell Differentiation; Cells, Cultured; Dependovirus; Disease Models, Animal; DNA; Gene Expression; Gene Order; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Mice; Mice, Knockout; Neural Stem Cells; Photoreceptor Cells; Plasmids; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Transduction, Genetic; Transgenes

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In

Topics: Animals; Biosynthetic Pathways; Drosophila; Drosophila Proteins; Gene Deletion; GTP-Binding Protein alpha Subunits, Gq-G11; Light Signal Transduction; Mutation; Protein Isoforms; Retina; Retinal Degeneration; Rhodopsin

Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.

Topics: Animals; Autophagy; Cell Death; CRISPR-Cas Systems; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Mice; Mice, Knockout; Protein Disulfide-Isomerases; Proteoglycans; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Transcription Factor CHOP

Neurons carry electrical signals and communicate via electrical activities. The therapeutic potential of electrical stimulation (ES) for the nervous system, including the retina, through improvement of cell survival and function has been noted. Here we investigated the neuroprotective and regenerative potential of ES in a mouse model of inherited retinal degeneration.. Rhodopsin-deficient (Rho-/-) mice received one or two sessions of transpalpebral ES or sham treatments for 7 consecutive days. Intraperitoneal injection of 5-ethynyl-2'-deoxyuridine was used to label proliferating cells. Weekly electroretinograms were performed to monitor retinal function. Retinal morphology, photoreceptor survival, and regeneration were evaluated in vivo using immunohistochemistry and genetic fate-mapping techniques. Müller cell (MC) cultures were employed to further define the optimal conditions of ES application.. Noninvasive transpalpebral ES in Rho-/- mice improved photoreceptor survival and electroretinography function in vivo. ES also triggered residential retinal progenitor-like cells such as MCs to reenter the cell cycle, possibly producing new photoreceptors, as shown by immunohistochemistry and genetic fate-mapping techniques. ES directly stimulated cell proliferation and the expression of progenitor cell markers in MC cultures, at least partially through bFGF signaling.. Our study showed that transpalpebral ES improved photoreceptor survival and retinal function and induced the proliferation, probably photoreceptor regeneration, of MCs; this occurs via stimulation of the bFGF pathways. These results suggest the exciting possibility of applying noninvasive ES as a versatile tool for preventing photoreceptor loss and mobilizing endogenous progenitors for reversing vision loss in patients with photoreceptor degeneration.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Electric Stimulation Therapy; Electroretinography; Ependymoglial Cells; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of

Topics: Animals; Armadillo Domain Proteins; Cytoskeletal Proteins; Disease Models, Animal; Electroretinography; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NAD; Photoreceptor Cells; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Mutations in the rhodopsin gene may cause photoreceptor degeneration in autosomal dominant retinitis pigmentosa (ADRP) by dominant negative or toxic gain-of-function mechanisms. Controversy exists as to the mechanism by which the widely studied P23H mutation induces rod cell dysfunction and death. Inherited disease caused by dominant negative mutations may be amenable to treatment using wild-type gene augmentation. Indeed, prior studies in the

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Knock-In Techniques; Genetic Therapy; Genetic Vectors; Mice; Mice, Transgenic; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Mutations in TMEM216, a ciliary transition zone tetraspan transmembrane protein, are linked to Joubert syndrome and Meckel syndrome. Photoreceptor degeneration is a prominent phenotype in Joubert syndrome. How TMEM216 contributes to photoreceptor health is poorly understood.. We have generated tmem216 knockout zebrafish by CRISPR genome editing. The impact of TMEM216 deletion on photoreceptors was evaluated by immunofluorescence staining and electron microscopy.. Homozygous tmem216 knockout zebrafish died before 21 days after fertilization. Their retina exhibited reduced immunoreactivity to rod photoreceptor outer segment marker 4D2 and cone photoreceptor outer segment marker G protein subunit α transducin 2 (GNAT2). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed an increase in TUNEL-positive nuclei in the knockout retina, indicating photoreceptor degeneration. The tmem216 mutation resulted in shortened photoreceptor ciliary axoneme, as revealed by acetylated α-tubulin immunostaining. Photoreceptors in knockout zebrafish exhibited mislocalization of outer segment proteins such as rhodopsin, GNAT2, and red opsin to the inner segment and cell bodies. Additionally, electron microscopy revealed that the mutant photoreceptors elaborated outer segment with abnormal disc morphology such as shortened discs and vesicles/vacuoles within the outer segment.. Our results indicate that TMEM216 is essential for normal genesis of outer segment disc structures, transport of outer segment materials, and survival of photoreceptors in zebrafish. These tmem216 knockout zebrafish will be useful in studying how transition zone proteins regulate photoreceptor outer segment formation and maintenance.

Topics: Animals; Cone Opsins; Gene Knockout Techniques; Humans; Membrane Proteins; Mutation; Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Zebrafish; Zebrafish Proteins

Numerous rhodopsin mutations have been implicated in night blindness and retinal degeneration, often with unclear etiology. D190N-rhodopsin (D190N-Rho) is a well-known inherited human mutation causing retinitis pigmentosa. Both higher-than-normal spontaneous-isomerization activity and misfolding/mistargeting of the mutant protein have been proposed as causes of the disease, but neither explanation has been thoroughly examined. We replaced wild-type rhodopsin (WT-Rho) in

Topics: Animals; Cell Line; Disease Models, Animal; HEK293 Cells; Humans; Light Signal Transduction; Mice; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

Rhodopsin S334ter-3 retinal degeneration rats have been widely used to investigate degenerative diseases of the retina. In this model, morphological and electrophysiological changes have been observed in the retina, superior colliculus and primary visual cortex (V1). However, no study so far has examined rhodopsin S334ter-3 rats with regards to their contrast response in V1 - a fundamental property of visual information processing. In this study, experimental rats (S334ter-3) carried one copy of the mutant transgene. We compared responses to spatio-temporal variations in luminance contrast in the primary visual cortex of these rats with those in Long-Evans (LE) rats to elucidate the degeneration-specific activity changes in this part of the visual pathway. We measured extracellular responses to different stimulus contrasts at the preferred parameters of each recorded cell under classical receptive field (CRF) stimulation. Our results show that V1 cells in the S334ter-3 group exhibit stronger spontaneous activity but weaker stimulus-evoked responses at medium and high contrasts. By fitting responses to a sigmoid function, we found that the S334ter-3 group had a lower R

Topics: Animals; Rats; Rats, Long-Evans; Retina; Retinal Degeneration; Rhodopsin; Visual Cortex

Retinitis pigmentosa (RP) is a group of blinding disorders caused by diverse mutations, including in rhodopsin (RHO). Effective therapies have yet to be discovered. The I307N Rho mouse is a light-inducible model of autosomal dominant RP. Our purpose was to describe the glial response in this mouse model to educate future experimentation. I307N Rho mice were exposed to 20,000 lx of light for thirty minutes to induce retinal degeneration. Immunofluorescence staining of cross-sections and flat-mounts was performed to visualize the response of microglia and Müller glia. Histology was correlated with spectral-domain optical coherence tomography imaging (SD-OCT). Microglia dendrites extended between photoreceptors within two hours of induction, withdrew their dendrites between twelve hours and one day, appeared ameboid by three days, and assumed a ramified morphology by one month. Glial activation was more robust in the inferior retina and modulated across the boundary of light damage. SD-OCT hyper-reflectivity overlapped with activated microglia. Finally, microglia transiently adhered to the RPE before which RPE cells appeared dysmorphic. Our data demonstrate the spatial and temporal pattern of glial activation in the I307N Rho mouse, and correlate these patterns with SD-OCT images, assisting in interpretation of SD-OCT images in preclinical models and in human RP.

Topics: Animals; Dendrites; Disease Models, Animal; Genes, Dominant; Humans; Mice; Microglia; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

Dark-rearing has been found to slow the rate of retinal degeneration in albino P23H but not S334ter mutant rhodopsin transgenic (Tg) rats. Since eye pigmentation has the same protective slowing effect as dark-rearing in RCS rats, we examined whether eye pigmentation has a comparable slowing effect in the different mutant rhodopsin Tg rats. Different lines of albino P23H and S334ter Tg rats on the Sprague-Dawley (SD) background were bred to Long-Evans (LE) rats to produce pigmented Tg rats. These were compared to albino Tg rats at postnatal days of different ages using the outer nuclear layer (ONL) as a morphological measure of photoreceptor number and electroretinogram (ERG) a- and b-wave amplitudes as a measure of retinal function. When compared to albino P23H rats, pigmented P23H rats had a slower rate of degeneration as measured by greater ONL thicknesses and greater ERG a- and b-wave amplitudes. By contrast, pigmented S334ter rats showed no difference in ONL thicknesses or ERG a- and b-wave amplitudes when compared to their albino equivalents. Thus, degeneration of photoreceptors in P23H Tg rats is slowed by eye pigmentation as measured by ONL thickness, while it is not in the S334ter Tg rats. Eye pigmentation also protects functional changes in ERG a- and b-waves for the P23H lines, but not for the S334ter lines.

Topics: Animals; Electroretinography; Eye Color; Mutation; Phenotype; Photoreceptor Cells, Vertebrate; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Degeneration; Rhodopsin

We previously reported that modest running exercise protects photoreceptors in mice undergoing light-induced retinal degeneration and in the rd10 mouse model of autosomal recessive retinitis pigmentosa (arRP). We hypothesized that exercise would protect against other types of retinal degeneration, specifically, in autosomal dominant inherited disease. We tested whether voluntary running wheel exercise is protective in a retinal degeneration mouse model of class B1 autosomal dominant RP (adRP).. C57BL/6J mice heterozygous for the mutation in I307N rhodopsin (. In vivo measures revealed that induction of the I307N. Voluntary wheel running partially protected against retinal degeneration and inflammation, and RPE disruption in a model of inducible adRP. This is the first report of exercise protection in an adult adRP animal model. It is also the first report of an RPE phenotype in the I307N

Topics: Animals; Disease Models, Animal; Genes, Dominant; Inflammation; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Physical Conditioning, Animal; Retinal Degeneration; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.

Topics: Animals; Animals, Newborn; Cell Culture Techniques; Cell Engineering; Dependovirus; Disease Models, Animal; Female; Genetic Vectors; Halorhodopsins; HEK293 Cells; Humans; Induced Pluripotent Stem Cells; Male; Mice; Mice, Knockout; Optogenetics; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Rhodopsin; Transfection; Treatment Outcome

Clinical manifestations of photoreceptor degeneration include gradual thinning of the outer nuclear layer (ONL) and progressive reduction of electroretinogram (ERG) amplitudes and vision loss. Although preclinical evaluations of treatment strategies greatly depend on rodent models, the courses of these changes in mice remain unclear. We thus sought to investigate the temporal correlations in changes of spatial vision, ERG response, and ONL thickness in mice with progressive photoreceptor degeneration.. Adult wild-type (WT) mice and mice carrying rhodopsin deficiency (Rho-/-), a frequently used mouse model of human retinitis pigmentosa, were selected for investigation. Mouse spatial vision, including visual acuity (VA) and contrast sensitivity (CS), was determined using optomotor response (OMR) assays; ONL thickness was quantified by spectral-domain optical coherence tomography (SD-OCT), and ERG was performed to evaluate retinal functions. The mice were killed when they were 14 weeks old, and the cone photoreceptors in retinal sections were counted.. Spatial vision, ONL thickness, and ERG amplitudes remained stable in WT mice at all examined time points. While 6-week-old Rho-/- mice had VA, CS, as well as ERG responses similar to those of WT mice, progressive reductions in the spatial vision and retinal functions were recorded thereafter. Most tested 12-week-old Rho-/- mice had no visual-evoked OMR and ERG responses. Moreover, CS, but not VA, displayed a linear decline that was closely associated with ONL thinning, reduction of ERG amplitudes, and loss of cones.. We presented a comprehensive study of the relation between the changes of spatial vision, retinal function, and ONL thickness in postnatal week (PW)6 to PW12 Rho-/- mice. CS is a more sensitive indicator of spatial vision compared to VA, although both are required as separate parameters for monitoring the visual changes in retina undergoing photoreceptor degeneration.

Topics: Animals; Contrast Sensitivity; Disease Models, Animal; Electroretinography; Mice; Mice, Knockout; Retinal Degeneration; Rhodopsin; Vision Disorders; Visual Fields

We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain.. C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice.. Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice.. Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anisomycin; Cell Cycle Proteins; Cell Death; Dependovirus; Electroretinography; Eukaryotic Initiation Factors; Gene Expression Regulation; In Situ Nick-End Labeling; Injections, Subcutaneous; Mice; Mice, Inbred C57BL; Mice, Knockout; Parvovirinae; Protein Biosynthesis; Protein Synthesis Inhibitors; Retina; Retinal Degeneration; Rhodopsin; Transfection

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.

Topics: Animals; Calpain; Cell Death; Disease Models, Animal; Endoplasmic Reticulum Stress; Gene Knock-In Techniques; Mice; Mice, Transgenic; Protein Folding; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

We previously found that valproic acid (VPA) and other histone deacetylase inhibitors (HDACis) ameliorate retinal degeneration (RD) caused by P23H rhodopsin in Xenopus laevis larvae and hypothesized that this may be due to enhancement of autophagy. Here we use X. laevis expressing an autophagy marker to assess effects of HDACis on autophagy. We also assess the effects of non-HDACi activators and inducers of autophagy on RD caused by P23H rhodopsin.

Topics: Animals; Autophagy; Disease Models, Animal; Histone Deacetylase Inhibitors; Larva; Retinal Degeneration; Rhodopsin; Xenopus laevis

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Topics: Animals; Cell Death; Disease Models, Animal; Genotype; Rats; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Disease Models, Animal; Rats; Rats, Mutant Strains; Rats, Transgenic; Retinal Degeneration; Rhodopsin

We have previously reported that xeno-transplanted human ESC-derived retinas are able to mature in the immunodeficient retinal degeneration rodent models, similar to allo-transplantations using mouse iPSC-derived retina. The photoreceptors in the latter developed outer segments and formed synapses with host bipolar cells, driving light responses of host retinal ganglion cells. In view of clinical application, here we further confirmed the competency of human iPSC-derived retina (hiPSC-retina) to mature in the degenerated retinas of rat and monkey models.. Human iPSC-retinas were transplanted in rhodopsin mutant SD-Foxn1 Tg(S334ter)3LavRrrc nude rats and two monkeys with laser-induced photoreceptor degeneration. Graft maturation was studied by immunohistochemistry and its function was examined by multi-electrode array (MEA) recording in rat retinas and visually-guided saccade (VGS) in a monkey.. A substantial amount of mature photoreceptors in hiPSC-retina graft survived well in the host retinas for at least 5 months (rat) to over 2 years (monkey). In 4 of 7 transplanted rat retinas, RGC light responses were detected at the grafted area. A mild recovery of light perception was also suggested by the VGS performance 1.5 years after transplantation in that monkey.. Our results support the competency of hiPSC-derived retinas to be clinically applied for transplantation therapy in retinal degeneration, although the light responses observed in the present models were not conclusively distinguishable from residual functions of degenerating host retinas. The functional analysis may be further elaborated using other models with more advanced retinal degeneration.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Electroretinography; Forkhead Transcription Factors; Haplorhini; Humans; Induced Pluripotent Stem Cells; Lasers; Mutation; Rats; Rats, Nude; Retina; Retinal Degeneration; Rhodopsin

Preserving of vision is the main goal in vision research. The presented research evaluates the preservation of visual function in Royal College of Surgeon (RCS) rats using a depth perception test. Rats were placed on a stage with one side containing an illusory steep drop ("cliff") and another side with a minimal drop ("table"). Latency of stage dismounting and the percentage of rats that set their first foot on the "cliff" side were determined. Nondystrophic Long-Evans (LE) rats were tested as control. Electroretinogram and histology analysis were used to determine retinal function and structure. Four-week-old RCS rats presented a significantly shorter mean latency to dismount the stage compared with 6-week-old rats (mean ± standard error, 13.7 ± 1.68 vs. 20.85 ± 6.5 s, P = 0.018). Longer latencies were recorded as rats aged, reaching 45.72 s in 15-week-old rats (P < 0.00001 compared with 4-week-old rats). All rats at the age of 4 weeks placed their first foot on the table side. By contrast, at the age of 8 weeks, 28.6% rats dismounted on the cliff side and at the age of 10 and 15 weeks, rats randomly dismounted the stage to either table or cliff side. LE rats dismounted the stage faster than 4-week-old RCS rats, but the difference was not statistically significant (7 ± 1.58 s, P = 0.057) and all LE rats dismounted on the table side. The latency to dismount the stage in RCS rats correlated with maximal electroretinogram b-wave under dark and light adaptation (Spearman's rho test = -0.603 and -0.534, respectively, all P < 0.0001), outer nuclear layer thickness (Spearman's rho test = -0.764, P = 0.002), and number of S- and M-cones (Spearman's rho test = -0.763 [P = 0.002], and -0.733 [P = 0.004], respectively). The cliff avoidance test is an objective, quick, and readily available method for the determination of RCS rats' visual function.

Topics: Aging; Animals; Cone Opsins; Depth Perception; Electroretinography; Microscopy, Fluorescence; Rats; Rats, Long-Evans; Rats, Mutant Strains; Retina; Retinal Degeneration; Rhodopsin; Vision Tests; Visual Acuity

Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensitivity and adaptation needed to restore patterned vision.

Topics: Animals; Blindness; Cell Line; Cone Opsins; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Humans; Intravitreal Injections; Keratinocytes; Mice; Mice, Inbred C57BL; Optogenetics; Patch-Clamp Techniques; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Treatment Outcome

Retinal degenerations are a major cause of blindness in human patients. The identification of endogenous mechanisms involved in neurodegeneration or neuroprotection helps to understand the response of the retina to stress and provides essential information not only for basic retinal physiology but also for defining molecular targets for neuroprotective strategies. Here we used excessive light exposure as a model system to study mechanisms of photoreceptor degeneration in mice. Using one wild type and four genetically modified mouse strains, we demonstrate that light exposure resulted not only in the degeneration of rods but also in an early but transient repression of several cone-specific genes, in a reversible hyperreflectivity of the outer retina including the outer plexiform layer, and in the loss of horizontal cells. The effects on cones, horizontal cells and the inner retina depended on light absorption by rhodopsin and, at least partially, on leukemia inhibitory factor. This demonstrates the existence of intercellular communication routes that transduce rod stress to other cells, likely to provide support for photoreceptors and increase cell survival in the injured retina.

Topics: Animals; Electroretinography; Light; Mice; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Stress, Physiological

Cilia are highly conserved and ubiquitously expressed organelles. Ciliary defects of genetic origins lead to ciliopathies, in which retinal degeneration (RD) is one cardinal clinical feature. In order to efficiently find and design new therapeutic strategies the underlying mechanism of retinal degeneration of three murine model was compared. The rodent models correspond to three emblematic ciliopathies, namely: Bardet-Biedl Syndrome (BBS), Alström Syndrome (ALMS) and CEP290-mediated Leber Congenital Amaurosis (LCA). Scotopic rodent electroretinography (ERG) was used to test the retinal function of mice, Transmitted Electron microscopy (T.E.M) was performed to assess retinal structural defects and real-time PCR for targeted genes was used to monitor the expression levels of the major apoptotic Caspase-related pathways in retinal extracts to identify pathological pathways driving the RD in order to identify potential therapeutic targets. We found that BBS and CEP290-mediated LCA mouse models exhibit perinatal retinal degeneration associated with rhodopsin mislocalization in the photoreceptor and the induction of an Endoplasmic Reticulum (ER) stress. On the other hand, the tested ALMS mouse model, displayed a slower degeneration phenotype, with no Rhodopsin mislocalization nor ER-stress activity. Our data points out that behind the general phenotype of vision loss associated with these ciliopathies, the mechanisms and kinetics of disease progression are different.

Topics: Animals; Bardet-Biedl Syndrome; Ciliopathies; Disease Models, Animal; Electroretinography; Leber Congenital Amaurosis; Mice; Retina; Retinal Degeneration; Rhodopsin

The P23H variant of rhodopsin results in misfolding of the protein, and is a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). We have recently demonstrated that degeneration of photoreceptor cells in retinas of P23H mice is due to the endoplasmic reticulum stress (ERS)-induced activation of autophagy that leads to a secondary proteasome insufficiency and activation of cell death pathways. We propose that this increased level of autophagy flux relative to proteasome activity, which we term the A:P ratio, represents a marker of altered photoreceptor cell homeostasis, and that therapies aimed at normalizing this ratio will result in increased photoreceptor cell survival. To test this postulate, we treated P23H mice with a chemical chaperone (4-phenylbutyric acid) to improve rhodopsin folding, or with a selective phosphodiesterase-4 inhibitor (rolipram) to increase proteasome activity. P23H mice treated with either of these agents exhibited reduced ERS, decreased autophagy flux, increased proteasome activity, and decreased activation of cell death pathways. In addition, rates of retinal degeneration were decreased, and photoreceptor morphology and visual function were preserved. These findings support the conclusion that normalizing the A:P ratio, either by reducing the ERS-induced activation of autophagy, or by increasing proteasome activity, improves photoreceptor survival, and suggest a potential new therapeutic strategy for the treatment of adRP caused by protein folding defects.

Topics: Animals; Autophagy; Cell Death; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Homeostasis; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phenylbutyrates; Phosphodiesterase 4 Inhibitors; Proteasome Endopeptidase Complex; Protein Folding; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7

Topics: Animals; Cytoskeletal Proteins; Disease Models, Animal; DNA-Binding Proteins; Electroretinography; Mice; Mice, Knockout; Proteins; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

Topics: Animals; Disease Models, Animal; Electroretinography; Microscopy; Microscopy, Electron; Phenotype; Photoreceptor Cells, Vertebrate; Point Mutation; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative conditions and a leading cause of irreversible blindness. 25%-30% of RP cases are caused by inherited autosomal dominant (ad) mutations in the rhodopsin (Rho) protein of the retina, which impose a barrier for developing therapeutic treatments for this genetically heterogeneous disorder, as simple gene replacement is not sufficient to overcome dominant disease alleles. Previously, we have explored using the genomic short-form of Rho (sgRho) for gene augmentation therapy of RP in a Rho knockout mouse model. We have shown improved gene expression and fewer epigenetic modifications compared with the use of a Rho cDNA expression construct. In the current study, we altered our strategy by delivering a codon-optimized genomic form of Rho (co-sgRho) (for gene replacement) in combination with an RNAi-based inactivation of endogenous Rho alleles (gene suppression of both mutant Rho alleles, but mismatched with the co-sgRho) into a homozygous Rho

Topics: Animals; Disease Models, Animal; DNA; Gene Knock-In Techniques; Genes, Dominant; Genetic Therapy; Mice; Mice, Inbred C57BL; Nanoparticles; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

Topics: Alleles; Animals; CRISPR-Cas Systems; Electroporation; Fibroblasts; Gene Targeting; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Mutation; Retina; Retinal Degeneration; Rhodopsin; RNA, Guide, Kinetoplastida

Müller cells provide support to photoreceptors under many conditions of stress and degeneration. Leukemia inhibitory factor is known to be expressed in Müller cells, which is necessary to promote photoreceptor survival in stress. We hypothesize that Müller cells that express LIF are undergoing other biological processes or functions which may benefit photoreceptors in disease. In this study, we analyze an existing single Müller cell microarray dataset to determine which processes are upregulated in Müller cells that express LIF, by correlating LIF expression to the expression of other genes using a robust correlation method. Some enriched processes include divalent inorganic cation homeostasis, negative regulation of stem cell proliferation, and gamma-glutamyl transferase activity.

Topics: 3' Untranslated Regions; Animals; Calcium; Cations; Cell Self Renewal; Datasets as Topic; Ependymoglial Cells; gamma-Glutamyltransferase; Gene Expression Regulation; Leukemia Inhibitory Factor; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Receptors, Cell Surface; Retinal Degeneration; Rhodopsin; Single-Cell Analysis; Tissue Array Analysis; Up-Regulation

Synthesis and maturation of the light sensor, rhodopsin, are critical for the maintenance of light sensitivity and for photoreceptor homeostasis. In Drosophila, the main rhodopsin, Rh1, is synthesized in the endoplasmic reticulum and transported to the rhabdomere through the secretory pathway. In an unbiased genetic screen for factors involved in rhodopsin homeostasis, we identified mutations in vha68-1, which encodes the vacuolar proton-translocating ATPase (V-ATPase) catalytic subunit A isoform 1 of the V1 component. Loss of vha68-1 in photoreceptor cells disrupted post-Golgi anterograde trafficking of Rh1, reduced light sensitivity, increased secretory vesicle pH, and resulted in incomplete Rh1 deglycosylation. In addition, vha68-1 was required for activity-independent photoreceptor cell survival. Importantly, vha68-1 mutants exhibited phenotypes similar to those exhibited by mutations in the V0 component of V-ATPase, vha100-1. These data demonstrate that the V1 and V0 components of V-ATPase play key roles in post-Golgi trafficking of Rh1 and that Drosophila may represent an important animal model system for studying diseases associated with V-ATPase dysfunction.

Topics: Animals; Drosophila melanogaster; Drosophila Proteins; Glycosylation; Homeostasis; Hydrogen-Ion Concentration; Light Signal Transduction; Models, Biological; Mutation; Photoreceptor Cells, Invertebrate; Protein Transport; Retinal Degeneration; Rhodopsin; Secretory Vesicles; Vacuolar Proton-Translocating ATPases; Visual Pathways

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC

Topics: Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Cell Line, Tumor; Disease Models, Animal; Diterpenes; Female; HEK293 Cells; High-Throughput Screening Assays; Humans; Light; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Neuroprotective Agents; NIH 3T3 Cells; Protein Folding; Protein Transport; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinaldehyde; Rhodopsin; Thiophenes; Treatment Outcome

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Beclin-1; Hydroxychloroquine; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Proteasome Endopeptidase Complex; Protein Folding; Proteolysis; Retinal Degeneration; Rhodopsin; Sirolimus

Retinitis pigmentosa (RP) comprises a group of inherited retinal degenerative conditions characterized by primary degeneration of the rod photoreceptors. Increased oxidative damage is observed in the retina, aqueous humor, and plasma of RP animal models and patients. The hepatic oxidative status may also be affected in RP due to oxidative damage influencing soluble macromolecules exiting the retina or to alterations in the melanopsin system resulting in chronic circadian desynchronization that negatively alters the oxidative stress defense system. P23H rats were crossed with pigmented Long Evans rats to produce offspring exhibiting the clinical conditions of RP. We measured hepatic malondialdehyde and 4-hydroxyalkenal concentrations as oxidative stress markers; nitrite level as a total nitrosative damage marker; total antioxidant capacity; and the activities of catalase, superoxide dismutase (SOD), and glutathione S-transferase. Retinal visual function was assessed based on optomotor and electroretinogram responses. P23H transgenic rats exhibited diminished visual acuity, contrast sensitivity, and electroretinographic responses according to the level of retinal degeneration. P23H rats at 30 days of age already demonstrated only 47% of the hepatic total antioxidant capacity of wild-type animals. Hepatic catalase and SOD activities were also reduced in P23H rats after 120 days, but we detected no difference in glutathione S-transferase activity. P23H rats had increased hepatic oxidative and nitrosative damage markers. GSH/GSSG ratio showed a significant diminution in P23H rats at P120 compared to WT. We conclude that the liver is under increased oxidative stress in P23H rats. Further studies are required, however, to clarify the contribution of systemic oxidative damage to the pathogenesis of RP.

Topics: Animals; Antioxidants; Free Radicals; Liver; Oxidative Stress; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic

Topics: Adenosine Triphosphatases; Animals; Archaeal Proteins; Disease Models, Animal; Female; Genes, Synthetic; Genetic Therapy; GTP-Binding Protein gamma Subunits; HEK293 Cells; Humans; Methanocaldococcus; Mice; Mice, Knockout; Mice, Transgenic; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Folding; Proteostasis Deficiencies; Recombinant Fusion Proteins; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Transfection; Transgenes

The retinal pigment epithelium (RPE)-dependent visual cycle provides 11-

Topics: Acyltransferases; Animals; Ciliary Neurotrophic Factor; cis-trans-Isomerases; Disease Models, Animal; Electroretinography; Humans; Mice; Mice, Knockout; Protein Transport; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinaldehyde; Rhodopsin

Xenopus is an attractive model system for regeneration studies, as it exhibits an extraordinary regenerative capacity compared to mammals. It is commonly used to study body part regeneration following amputation, for instance of the limb, the tail, or the retina. Models with more subtle injuries are also needed for human degenerative disease modeling, allowing for the study of stem cell recruitment for the regeneration of a given cellular subtype. We present here a model to ablate photoreceptor cells in the Xenopus retina. This method is based on the nitroreductase/metronidazole (NTR/MTZ) system, a combination of chemical and genetic tools, allowing for the conditional ablation of targeted cells. This type of approach establishes Xenopus as a powerful model to study cellular regeneration and stem cell regulation.

Topics: Animals; Animals, Genetically Modified; Green Fluorescent Proteins; Metronidazole; Nitroreductases; Photoreceptor Cells, Vertebrate; Promoter Regions, Genetic; Recombinant Fusion Proteins; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Transgenes; Xenopus

Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish. One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes.. Using gRNAs that targeted highly conserved regions of. The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.

Topics: Animals; Animals, Genetically Modified; Codon, Terminator; CRISPR-Cas Systems; Disease Models, Animal; Frameshift Mutation; Gene Targeting; Immunoblotting; Polymorphism, Restriction Fragment Length; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; RNA, Messenger; Zebrafish; Zebrafish Proteins

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the

Topics: Animals; ATP-Binding Cassette Transporters; c-Mer Tyrosine Kinase; Cells, Cultured; Disease Models, Animal; Lipofuscin; Lysosomes; Macular Degeneration; Mice; Mice, Inbred BALB C; Mice, Knockout; Phagocytosis; Photoreceptor Cells; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinaldehyde; Rhodopsin; Stargardt Disease

The I307N rhodopsin (Rho) mouse is a light-inducible model of autosomal dominant retinitis pigmentosa (adRP) that may be useful in testing therapies. We investigated the time-course of retinal changes of the I307N Rho mouse with spectral-domain optical coherence tomography (SD-OCT).. SD-OCT was performed up to day 30 after light damage; electroretinography (ERG) was employed to evaluate photoreceptor function. We utilized ImageJ to analyze reflectivity of the retina. We used light and electron microscopy to assess retinal organization. We stained synaptophysin and zonula occludins-1 with immunohistochemistry to determine injury to the plexiform layers and retinal pigment epithelium (RPE). We performed lectin staining to evaluate retinal blood vessels.. Retinal degeneration increased with longer exposures to light. An increase in retinal thickness was detected by SD-OCT on day 1 after light challenge followed by loss of the outer nuclear layer (ONL) by day 8. Degeneration was most severe in the nasal and inferior retina. Hyper-reflectivity on SD-OCT developed as early as 1 day after light exposure. Disorganization of the ONL, condensation of photoreceptor chromatin, disruption of the outer limiting membrane, and disarray of outer segments were associated with the hyper-reflectivity. Retraction of the outer plexiform synapses and resorption of the subretinal detachment contributed to retinal thinning. The RPE remained intact, whereas atrophied major retinal vessels were evident after light damage.. Our time-course analysis of retinal degeneration in the I307N Rho mouse with SD-OCT and other outcome measures should enable the use of the mouse model in preclinical efficacy studies and mechanistic studies.

Topics: Animals; Disease Models, Animal; Genes, Dominant; Immunohistochemistry; Mice; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Ganglion Cells; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Synaptophysin; Tomography, Optical Coherence; Zonula Occludens-1 Protein

To evaluate the potential of a poly(lactic-co-glycolic acid) (PLGA)-based slow release formulation of glial cell line-derived neurotrophic factor (GDNF) alone or in combination with melatonin to rescue photoreceptors in a mouse model of retinal degeneration.. The microspheres were able to deliver GDNF or to codeliver GDNF and melatonin in a sustained manner. Intravitreal injection of GDNF or GDNF/melatonin-loaded MSs led to partial functional and structural rescue of photoreceptors compared to blank microspheres or vehicle. No significant intraocular inflammatory reaction was observed after intravitreal injection of the microspheres.

Topics: Animals; Delayed-Action Preparations; Disease Models, Animal; Drug Combinations; Drug Compounding; Drug Liberation; Electroretinography; Gene Expression; Glial Cell Line-Derived Neurotrophic Factor; Intravitreal Injections; Melatonin; Mice; Mice, Knockout; Microspheres; Polylactic Acid-Polyglycolic Acid Copolymer; Retina; Retinal Degeneration; Rhodopsin; Vitamin E; Vitreous Body

To characterize the spectral-domain optical coherence tomography (SD-OCT) findings of the rhodopsin S334ter transgenic rats (line 4) in relation to the morphologic and electroretinographic features.. Rhodopsin S334ter transgenic rats (line 4) were employed as a model of retinal degeneration. The Sprague-Dawley (SD) rats were used as a wild-type control. SD-OCT (Micron IV®; Phoenix Research Labs, Pleasanton, CA, USA) was performed on the S334ter rats (line 4) from postnatal days (P) 13-110. The longitudinal changes of the SD-OCT images were analyzed both qualitatively and quantitatively in comparison to those of SD rats. The SD-OCT images were also compared to the histological and electron microscopic findings from examination performed on P 22, 36, and 61. Full field combined rod and cone electroretinography (ERG) was performed and the relationship between the thickness of the retinal sublayers and the amplitudes of the a- and b-waves was further analyzed.. The photoreceptor inner and outer segment layer became diffusely hyperreflective in the SD-OCT images of the S334ter rats; these findings were not observed in the SD rats. This hyperreflective change corresponded to the degenerated inner and outer segments and the accumulation of the extracellular vesicles in the interphotoreceptor matrix. Quantitatively, the retinal outer sublayer and the photoreceptor sublayer in the S334ter rats became progressively thinner in comparison to those in the SD rats; the difference was statistically significant. The amplitudes of both the a- and b-waves on ERG were severely deteriorated in the S334ter rats.. The SD-OCT images in the S334ter rats noninvasively provided information regarding the pathological changes in the photoreceptors and the longitudinal changes of both qualitative and quantitative changes during retinal degeneration in the S334ter rats (line 4). The pathological features of the photoreceptor inner and outer segments can be detected on SD-OCT as diffuse hyperreflective changes in the photoreceptor layer.

Topics: Animals; Disease Models, Animal; Electroretinography; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Tomography, Optical Coherence

Cyclic-GMP is a second messenger in phototransduction, a G-protein signaling cascade that conveys photon absorption by rhodopsin to a change in current at the rod photoreceptor outer segment plasma membrane. Basal cGMP level is strictly controlled by the opposing actions of phosphodiesterase (PDE6) and retinal guanylyl cyclases (GCs), and mutations in genes that disrupt cGMP homeostasis leads to retinal degeneration in humans through mechanisms that are incompletely understood. The purpose of this study is to examine two distinct cellular targets of cGMP: the cGMP-gated (CNG) channels and protein kinase G (PRKG), and how each may contribute to rod cell death. Using a mouse genetic approach, we found that abolishing expression of CNG channels prolongs rod survival caused by elevated cGMP in a PDE6 mutant mouse model. This observation supports the use of channel blockers to delay rod death, which is expected to prolong useful vision through enhanced cone survival. However, the absence of CNG channel alone also caused abnormal cGMP accumulation. In a mouse model of CNG channel loss-of-function, abolishing PRKG1 expression had a long-lasting effect in promoting rod cell survival. Our data strongly implicate two distinct cGMP-mediated cell death pathways, and suggest that therapeutic designs targeting both pathways will be more effective at slowing photoreceptor cell death caused by elevated cGMP.

Topics: Animals; Cell Death; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide-Gated Cation Channels; Guanylate Cyclase; Ion Channels; Mice; Mice, Knockout; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Cell Outer Segment; Signal Transduction

Metabolic syndrome (MetS) is associated with several degenerative diseases, including retinal degeneration. Previously, we reported on progressive retinal degeneration in a spontaneous obese rat (WNIN/Ob) model. In this study, we investigated the additional effect of impaired glucose tolerance (IGT), an essential component of MetS, on retinal degeneration using the WNIN/GR-Ob rat model.. The retinal morphology and ultrastructure of WNIN/GR-Ob and age-matched littermate lean rats were studied by microscopy and immunohistochemistry. The retinal transcriptome of WNIN/GR-Ob was compared with the respective lean controls and with the WNIN/Ob model using microarray analysis. Expression of selected retinal marker genes was studied via real-time PCR.. Progressive loss of photoreceptor cells was observed in WNIN/GR-Ob rats with an onset as early as 3 months. Similarly, thinning of the inner nuclear layer was observed from 6 months in these rats. Immunohistochemical analysis showed decreased levels of rhodopsin and postsynaptic density protein-95 (PSD-95) proteins and increased levels of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and calretinin in WNIN/GR-Ob rats compared with the age-matched lean controls, further supporting cellular stress/damage and retinal degeneration. The retinal transcriptome analysis indicated altered expression profiles in both the WNIN/GR-Ob and WNIN/Ob rat models compared to their respective lean controls; these pathways are associated with activation of pathways like cellular oxidative stress response, inflammation, apoptosis, and phototransduction, although the changes were more prominent in WNIN/GR-Ob than in WNIN/Ob animals.. WNIN/GR-Ob rats with added glucose intolerance developed retinal degeneration similar to the parent line WNIN/Ob. The severity of retinal degeneration was greater in WNIN/GR-Ob rats compared to WNIN/Ob, suggesting a possible role for IGT in this model. Hence, the WNIN/GR-Ob model could be a valuable tool for investigating the impact of MetS on retinal degeneration pathology.

Topics: Animals; Calbindin 2; Disease Models, Animal; Disks Large Homolog 4 Protein; Glial Fibrillary Acidic Protein; Glucose Intolerance; Male; Metabolic Syndrome; Obesity; Photoreceptor Cells, Vertebrate; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Retinal Degeneration; Rhodopsin; Vascular Endothelial Growth Factor A

Topics: Animals; Cell Polarity; DNA Mutational Analysis; Drosophila melanogaster; Drosophila Proteins; Ependymoglial Cells; Eye Proteins; Genetic Predisposition to Disease; Humans; Membrane Proteins; Morphogenesis; Mutation, Missense; Nerve Tissue Proteins; Retinal Degeneration; Retinal Diseases; Rhodopsin

The purpose of this work was to evaluate a potentially useful animal model, Meriones shawi (M.sh)-developing metabolic X syndrome, diabetes and possessing a visual streak similar to human macula-in the study of diabetic retinopathy and diabetic macular edema (DME). Type 2 diabetes (T2D) was induced by high fat diet administration in M.sh. Body weights, blood glucose levels were monitored throughout the study. Diabetic retinal histopathology was evaluated 3 and 7 months after diabetes induction. Retinal thickness was measured, retinal cell types were labeled by immunohistochemistry and the number of stained elements were quantified. Apoptosis was determined with TUNEL assay. T2D induced progressive changes in retinal histology. A significant decrease of retinal thickness and glial reactivity was observed without an increase in apoptosis rate. Photoreceptor outer segment degeneration was evident, with a significant decrease in the number of all cones and M-cone subtype, but-surprisingly-an increase in S-cones. Damage of the pigment epithelium was also confirmed. A decrease in the number and labeling intensity of parvalbumin- and calretinin-positive amacrine cells and a loss of ganglion cells was detected. Other cell types showed no evident alterations. No DME-like condition was noticed even after 7 months. M.sh could be a useful model to study the evolution of diabetic retinal pathology and to identify the role of hypertension and dyslipidemia in the development of the reported alterations. Longer follow up would be needed to evaluate the potential use of the visual streak in modeling human macular diseases.

Topics: Animals; Apoptosis; cis-trans-Isomerases; Diabetic Retinopathy; Disease Models, Animal; Gerbillinae; Macular Degeneration; Male; Microfilament Proteins; Nerve Tissue Proteins; Opsins; Retina; Retinal Degeneration; Rhodopsin; Transcription Factor Brn-3A

A striking aspect of tissue regeneration is its uneven distribution among different animal classes, both in terms of modalities and efficiency. The retina does not escape the rule, exhibiting extraordinary self-repair properties in anamniote species but extremely limited ones in mammals. Among cellular sources prone to contribute to retinal regeneration are Müller glial cells, which in teleosts have been known for a decade to re-acquire a stem/progenitor state and regenerate retinal neurons following injury. As their regenerative potential was hitherto unexplored in amphibians, we tackled this issue using two Xenopus retinal injury paradigms we implemented: a mechanical needle poke injury and a transgenic model allowing for conditional photoreceptor cell ablation. These models revealed that Müller cells are indeed able to proliferate and replace lost cells following damage/degeneration in the retina. Interestingly, the extent of cell cycle re-entry appears dependent on the age of the animal, with a refractory period in early tadpole stages. Our findings pave the way for future studies aimed at identifying the molecular cues that either sustain or constrain the recruitment of Müller glia, an issue of utmost importance to set up therapeutic strategies for eye regenerative medicine.

Topics: Age Factors; Animals; Animals, Genetically Modified; Animals, Newborn; Bromodeoxyuridine; Cell Proliferation; Diamines; Disease Models, Animal; Ependymoglial Cells; Gene Expression Regulation; Green Fluorescent Proteins; Metronidazole; Proliferating Cell Nuclear Antigen; Radiation-Sensitizing Agents; Regeneration; Retinal Degeneration; Rhodopsin; SOX9 Transcription Factor; Thiazoles; Urea; Xenopus laevis

In this work we study the effects of an acute light-induced retinal degeneration on the population of melanopsin positive retinal ganglion cells (m

Topics: Animals; Blotting, Western; Disease Models, Animal; Down-Regulation; Female; Fluorescent Antibody Technique, Indirect; Light; Microscopy, Fluorescence; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Rod Opsins; Transcription Factor Brn-3A

The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations.

Topics: Animals; Cell Membrane; Environment; Light; Membranes; Mice; Microscopy, Atomic Force; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Cell Outer Segment

Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage.. Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation.. Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions.. Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.

Topics: Animals; Antioxidants; Blotting, Western; Cell Survival; Dietary Supplements; Drug Evaluation, Preclinical; Eye Proteins; Light; Male; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin

Mutations in the RPGR-interacting protein 1 (RPGRIP1) gene cause recessive Leber congenital amaurosis (LCA), juvenile retinitis pigmentosa (RP) and cone-rod dystrophy. RPGRIP1 interacts with other retinal disease-causing proteins and has been proposed to have a role in ciliary protein transport; however, its function remains elusive. Here, we describe a new zebrafish model carrying a nonsense mutation in the rpgrip1 gene. Rpgrip1homozygous mutants do not form rod outer segments and display mislocalization of rhodopsin, suggesting a role for RPGRIP1 in rhodopsin-bearing vesicle trafficking. Furthermore, Rab8, the key regulator of rhodopsin ciliary trafficking, was mislocalized in photoreceptor cells of rpgrip1 mutants. The degeneration of rod cells is early onset, followed by the death of cone cells. These phenotypes are similar to that observed in LCA and juvenile RP patients. Our data indicate RPGRIP1 is necessary for rod outer segment development through regulating ciliary protein trafficking. The rpgrip1 mutant zebrafish may provide a platform for developing therapeutic treatments for RP patients.

Topics: Animals; Cilia; Codon, Nonsense; Protein Transport; rab GTP-Binding Proteins; Retina; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Zebrafish; Zebrafish Proteins

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Topics: AMP-Activated Protein Kinases; Animals; Disease Models, Animal; Humans; Metformin; Mice; Mutant Proteins; Photoreceptor Cells; Protein Folding; Proteostasis Deficiencies; Rats; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Transcriptional Activation

Oxidative stress alters physiological function in most biological tissues and can lead to cell death. In the retina, oxidative stress initiates a cascade of events leading to focal loss of RPE and photoreceptors, which is thought to be a major contributing factor to geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative stress under normal and pathological conditions remains largely unknown. A better understanding of the mechanisms involved in regulating RPE and photoreceptors oxidative stress response is greatly needed. To this end we evaluated photoreceptor and RPE changes in mice deficient in DJ-1, a protein that is thought to be important in protecting cells from oxidative stress. Young (3 months) and aged (18 months) DJ-1 knockout (DJ-1 KO) and age-matched wild-type mice were examined. In both group of aged mice, scanning laser ophthalmoscopy (SLO) showed the presence of a few autofluorescent foci. The 18 month-old DJ-1 KO retinas were also characterized by a noticeable increase in RPE fluorescence to wild-type. Optical coherence tomography (OCT) imaging demonstrated that all retinal layers were present in the eyes of both DJ-1 KO groups. ERG comparisons showed that older DJ-1 KO mice had reduced sensitivity under dark- and light-adapted conditions compared to age-matched control. Histologically, the RPE contained prominent vacuoles in young DJ-1 KO group with the appearance of enlarged irregularly shaped RPE cells in the older group. These were also evident in OCT and in whole mount RPE/choroid preparations labeled with phalloidin. Photoreceptors in the older DJ-1 KO mice displayed decreased immunoreactivity to rhodopsin and localized reduction in cone markers compared to the wild-type control group. Lower levels of activated Nrf2 were evident in retina/RPE lysates in both young and old DJ-1 KO mouse groups compared to wild-type control levels. Conversely, higher levels of protein carbonyl derivatives and iNOS immunoreactivity were detected in retina/RPE lysates from both young and old DJ-1 KO mice. These results demonstrate that DJ-1 KO mice display progressive signs of retinal/RPE degeneration in association with higher levels of oxidative stress markers. Collectively this analysis indicates that DJ-1 plays an important role in protecting photoreceptors and RPE from oxidative damage during aging.

Topics: Aging; Animals; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Photoreceptor Cells, Vertebrate; Protein Deglycase DJ-1; Retinal Degeneration; Retinal Pigment Epithelium; Rhodopsin

Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are non-motile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina.

Topics: Animals; Apoptosis; Cilia; Glutamic Acid; Glycosylation; Mice, Inbred C57BL; Neuroglia; Peptide Synthases; Phenotype; Photoreceptor Cells, Vertebrate; Purkinje Cells; Retina; Retinal Degeneration; Rhodopsin; Time Factors; Tubulin

To characterize a light damage paradigm and establish structural and immunocytochemical measures of acute and protracted light-induced retinal degeneration in the rhodopsin (RHO) T4R dog model of RHO-autosomal dominant retinitis pigmentosa (ADRP).. Retinal light damage was induced in mutant dogs with a 1-minute exposure to various light intensities (0.1-1.0 mW/cm2) delivered with a Ganzfeld stimulator, or by fundus photography. Photoreceptor cell death was assessed by TUNEL assay, and alterations in retinal layers were examined by histology and immunohistochemistry 24 hours and 2 weeks after light exposure. Detailed topographic maps were made to document changes in the outer retinal layers of all four retinal quadrants 2 weeks post exposure.. Twenty-four hours post light exposure, the severity of photoreceptor cell death was dose dependent. Immunohistochemical analysis revealed disruption of rod outer segments, focal loss of the RPE integrity, and an increase in expression of endothelin receptor B in Müller cells with the two highest doses of light and fundus photography. Two weeks after light exposure, persistence of photoreceptor death, thinning of the outer nuclear layer, and induction of Müller cell gliosis occurred with the highest doses of light.. We have characterized outcome measures of acute and continuing retinal degeneration in the RHO T4R dog following light exposure. These will be used to assess the molecular mechanisms of light-induced damage and rescue strategies in this large animal model of RHO-ADRP.

Topics: Animals; Animals, Genetically Modified; Cell Death; DNA; DNA Mutational Analysis; Dogs; Immunohistochemistry; In Situ Nick-End Labeling; Light; Mutation; Photoreceptor Cells, Vertebrate; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

To determine the contribution of second- and third-order retinal neurons to the photopic electroretinograms (ERGs) after the degeneration of the rods in rhodopsin P347L transgenic rabbits (Tg).. Four wild-type (WT) rabbits and four Tg rabbits were studied at 18 months of age. The photopic ERGs elicited at stimulus onset and offset were analyzed. To block different retinal pathways, 2-amino-4-phosphonobutyric acid (APB), 6-cyano-7-nitroquinoxaline-2, 3 (1H,4H)-dione (CNQX), tetrodotoxin (TTX), and N-methyl-DL-aspartic acid (NMDA) were injected intravitreally. Digital subtraction of the postdrug ERGs from the predrug ERGs was used to determine the contributions of the ON-components blocked by APB, the OFF-components blocked by CNQX, and the third-order neurons blocked by TTX+NMDA.. Contribution of the cone photoreceptors to the photopic ERGs in Tg rabbits was approximately 10% of that in WT rabbits. The amplitudes of the positive waves of the ON-components at stimulus onset in Tg rabbits were approximately one-half as large as those in WT. On the other hand, the amplitudes of the positive waves of the OFF-components at stimulus offset in Tg rabbits were approximately 1.4 to 2.3 times larger than those in WT. Transgenic rabbits had a positive wave at stimulus offset, which was reduced after the TTX+NMDA injection.. A reduced ON-component and an augmented OFF-component with abnormal responses of the third-order neurons contributed to the cone ERGs after the loss of rod function in Tg rabbits. Our results suggest a complex synaptic remodeling of the residual retinal cells in the advanced stage in Tg rabbits.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Photic Stimulation; Rabbits; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Neurons; Rhodopsin

The culture of retinal progenitors from an accessible adult stem cell source such as the limbus could provide a useful autologous source of retinal cell therapies. The human corneoscleral limbus contains multipotent stem cells that can be cultured as floating neurospheres. Previous work in rodents has demonstrated neuronal and photoreceptor differentiation from limbal neurosphere cultures. Here, this study has examined undifferentiated cultured adult human limbal neurospheres as donor cells for retinal cell therapies by transplantation into a rat model of retinal degeneration.. Gene expression in limbal neurospheres was examined by immunostaining and western blot. Human limbal neurospheres were transplanted into the subretinal space of Royal College of Surgeon's rats. Rats were monitored by optical coherence tomography for 6 weeks then processed for retinal histology.. Human limbal neurospheres expressed the neural lineage markers, Nestin, sex determining region box-2 and N-cadherin, and the retinal transcription factors microphthalmia-associated transcription factor, sex determining region box-2 and orthodentical homeobox-2. Human limbal neurospheres could be cultured to express NeuN, neurofilament and rhodopsin. Rats receiving saline or no injection underwent complete degeneration of the retinal outer nuclear layer after 3 weeks. In contrast, rats injected with human limbal neurospheres or retinal pigment epithelial cells maintained the outer nuclear layer for up to 6 weeks. Gene expression in transplanted limbal neurospheres was inconsistent with the production of mature retinal pigment epithelial or photoreceptor cells.. Human limbal neurospheres represent an accessible source of autologous donor cells for the treatment of retinal diseases.

Topics: Animals; Antigens, Nuclear; Blotting, Western; Cell Death; Cell Differentiation; Cells, Cultured; Disease Models, Animal; DNA; Gene Expression Regulation; Genes, Homeobox; Humans; Injections, Intraocular; Limbus Corneae; Nerve Tissue Proteins; Pigment Epithelium of Eye; Rats; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Stem Cells; Tomography, Optical Coherence

Topics: Animals; Cell Death; cis-trans-Isomerases; Digoxin; Eye Proteins; Inflammation; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Stress, Physiological; Vision, Ocular

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

Topics: Animals; Cell Survival; Electroretinography; Gene Expression; Humans; Mice, Knockout; Mice, Transgenic; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Transcription Factor CHOP; Transgenes

Oxidative stress and inflammation play key roles in the light damage (LD) model of photoreceptor degeneration, as well as in age-related macular degeneration (AMD). We sought to investigate whether Berberine (BBR), an antioxidant herb extract, would protect the retina against light-induced degeneration. To accomplish this, Balb/c mice were treated with BBR or PBS via gavage for 7 days, and then were placed in constant cool white light-emitting diode (LED) light (10,000 lux) for 4 h. Retinal function and degeneration were evaluated by histology, electroretinography (ERG) and optical coherence tomography (OCT) at 7d after LD. Additionally, mRNA levels of cell-type specific, antioxidant, and inflammatory genes were compared 7d after LD. Photoreceptor DNA fragmentation was assessed via the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay. LD resulted in substantial photoreceptor-specific cell death. Histological analysis using plastic sections showed dosing with BBR preserved photoreceptors. The ERG analysis demonstrated functional protection by BBR in rod-b, -a, and cone-b waves. In OCT images, mice receiving PBS showed severe thinning and disorganization of the photoreceptor layer 7 days after LD, whereas mice treated with BBR had significantly less thinning and disorganization. Consistent with OCT results, the mRNA levels of Rho in the NSR, and Rpe65 and Mct3 in the RPE, were significantly higher in mice treated with BBR. The numbers of TUNEL-positive photoreceptors were significantly decreased in BBR-treated mice. The retinal mRNA levels of oxidative stress genes, the number of microglia/macrophages, and the malondialdehyde (MDA) immunolabeling were significantly lower in BBR-treated mice compared to controls 48 h after LD, which indicates oxidative stress was reduced by BBR in light-damaged eyes. In conclusion, systemic BBR is protective against light-induced retinal degeneration associated with diminished oxidative stress in the retina. These results suggest that BBR may be protective against retinal diseases involving oxidative stress.

Topics: Animals; Berberine; Cell Death; Disease Models, Animal; Electroretinography; In Situ Nick-End Labeling; Light; Male; Mice; Mice, Inbred BALB C; Oxidative Stress; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Rhodopsin; Tomography, Optical Coherence

Photoreceptor death leads to vision impairment in several retinal degenerative disorders. Therapies protecting photoreceptor from degeneration remain to be developed. Anti-inflammation, anti-oxidative stress, and neuroprotective effects of celastrol have been demonstrated in a variety of disease models. The current study aimed to investigate the photoreceptor protective effect of celastrol.. Bright light-induced retinal degeneration in BALB/c mice was used, and morphological, functional, and molecular changes of retina were evaluated in the absence and presence of celastrol treatment.. Significant morphological and functional protection was observed as a result of celastrol treatment in bright light-exposed BALB/c mice. Celastrol treatment resulted in suppression of cell death in photoreceptor cells, alleviation of oxidative stress in the retinal pigment epithelium and photoreceptors, downregulation of retinal expression of proinflammatory genes, and suppression of microglia activation and gliosis in the retina. Additionally, leukostasis was found to be induced in the retinal vasculature in light-exposed BALB/c mice, which was significantly attenuated by celastrol treatment. In vitro, celastrol attenuated all-trans-retinal-induced oxidative stress in cultured APRE19 cells. Moreover, celastrol treatment significantly suppressed lipopolysaccharides-stimulated expression of proinflammatory genes in both APRE19 and RAW264.7 cells.. The results demonstrated for the first time that celastrol prevents against light-induced retinal degeneration through inhibition of retinal oxidative stress and inflammation.

Topics: Animals; Cells, Cultured; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Inflammation; Light; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Neuroprotective Agents; Opsins; Oxidative Stress; Pentacyclic Triterpenes; Retinal Degeneration; Rhodopsin; Triterpenes

Rod photoreceptor outer segment (OS) morphogenesis, structural integrity, and proper signal transduction rely on critical proteins found in the different OS membrane domains (e.g., plasma, disc, and disc rim membrane). Among these key elements are retinal degeneration slow (RDS, also known as peripherin-2), rhodopsin, and the beta subunit of the cyclic nucleotide gated channel (CNGB1a), which have been found to interact in a complex. The purpose of this study was to evaluate the potential interplay between these three proteins by examining retinal disease phenotypes in animal models expressing varying amounts of CNGB1a, rhodopsin, and RDS.. Outer segment trafficking, retinal function, and photoreceptor structure were evaluated using knockout mouse lines.. Eliminating Cngb1 and reducing RDS leads to additive defects in RDS expression levels and rod electroretinogram (ERG) function, (e.g., Cngb1-/-/rds+/- versus rds+/- or Cngb1-/-) but not to additive defects in rod ultrastructure. These additive effects also manifested in cone function: Photopic ERG responses were significantly lower in the Cngb1-/-/rds+/- versus rds+/- or Cngb1-/-, suggesting that eliminating Cngb1 can accelerate the cone degeneration that usually presents later in the rds+/-. This was not the case with rhodopsin; reducing rhodopsin levels in concert with eliminating CNGB1a did not lead to phenotypes more severe than those observed in the Cngb1 knockout alone.. These data support a role for RDS as the core component of a multiprotein plasma membrane-rim-disc complex that has both a structural role in photoreceptor OS formation and maintenance and a functional role in orienting proteins for optimal signal transduction.

Topics: Animals; Cyclic Nucleotide-Gated Cation Channels; Electroretinography; Gene Expression Regulation; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Nerve Tissue Proteins; Peripherins; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; RNA

The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1β and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

Topics: Animals; Bystander Effect; Cell Death; Cell Survival; Disease Models, Animal; Gene Expression Regulation; Humans; Imidazoles; Indoles; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction

Peripherin-2 is a glycomembrane protein exclusively expressed in the light-sensing compartments of rod and cone photoreceptors designated as outer segments (OS). Mutations in peripherin-2 are associated with degenerative retinal diseases either affecting rod or cone photoreceptors. While peripherin-2 has been extensively studied in rods, there is only little information on its supramolecular organization and function in cones. Recently, we have demonstrated that peripherin-2 interacts with the light detector rhodopsin in OS of rods. It remains unclear, however, if peripherin-2 also binds to cone opsins. Here, using a combination of co-immunoprecipitation analyses, transmission electron microscopy (TEM)-based immunolabeling experiments, and quantitative fluorescence resonance energy transfer (FRET) measurements in cone OS of wild type mice, we demonstrate that peripherin-2 binds to both, S-opsin and M-opsin. However, FRET-based quantification of the respective interactions indicated significantly less stringent binding of peripherin-2 to S-opsin compared to its interaction with M-opsin. Subsequent TEM-studies also showed less co-localization of peripherin-2 and S-opsin in cone OS compared to peripherin-2 and M-opsin. Furthermore, quantitative FRET analysis in acutely isolated cone OS revealed that the cone degeneration-causing V268I mutation in peripherin-2 selectively reduced binding to M-opsin without affecting the peripherin-2 interaction to S-opsin or rhodopsin. The differential binding of peripherin-2 to cone opsins and the mutant-specific interference with the peripherin-2/M-opsin binding points to a novel role of peripherin-2 in cones and might contribute to understanding the differential penetrance of certain peripherin-2 mutations in rods and cones. Finally, our results provide a proof-of-principle for quantitative FRET measurements of protein-protein interactions in cone OS.

Topics: Animals; Antigens, Neoplasm; Cone Opsins; Fluorescence Resonance Energy Transfer; Humans; Mice; Microscopy, Electron, Transmission; Mutation; Protein Binding; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin

Calcium has an important role in regulating numerous cellular activities. However, extremely high levels of intracellular calcium can lead to neurotoxicity, a process commonly associated with degenerative diseases. Despite the clear role of calcium cytotoxicity in mediating neuronal cell death in this context, the pathological mechanisms remain controversial. We used a well-established Drosophila model of retinal degeneration, which involves the constitutively active TRP(P365) channels, to study calcium-induced neurotoxicity. We found that the disruption of mitochondrial function was associated with the degenerative process. Further, increasing autophagy flux prevented cell death in Trp(P365) mutant flies, and this depended on the PINK1/Parkin pathway. In addition, the retinal degeneration process was also suppressed by the coexpression of PINK1 and Parkin. Our results provide genetic evidence that mitochondrial dysfunction has a key role in the pathology of cellular calcium neurotoxicity. In addition, the results demonstrated that maintaining mitochondrial homeostasis via PINK1/Parkin-dependent mitochondrial quality control can potentially alleviate cell death in a wide range of neurodegenerative diseases.

Topics: Animals; Antiporters; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Blotting, Western; Calcium; Calcium-Binding Proteins; Disease Models, Animal; Drosophila; Drosophila Proteins; Microscopy, Electron, Transmission; Mitochondria; Mitochondrial Dynamics; Mutagenesis; Neurons; Photoreceptor Cells; Protein Serine-Threonine Kinases; Retinal Degeneration; Rhodopsin; Transient Receptor Potential Channels; Ubiquitin-Protein Ligases

The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4-5.5 months) were used in this study: 3 homozygous mutant RHO(T4R/T4R), 8 heterozygous mutant RHO(T4R/+), and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHO(T4R/T4R)) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm(2), 170 lux; 0.5 mW/cm(2), 820 lux; or 1 mW/cm(2), 1590 lux) for 1 min. All 8 heterozygous (RHO(T4R/+)) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHO(T4R/T4R) and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2-36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHO(T4R/T4R) retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHO(T4R/T4R) retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHO(T4R/T4R) retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing ce

Topics: Animals; Disease Models, Animal; Dogs; Light; Photophobia; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Visual Perception

T17M rhodopsin expression in rod photoreceptors leads to severe retinal degeneration and is associated with the activation of ER stress related Unfolded Protein Response (UPR) signaling. Here, we show a novel role of a UPR transcription factor, ATF4, in photoreceptor cellular pathology. We demonstrated a pro-death role for ATF4 overexpression during autosomal dominant retinitis pigmentosa (ADRP). Based on our results in ATF4 knockout mice and adeno-associated viral (AAV) delivery of ATF4 to the retina, we validated a novel therapeutic approach targeting ATF4 over the course of retinal degeneration. In T17M rhodopsin retinas, we observed ATF4 overexpression concomitantly with reduction of p62 and elevation of p53 levels. These molecular alterations, together with increased CHOP and caspase-3/7 activity, possibly contributed to the mechanism of photoreceptor cell loss. Conversely, ATF4 knockdown retarded retinal degeneration in 1-month-old T17M Rhodopsin mice and promoted photoreceptor survival, as measured by scotopic and photopic ERGs and photoreceptor nuclei row counts. Similarly, ATF4 knockdown also markedly delayed retinal degeneration in 3-month-old ADRP animals. This delay was accompanied by a dramatic decrease in UPR signaling, the launching of anti-oxidant defense, initiation of autophagy, and improvement of rhodopsin biosynthesis which together perhaps combat the cellular stress associated with T17M rhodopsin. Our data indicate that augmented ATF4 signals during retinal degeneration plays a cytotoxic role by triggering photoreceptor cell death. Future ADRP therapy regulating ATF4 expression can be developed to treat retinal degenerative disorders associated with activated UPR.

Topics: Activating Transcription Factor 4; Animals; Autophagy; Caspase 3; Caspase 7; Disease Models, Animal; Endoplasmic Reticulum; Mice; Mice, Inbred C57BL; Mice, Knockout; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Stress, Physiological; Transcription Factors; Unfolded Protein Response

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Mice; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration.

Topics: Action Potentials; Afferent Pathways; Animals; Animals, Genetically Modified; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Form Perception; Rats; Rats, Long-Evans; Reaction Time; Retina; Retinal Degeneration; Retinal Neurons; Retinitis Pigmentosa; Rhodopsin; Visual Cortex; Visual Fields

Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.

Topics: Animals; Autophagy; Autophagy-Related Proteins; Cell Survival; Class III Phosphatidylinositol 3-Kinases; Endosomes; Gene Expression Regulation; Light; Light Signal Transduction; Lysosomes; Membrane Fusion; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphatidylinositol Phosphates; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Bardet-Biedl syndrome (BBS) is a heterogeneous disease characterized by deficiencies in various organs that are caused by defects in genes involved in the genesis, structural maintenance, and protein trafficking of cilia. Leucine zipper transcription factor-like 1 (LZTFL1) has been identified as a BBS protein (BBS17), because patients with mutations in this gene exhibit the common BBS phenotypes. In this study, we generated a knockout mouse model to investigate the effects of LZTFL1 depletion. Lztfl1 knockout mice were born with low birth weight, reached similar weight to those of wild-type mice at 10 weeks of age, and later gained more weight than their wild-type counterparts. LZTFL1 was localized to the primary cilium of kidney cells, and the absence of LZTFL1 increased the ciliary localization of BBS9. Moreover, in the retinas of Lztfl1 knockout mice, the photoreceptor outer segment was shortened, the distal axoneme of photoreceptor connecting cilium was significantly enlarged, and rhodopsin was targeted to the outer nuclear layer. TUNEL assay showed that many of these abnormal photoreceptor cells in Lztfl1 knockout mice underwent apoptosis. Interestingly, the absence of LZTFL1 caused an abnormal increase of the adaptor protein complex 1 (AP1) in some photoreceptor cells. Based on these data, we conclude that LZTFL1 is a cilium protein and regulates animal weight and photoreceptor connecting cilium function probably by controlling microtubule assembly and protein trafficking in cilia.

Topics: Adaptor Protein Complex 1; Animals; Axoneme; Bardet-Biedl Syndrome; Body Weight; Cell Death; Cilia; Cytoskeletal Proteins; Disease Progression; Gene Knockout Techniques; Growth and Development; Kidney; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Phenotype; Photoreceptor Cells; Protein Transport; Retina; Retinal Degeneration; Rhodopsin; Transcription Factors

Objective To investigate the potential of the treatment of growth-associated protein 43 (GAP43) gene-modified bone marrow-derived mesenchymal stem cells (BMSCs) for retinitis pigmentosa (RP). Methods BMSCs were isolated and cultured by adherence method. By transfecting GAP43 gene into BMSCs via a lentivirus vector, we got GAP43 gene-modified BMSCs. Sixty-three Royal College of Surgeons (RCS) rats were randomly divided into three groups: experimental group, negative control group and blank control group. The experimental rats received subretinal injection of GAP43 gene-modified BMSCs. The negative control rats received subretinal injection of BMSCs. The control rats received subretinal injection of PBS. Thirty days after transplanting, the retinal thickness was detected by optical coherence tomography (OCT), and the expression of rhodopsin in RCS rat retinas was examined by Western blotting. Results Compared with the blank control group and the negative control group, 30 days after GAP43 gene-modified BMSC transplantation, the retinal thickness of the experimental group remarkably increased and the expression of rhodopsin significantly rose. Conclusion GAP43 gene-modified BMSC transplantation can increase survival photoreceptor cells and delay retinal degeneration.

Topics: Animals; Blotting, Western; Bone Marrow Cells; Cell Survival; Cells, Cultured; Flow Cytometry; GAP-43 Protein; Genetic Therapy; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Random Allocation; Rats; Retina; Retinal Degeneration; Rhodopsin; Time Factors; Tomography, Optical Coherence; Transfection

Using a previously described retinal explant culture system as an acute injury model, we here explore the role of C1q, the initiator of the classical complement pathway, in neuronal cell survival and retinal homeostasis. Full-thickness adult rat retinal explants were divided into four groups, receiving the following supplementation: C1q (50nM), C1-inhibitor (C1-inh; Berinert; 500mg/l), C1q+C1-inh, and no supplementation (culture controls). Explants were kept for 12h or 2days after which they were examined morphologically and with a panel of immunohistochemical markers. C1q supplementation protects ganglion cells from degeneration within the explant in vitro system. This effect is correlated to an attenuated endogenous production of C1q, and a quiesced gliotic response.

Topics: Aluminum Silicates; Animals; Calcium-Binding Proteins; Cell Survival; Complement C1q; Disease Models, Animal; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; In Situ Nick-End Labeling; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Microfilament Proteins; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Time Factors

Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show that a red-shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV-ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV- and lentivirus-mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

Topics: Animals; Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Light; Macaca; Mice; Phototherapy; Retina; Retinal Degeneration; Rhodopsin; Transduction, Genetic; Treatment Outcome

Mutations in the ORF15 exon of the RPGR gene cause a common form of X-linked retinitis pigmentosa, which often results in severe loss of vision. In dogs and mice, gene augmentation therapy has been shown to arrest the progressive degeneration of rod and cone photoreceptors. However, the distribution of potentially treatable photoreceptors across the human retinas and the rate of degeneration are not known. Here, we have defined structural and functional features of the disease in 70 individuals with ORF15 mutations. We also correlated the features observed in patients with those of three Rpgr-mutant (Rpgr-ko, Rd9, and Rpgr-cko) mice. In patients, there was pronounced macular disease. Across the retina, rod and cone dysfunction showed a range of patterns and a spectrum of severity between individuals, but a high symmetry was observed between eyes of each individual. Genotype was not related to disease expression. In the Rpgr-ko mice, there were intra-retinal differences in rhodopsin and cone opsin trafficking. In Rd9 and Rpgr-cko mice, retinal degeneration showed inter-ocular symmetry. Longitudinal results in patients revealed localized rod and cone dysfunction with progression rates of 0.8 to 1.3 log per decade in sensitivity loss. Relatively retained rod and cone photoreceptors in mid- and far-peripheral temporal-inferior and nasal-inferior visual field regions should be good targets for future localized gene therapies in patients.

Topics: Adolescent; Adult; Aged; Animals; Child; Eye Proteins; Heterozygote; Humans; Mice; Mice, Knockout; Middle Aged; Mutation; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinoschisis; Rhodopsin; Young Adult

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.

Topics: Animals; CD59 Antigens; Cytokines; Disease Models, Animal; Electroretinography; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Ependymoglial Cells; Eye Enucleation; Heat-Shock Proteins; Light; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Nerve Growth Factors; Neuroglia; Phagocytosis; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinaldehyde; Rhodopsin; RNA, Messenger; Up-Regulation

To evaluate the long-term protective effects of transscleral unoprostone (UNO) against retinal degeneration in transgenic (Tg) rabbits (Pro347Leu rhodopsin mutation).. The UNO release devices (URDs) were implanted into the sclerae of Tg rabbits and ERG, optical coherence tomography (OCT), and ophthalmic examinations were conducted for 40 weeks. Unoprostone metabolites in retina, choroid/RPE, aqueous humor, and plasma from wild-type (Wt) rabbits were measured using liquid chromatography-tandem mass spectrometry. In situ hybridization and immunohistochemistry evaluated the retinal distribution of big potassium (BK) channels, and RT-PCR evaluated the expressions of BK channels and m-opsin at 1 week after URD treatment.. The URD released UNO at a rate of 10.2 ±1.0 μg/d, and the release rate and amount of UNO decreased during 32 weeks. Higher ERG amplitudes were observed in the URD-treated Tg rabbits compared with the placebo-URD, or nontreated controls. At 24 weeks after implantation into the URD-treated Tg rabbits, OCT images showed preservation of retinal thickness, and histologic examinations (44 weeks) showed greater thickness of outer nuclear layers. Unoprostone was detected in the retina, choroid, and plasma of Wt rabbits. Retina/plasma ratio of UNO levels were 38.0 vs. 0.68 ng UNO*hour/mL in the URD-treated group versus control (topical UNO), respectively. Big potassium channels were observed in cone, cone ON-bipolar, and rod bipolar cells. Reverse-transcriptase PCR demonstrated BK channels and m-opsins increased in URD-treated eyes.. In Tg rabbits, URD use slowed the decline of retinal function for more than 32 weeks, and therefore provides a promising tool for long-term treatment of RP.

Topics: Animals; Animals, Genetically Modified; Aqueous Humor; Choroid; Chromatography, Liquid; Delayed-Action Preparations; Dinoprost; Disease Models, Animal; DNA; DNA Mutational Analysis; Drug Implants; Electroretinography; Follow-Up Studies; Gene Expression Regulation; Immunohistochemistry; In Situ Hybridization; Large-Conductance Calcium-Activated Potassium Channels; Mutation; Rabbits; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Sclera; Time Factors; Tomography, Optical Coherence

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

Topics: Animals; Animals, Newborn; Apoptosis; Endoplasmic Reticulum Stress; Endoplasmic Reticulum-Associated Degradation; Gene Knock-In Techniques; Immunoprecipitation; Membrane Proteins; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Proteolysis; Retina; Retinal Degeneration; Retinal Photoreceptor Cell Inner Segment; Rhodopsin; RNA, Messenger; Signal Transduction; Transcription Factor CHOP; Ubiquitination

Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy. We used adeno-associated virus (AAV) to express these isoforms in mice and found that both systemic and intravitreal injection of engineered AAV vectors resulted in RdCVF and RdCVFL expression in the eye. Systemic delivery of AAV92YF vectors in neonates resulted in earlier onset of RdCVF and RdCVFL expression compared with that observed with intraocular injection using the same vectors at P14. We also evaluated the efficacy of intravitreal injection using a recently developed photoreceptor-transducing AAV variant (7m8) at P14. Systemic administration of AAV92YF-RdCVF improved cone function and delayed cone loss, while AAV92YF-RdCVFL increased rhodopsin mRNA and reduced oxidative stress by-products. Intravitreal 7m8-RdCVF slowed the rate of cone cell death and increased the amplitude of the photopic electroretinogram. Together, these results indicate different functions for Nxnl1 isoforms in the retina and suggest that RdCVF gene therapy has potential for treating retinal degenerative disease.

Topics: Animals; Cell Survival; Dependovirus; Evoked Potentials, Visual; Eye Proteins; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Mice, Inbred C57BL; Photic Stimulation; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Thioredoxins; Transduction, Genetic

Genetic mapping was recently used to identify the underlying cause for a previously uncharacterized cohort of autosomal recessive retinitis pigmentosa cases. Genetic mapping of affected individuals resulted in the identification of an uncharacterized gene, C2Orf71, as the causative locus. However, initial homology searches failed to reveal similarities to any previously characterized protein or domain. To address this issue, we characterized the mouse homolog, BC027072. Immunohistochemistry with a custom polyclonal antibody showed staining localized to the inner segments (IS) of photoreceptor cells, as well as the outer segments (OS) of cone cells. A knockout mouse line (BC(-/-)) was generated and demonstrated that loss of this gene results in a severe, early-onset retinal degeneration. Histology and electron microscopy (EM) revealed disorganized OS as early as 3 weeks with complete loss by 24 weeks of age. EM micrographs displayed packets of cellular material containing OS discs or IS organelles in the OS region and abnormal retinal pigmented epithelium cells. Analyses of retinoids and rhodopsin levels showed <20% in BC(-/-) versus wild-type mice early in development. Electroretinograms demonstrated that affected mice were virtually non-responsive to light by 8 weeks of age. Lastly, RNAseq analysis of ocular gene expression in BC(-/-) mice revealed clues to the causes of the progressive retinal degenerations. Although its function remains unknown, this protein appears essential for normal OS development/maintenance and vision in humans and mice. RNAseq data are available in the GEO database under accession: GSE63810.

Topics: Animals; Disease Models, Animal; Electroretinography; Eye Proteins; Gene Expression; Gene Expression Profiling; Gene Order; Gene Targeting; Genes, Recessive; Genetic Association Studies; Homozygote; Mice; Mice, Knockout; Protein Biosynthesis; Quantitative Trait Loci; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Retinal gene therapy has proven safe and at least partially successful in clinical trials and in numerous animal models. Gene therapy requires characterization of the progression of the disease and understanding of its genetic cause. Testing gene therapies usually requires an animal model that recapitulates the key features of the human disease, though photoreceptors and cells of the retinal pigment epithelium produced from patient-derived stem cells may provide an alternative test system for retinal gene therapy. Gene therapy also requires a delivery system that introduces the therapeutic gene to the correct cell type and does not cause unintended damage to the tissue. Current systems being tested in the eye are nanoparticles, pseudotyped lentiviruses, and adeno-associated virus (AAV) of various serotypes. Here, we describe the techniques of AAV vector design as well as the in vivo and ex vivo tests necessary for assessing the efficacy of retinal gene therapy to treat retinal degeneration caused by mutations in the rhodopsin gene.

Topics: Animals; Cell Line; Dependovirus; Disease Models, Animal; Genetic Therapy; Humans; Retina; Retinal Degeneration; Rhodopsin

Retinal ischemia results in a progressive degeneration of neurons and a pathological activation of glial cells, resulting in vision loss. In the brain, progressive damage after ischemic insult has been correlated to neuroinflammatory processes involving microglia. Galectin-3 has been shown to mediate microglial responses to ischemic injury in the brain. Therefore, we wanted to explore the contribution of Galectin-3 (Gal-3) to hypoperfusion-induced retinal degeneration in mice.. Gal-3 knockout (Gal-3 KO) and wildtype (WT) C57BL/6 mice were subjected to chronic cerebral hypoperfusion by bilateral narrowing of the common carotid arteries using metal coils resulting in a 30% reduction of blood flow. Sham operated mice served as controls. After 17 weeks, the mice were sacrificed and the eyes were analyzed for retinal architecture, neuronal cell survival, and glial reactivity using morphological staining and immunohistochemistry.. Hypoperfusion caused a strong increase in Gal-3 expression and microglial activation in WT mice, coupled with severe degenerative damage to all retinal neuronal subtypes, remodeling of the retinal lamination and Müller cell gliosis. In contrast, hypoperfused Gal-3 KO mice displayed a retained laminar architecture, a significant preservation of photoreceptors and ganglion cell neurons, and an attenuation of microglial and Müller cell activation.. Moderate cerebral blood flow reduction in the mouse results in severe retinal degenerative damage. In mice lacking Gal-3 expression, pathological changes are significantly attenuated. Gal-3 is thereby a potential target for treatment and prevention of hypoperfusion-induced retinal degeneration and a strong candidate for further research as a factor behind retinal degenerative disease.

Topics: Animals; Calbindins; Calcium-Binding Proteins; Carotid Artery Diseases; Galectin 3; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Neurons; Phosphopyruvate Hydratase; Protein Kinase C; Recoverin; Retinal Degeneration; Rhodopsin; Time Factors

In the present study, we have evaluated one of the dietary supplements enriched with antioxidants and fish oil used in clinical care for patient with age-related macular degeneration. Rats were orally fed by a gastric canula daily with 0.2 ml of water or dietary supplement until they were sacrificed. After one week of treatment, animals were either sacrificed for lipid analysis in plasma and retina, or used for evaluation of rod-response recovery by electroretinography (ERG) followed by their sacrifice to measure rhodopsin content, or used for progressive light-induced retinal degeneration (PLIRD). For PLIRD, animals were transferred to bright cyclic light for one week. Retinal damage was quantified by ERG, histology and detection of apoptotic nuclei. Animals kept in dim-cyclic-light were processed in parallel. PLIRD induced a thinning of the outer nuclear layer and a reduction of the b-wave amplitude of the ERG in the water group. Retinal structure and function were preserved in supplemented animals. Supplement induced a significant increase in omega-3 fatty acids in plasma by 168% for eicosapentaenoic acid (EPA), 142% for docosapentaenoic acid (DPA) and 19% for docosahexaenoic acid (DHA) and a decrease in the omega-6 fatty acids, DPA by 28%. In the retina, supplement induced significant reduction of linolenic acid by 67% and an increase in EPA and DPA by 80% and 72%, respectively, associated with significant decrease in omega-6 DPA by 42%. Supplement did not affect rhodopsin content or rod-response recovery. The present data indicate that supplement rapidly modified the fatty acid content and induced an accumulation of EPA in the retina without affecting rhodopsin content or recovery. In addition, it protected the retina from oxidative stress induced by light. Therefore, this supplement might be beneficial to slow down progression of certain retinal degeneration.

Topics: Animals; Antioxidants; Apoptosis; Biosynthetic Pathways; Dietary Supplements; Disease Progression; Electroretinography; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Female; Light; Male; Neuroprotective Agents; Plasmalogens; Rats, Sprague-Dawley; Regeneration; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residue at this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant.

Topics: Animals; Aspartic Acid; Blotting, Western; Conserved Sequence; Drosophila melanogaster; Drosophila Proteins; Glutamic Acid; Microspectrophotometry; Mutation; Opsins; Phylogeny; Protein Structure, Secondary; Retinal Degeneration; Rhodopsin

Many retinal dystrophies result in photoreceptor loss, but the inner retinal neurons can survive, making them potentially amenable to emerging optogenetic therapies. Here, we show that ectopically expressed human rod opsin, driven by either a non-selective or ON-bipolar cell-specific promoter, can function outside native photoreceptors and restore visual function in a mouse model of advanced retinal degeneration. Electrophysiological recordings from retinal explants and the visual thalamus revealed changes in firing (increases and decreases) induced by simple light pulses, luminance increases, and naturalistic movies in treated mice. These responses could be elicited at light intensities within the physiological range and substantially below those required by other optogenetic strategies. Mice with rod opsin expression driven by the ON-bipolar specific promoter displayed behavioral responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at levels of contrast and illuminance (≈50-100 lux) typical of natural indoor environments. These data reveal that virally mediated ectopic expression of human rod opsin can restore vision under natural viewing conditions and at moderate light intensities. Given the inherent advantages in employing a human protein, the simplicity of this intervention, and the quality of vision restored, we suggest that rod opsin merits consideration as an optogenetic actuator for treating patients with advanced retinal degeneration.

Topics: Animals; Ectopic Gene Expression; Humans; Mice; Retinal Degeneration; Rhodopsin

Outer retinal degeneration is the leading cause of blindness in the developed world. A new study now demonstrates that ectopic expression of human rhodopsin in the inner retina, mediated by viral gene therapy, can restore light sensitivity and some vision to mice blind from outer retinal degeneration.

Topics: Animals; Ectopic Gene Expression; Humans; Retinal Degeneration; Rhodopsin

The Drosophila visual system has been proved to be a powerful genetic model to study eye disease such as retinal degeneration. Here, we describe a genetic method termed "Rh1::GFP ey-flp/hid" that is based on the fluorescence of GFP-tagged major rhodopsin Rh1 in the eyes of living flies and can be used to monitor the integrity of photoreceptor cells. Through combination of this method and ERG recording, we examined a collection of 667 mutants and identified 18 genes that are required for photoreceptor cell maintenance, photoresponse, and rhodopsin synthesis. Our findings demonstrate that this "Rh1::GFP ey-flp/hid" method enables high-throughput F1 genetic screens to rapidly and precisely identify mutations of retinal degeneration.

Topics: Animals; Animals, Genetically Modified; Drosophila; Drosophila Proteins; Electroretinography; Genetic Testing; Green Fluorescent Proteins; Microscopy, Fluorescence; Mutation; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin; Synaptic Transmission

Ambient light is both a stimulus for visual function and a regulator of photoreceptor physiology. However, it is not known if light can regulate any aspect of photoreceptor development. The purpose of this study was to investigate whether ambient light is required for the development of mouse rod photoreceptors.. Newborn mouse pups (C57BL/6) were reared in either cyclic light (LD) or constant dark (DD). Pups were collected at postnatal day (P)5, P10, P17, or P24. We performed retinal morphometric and cell death analysis at P5, P10, and P17. Rhodopsin expression was assessed using immunofluorescence, Western blot, and quantitative RT-PCR analysis. Electroretinograms were performed at P17 and P24. Radioimmunoassay and ELISA were used to follow changes in thyroid hormone levels in the serum and vitreous.. In the DD pups, the outer nuclear layer was significantly thinner at P10 and there were higher numbers of apoptotic cells at P5 compared to the LD pups. Rhodopsin expression was lower at P10 and P17 in DD pups. Electroretinogram a-waves were reduced in amplitude at P17 in the DD pups. The DD animals had lower levels of circulating thyroid hormones at P10. Light-mediated changes in thyroid hormones occur as early as P5, as we detected lower levels of total triiodothyronine in the vitreous from the DD animals. Drug-induced developmental hypothyroidism resulted in lower rhodopsin expression at P10.. Our data demonstrate that light exposure during postnatal development is required for rod photoreceptor development and that this effect could be mediated by thyroid hormone signaling.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Death; Dark Adaptation; Disease Models, Animal; DNA; Electroretinography; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Developmental; Light; Light Signal Transduction; Mice; Mice, Inbred C57BL; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Thyroid Hormones

Retinal rod photoreceptor cells have double membrane discs located in their outer segments (ROS) that are continuously formed proximally from connecting cilia (CC) and phagocytized distally by the retinal pigmented epithelium. The major component of these rod discs, the light-sensitive visual pigment rhodopsin (Rho), consists of an opsin protein linked to 11-cis-retinal. The P23H mutation of rod opsin (P23H opsin) is the most common cause of human blinding autosomal dominant retinitis pigmentosa (adRP). A mouse model of adRP with this mutation (Rho(P23H/+)) shows low levels of P23H opsin protein, partial misalignment of discs and progressive retinal degeneration. However, the impact of mutant P23H opsin on the formation of abnormal discs is unclear and it is still unknown whether this mutant pigment can mediate phototransduction. Using transretinal ERG recordings, we demonstrate that P23H mutant Rho can trigger phototransduction but Rho(P23H/P23H) rods are ∼17 000-fold less sensitive to light than Rho(+/+) rods and produce abnormally fast photo-responses. By analyzing homozygous Rho(P23H/P23H) knock-in mice, we show that P23H opsin is transported to ciliary protrusions where it forms sagittally elongated discs. Transmission electron microscopy of postnatal day (PND) 14 Rho(P23H/+) mouse retina revealed disordered sagittally oriented discs before the onset of retinal degeneration. Surprisingly, we also observed smaller, immature sagittally oriented discs in PND14 Rho(+/)(-) and Rho(+/+) mice that were not seen in older animals. These findings provide fundamental insights into the pathogenesis of the P23H mutant opsin and reveal a novel early sagittally aligned disc formation step in normal ROS disc expansion.

Topics: Animals; Disease Models, Animal; Gene Knock-In Techniques; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morphogenesis; Mutation; Retinal Degeneration; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Vision, Ocular

Müller glia possess stem cell characteristics that have been recognized to be responsible for the regeneration of injured retina in fish and amphibians. Although these cells are present in the adult human eye, they are not known to regenerate human retina in vivo. Human Müller glia with stem cell characteristics (hMSCs) can acquire phenotypic and genotypic characteristics of rod photoreceptors in vitro, suggesting that they may have potential for use in transplantation strategies to treat human photoreceptor degenerations. Much work has been undertaken in rodents using various sources of allogeneic stem cells to restore photoreceptor function, but the effect of human Müller glia-derived photoreceptors in the restoration of rod photoreceptor function has not been investigated. This study aimed to differentiate hMSCs into photoreceptor cells by stimulation with growth and differentiation factors in vitro to upregulate gene and protein expression of CRX, NR2E3, and rhodopsin and various phototransduction markers associated with rod photoreceptor development and function and to examine the effect of subretinal transplantation of these cells into the P23H rat, a model of primary photoreceptor degeneration. Following transplantation, hMSC-derived photoreceptor cells migrated and integrated into the outer nuclear layer of the degenerated retinas and led to significant improvement in rod photoreceptor function as shown by an increase in a-wave amplitude and slope using scotopic flash electroretinography. These observations suggest that hMSCs can be regarded as a cell source for development of cell-replacement therapies to treat human photoreceptor degenerations and may also offer potential for the development of autologous transplantation.

Topics: Animals; Cell Differentiation; Cell- and Tissue-Based Therapy; Disease Models, Animal; Electroretinography; Ependymoglial Cells; Gene Expression Regulation; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Orphan Nuclear Receptors; Rats; Recovery of Function; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Trans-Activators; Transplantation, Heterologous; Vision, Ocular

Inherited mutations that lead to misfolding of the visual pigment rhodopsin (Rho) are a prominent cause of photoreceptor neuron (PN) degeneration and blindness. How Rho proteotoxic stress progressively impairs PN viability remains unknown. To identify the pathways that mediate Rho toxicity in PNs, we performed a comprehensive proteomic profiling of retinas from Drosophila transgenics expressing Rh1(P37H), the equivalent of mammalian Rho(P23H), the most common Rho mutation linked to blindness in humans. Profiling of young Rh1(P37H) retinas revealed a coordinated upregulation of energy-producing pathways and attenuation of energy-consuming pathways involving target of rapamycin (TOR) signaling, which was reversed in older retinas at the onset of PN degeneration. We probed the relevance of these metabolic changes to PN survival by using a combination of pharmacological and genetic approaches. Chronic suppression of TOR signaling, using the inhibitor rapamycin, strongly mitigated PN degeneration, indicating that TOR signaling activation by chronic Rh1(P37H) proteotoxic stress is deleterious for PNs. Genetic inactivation of the endoplasmic reticulum stress-induced JNK/TRAF1 axis as well as the APAF-1/caspase-9 axis, activated by damaged mitochondria, dramatically suppressed Rh1(P37H)-induced PN degeneration, identifying the mitochondria as novel mediators of Rh1(P37H) toxicity. We thus propose that chronic Rh1(P37H) proteotoxic stress distorts the energetic profile of PNs leading to metabolic imbalance, mitochondrial failure, and PN degeneration and therapies normalizing metabolic function might be used to alleviate Rh1(P37H) toxicity in the retina. Our study offers a glimpse into the intricate higher order interactions that underlie PN dysfunction and provides a useful resource for identifying other molecular networks that mediate Rho toxicity in PNs.

Topics: Animals; Animals, Genetically Modified; Blotting, Western; Caspase 9; Coloring Agents; Drosophila melanogaster; Drosophila Proteins; Electroretinography; Endoplasmic Reticulum Stress; Endoplasmic Reticulum-Associated Degradation; Energy Metabolism; Mass Spectrometry; Microscopy, Electron; Mitochondria; Mutation; Oxidative Stress; Protein Folding; Protein Hydrolysates; Proteomics; Retinal Degeneration; Rhodopsin; Signal Transduction; Tolonium Chloride; TOR Serine-Threonine Kinases

Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.

Topics: Adenosine Triphosphate; Animals; Calbindin 1; Cell Death; Cell Movement; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Neuroglia; Neurons; Optic Nerve; Photoreceptor Cells, Vertebrate; Rats; Rats, Transgenic; Retina; Retinal Degeneration; Rhodopsin; Time Factors; Vesicular Glutamate Transport Protein 1; Visual Acuity

The goal of this study is to validate whether reprogramming of the UPR via modulation of pro-apoptotic caspase-7 and CHOP proteins could be an effective approach to slow down the rate of retinal degeneration in ADRP mice. In order to pursue our goal we created the T17M RHO CASP7 and T17M RHO CHOP mice to study the impact of the CASP7 or CHOP ablations in T17M RHO retina by ERG, SD-OCT, histology and western blot analysis. The scotopic ERG demonstrated that the ablation of the CASP7 in T17M RHO retina leads to significant preservation of the function of photoreceptors compared to control. Surprisingly, the ablation of pro-apoptotic CHOP protein in T17M RHO mice led to a more severe form of retinal degeneration. Results of the SD-OCT and histology were in agreement with the ERG data. The further analysis demonstrated that the preservation of the structure and function or the acceleration of the onset of the T17M RHO photoreceptor degeneration occurred via reprogramming of the UPR. In addition, the CASP7 ablation leads to the inhibition of cJUN mediated apoptosis, while the ablation of CHOP induces an increase in the HDAC. Thus, manipulation with the UPR requires careful examination in order to achieve a therapeutic effect.

Topics: Animals; Apoptosis; Caspase 7; Disease Models, Animal; Electroretinography; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transcription Factor CHOP; Unfolded Protein Response

Rhodopsin mutations cause many types of heritable retinitis pigmentosa (RP). Biochemical and in vitro studies have demonstrated that many RP-linked mutant rhodopsins produce misfolded rhodopsin proteins, which are prone to aggregation and retention within the endoplasmic reticulum, where they cause endoplasmic reticulum stress and activate the Unfolded Protein Response signaling pathways. Many vertebrate models of retinal degeneration have been created through expression of RP-linked rhodopsins in photoreceptors including, but not limited to, VPP/GHL mice, P23H Rhodopsin frogs, P23H rhodopsin rats, S334ter rhodopsin rats, C185R rhodopsin mice, T17M rhodopsin mice, and P23H rhodopsin mice. These models have provided many opportunities to test therapeutic strategies to prevent retinal degeneration and also enabled in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death. Here, we examine and compare the contribution of endoplasmic reticulum stress to retinal degeneration in several vertebrate models of RP generated through expression of mutant rhodopsins.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Mice; Mice, Transgenic; Rats; Rats, Transgenic; Retinal Degeneration; Rhodopsin; Species Specificity; Unfolded Protein Response; Vertebrates; Xenopus laevis

Many mutations in rhodopsin gene linked to retinitis pigmentosa (RP) cause rhodopsin misfolding. Rod photoreceptor cells expressing misfolded rhodopsin eventually die. Identifying mechanisms to prevent rhodopsin misfolding or to remove irreparably misfolded rhodopsin could provide new therapeutic strategies. IRE1, ATF6, and PERK signaling pathways, collectively called the unfolded protein response (UPR), regulate the functions of endoplasmic reticulum, responsible for accurate folding of membrane proteins such as rhodopsin. We used chemical and genetic approaches to selectively activate IRE1, ATF6, or PERK signaling pathways one at a time and analyzed their effects on mutant rhodopsin linked to RP. We found that both artificial IRE1 and ATF6 signaling promoted the degradation of mutant rhodopsin with lesser effects on wild-type rhodopsin. Furthermore, IRE1 and ATF6 signaling preferentially reduced levels of aggregated rhodopsins. By contrast, PERK signaling reduced levels of wild-type and mutant rhodopsin. These studies indicate that activation of either IRE1, ATF6, or PERK prevents mutant rhodopsin from accumulating in the cells. In addition, activation of IRE1 or ATF6 can selectively remove aggregated or mutant rhodopsin from the cells and may be useful in treating RP associated with rhodopsin protein misfolding.

Topics: Activating Transcription Factor 6; eIF-2 Kinase; Endoribonucleases; Humans; Protein Serine-Threonine Kinases; Proteostasis Deficiencies; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Retinal degenerations are a heterogeneous group of diseases in which there is slow but progressive loss of photoreceptors (PR). There are currently no approved therapies for treating retinal degenerations. In an effort to identify novel small molecules that are (1) neuroprotective and (2) promote PR differentiation, we have developed microscale (1,536 well) cell culture assays using primary retinal neurons.. Primary murine retinal cells are isolated, seeded, treated with a 1,280 compound chemical library in a 7 point titration and then cultured under conditions developed to assay protection against an introduced stress or enhance PR differentiation. In the protection assays a chemical insult is introduced and viability assessed after 72 h using CellTiterGlo, a single-step chemiluminescent reagent. In the differentiation assay, cells are isolated from the rhodopsin-GFP knock-in mouse and PR differentiation is assessed by fixing cells after 21 days in culture and imaging with the Acumen plate-based laser cytometer (TTP Labtech) to determine number and intensity of GFP-expressing cells. Positive wells are re-imaged at higher resolution with an INCell2000 automated microscope (GE). Concentration-response curves are generated to pharmacologically profile each compound and hits identified by xx.. We have developed PR differentiation and neuroprotection assays with a signal to background (S/B) ratios of 11 and 3, and a coefficient of variation (CV) of 20 and 9 %, suitable for chemical screening. Staurosporine has been shown in our differentiation assay to simultaneously increase the number of rhodopsin positive objects while decreasing the mean rhodopsin intensity and punctate rhodopsin fluorescent objects.. Using primary murine retinal cells, we developed high throughput assays to identify small molecules that influence PR development and survival. By screening multiple compound concentrations, dose-response curves can be generated, and the false negative rate minimized. It is hoped that this work will identify both potential preclinical candidates as well as molecular probes that will be useful for analysis of the molecular mechanisms that promote PR differentiation and survival.

Topics: Animals; Cell Count; Cell Culture Techniques; Drug Discovery; Green Fluorescent Proteins; High-Throughput Screening Assays; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Photoreceptor Cells, Vertebrate; Primary Cell Culture; Retinal Degeneration; Rhodopsin

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.

Topics: Animals; Drosophila melanogaster; Drosophila Proteins; Endocytosis; Light; Lysosomes; Mutation; Photoreceptor Cells, Invertebrate; Protein Transport; Retinal Degeneration; Rhodopsin; Vesicular Transport Proteins

Spectral-Domain Optical Coherence Tomography (SD-OCT) is a widely used method to observe retinal layers and follow pathological events in human. Recently, this technique has been adapted for animal imaging. This non-invasive technology brings a cross-sectional visualization of the retina, which permits to observe precisely each layer. There is a clear expansion of the use of this imaging modality in rodents, thus, a precise characterization of the different outer retinal layers observed by SD-OCT is now necessary to make the most of this technology. The identification of the inner strata until the outer nuclear layer has already been clearly established, while the attribution of the layers observed by SD-OCT to the structures corresponding to photoreceptors segments and retinal pigment epithelium is much more questionable. To progress in the understanding of experimental SD-OCT imaging, we developed a method for averaging SD-OCT data to generate a mean image allowing to better delineate layers in the retina of pigmented and albino strains of mice and rats. It allowed us to locate precisely the interface between photoreceptors and retinal pigment epithelium and to identify unambiguously four layers corresponding to the inner and outer parts of photoreceptors segments. We show that the thickness of the various layers can be measured as accurately in vivo on SD-OCT images, than post-mortem by a morphometric analysis of histological sections. We applied SD-OCT to different models and demonstrated that it allows analysis of focal or diffuse retinal pathological processes such as mutation-dependent damages or light-driven modification of photoreceptors. Moreover, we report a new method of combined use of SD-OCT and integration to quantify laser-induced choroidal neovascularization. In conclusion, we clearly demonstrated that SD-OCT represents a valuable tool for imaging the rodent retina that is at least as accurate as histology, non-invasive and allows longitudinal follow-up of the same animal.

Topics: Animals; Choroidal Neovascularization; Laser Coagulation; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Reproducibility of Results; Retina; Retinal Degeneration; Rhodopsin; Sensitivity and Specificity; Tomography, Optical Coherence

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.

Topics: Animals; Cell Line; Cell Survival; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Electroretinography; Heat-Shock Response; Humans; Hydroxylamines; Mutation; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Time Factors; Transfection; Unfolded Protein Response; Vision, Ocular

Rod outer segment (OS) morphogenesis involves assembly of flattened discs circumscribed by a hairpin-like rim, however, the role of the rim and rim proteins such as retinal degeneration slow (RDS) and its homologue rod OS membrane protein-1 (ROM-1) in this process remains unclear. Here we show that without RDS, no disc/OS formation occurs, while without rhodopsin, small OS structures form containing aligned nascent discs. In the absence of both rhodopsin and RDS, RDS-associated degeneration is slowed, and ROM-1 is stabilized and trafficked to the OS. These animals (rho-/-/rds-/-) exhibit OSs slightly better than those lacking only RDS, but still without signs of disc formation. These results clearly demonstrate that OS morphogenesis is initiated by RDS-mediated rim formation, a process ROM-1 cannot recapitulate, with subsequent disc growth mediated by rhodopsin. The critical role of RDS in this process helps explain why photoreceptors are so sensitive to varied RDS levels, and why mutations in RDS cause debilitating retinal disease.

Topics: Animals; Eye Proteins; Gene Expression; Membrane Proteins; Mice; Mice, Knockout; Morphogenesis; Peripherins; Photoreceptor Cells; Protein Transport; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Tetraspanins

To investigate the retinal-protective effects of the oral iron chelator deferiprone (DFP) in mice lacking the iron regulatory hormone hepcidin (Hepc). These Hepc knockout (KO) mice have age-dependent systemic and retinal iron accumulation leading to retinal degeneration.. Hepc KO mice were given DFP in drinking water from age 6 to 18 months. They were then compared to Hepc KO mice not receiving DFP by fundus imaging, electroretinography (ERG), histology, immunofluorescence, and quantitative PCR to investigate the protective effect of DFP against retinal and retinal pigment epithelial (RPE) degeneration.. In Hepc KO mice, DFP diminished RPE depigmentation and autofluorescence on fundus imaging. Autofluorescence in the RPE layer in cryosections was significantly diminished by DFP, consistent with the fundus images. Immunolabeling with L-ferritin and transferrin receptor antibodies showed a decreased signal for L-ferritin in the inner retina and RPE cells and an increased signal for transferrin receptor in the inner retina, indicating diminished retinal iron levels with DFP treatment. Plastic sections showed that photoreceptor and RPE cells were well preserved in Hepc KO mice treated with DFP. Consistent with photoreceptor protection, the mRNA level of rhodopsin was significantly higher in retinas treated with DFP. The mRNA levels of oxidative stress-related genes heme oxygenase-1 and catalase were significantly lower in DFP-treated Hepc KO retinas. Finally, ERG rod a- and b- and cone b-wave amplitudes were significantly higher in DFP-treated mice.. Long-term treatment with the oral iron chelator DFP diminished retinal and RPE iron levels and oxidative stress, providing significant protection against retinal degeneration caused by chronic systemic iron overload in Hepc KO mice. This indicates that iron chelation could be a long-term preventive treatment for retinal disease involving iron overload and oxidative stress.

Topics: Administration, Oral; Animals; Deferiprone; Disease Models, Animal; Electroretinography; Ferritins; Fluorescein Angiography; Fluorescent Antibody Technique, Indirect; Heme Oxygenase-1; Hepcidins; Iron Chelating Agents; Iron Overload; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Optical Imaging; Oxidative Stress; Pyridones; Real-Time Polymerase Chain Reaction; Receptors, Transferrin; Retinal Degeneration; Retinal Pigment Epithelium; Rhodopsin; RNA, Messenger

RPE65, a retinal pigment epithelium-specific 65-kDa protein, plays a critical role in the visual cycle of the eye. Rpe65(-/-) mice develop vision loss due to a lack of 11-cis-retinal, degradation of M-opsin and mislocalization of S-opsin. Several studies have suggested that 9-cis-β-carotene, a precursor of 9-cis-retinal and all-trans-retinal, could have therapeutic applications in vision loss. We therefore examined whether Dunaliella bardawil, a 9-cis-β-carotene-rich alga, protects against the degradation of M-opsin using Rpe65(-/-) mouse retinal explant cultures.. The eyes of three-week-old Rpe65(-/-) and C57BL/6 J mice were enucleated, and the corneas were removed. The eyecups were incubated with culture medium in the absence or presence of D. bardawil for 6 h to 4 days. Localizations of M-opsin proteins in the retina were observed immunohistochemically. Expression levels of M-opsin, S-opsin and rhodopsin proteins were evaluated by Western blot analysis.. In C57BL/6 J mouse retina, no change was observed in localization and expression levels of M-opsin in the explant culture system. In Rpe65(-/-) mouse retina, the amount of M-opsin protein was decreased in the photoreceptor outer segment after 6 h to 4 days of culture. However, the presence of D. bardawil significantly ameliorated this decrease. In contrast, expression levels of S-opsin and rhodopsin were unchanged in the presence of the explant culture.. These results demonstrate that D. bardawil treatment protects against M-opsin degradation in Rpe65(-/-) mouse retina and suggest that D. bardawil has therapeutic potential for retinal degeneration caused by Rpe65 gene mutation, such as Leber congenital amaurosis and retinitis pigmentosa.

Topics: Animals; beta Carotene; Blotting, Western; Chlorophyta; cis-trans-Isomerases; Cone Opsins; Fluorescent Antibody Technique, Indirect; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Culture Techniques; Plant Extracts; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Rod Opsins

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative human diseases characterized by the loss of photoreceptor cells by apoptosis and eventual blindness. A single intraperitoneal (ip) injection of N-methyl-N-nitrosourea (MNU) causes photoreceptor cell apoptosis within 7 days in rats. Green tea extract (THEA-FLAN 90S; GTE) is a common herbal supplement with pluripotent properties including antioxidant activity. The purpose of the present study was to evaluate the efficacy of GTE against photoreceptor apoptosis in 7-week-old female Sprague-Dawley rats that received a single ip injection of 40 mg/kg MNU.. The oral administration of 250 mg/kg/day GTE was initiated 3 days prior to MNU injection and continued once daily throughout the experiment. Rats were sacrificed at 12, 24, and 72 h and 7 days after MNU injection, and the eyes were examined morphologically and morphometrically. The photoreceptor cell ratio, retinal damage ratio, and retinal preservation ratio were used to determine the structural and functional alterations. The number of apoptotic photoreceptor cells per mm(2) was determined in situ by TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL). Our results indicated that oral administration of GTE significantly suppressed the loss of photoreceptor cells morphometrically 7 days after MNU injection. The number of TUNEL-positive cells per mm(2) in MNU-exposed rat central retina with or without GTE administration was 981 vs. 2056 at 24 h after MNU injection.. GTE structurally and functionally suppressed MNU-induced photoreceptor cell apoptosis. These findings indicate that GTE may help to ameliorate the onset and progression of human RP.

Topics: Administration, Oral; Alkylating Agents; Animals; Apoptosis; Catechin; Chromatography, Liquid; Cyclic Nucleotide Phosphodiesterases, Type 6; Female; In Situ Nick-End Labeling; Injections, Intraperitoneal; Methylnitrosourea; Photoreceptor Cells, Vertebrate; Phytotherapy; Plant Extracts; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; Tandem Mass Spectrometry; Tea

Efficient regeneration of visual pigment following its destruction by light is critical for the function of mammalian photoreceptors. Here, we show that misexpression of a subset of cone genes in the rd7 mouse hybrid rods enables them to access the normally cone-specific retina visual cycle. The rapid supply of chromophore by the retina visual cycle dramatically accelerated the mouse rod dark adaptation. At the same time, the competition between rods and cones for retina-derived chromophore slowed cone dark adaptation, indicating that the cone specificity of the retina visual cycle is key for rapid cone dark adaptation. Our findings demonstrate that mammalian photoreceptor dark adaptation is dominated by the supply of chromophore. Misexpression of cone genes in rods may represent a novel approach to treating visual disorders associated with mutations of visual cycle proteins or with reduced retinal pigment epithelium function due to aging.

Topics: Action Potentials; Animals; Dark Adaptation; Female; GTP-Binding Protein alpha Subunits; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroglia; Orphan Nuclear Receptors; Photic Stimulation; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Time Factors; Transducin; Vitamin A; Vitamins

Oxidative stress and inflammation have key roles in the light damage (LD) model of retinal degeneration as well as in age-related macular degeneration (AMD). We sought to determine if lipoic acid (LA), an antioxidant and iron chelator, protects the retina against LD.. Balb/c mice were treated with LA or control saline via intraperitoneal injection, and then were placed in constant cool white light-emitting diode (LED) light (10,000 lux) for 4 hours. Retinas were evaluated at several time points after LD. Photoreceptor apoptosis was assessed using the TUNEL assay. Retinal function was analyzed via electroretinography (ERG). Retinal degeneration was assessed after LD by optical coherence tomography (OCT), TUNEL analysis, and histology. The mRNAs of several oxidative stress, inflammation, and iron-related genes were quantified by quantitative PCR (qPCR).. The LD resulted in substantial photoreceptor-specific cell death. Dosing with LA protected photoreceptors, decreasing the numbers of TUNEL-positive photoreceptors and increasing the number of surviving photoreceptors. The retinal mRNA levels of genes indicating oxidative stress, inflammation, and iron accumulation were lower following LD in mice treated with LA than in control mice. The ERG analysis demonstrated functional protection by LA.. Systemic LA is protective against light-induced retinal degeneration. Since this agent already has proven protective in other retinal degeneration models, and is safe and protective against diabetic neuropathy in patients, it is worthy of consideration for a human clinical trial against retinal degeneration or AMD.

Topics: Animals; Antioxidants; Apoptosis; cis-trans-Isomerases; Electroretinography; Gene Expression; In Situ Nick-End Labeling; Iron Chelating Agents; Light; Male; Mice; Mice, Inbred BALB C; Oxidative Stress; Retina; Retinal Degeneration; Retinitis; Rhodopsin; Thioctic Acid; Tomography, Optical Coherence

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28's SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.

Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Biological Transport; Blotting, Western; Cell Survival; Drosophila melanogaster; Drosophila Proteins; Golgi Apparatus; Humans; Microscopy, Confocal; Microscopy, Electron; Molecular Sequence Data; Mutation; Photoreceptor Cells, Invertebrate; Qb-SNARE Proteins; Retinal Degeneration; Rhodopsin; Sequence Homology, Amino Acid

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.

Topics: Analysis of Variance; Animals; Animals, Genetically Modified; Chlorocebus aethiops; COS Cells; Disease Models, Animal; Humans; Light; Microscopy, Confocal; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Statistics, Nonparametric; Transfection; Vitamin A; Wheat Germ Agglutinins; Xenopus laevis

The histone-deacetylase inhibitor activity of valproic acid (VPA) was discovered after VPA's adoption as an anticonvulsant. This generated speculation for VPA's potential to increase the expression of neuroprotective genes. Clinical trials for retinitis pigmentosa (RP) are currently active, testing VPA's potential to reduce photoreceptor loss; however, we lack information regarding the effects of VPA on available mammalian models of retinal degeneration, nor do we know if retinal gene expression is perturbed by VPA in a predictable way. Thus, we examined the effects of systemic VPA on neurotrophic factor and Nrl-related gene expression in the mouse retina and compared VPA's effects on the rate of photoreceptor loss in two strains of mice, Pde6b(rd1/rd1) and Pde6b(rd10/rd10) .. The expression of Bdnf, Gdnf, Cntf, and Fgf2 was measured by quantitative PCR after single and multiple doses of VPA (intraperitoneal) in wild-type and Pde6b(rd1/rd1) mice. Pde6b(rd1/rd1) mice were treated with daily doses of VPA during the period of rapid photoreceptor loss. Pde6b(rd10/rd10) mice were also treated with systemic VPA to compare in a partial loss-of-function model. Retinal morphology was assessed by virtual microscopy or spectral-domain optical coherence tomography (SD-OCT). Full-field and focal electroretinography (ERG) analysis were employed with Pde6b(rd10/rd10) mice to measure retinal function.. In wild-type postnatal mice, a single VPA dose increased the expression of Bdnf and Gdnf in the neural retina after 18 h, while the expression of Cntf was reduced by 70%. Daily dosing of wild-type mice from postnatal day P17 to P28 resulted in smaller increases in Bdnf and Gdnf expression, normal Cntf expression, and reduced Fgf2 expression (25%). Nrl gene expression was decreased by 50%, while Crx gene expression was not affected. Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28. Daily injections with VPA (P9-P21) dramatically slowed the loss of rod photoreceptors in Pde6b(rd1/rd1) mice. At age P21, VPA-treated mice had several extra rows of rod photoreceptor nuclei compared to PBS-injected littermates. Dosing started later (P14) or dosing every second day also rescued photoreceptors. In contrast, systemic VPA treatment of Pde6b(rd10/rd10) mice (P17-P28) reduced visual function that correlated with a slight increase in photoreceptor loss. Treating Pde6b(rd10/rd10) mice earlier (P9-P21) also failed to rescue photoreceptors. Treating wild-type mice earlier (P9-P21) reduced the number of photoreceptors in VPA-treated mice by 20% compared to PBS-treated animals.. A single systemic dose of VPA can change retinal neurotrophic factor and rod-specific gene expression in the immature retina. Daily VPA treatment from P17 to P28 can also alter gene expression in the mature neural retina. While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model. The apparent rescue of photoreceptors in the rd1 model was not the result of producing more photoreceptors before degeneration. In fact, daily systemic VPA was toxic to wild-type photoreceptors when started at P9. However, the effective treatment period for Pde6b(rd1/rd1) mice (P9-P21) has significant overlap with the photoreceptor maturation period, which complicates the use of the rd1 model for testing of VPA's efficacy. In contrast, VPA treatment started after P17 did not cause photoreceptor loss in wild-type mice. Thus, the acceleration of photoreceptor loss in the rd10 model may be more relevant where both photoreceptor loss and VPA treatment (P17-P28) started when the central retina was mature.

Topics: Animals; Basic-Leucine Zipper Transcription Factors; Brain-Derived Neurotrophic Factor; Cyclic Nucleotide Phosphodiesterases, Type 6; Enzyme Inhibitors; Eye Proteins; Fibroblast Growth Factor 2; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Homeodomain Proteins; Injections, Intraperitoneal; MEF2 Transcription Factors; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Growth Factors; Orphan Nuclear Receptors; Protective Agents; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Species Specificity; Time Factors; Trans-Activators; Valproic Acid

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.

Topics: Animals; Apoptosis; Caspase 7; Gene Expression; Hypoxia-Inducible Factor 1, alpha Subunit; JNK Mitogen-Activated Protein Kinases; Light; Mice; Mice, Transgenic; Poly(ADP-ribose) Polymerases; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA Interference; RNA, Small Interfering; TNF Receptor-Associated Factor 2; TOR Serine-Threonine Kinases; Unfolded Protein Response

The proline-23-histidine line 3 (P23H-3) transgenic rat carries a human opsin gene mutation leading to progressive photoreceptor loss characteristic of human autosomal dominant retinitis pigmentosa. The aim of the present study was to evaluate neurochemical modifications in the P23H-3 retina as a function of development and degeneration. Specifically, we investigated the ion channel permeability of photoreceptors by tracking an organic cation, agmatine (1-amino-4-guanidobutane, AGB), which permeates through nonspecific cation channels. We also investigated the activity of ionotropic glutamate receptors in distinct populations of bipolar, amacrine, and ganglion cells using AGB tracking in combination with macromolecular markers. We found elevated cation channel permeation in photoreceptors as early as postnatal day 12 (P12) suggesting that AGB labeling is an early indicator of impending photoreceptor degeneration. However, bipolar, amacrine, or ganglion cells displayed normal responses secondary to ionotropic glutamate receptor activation even at P138 when about one half of the photoreceptor layer was lost and apoptosis and gliosis were observed. These results suggest that possible therapeutic windows as downstream neurons in inner retina appear to retain normal function with regard to AGB permeation when photoreceptors are significantly reduced but not lost.

Topics: Age Factors; Agmatine; Animals; Apoptosis; Gene Expression Regulation, Developmental; Histidine; Humans; Mutation; Nerve Tissue Proteins; Neurons; Photoreceptor Cells; Proline; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Degeneration; Rhodopsin; Silver Staining

Outer retinal injury has been well described in glaucoma. To better understand the source of this injury, we wanted to develop a reliable model of partial retinal ganglion cell (RGC) axotomy.. Endodiathermy spots were placed along the inferior 180° adjacent to the optic nerve margin in the right eyes of four cynomolgus monkeys. Fluorescein angiography, spectral domain optical coherence tomography (SD-OCT), and multifocal electroretinography (mfERG) were performed at various intervals. Two animals were sacrificed at 3 months. Two animals were sacrificed at 4 months, at which time they underwent an injection of fluorescent microspheres to measure regional choroidal blood flow. Retinal immunohistochemistry for glial fibrillary acidic protein (GFAP), rhodopsin, S-cone opsin, and M/L-cone opsin were performed, as were axon counts of the optic nerves.. At 3 months, there was marked thinning of the inferior nerve fiber layer on SD-OCT. The mfERG waveforms were consistent with inner but not outer retinal injury. Greater than 95% reduction in axons was seen in the inferior optic nerves but no secondary degeneration superiorly. There was marked thinning of the nerve fiber and ganglion cell layers in the inferior retinas. However, the photoreceptor histology was similar in the axotomized and nonaxotomized areas. Regional choroidal blood flow was not affected by the axotomy.. Unlike experimental glaucoma, hemiretinal endodiathermy axotomy (HEA) of the RGCs produces no apparent anatomic, functional, or blood flow effects on the outer retina and choroid.

Topics: Animals; Axotomy; Choroid; Disease Models, Animal; Electrocoagulation; Electroretinography; Female; Fluorescein Angiography; Glial Fibrillary Acidic Protein; Immunohistochemistry; Macaca fascicularis; Male; Nerve Fibers; Opsins; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Ganglion Cells; Retinal Vessels; Rhodopsin; Tomography, Optical Coherence

Despite the proposed link between ablation of the CHOP protein and delay of the onset of ER stress-mediated disorders including diabetes, Alzheimer Disease, and cardiac hypertrophy, the role of CHOP protein in photoreceptor cell death associated with Autosomal Dominant Retinitis Pigmentosa (ADRP) has not been investigated. T17M RHO transgenic mice carry a mutated human rhodopsin transgene, the expression of which in retina leads to protein misfolding, activation of UPR and progressive retinal degeneration. The purpose of this study is to investigate the role of CHOP protein in T17M RHO retina. Wild-type, CHOP-/-, T17M RHO and T17M RHO CHOP-/-mice were used in the study. Evaluation of the impact of CHOP ablation was performed using electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), quantitative Real-Time PCR (qRT-PCR) and western blot analysis. Dark-adapted ERG analysis demonstrated that by 1 month, the T17M RHO CHOP-/- mice had a 70% reduction of the a-wave amplitude compared to the T17M RHO mice. The loss of function in T17M RHO CHOP-/- photoreceptors was associated with a 22-24% decline in the thickness of the outer nuclear layer. These mice had significant reduction in the expression of transcription factors, Crx and Nrl, and also in mouse Rho, and human RHO. The reduction was associated with an 8-fold elevation of the UPR marker, p-eIf2α protein and 30% down-regulation of sXbp1 protein. In addition, the histone deacetylase 1 (Hdac1) protein was 2-fold elevated in the T17M RHO CHOP-/- retina. The ablation of CHOP led to a reduction in the expression of photoreceptor-specific transcriptional factors, and both endogenous and exogenous RHO mRNA. Thus, despite its role in promoting apoptosis, CHOP protects rod photoreceptors carrying an ADRP mutation.

Topics: Animals; Basic-Leucine Zipper Transcription Factors; Disease Models, Animal; DNA-Binding Proteins; Electroretinography; Eye Proteins; Gene Expression Regulation; Histone Deacetylase 1; Homeodomain Proteins; Humans; Mice; Mice, Knockout; p300-CBP Transcription Factors; Regulatory Factor X Transcription Factors; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger; Tomography, Optical Coherence; Trans-Activators; Transcription Factor CHOP; Transcription Factors

Arrestins bind ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) and terminate the activation of G proteins. Additionally, nonvisual arrestin/GPCR complex can initiate G protein-independent intracellular signals through their ability to act as scaffolds that bring other signaling molecules to the internalized GPCR. Like nonvisual arrestins, vertebrate visual arrestin (ARR1) terminates G protein signaling from light-activated, phosphorylated GPCR, rhodopsin. Unlike nonvisual arrestins, its role as a transducer of signaling from internalized rhodopsin has not been reported in the vertebrate retina. Formation of signaling complexes with arrestins often requires recruitment of the endocytic adaptor protein, AP-2. We have previously shown that Lys296 → Glu (K296E), which is a naturally occurring rhodopsin mutation in certain humans diagnosed with autosomal dominant retinitis pigmentosa, causes toxicity through forming a stable complex with ARR1. Here we investigated whether recruitment of AP-2 by the K296E/ARR1 complex plays a role in generating the cell death signal in a transgenic mouse model of retinal degeneration. We measured the binding affinity of ARR1 for AP-2 and found that, although the affinity is much lower than that of the other arrestins, the unusually high concentration of ARR1 in rods would favor this interaction. We further demonstrate that p44, a splice variant of ARR1 that binds light-activated, phosphorylated rhodopsin but lacks the AP-2 binding motif, prevents retinal degeneration and rescues visual function in K296E mice. These results reveal a unique role of ARR1 in a G protein-independent signaling cascade in the vertebrate retina.

Topics: Adaptor Protein Complex 2; Analysis of Variance; Animals; Arrestins; beta-Arrestin 1; beta-Arrestins; Blotting, Western; Cell Survival; Electron Spin Resonance Spectroscopy; Electroretinography; GTP-Binding Proteins; Immunohistochemistry; Mice; Mice, Transgenic; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Rhodopsin; Signal Transduction

Retinal degeneration caused by a defect in the phototransduction cascade leads to the apoptosis of photoreceptor cells, although the precise molecular mechanism is still unknown. In addition, constant low light exposure produces photoreceptor cell death through the activation of downstream phototransduction. The authors investigated the time course and molecular mechanisms of death and the rhodopsin phosphorylation occurring during retinal degeneration after exposure to continuous low-intensity light.. Wistar rats were exposed to constant cool white 200 lx intensity LED light (LL) for one to ten days and compared with animals kept in the dark (DD) or controls exposed to a regular 12:12 h (LD) cycle. One eye from each rat was used for histological and quantitative outer nuclear layer (ONL) analysis and the other for biochemical assays.. The histological analysis showed a significant reduction in the ONL of LL-exposed rats after seven days compared with LD- or DD-exposed rats. Retinal analysis by flow cytometer and the TUNEL assay revealed an increase in cell death in the ONL, the in vitro enzymatic activity assay and western blot analysis showing no caspase-3 activation. The rhodopsin analysis demonstrated more phosphorylation in serine 334 residues (Ser(334)) in LL-exposed than in LD- or DD-exposed rats. However, for all times studied, rhodopsin was completely dephosphorylated after four days of DD treatment.. Constant light exposure for seven days produces ONL reduction by photoreceptor cell death through a capase-3-independent mechanism. Increases in rhodopsin-phospho-Ser(334) levels were observed, supporting the notion that changes in the regulation of the phototransduction cascade occur during retinal degeneration.

Topics: Animals; Annexin A5; Caspase 3; Cell Death; Disease Models, Animal; In Situ Nick-End Labeling; Light; Mammals; Phosphorylation; Phosphoserine; Photoreceptor Cells, Vertebrate; Propidium; Rats; Rats, Wistar; Retinal Degeneration; Rhodopsin

To investigate the spatiotemporal relationship between rod and cone degeneration in the P23H-1 rat.. Control Sprague-Dawley (SD) and P23H-1 rats of ages ranging from P30 to P365 were used. Retinas were processed for whole mounts or cross sections and rods and cones were immunodetected. We used newly developed image analysis techniques to quantify the total population of L/M cones (the most abundant cones in the rat) and analyzed the rings of rod-cone degeneration.. In P23H-1 rats, rod degeneration occurs rapidly: first the rod outer segment shortens, at P30 there is extensive rod loss, and by P180 rod loss is almost complete except for the most peripheral retina. The numbers of L/M cones are, at all postnatal ages, lower in P23H-1 rats than in control SD rats, and decrease significantly with age (by P180). Rod and cone degeneration is spatiotemporally related and occurs in rings that appear already at P90 and spread throughout the entire retina. At P180, the rings of rod-cone degeneration are more abundant in the equatorial retina and are larger in the dorsal retina.. This work describes for the first time that in the P23H-1 rat, rod and cone degeneration is spatiotemporally related and occurs in rings. Cone loss follows rod loss and starts very soon, even before P30, the first age analyzed here. The characteristics of the rings suggest that secondary cone degeneration is influenced by retinal position and/or other intrinsic or extrinsic factors.

Topics: Animals; Disease Models, Animal; Female; Immunohistochemistry; Opsins; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

Topics: Animals; Apoptosis; Cell Proliferation; Disease Models, Animal; Methylnitrosourea; Neuroglia; Regeneration; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Zebrafish

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.

Topics: Age of Onset; Humans; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA

Obesity is associated with various health afflictions, including ocular complications such as diabetic retinopathy, high intraocular pressure, cataracts, and macular degeneration. We previously reported progressive retinal degeneration after the onset of obesity in the spontaneously obese rat (WNIN/Ob) model. In the present study, we investigated vitamin A supplementation to ameliorate obesity-associated retinal degeneration in the WNIN/Ob rat.. Five-month-old male WNIN/Ob obese (O) and lean (L) control rats were fed with vitamin A 2.6 mg (L/O-I), 26 mg (L/O-II), 52 mg (L/O-III), and 129 mg (L/O-IV) per kilogram of diet as retinyl palmitate for 4 mo 2 wk. Retinal morphology and retinal gene expression were assessed by histologic, immunohistochemical, and real-time polymerase chain reaction methods.. Supplementation of vitamin A at 26 or 52 mg significantly modulated the expression of retinal genes in the O but not in the L phenotype. Vitamin A supplementation significantly upregulated the expression of genes, such as rhodopsin, rod arrestin, phosphodiesterase, transducins, and fatty acid elongase-4, that were otherwise downregulated in O rat retina. The expression of glial fibrillary acidic protein was downregulated by vitamin A feeding in O rat retina. The immunohistochemical and histologic findings corroborated the gene expression data. The effects were significant at a 26- or 52-mg dose of vitamin A.. Vitamin A supplementation alleviated obesity-associated retinal degeneration in the WNIN/Ob rat.

Topics: Acetyltransferases; Animals; Arrestin; Base Sequence; DNA Primers; Fatty Acid Elongases; Gene Expression; Glial Fibrillary Acidic Protein; Immunohistochemistry; Male; Obesity; Rats; Retina; Retinal Degeneration; Rhodopsin; Vitamin A

Our previous study on retinal light exposure suggests the involvement of zinc (Zn(2+)) toxicity in the death of RPE and photoreceptors (LD) which could be attenuated by pyruvate and nicotinamide, perhaps through restoration of NAD(+) levels. In the present study, we examined Zn(2+) toxicity, and the effects of NAD(+) restoration in primary retinal cultures. We then reduced Zn(2+) levels in rodents by reducing Zn(2+) levels in the diet, or by genetics and measured LD. Sprague Dawley albino rats were fed 2, or 61 mg Zn(2+)/kg of diet for 3 weeks, and exposed to 18 kLux of white light for 4 h. We light exposed (70 kLux of white light for 50 h) Zn(2+) transporter 3 knockout (ZnT3-KO, no synaptic Zn(2+)), or RPE65 knockout mice (RPE65-KO, lack rhodopsin cycling), or C57/BI6/J controls and determined light damage and Zn(2+) staining. Retinal Zn(2+) staining was examined at 1 h and 4 h after light exposure. Retinas were examined after 7 d by optical coherence tomography and histology. After LD, rats fed the reduced Zn(2+) diet showed less photoreceptor Zn(2+) staining and degeneration compared to a normal Zn(2+) diet. Similarly, ZnT3-KO and RPE65-KO mice showed less Zn(2+) staining, NAD(+) loss, and RPE or photoreceptor death than C57/BI6/J control mice. Dietary or ZnT3-dependent Zn(2+) stores, and intracellular Zn(2+) release from rhodopsin recycling are suggested to be involved in light-induced retinal degeneration. These results implicate novel rhodopsin-mediated mechanisms and therapeutic targets for LD. Our companion manuscript demonstrates that pharmacologic, circadian, or genetic manipulations which maintain NAD(+) levels reduce LD.

Topics: Animals; Carrier Proteins; Cation Transport Proteins; Cell Death; Cells, Cultured; cis-trans-Isomerases; Diet; Disease Models, Animal; Light; Membrane Proteins; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; NAD; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; Time Factors; Tomography, Optical Coherence; Zinc

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

Topics: Animals; Basic-Leucine Zipper Transcription Factors; CpG Islands; DNA Methylation; Electroretinography; Eye Proteins; Gene Expression; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Autophagy is a conserved feature of lysosome-mediated intracellular degradation. Dysregulated autophagy is implicated as a contributor in neurodegenerative diseases; however, the role of autophagy in retinal degeneration remains largely unknown. Here, we report that the photo-activated visual chromophore, all-trans-retinal, modulated autophagosome formation in ARPE19 retinal cells. Increased formation of autophagosomes in these cells was observed when incubated with 2.5 μM all-trans-retinal, a condition that did not cause cell death after 24 h in culture. However, autophagosome formation was decreased at concentrations, which caused cell death. Increased expression of activating transcription factor 4 (Atf4), which indicates the activation of oxidative stress, was recorded in response to light illumination in retinas of Abca4(-/-)Rdh8(-/-) mice, which showed delayed clearance of all-trans-retinal after light exposure. Expression of autophagosome marker LC3B-II and mitochondria-specific autophagy, mitophagy, regulator Park2, were significantly increased in the retinas of Abca4(-/-)Rdh8(-/-) mice after light exposure, suggesting involvement of autophagy and mitophagy in the pathogenesis of light-induced retinal degeneration. Deletion of essential genes required for autophagy, including Beclin1 systemically or Atg7 in only rod photoreceptors resulted in increased susceptibility to light-induced retinal damage. Increased photoreceptor cell death was observed when retinas lacking the rod photoreceptor-specific Atg7 gene were coincubated with 20 μM all-trans-retinal. Park2(-/-) mice also displayed light-induced retinal degeneration. Ultra-structural analyses showed mitochondrial and endoplasmic reticulum impairment in retinas of these model animals after light exposure. Taken together, these observations provide novel evidence implicating an important role of autophagy and mitophagy in protecting the retina from all-trans-retinal- and light-induced degeneration.

Topics: Alcohol Dehydrogenase; Alcohol Oxidoreductases; Animals; Apoptosis; Apoptosis Regulatory Proteins; ATP-Binding Cassette Transporters; Autophagy; Autophagy-Related Protein 7; Beclin-1; Cell Death; Cell Line; Humans; Light; Lysosomes; Membrane Proteins; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Photic Stimulation; Retina; Retinal Degeneration; Rhodopsin; Ubiquitin-Activating Enzymes; Ubiquitin-Protein Ligases; Vitamin A

The prototypical transient receptor potential (TRP) channel is the major light-sensitive, and Ca(2+)-permeable channel in the microvillar photoreceptors of Drosophila. TRP channels are activated following hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] by the key effector enzyme phospholipase C (PLC). Mutants lacking TRP channels undergo light-dependent retinal degeneration, as a consequence of the reduced Ca(2+) influx. It has been proposed that degeneration is caused by defects in the Ca(2+)-dependent visual pigment cycle, which result in accumulation of toxic phosphorylated metarhodopsin-arrestin complexes (MPP-Arr2). Here we show that two interventions, which prevent accumulation of MPP-Arr2, namely rearing under red light or eliminating the C-terminal rhodopsin phosphorylation sites, failed to rescue degeneration in trp mutants. Instead, degeneration in trp mutants reared under red light was rescued by mutation of PLC. Degeneration correlated closely with the light-induced depletion of PtdIns(4,5)P₂ that occurs in trp mutants due to failure of Ca(2+)-dependent inhibition of PLC. Severe retinal degeneration was also induced in the dark in otherwise wild-type flies by overexpression of a bacterial PtdInsPn phosphatase (SigD) to deplete PtdIns(4,5)P₂. In degenerating trp photoreceptors, phosphorylated Moesin, a PtdIns(4,5)P₂-regulated membrane-cytoskeleton linker essential for normal microvillar morphology, was found to delocalize from the rhabdomere and there was extensive microvillar actin depolymerisation. The results suggest that compromised light-induced Ca(2+) influx, due to loss of TRP channels, leads to PtdIns(4,5)P₂ depletion, resulting in dephosphorylation of Moesin, actin depolymerisation and disintegration of photoreceptor structure.

Topics: Actins; Animals; Drosophila; Microfilament Proteins; Phosphatidylinositol 4,5-Diphosphate; Retinal Degeneration; Rhodopsin; Transient Receptor Potential Channels; Type C Phospholipases

Retinal degeneration is a devastating complication of diabetes and other disorders. Stem cell therapy for retinal degeneration has shown encouraging results but functional regeneration has not been yet achieved. Our study was undertaken to evaluate the localization of stem cells delivered to the retina by intravenous versus intravitreal infusion, because stem cell localization is a key factor in ultimate in vivo function. We used lineage-negative bone marrow-derived stem cells in a model wherein retina of mice was induced by precise and reproducible laser injury. Lin(-ve) bone marrow cells (BMCs) were labeled with a tracking dye and their homing capacity was analyzed at time points after infusion. We found that Lin(-ve) BMCs get incorporated into laser-injured retina when transplanted through either the intravitreal or intravenous route. The intravenous route resulted in optimal localization of donor cells at the site of injury. These cells incorporated into injured retina in a dose-dependent manner. The data presented in this study reflect the importance of dose and route for stem cell-based treatment designed to result in retinal regeneration.

Topics: Angiography; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Disease Models, Animal; Female; Fluorescent Dyes; Injections, Intravenous; Intravitreal Injections; Lasers; Mice; Mice, Inbred C57BL; Regeneration; Retina; Retinal Degeneration; Rhodopsin; Staining and Labeling

Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.

Topics: Animals; Central Nervous System; Dependovirus; Disease Models, Animal; Gene Expression Regulation, Developmental; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; Humans; Mice; Mice, Inbred C57BL; Mutation; Promoter Regions, Genetic; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Topics: Animals; Caspase 3; Cell Survival; Dexamethasone; Drug Interactions; Glucocorticoids; Hormone Antagonists; Light; Male; Mice; Mice, Inbred BALB C; Mifepristone; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Rhodopsin

ENU mutagenesis is an efficient method to identify new animal models of ocular disease. The new alleles described herein will be a useful resource to further examine the role of the affected molecules and the effects of their disruption within the retina.

Topics: Alkylating Agents; Animals; Chromosome Mapping; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; DNA Mutational Analysis; Ethylnitrosourea; Eye Proteins; Humans; Mice; Mice, Inbred C57BL; Mutagenesis; Retinal Degeneration; Rhodopsin; Translational Research, Biomedical

Topics: Animals; Carrier Proteins; cis-trans-Isomerases; Disease Models, Animal; Eye Proteins; Light; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Retina; Retinal Degeneration; Rhodopsin; Tomography, Optical Coherence

Topics: Animals; Cell Compartmentation; Eye Proteins; Laser Capture Microdissection; Mice; Mice, Mutant Strains; Photoreceptor Cells, Vertebrate; Protein Transport; Proteomics; Retinal Degeneration; Rhodopsin

Topics: Animals; Chaperonins; Eye Proteins; Intermediate Filament Proteins; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells, Vertebrate; Protein Transport; Retinal Degeneration; Retinal Photoreceptor Cell Outer Segment; Rhodopsin

To investigate rod function and survival after cone dysfunction and degeneration in a mouse model of cone cyclic nucleotide-gated (CNG) channel deficiency.. Rod function and survival in mice with cone CNG channel subunit CNGA3 deficiency (CNGA3-/- mice) were evaluated by electroretinographic (ERG), morphometric, and Western blot analyses. The arrangement, integrity, and ultrastructure of photoreceptor terminals were investigated by immunohistochemistry and electron microscopy.. The authors found loss of cone function and cone death accompanied by impairment of rods and rod-driven signaling in CNGA3-/- mice. Scotopic ERG b-wave amplitudes were reduced by 15% at 1 month, 30% at 6 months, and 40% at 9 months and older, while scotopic a-wave amplitudes were decreased by 20% at 9 months, compared with ERGs of age-matched wild-type mice. Outer nuclear layer thickness in CNGA3-/- retina was reduced by 15% at 12 months compared with age-matched wild-type controls. This was accompanied by a 30%-40% reduction in expression of rod-specific proteins, including rhodopsin, rod transducin α-subunit, and glutamic acid-rich protein (GARP). Cone terminals in the CNGA3-/- retina showed a progressive loss of neurochemical and ultrastructural integrity. Abnormalities were observed as early as 1 month. Disorganized rod terminal ultrastructure was noted by 12 months.. These findings demonstrate secondary rod impairment and degeneration after cone degeneration in mice with cone CNG channel deficiency. Loss of cone phototransduction accompanies the compromised integrity of cone terminals. With time, rod synaptic structure, function, and viability also become compromised.

Topics: Animals; Arrestin; Blotting, Western; Cell Survival; Cyclic Nucleotide-Gated Cation Channels; Electroretinography; Heterotrimeric GTP-Binding Proteins; Immunohistochemistry; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron; Presynaptic Terminals; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Opsins; Transducin; Vision, Ocular

The term retinitis pigmentosa (RP) comprises a heterogeneous group of hereditary and sporadic human retinal degenerative diseases. The molecular and cellular events still remain obscure, thus hiding effective therapies. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a hematopoietic factor which plays a crucial role in protecting neuronal cells. Binding of GM-CSF to its receptor induces several intracellular signaling pathways and kinases. Here we examined whether GM-CSF has a neuroprotective effect on photoreceptor degeneration in Royal College of Surgeons (RCS) rats.. GM-CSF was injected into the vitreous body of RCS rats either once at the onset of photoreceptor degeneration at day 21, or twice at day 21 and day 42. At day 84, when photoreceptor degeneration is completed, the rats were sacrificed, their eyes enucleated and processed for histological staining and counting the surviving photoreceptor nuclei. The expression of apoptosis-related factors, such as BAD, APAF1 and BCL-2 was examined by Western blot analysis. The expression of neurotrophins such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic actor (GDNF), as well as glial fibrillary acidic protein (GFAP) was analysed by Western blots and immunohistochemistry. The expression of JAK/STAT, ERK1/2 and SRC pathway proteins was assessed by Western blot analysis.. GM-CSF protects significantly against photoreceptor degeneration in comparison to control group. After a single injection of GM-CSF at P21, a 4-fold increase of photoreceptors was observed, whereas eyes which received a repeated injection of GM-CSF at P42 showed a 10-fold increase of photoreceptors. Western blot analysis revealed a decreased BAD and an increased pBAD and BCL-2 expression, indicating changed expression profiles of apoptosis-related proteins. Neurotrophic factors examined are up-regulated, whereas GFAP was also modulated. At cell signalling levels, GM-CSF activates SRC-dependent STAT3 which is independent of JAK2, while proteins of the ERK1/2 pathway are not affected.. The data suggest that GM-CSF is a potent therapeutic agent in photoreceptor degeneration caused by mutation of the receptor tyrosine kinase gene (Mertk), and may be also effective in other photoreceptor degeneration.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; bcl-Associated Death Protein; Blotting, Western; Glial Fibrillary Acidic Protein; Granulocyte-Macrophage Colony-Stimulating Factor; Immunohistochemistry; Intravitreal Injections; Nerve Growth Factors; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Mutant Strains; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Retinal Degeneration; Rhodopsin; Signal Transduction; src-Family Kinases; STAT3 Transcription Factor

This study evaluated the capacity of Xenopus laevis retina to regenerate photoreceptor cells after cyclic light-mediated acute rod photoreceptor degeneration in a transgenic P23H mutant rhodopsin model of retinits pigmentosa. After discontinuation of cyclic light exposure, we monitored histologic progression of retinal regeneration over a 3 week recovery period. To assess their metabolomic states, contralateral eyes were processed for computational molecular phenotyping. We found that retinal degeneration in the P23H rhodopsin mutation could be partially reversed, with regeneration of rod photoreceptors recovering normal morphology (including full-length rod outer segments) by the end of the 3 week recovery period. In contrast, retinal degeneration mediated by directly induced apoptosis did not recover in the 3 week recovery period. Dystrophic rod photoreceptors with truncated rod outer segments were identified as the likely source of rod photoreceptor regeneration in the P23H retinas. These dystrophic photoreceptors remain metabolically active despite having lost most of their outer segments.

Topics: Amino Acid Substitution; Animals; Animals, Genetically Modified; Disease Models, Animal; Histidine; Mutation; Nerve Regeneration; Proline; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis

Appropriate termination of the phototransduction cascade is critical for photoreceptors to achieve high temporal resolution and to prevent excessive Ca(2+)-induced cell toxicity. Using a genetic screen to identify defective photoresponse mutants in Drosophila, we isolated and identified a novel Gα(q) mutant allele, which has defects in both activation and deactivation. We revealed that G(q) modulates the termination of the light response and that metarhodopsin/G(q) interaction affects subsequent arrestin-rhodopsin (Arr2-Rh1) binding, which mediates the deactivation of metarhodopsin. We further showed that the Gα(q) mutant undergoes light-dependent retinal degeneration, which is due to the slow accumulation of stable Arr2-Rh1 complexes. Our study revealed the roles of G(q) in mediating photoresponse termination and in preventing retinal degeneration. This pathway may represent a general rapid feedback regulation of G protein-coupled receptor signaling.

Topics: Alleles; Animals; Animals, Genetically Modified; Arrestins; Drosophila melanogaster; Drosophila Proteins; Electrophysiology; GTP-Binding Protein alpha Subunits, Gq-G11; Light; Light Signal Transduction; Models, Genetic; Mutation; Photoreceptor Cells, Invertebrate; Receptors, G-Protein-Coupled; Retinal Degeneration; Rhodopsin

Endocytosis-mediated cell death is a form of degeneration displayed in several Drosophila mutants. This form of degeneration is displayed in several Drosophila mutant lines including flies lacking the eye-specific PLC (norpA). The cell death pathway is initiated by the stabilization of complexes between rhodopsin and arrestin which undergo massive endocytosis into the cell body. The internalized rhodopsin becomes insoluble and builds up in the late endosomal system, wherein it triggers cell death. Cathepsins are resident late endosome/lysosome proteases that have been shown to mediate apoptosis in many disease models. Therefore we sought to test the involvement of cathepsins in endocytosis-mediated retinal degeneration. Here we show that cathepsins mediate cell death in light-exposed norpA eyes. Moreover, we show that the cathepsin L-like cysteine protease, CP1, specifically mediates retinal degeneration, while the aspartyl protease, cathepsin D, does not. Furthermore, eye-specific expression of pan-cathepsin inhibitors also blocks cell death. Western blot analysis demonstrates that cathepsin L levels remain unchanged during retinal degeneration. However, whole mount immunohistochemistry performed on light-exposed retinas revealed a decrease in cathepsin L levels and a loss of rhodopsin/ CP1 colocalization, suggesting that cathepsin L translocates during the degeneration process. Lastly, we show that the retinal degeneration can be enhanced by the overexpression of cathepsin L in the sensitized norpA background. Together these data show that cathepsins play a crucial role in endocytosis-mediated retinal degeneration and are consistent with a model where rhodopsin internalization and accumulation in the endosomal/lysosomal system triggers cathepsin translocation to the cytosol.

Topics: Animals; Blotting, Western; Cathepsin L; Cathepsins; Cell Death; Cytosol; Drosophila; Endocytosis; Endosomes; Immunohistochemistry; Lysosomes; Photoreceptor Cells, Invertebrate; Protein Transport; Retina; Retinal Degeneration; Rhodopsin

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

Topics: Aging; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Autophagy; Biomarkers; Calpain; Endoplasmic Reticulum Stress; Endoplasmic Reticulum-Associated Degradation; Extracellular Signal-Regulated MAP Kinases; Eye Proteins; Gene Expression Regulation; Humans; JNK Mitogen-Activated Protein Kinases; Mitochondria; Mutation; Oxidative Stress; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Rats; Retina; Retinal Degeneration; Rhodopsin; Time Factors; Transcription Factors

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Disease Models, Animal; Electroretinography; Exons; Eye Proteins; Female; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Mutant Strains; Molecular Sequence Data; Mutation; Retina; Retinal Degeneration; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid

The human rhodopsin (Rho) mutation T17M leads to autosomal dominant retinitis pigmentosa (adRP). The goal of our study was to elucidate the role of endoplasmic reticulum (ER) stress in retinal degeneration in hT17M Rho mice and identify potential candidates for adRP gene therapy.. We used transgenic mice expressing the ER stress-activated indicator (ERAI) and hT17M Rho to evaluate the activation of ER stress responses. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze changes in the expression of 30 unfolded protein response (UPR)-associated genes at P12, 15, 18, 21, and 25. The cytosolic fraction of hT17M Rho retinal cells was used to measure the release of cytochrome C and apoptotic inducing factor-1 (AIF1) by Western blotting. Optical coherence tomography (OCT) analysis was performed for 1-month-old hT17M Rho mice.. hT17M Rho was localized in the outer nuclear layer (ONL) of T17M(+/-)ERAI(+/-) photoreceptors as well as C57BL/6 retinas injected with AAV-hT17M Rho-GFP. In P15 hT17M Rho retinas, we observed an up-regulation of UPR genes (Atf4, Eif2α, Xbp1, Bip, Canx, and Hsp90), autophagy genes and proapoptotic Bcl2 genes. OCT, and the downregulation of Nrl and Crx gene expression confirmed that cell death occurs in 55% of photoreceptors via the up-regulation of caspase-3 and caspase-12, and the release of AIF1 from the mitochondria.. The ER stress response is involved in retinal degeneration in hT17M Rho mice. The final demise of photoreceptors occurs via apoptosis involving ER stress-associated and mitochondria-induced caspase activation. We identified Atg5, Atg7, Bax, Bid, Bik, and Noxa as potential therapeutic targets for adRP treatment.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Autophagy; Blotting, Western; Caspase 12; Caspase 3; Cytochrome c Group; Endoplasmic Reticulum Stress; Genes, bcl-2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Tomography, Optical Coherence; Unfolded Protein Response

The rhodopsin(-/-) C57Bl/6 (rho(-/-)) mouse is a very important model for understanding retinal degenerative diseases. In this study, spectral domain optical coherence tomography (SD-OCT) was used to monitor the dynamic morphological changes in retina of rho(-/-) mice.. Rho(-/-) mice and wild type C57Bl/6 (B6) mice at the age of 3, 6, 9, and 12 weeks were investigated using SD-OCT to obtain cross-sectional images of the retina. The outer nuclear layer (ONL) thickness was measured. Histological sections were used to compare to the OCT data. Electroretinograms (ERG) were performed to evaluate the physiological change for establishing the relationship between retinal morphology and its functional changes.. There was no apparent morphological or functional change in B6 mice at any time point. The SD-OCT measurement showed that the ONL thickness in rho(-/-) mice was significantly decreased from 40.6 μm to 6.0 μm from week 3 to week 12 postnatal. Histological examinations identified a similar trend, although the average thickness of ONL from histological sections at these time points was slightly larger (ranging from 55.4 μm to 14.9 μm). The ERG in rho(-/-) mice indicated functional changes that were in concordant with morphological changes; a significant linear positive association was identified between the amplitude of b-wave and the ONL thickness.. Our findings confirmed that the functional changes in retina were concordant with morphological changes measured by SD-OCT in vivo, which indicates that SD-OCT can be used as a reliable noninvasive method to monitor the degenerative progression in retinal disease models.

Topics: Animals; Disease Models, Animal; Electroretinography; Gene Silencing; Mice; Mice, Inbred C57BL; Mice, Knockout; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Rhodopsin; Tomography, Optical Coherence

Retinitis pigmentosa (RP) is a progressive retinal degeneration in which the retina loses nearly all of its photoreceptor cells and undergoes major structural changes. Little is known regarding the role the resident glia, the Müller glia, play in the progression of the disease. In this article, we define gene expression changes in Müller glial cells (MGCs) from two different mouse models of RP, the retinal degeneration 1 (rd1) and rhodopsin knockout (Rhod-ko) models. The RNA repertoire of single MGCs was comprehensively profiled, and a comparison was made between MGCs from wild-type (WT) and mutant retinas. Two time points were chosen for analysis, one at the peak of rod photoreceptor death and one during the period of cone photoreceptor death.. Retinas were dissociated, and single MGCs were chosen under a dissecting microscope using a micropipette. Single cell cDNAs were generated and genome-wide profiles were obtained by hybridization to Affymetrix arrays. A comparison was made among all samples to discover the changes in gene expression during the periods of rod and cone photoreceptor death.. MGCs respond to retinal degeneration by undergoing gliosis, a process marked by the upregulation of glial fibrillary acidic protein (Gfap). Many additional transcripts were found to change. These can be placed into functional clusters, such as retinal remodeling, stress response, and immune-related response.. A high degree of heterogeneity among the individual cells was observed, possibly due to their different spatial proximities to dying cells and/or inherent heterogeneity among MGCs.

Topics: Animals; Cell Death; Gene Expression; Gene Expression Profiling; Gene Knockout Techniques; Glial Fibrillary Acidic Protein; Mice; Nerve Tissue Proteins; Neuroglia; Oligonucleotide Array Sequence Analysis; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Single-Cell Analysis; Up-Regulation

In experimental eye research, zebrafish has become a powerful model for human retina disorders. The purpose of the present study is the characterization of antibodies commonly employed in zebrafish models for rod photoreceptor degeneration.. The 1D4 monoclonal antibody, developed against bovine rhodopsin, has been widely used in studies addressing structural and functional features of rhodopsin and was reported as an informative marker to stain rod outer segments in both mice and zebrafish. We have used transgenic reporter lines and histologic analysis to determine the photoreceptor types identified by 1D4 and other antibodies in zebrafish.. We demonstrate that 1D4, in contrast to what has been reported previously, does not recognize rod outer segments in zebrafish, but instead labels long double cone outer segments consistent with sequence conservation of the respective epitope. As an alternative marker for zebrafish rods, we characterized the monoclonal antibody zpr-3, which was found to stain outer segments of both rods, as well as double cones.. Our findings highlight the importance to confirm specificity of antibodies in cross-species experiments for correct interpretation of experimental data. Our findings clarify conflicting published information arising from studies using 1D4 and zpr-3 antibodies in zebrafish.

Topics: Animals; Antibodies, Monoclonal; Biomarkers; Blotting, Western; Cattle; Disease Models, Animal; Immunohistochemistry; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Sensitivity and Specificity; Zebrafish

The Dbp21E2 (DEAD box protein 21E2) is a member of a family of DEAD box helicases active in RNA processing and stability. The authors used genetic mosaics to identify mutants in Dbp21E2 that affect rhodopsin biogenesis and the maintenance of photoreceptor structure. Analysis of a green fluorescent protein (GFP)-tagged Rh1 rhodopsin construct placed under control of a heat shock promoter showed that Dbp21E21 fails to efficiently transport Rh1 from the photoreceptor cell body to the rhabdomere. Retinal degeneration is not dependent on the Rh1 transport defects. The authors also showed that GFP- and red fluorescent protein (RFP)-tagged Dbp21E2 proteins are localized to discrete cytoplasmic structures that are not associated with organelles known to be active in rhodopsin transport. The molecular genetic analysis described here reveals an unexpected role for the Dbp21E2 helicase and provides an experimental system to further characterize its function.

Topics: Animals; Animals, Genetically Modified; DEAD-box RNA Helicases; Drosophila; Drosophila Proteins; Electroretinography; Green Fluorescent Proteins; Humans; Microscopy, Electron, Transmission; Molecular Sequence Data; Mutagenesis; Mutation; Photoreceptor Cells, Invertebrate; Protein Transport; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

Topics: Animals; Cyclic Nucleotide-Gated Cation Channels; Dependovirus; Disease Models, Animal; DNA, Complementary; Electroretinography; Genetic Therapy; Genetic Vectors; Mice; Mice, Knockout; Nerve Tissue Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

To examine the effects of transcorneal electrical stimulation (TES) on retinal degeneration of light-exposed rats.. Thirty-three Sprague Dawley albino rats were divided into three groups: STIM (n = 15) received 60 minutes of TES, whereas SHAM (n = 15) received identical sham stimulation 2 hours before exposure to bright light with 16,000 lux; healthy animals (n = 3) served as controls for histology. At baseline and weekly for 3 consecutive weeks, dark- and light-adapted electroretinography was used to assess retinal function. Analysis of the response versus luminance function retrieved the parameters Vmax (saturation amplitude) and k (luminance to reach ½Vmax). Retinal morphology was assessed by histology (hematoxylin-eosin [HE] staining; TUNEL assay) and immunohistochemistry (rhodopsin staining).. Vmax was higher in the STIM group compared with SHAM 1 week after light damage (mean intra-individual difference between groups 116.06 μV; P = 0.046). The b-wave implicit time for the rod response (0.01 cd.s/m²) was lower in the STIM group compared with the SHAM group 2 weeks after light damage (mean intra-individual difference between groups 5.78 ms; P = 0.023); no other significant differences were found. Histological analyses showed photoreceptor cell death (TUNEL and HE) in SHAM, most pronounced in the superior hemiretina. STIM showed complete outer nuclear layer thickness preservation, reduced photoreceptor cell death, and preserved outer segment length compared with SHAM (HE and rhodopsin).. This sham-controlled study shows that TES can protect retinal cells against mild light-induced degeneration in Sprague Dawley rats. These findings could help to establish TES as a treatment in human forms of retinal degenerative disease.

Topics: Adaptation, Ocular; Animals; Electric Stimulation Therapy; Electroretinography; Immunohistochemistry; Light; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; Staining and Labeling

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

Topics: Animals; Cell Death; Drosophila melanogaster; Electroretinography; Fatty Acid Transport Proteins; Gene Expression; Mutation; Neurons; Photic Stimulation; Photoreceptor Cells, Invertebrate; Retina; Retinal Degeneration; Rhodopsin; Vitamin D

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. We used enhanced green fluorescent protein-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpA(P24) photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA(P24) is the failure to activate CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca(2+) entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. We investigated how reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration. To uncover the consequence underlying a lack of CaMKII activation, we characterized ala(1) flies in which CaMKII was suppressed by an inhibitory peptide, and showed that morphology of rhabdomeres was not affected. In contrast, we found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA(P24) photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration.

Topics: Animals; Animals, Genetically Modified; Arrestins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disease Models, Animal; Drosophila; Drosophila Proteins; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Mutation; Phospholipase C beta; Phosphorylation; Photoreceptor Cells, Vertebrate; Protein Binding; Retinal Degeneration; Rhodopsin; Sulfur Isotopes

To assess structural, functional, and visual behavioral relationships in mutant rhodopsin transgenic (Tg) rats and to determine whether early optokinetic tracking (OKT) visual experience, known to permanently elevate visual thresholds in normal rats, can enhance vision in rats with photoreceptor degeneration.. Eight lines of pigmented Tg rats and RCS rats were used in this study. OKT thresholds were tested at single ages (1, 2, 3, 4, and 6 months) in naïve groups of rats, or daily in groups that began at eye-opening (P15) or 10 days later (P25). Electroretinogram (ERG) response amplitudes were recorded after OKT testing, and outer nuclear layer (ONL) thickness measurements were then obtained.. OKT thresholds, when measured at a single time point in naïve Tg lines beginning at P30, did not decline until months after significant photoreceptor loss. Daily testing of Tg lines resulted mostly with OKT thresholds inversely related to photoreceptor degeneration, with rapid degenerations resulting in sustained OKT thresholds for long periods despite the rapid photoreceptor loss. Slower degenerations resulted in rapid decline of thresholds, long before the loss of most photoreceptors, which was even more pronounced when daily testing began at eye opening. This amplified loss of function was not a result of testing-induced damage to the rod or cone photoreceptors, as ERG amplitudes and ONL thicknesses were the same as untested controls.. The unexpected lack of correlation of OKT testing with photoreceptor degeneration in the Tg rats emphasizes the need in behavioral therapeutic studies for careful analysis of visual thresholds of experimental animals prior to therapeutic intervention.

Topics: Animals; Disease Models, Animal; Electroretinography; Mutation; Rats; Rats, Transgenic; Retinal Degeneration; Rhodopsin; Sensory Thresholds; Visual Perception

The Müller glial cells exhibit stem cell properties and express neuronal markers following experimentally induced retinal injury. However, it is not known whether Müller glia respond similarly to degenerative neuronal loss caused by genetic mutation. Here, we asked whether Müller cells dedifferentiate and express neuronal proteins in rd1 mouse, a naturally occurring mutant model of inherited retinal degeneration. Using immunohistochemistry and Western blotting, we studied expression patterns of glial fibrillary acidic protein (GFAP), nestin, rhodopsin, protein kinase C alpha (PKCα), β-III-tubulin and recoverin in Müller glia. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to detect any rhodopsin mRNA in the rd1 mouse retina. We found that Müller cell processes in rd1 mouse hypertrophied and overexpressed GFAP as early as postnatal day (P)-14, features that were maintained throughout development and in the adult stage. Furthermore, Müller cells continued to express nestin, a progenitor cell marker, up to 6 months of age, raising the possibility that they remain undifferentiated for several months in rd1 mouse. We did not find nestin expression in Müller cells in 1-year-old rd1 mouse. Interestingly, Müller cell processes in rd1 mouse also expressed rhodopsin, a rod-specific protein. The rhodopsin expression in Müller cells was evident at P-21, and remained so up to at least 1 year of age. The expression of rhodopsin by Müller cells was further supported by our finding of the rhodopsin transcript in the 9-month-old rd1 mouse retina. We did not find the expression of PKCα, β-III-tubulin or recoverin in Müller cells in adult rd1 mouse. These results suggested that Müller cells in rd1 mouse express proteins specific to retinal neurons that are the primary targets of the mutation in this mouse. Although the functional significance of rhodopsin expression by Müller cells is unclear, these results have implications for novel therapeutic strategies for retinal degeneration.

Topics: Age Factors; Animals; Animals, Newborn; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Gene Expression Regulation, Developmental; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Nerve Tissue Proteins; Neuroglia; Protein Kinase C; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger

The aim of this study was to examine the temporal relationship between behaviorally measured visual thresholds, photoreceptor degeneration and dysfunction, synaptic and neuronal morphology changes in the retina in the S334ter line 4 rat. Specifically, we examined the optokinetic tracking (OKT) behavior in S334ter rats daily and found that OKT thresholds reflected normal values at eye opening but quickly reduced to a non-response level by postnatal day (P) 22. By contrast, the scotopic electroretinogram (ERG) showed a much slower degeneration, with substantial scotopic function remaining after P90 as previously demonstrated for this line of rats. Photopic b-wave amplitudes revealed functional levels between 70 and 100% of normal between P30 and P90. Histological evidence demonstrated that photoreceptor degeneration occurred over many months, with an outer nuclear layer (ONL) roughly half the thickness of a normal age-matched control at P90. Immunohistochemical analysis revealed a number of changes in retinal morphology in the Tg S334ter line 4 rat that occur at or before P40 including: elevated levels of rod opsin expression in the ONL, cone photoreceptor morphology changes, glial cell activation, inner retinal neuron sprouting, and microglial cell activation. Many of these changes were evident at P30 and in some cases as early as eye opening (P15). Thus, the morphological changes occurred in concert with or before the very rapid loss of the behavioral (OKT) responses, and significantly before the loss of photoreceptors and photoreceptor function.

Topics: Animals; Biomarkers; Cell Survival; Electroretinography; Fluorescent Antibody Technique, Indirect; Microscopy, Confocal; Mutation; Neuroglia; Nystagmus, Optokinetic; Photoreceptor Cells, Vertebrate; Rats; Rats, Long-Evans; Rats, Transgenic; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Sensory Thresholds; Visual Perception

Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes.. Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses.. S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss.. Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

Topics: Animals; bcl-2-Associated X Protein; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum Stress; Environmental Exposure; Gene Expression Regulation; Mice; Mice, Inbred BALB C; Oligopeptides; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; RNA; Transcription Factor CHOP

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.

Topics: Aging; Animals; Cytoplasm; Drosophila melanogaster; Drosophila Proteins; Electroretinography; Female; Gene Knockdown Techniques; Genes, Insect; Guanine Nucleotide Exchange Factors; Light; Male; Mutation; Photoreceptor Cells, Invertebrate; Protein Binding; Protein Transport; rab GTP-Binding Proteins; Retinal Degeneration; Rhodopsin

The tubby (tub) and tubby-like protein (tulp) genes encode a small family of proteins found in many organisms. Previous studies have shown that TUB and TULP genes in mammalian involve in obesity, neural development, and retinal degeneration. The purpose of this study was to investigate the role of Drosophila king tubby (ktub) in rhodopsin 1 (Rh1) endocytosis and retinal degeneration upon light stimulation.. Drosophila ktub mutants were generated using imprecise excision. Wild type and mutant flies were raised in dark or constant light conditions. After a period of light stimulation, retinas were dissected, fixed and stained with anti-Rh1 antibody to reveal Rh1 endocytosis. Confocal and transmission electron microscope were used to examine the retinal degeneration. Immunocytochemical analysis shows that Ktub is expressed in the rhabdomere domain under dark conditions. When flies receive light stimulation, the Ktub translocates from the rhabdomere to the cytoplasm and the nucleus of the photoreceptor cells. Wild type photoreceptors form Rh1-immunopositive large vesicles (RLVs) shortly after light stimulation. In light-induced ktub mutants, the majority of Rh1 remains at the rhabdomere, and only a few RLVs appear in the cytoplasm of photoreceptor cells. Mutation of norpA allele causes massive Rh1 endocytosis in light stimulation. In ktub and norpA double mutants, however, Rh1 endocytosis is blocked under light stimulation. This study also shows that ktub and norpA double mutants rescue the light-induced norpA retinal degeneration. Deletion constructs further demonstrate that the Tubby domain of the Ktub protein participates in an important role in Rh1 endocytosis.. The results in this study delimit the novel function of Ktub in Rh1 endocytosis and retinal degeneration.

Topics: Animals; Arrestins; Drosophila melanogaster; Drosophila Proteins; Endocytosis; Gene Expression Regulation; Light; Mutation; Phospholipase C beta; Photoreceptor Cells, Invertebrate; Retina; Retinal Degeneration; Rhodopsin

The failure of the adult mammalian retina to regenerate can be partly attributed to the barrier formed by inhibitory extracellular matrix (ECM) and cell adhesion molecules, such as CD44 and neurocan, after degeneration. These molecules act to separate a sub-retinal graft from integrating into the host retina. It has been shown that matrix metalloproteinase 2 (MMP2) can promote host-donor integration by degrading these molecules. In order to enhance cellular integration and promote retinal repopulation, we co-transplanted biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres that have the ability to deliver active MMP2 with retinal progenitor cells (RPCs) to the sub-retinal space of adult retinal degenerative Rho-/- mice. Following delivery, significant degradation of CD44 and neurocan at the outer surface of the degenerative retina without disruption of the host retinal architecture was observed. Coincident with this, we observed a significant increase in the number of cells migrating beyond the barrier into the degenerative retina. No changes in the differentiation characteristics of RPCs were observed. Cells in the outer nuclear layer (ONL) could express the mature photoreceptor markers recoverin, make contacts with residual protein kinase C (PKC)-positive cells and express the ribbon synapse protein bassoon. Thus, co-transplantation of MMP2-PLGA microspheres with RPCs provides controlled release of active MMP2 to the site of retinal degeneration, stimulating inhibitory barrier removal and enhancing cell integration. This suggests a practical and effective strategy for retinal repair.

Topics: Animals; Biocompatible Materials; Drug Carriers; Drug Delivery Systems; Humans; Hyaluronan Receptors; Lactic Acid; Materials Testing; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Mice, Knockout; Microspheres; Neurocan; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Regeneration; Retina; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Stem Cells

This study focuses on the effects of retinal degeneration on the circadian patterns of P23H rats, as well as on the effect of exogenous melatonin administration. To this end, the body temperature of P23H and Sprague-Dawley rats was continuously monitored and their retinas examined at different stages of degeneration, by means of histological labeling and electroretinogram recordings. Melatonin (2 mg/kg BW/day) was supplied ad libitum throughout the experiment to a subset of animals. The body temperature recordings from wild-type and mutant animals showed no differences in the periodogram and the pattern of the mean waveform. However, a progressive decrease in the relative amplitude of the rhythm (RA), a decline in the coupling strength of the rhythm to environmental zeitgebers (interdaily stability, IS) and increased rhythm fragmentation (intradaily variability, IV) were observed in P23H rats, when compared to wild-type animals. The P23H animals showed a progressive decrease in light-induced retinal responses until reaching 18 months of age. By this age, all photoreceptors had already disappeared, and no responses were found in the EGRs. Exogenous administration of melatonin improved the visual response of P23H rats. In fact, the maximum b-wave recorded at 14 months of age was significantly higher in melatonin-treated P23H rats than in the control animals. Furthermore, the maximum b-wave recorded for P23H rats at the age of 14 months significantly correlated with RA, IS, and IV. This leads us to conclude that vision loss in P23H rats is correlated with a progressive fragmentation of their circadian patterns. Both effects are partially reversed by melatonin administration.

Topics: Animals; Animals, Genetically Modified; Antioxidants; Body Temperature; Circadian Rhythm; Electroretinography; Male; Melatonin; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin

XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model for investigating the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild-type and XOPS-mCFP retinas and identify genes that may contribute to the regeneration of the rods.. Adult wild-type and XOPS-mCFP retinal mRNA was subjected to microarray analysis. Pathway analysis was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization and immunohistochemistry and in a transgenic fluorescent reporter line.. More than 600 genes displayed significant expression changes in XOPS-mCFP retinas compared with expression in wild-type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, and cell development and death. RT-PCR analysis of a subset of these genes confirmed the microarray. Three transcription factors (sox11b, insm1a, and c-myb), displaying increased expression in XOPS-mCFP retinas, were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone.. This study identified numerous gene expression changes in response to rod degeneration in zebrafish and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration.

Topics: Animals; Animals, Genetically Modified; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Profiling; Gene Expression Regulation; Green Fluorescent Proteins; In Situ Hybridization; Male; Microarray Analysis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myb; Recombinant Fusion Proteins; Regeneration; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; SOX Transcription Factors; Transcription Factors; Transgenes; Zebrafish; Zebrafish Proteins

Chemokine receptors are reported to be involved in neuronal cell death and CNS neurodegenerative diseases. The aim of the current study was to investigate the expression of CCR1, a major chemokine receptor for CC chemokines in retinal dystrophy in rd (retinal degeneration) mice and further explore its role in photoreceptor degeneration.. The expression levels of CCR1 mRNA in the whole control and rd retinas at postnatal days (P) 8, 10, 12, 14, 16, and 18 were determined by RT-PCR assay. Location of CCR1 in the retina of rd mice at each age group was studied by immunohistochemical analysis. Expression of CCR1 in the photoreceptor cells and apoptotic cells was determined by double labeling.. Expression of CCR1 mRNA was noted in both control and rd retinas at each age group. CCR1-positive cells started to emerge in the outer nuclear layer (ONL) in rd retinas at P8 and reached a peak at P12 and P14. Double labeling of CCR1 with rhodopsin, CD11b, or TUNEL staining showed expression of CCR1 in the photoreceptor cells, rather than in the microglial cells. Partial CCR1 expression was observed in some of the apoptotic photoreceptor cells.. Expression of CCR1 in the photoreceptor cells was increased with the progress of retinal degeneration in rd mice. Activation of CCR1 may play a role in the photoreceptor apoptosis.

Topics: Aging; Animals; Animals, Newborn; Apoptosis; CD11b Antigen; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; In Situ Nick-End Labeling; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Photoreceptor Cells, Vertebrate; Receptors, CCR1; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger

Rhodopsin (Pro347Leu) transgenic pigs are recognized to be an excellent model for the human disease, retinitis pigmentosa. First published in 1997, the rhodopsin transgenic pigs have been maintained since that time at North Carolina State University by outcrossing hemizygous boars to unrelated sows. Nine generations of outcrossing have been completed. Since the genetic background of these pigs has undoubtedly changed, the question of the current phenotype of the transgenic pigs is relevant for their future use. Age-matched transgenic and non-transgenic eyes were submitted for histological analysis using hematoxylin and eosin staining. Even by 2 weeks of age, significant thinning of the outer nuclear layer of photoreceptors was observed. For ages 3 and 4 weeks, thinning was noted similar to that of 2 weeks of age. By 6 weeks of age outer nuclear layer thinning was greater than that of earlier age. At 11 weeks of age, most of the rods have degenerated leaving only a few layers of cones. In all, the phenotype, based on assessment of photoreceptor degeneration, is similar to that of the first description of the transgenic animals. As such the Pro347Leu rhodopsin transgenic pigs have exhibited phenotypic stability through generations of outcrossing and can be used confidently in future studies of the type of retinal degeneration seen with retinitis pigmentosa.

Topics: Age Factors; Animals; Animals, Genetically Modified; Eosine Yellowish-(YS); Eye; Female; Hematoxylin; Inbreeding; Inheritance Patterns; Male; Phenotype; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Rhodopsin; Staining and Labeling; Swine; Transgenes

Electrical stimulation threshold and retinal ganglion cell density were measured in a rat model of retinal degeneration. We performed in vivo electrophysiology and morphometric analysis on normal and S334ter line 3 (RD) rats (ages 84-782 days). We stimulated the retina in anesthetized animals and recorded evoked responses in the superior colliculus. Current pulses were delivered with a platinum-iridium (Pt-Ir) electrode of 75-μm diameter positioned on the epiretinal surface. In the same animals used for electrophysiology, SMI-32 immunolabeling of the retina enabled ganglion cell counting. An increase in threshold currents positively correlated with age of RD rats. SMI-32-labeled retinal ganglion cell density negatively correlated with age of RD rats. ANOVA shows that RD postnatal day (P)100 and P300 rats have threshold and density similar to normal rats, but RD P500 and P700 rats have threshold and density statistically different from normal rats (P < 0.05). Threshold charge densities were within the safety limits of Pt for all groups and pulse configurations, except at RD P600 and RD P700, where pulses were only safe up to 1- and 0.2-ms duration, respectively. Preservation of ganglion cells may enhance the efficiency and safety of electronic retinal implants.

Topics: Aging; Analysis of Variance; Animals; Cell Count; Disease Models, Animal; Electric Stimulation; Evoked Potentials; Light; Mutation; Neurofilament Proteins; Neurons; Rats; Rats, Mutant Strains; Rats, Transgenic; Retina; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Sensory Thresholds; Superior Colliculi

The eukaryotic endoplasmic reticulum operates multiple quality control mechanisms to ensure that only properly folded proteins are exported to their final destinations via the secretory pathway and those that are not are destroyed via the degradation pathway. However, molecular mechanisms underlying such regulated exportation to these distinct routes are unknown. In this article, we report the role of Drosophila arf72A--the fly homologue of the mammalian Arl1 - in the quality checks of proteins and in the autosomal-dominant retinopathy. ARF72A localizes to the Golgi membranes of Drosophila photoreceptor cells, consistent with mammalian Arl1 localization in cell culture systems. A loss of arf72A function changes the membrane character of the endoplasmic reticulum and shifts the membrane balance between the endoplasmic reticulum and the Golgi complex toward the Golgi complex, resulting in over-proliferated Golgi complexes and accelerated protein secretion. Interestingly, our study indicated that more ARF72A localized on the endoplasmic reticulum in the ninaE(D1) photoreceptor cell, a Drosophila model of autosomal-dominant retinitis pigmentosa, compared to that in the wild-type. In addition, arf72A loss was shown to rescue the ninaE(D1)-related membrane accumulation and the rhodopsin maturation defect, and suppress ninaE(D1)-triggered retinal degeneration, indicating that rhodopsin accumulated in the endoplasmic reticulum bypasses the quality checks. While previous studies of ARF small GTPases have focused on their roles in vesicular budding and transport between the specific organelles, our findings establish an additional function of arf72A in the quality check machinery of the endoplasmic reticulum distinguishing the cargoes for secretion from those for degradation.

Topics: ADP-Ribosylation Factors; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum; Feedback, Physiological; Golgi Apparatus; Green Fluorescent Proteins; Membrane Proteins; Photoreceptor Cells, Invertebrate; Protein Folding; Protein Transport; Recombinant Fusion Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

Phosphoinositide 3-kinase (PI3K) consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The biological significance of PI3K was investigated in vertebrate rod photoreceptors by deleting its regulatory p85α protein and examining its role in photoreceptor structure, function, and protein trafficking.. Mice that expressed Cre recombinase in rods were bred to mice with a floxed p85α (pik3r1) regulatory subunit of PI3K to generate a conditional deletion of pik3r1 in rods. Functional and structural changes were determined by ERG and morphometric analysis, respectively. PI3K activity was measured in retinal homogenates immunoprecipitated with an anti-PY antibody. Akt activation was determined by Western blot analysis with a pAkt antibody.. Light-induced stress increased PI3K activity in retinal immunoprecipitates and phosphorylation of Akt. There was no effect of pik3r1 deletion on retinal structure. However, twin flash electroretinography revealed a slight delay in recovery kinetics in pik3r1 knockout (KO) mice compared with wild-type controls. The movement of arrestin in the pik3r1 KO mice was slower than that in the wild-type mouse retinas at 5 minutes of exposure to light. At 10 minutes of exposure, the ROS localization of arrestin was almost identical between the wild-type and pik3r1 KO mice.. The results provide the first direct evidence that rods use PI3K-generated phosphoinositides for photoreceptor function. The lack of phenotype in pik3r1 KO rod photoreceptors suggests a redundant role in controlling PIP(3) synthesis.

Topics: Animals; Arrestin; Blotting, Western; Class Ia Phosphatidylinositol 3-Kinase; Electroretinography; Gene Deletion; Integrases; Light; Mice; Mice, Knockout; Mice, Transgenic; Microscopy, Fluorescence; Phosphatidylinositol 3-Kinase; Phosphorylation; Photic Stimulation; Protein Transport; Proto-Oncogene Proteins c-akt; Radiation Injuries, Experimental; Rats; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction; Transducin

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.

Topics: Animals; Chlorocebus aethiops; COS Cells; Curcumin; Immunohistochemistry; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Degeneration; Rhodopsin

Iodoacetic acid (IAA) induces photoreceptor (PR) degeneration in small animal models, however, eye size and anatomic differences detract from the usefulness of these models for studying retinal rescue strategies intended for humans. Porcine eyes are closer in size to human eyes and have a rich supply of rod and cones. This study investigated whether IAA also produced PR degeneration in the porcine retina, whether the damage was preferential for rods or cones, and whether IAA induced remodeling of the inner retina. Pigs were given a single i.v. injection of IAA and were euthanized 2-5 weeks later. Eyes were enucleated and immersed in fixative. Forty-six eyes were studied: Control (n = 13), and from pigs that had received the following IAA doses: 5.0 mg/kg (n = 7); 7.5 mg/kg (n = 10); 10.0 mg/kg (n = 6); 12.0 mg/kg (n = 6). Tissue was retrieved from four retinal locations: 8 mm and 2 mm above the dorsal margin of the optic disc, and 2 mm and 8 mm below the disc, and was processed for conventional histology, immunohistochemistry, and transmission electron microscopy. At 5.0 mg/kg IAA produced mild, variable cell loss, but remaining cells exhibited normal features. At doses above 5.0 mg/kg, a dose-dependent reduction was observed in the length of PR inner and outer segments, and in the number of PR nuclei. Specific labeling revealed a massive dropout of rod cell bodies with relative sparing of cone cell bodies, and electron microscopy revealed a reduction in the number of PR synaptic terminals. Mild dendritic retraction of rod bipolar cells and hypertrophy of Müller cell stalks was also observed, although the inner nuclear layer appeared intact. The porcine IAA model may be useful for developing and testing retinal rescue strategies for human diseases in which rods are more susceptible than cones, or are affected earlier in the disease process.

Topics: Animals; Biomarkers; Calbindins; Cell Count; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glial Fibrillary Acidic Protein; Immunohistochemistry; Injections, Intravenous; Iodoacetic Acid; Microscopy, Confocal; Microscopy, Electron, Transmission; Photoreceptor Cells, Vertebrate; Protein Kinase C-alpha; Retinal Degeneration; Rhodopsin; S100 Calcium Binding Protein G; Sus scrofa; Vimentin

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Topics: Acyltransferases; Alcohol Oxidoreductases; Animals; Carrier Proteins; cis-trans-Isomerases; Cyclic N-Oxides; Eye Proteins; Light; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)--the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself--as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model.. A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 μM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 μM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 μM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells.. Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsin⁺ photoreceptors and the calbindin⁺ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 μM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10⁺ or the Islet-1⁺ bipolar cells and the Pax-6⁺ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP⁺ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection.. Excessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.

Topics: Animals; Calbindins; Diabetic Retinopathy; Dose-Response Relationship, Drug; Eye Proteins; Female; Glial Fibrillary Acidic Protein; Homeodomain Proteins; Homocysteine; Hyperhomocysteinemia; Immunoblotting; Immunohistochemistry; Intravitreal Injections; LIM-Homeodomain Proteins; Mice; Mice, Inbred ICR; Nerve Tissue Proteins; Paired Box Transcription Factors; PAX6 Transcription Factor; Photoreceptor Cells; Repressor Proteins; Retina; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; S100 Calcium Binding Protein G; Transcription Factors

To determine how the different stages of retinal processing change after photoreceptor degeneration in rabbits carrying the Pro347Leu rhodopsin mutation (Tg rabbits).. Cone electroretinograms (ERGs) were elicited by 150-ms duration stimuli from 13 Tg rabbits at 12 and 24 weeks of age. The ERG recordings were made before and after an intravitreal injection of tetrodotoxin citrate (TTX) plus N-methyl-dl-aspartic acid (NMDA), with the addition of 2-amino-4-phosphonobutyric acid (APB) and then cis-2,3-piperidine-dicarboxylic acid (PDA) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Digital subtraction of the ERG after the injection from the ERG before the injection was used to extract the components that were blocked by these drugs. Thirteen age-matched, wild-type (WT) rabbits were studied with the same protocol.. In Tg rabbits, the cone ERGs elicited by intermediate intensities had a depolarizing pattern. At 12 weeks of age, the photoreceptor and OFF-bipolar/horizontal cell responses reflected in the ERG in the Tg rabbits did not differ significantly from those in the WT rabbits. The ON-bipolar cells and the third-order neuronal responses recorded after pharmacologic blockade were significantly enhanced in the Tg rabbits compared with those recorded in the WT rabbits. At 24 weeks of age, the ERG waveforms representing the photoreceptors and OFF-bipolar/horizontal cell responses were significantly decreased, but those representing the ON-bipolar cell and third-order neuronal responses were still preserved in the Tg rabbits.. A depolarizing pattern of the cone ERG responses was seen in Pro347Leu Tg rabbits. The enhancement or preservation of the ON-bipolar cell response in the ERGs contributed to shaping the waveform in the Tg rabbits. In this model, the functional alterations in the ON-pathway took place before the deterioration of cone photoreceptor function.

Topics: Aminobutyrates; Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Intravitreal Injections; Point Mutation; Rabbits; Retinal Bipolar Cells; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Tetrodotoxin; Vision, Ocular

Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51.. Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors.. Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons.. The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.

Topics: Arrestin; Bruch Membrane; Carrier Proteins; cis-trans-Isomerases; Electroretinography; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Humans; Male; Middle Aged; Mutation, Missense; Optic Nerve; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Pigment Epithelium; Rhodopsin; Vision Disorders; Visual Acuity; Visual Fields

In this study we investigated the biochemical and cell biologic characteristics of flies expressing two novel dominant alleles of the major rhodopsin encoding gene neither inactivation nor afterpotential E (ninaE) in a heterozygous background.. Presence of the deep pseudopupil in flies was assayed 5 days post eclosion. For structural analysis, 1-μm-retinal cross sections were obtained from fixed and resin-embedded Drosophila heads. Confocal microscopy was performed on dissected retinas stained with antibodies specific for rhodopsin, NinaA, and F-actin. Rhodopsin levels were determined by western and slot blot analysis.. Dominant rhodopsin mutants showed progressive age-dependent and light-independent loss of the deep pseudopupil, without any apparent retinal degeneration at the morphological level. Expression of mutant rhodopsin caused rhodopsin to mislocalize to the cell body and the endoplasmic reticulum compartment. Mutant rhodopsin also caused loss of solubility of wild-type rhodopsin and its accumulation presumably as a high molecular mass complex in the photoreceptor cell body.. In heterozygous mutant flies, there is loss of wild-type rhodopsin immunoreactivity on a western assay but less reduction using slot blot analysis. This suggests that mutant rhodopsin is likely inducing the misfolding and insolubility of wild-type rhodopsin. Localization of rhodopsin revealed that in mutant flies, wild-type rhodopsin is mislocalized to the cell body and the endoplasmic reticulum.

Topics: Actins; Aging; Alleles; Animals; Blotting, Western; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum; Genes, Dominant; Heterozygote; Humans; Microscopy, Confocal; Mutation; Photoreceptor Cells, Invertebrate; Protein Folding; Retina; Retinal Degeneration; Rhodopsin

To determine the efficacy of Fourier domain optical coherence tomography (FD-OCT) as a noninvasive, nonlethal method for detecting in vivo, pathologic signs of retinal degeneration in Xenopus laevis larvae.. A prototype OCT system using FD detection customized for tadpole imaging was used to noninvasively obtain retinal scans in two different transgenic X. laevis models of retinal degeneration. FD-OCT retinal scans were compared with laser scanning confocal micrographs of histologic sections of the same eye. Retinal thickness was measured in the histologic micrographs and compared with in vivo measurements acquired with FD-OCT.. In vivo retinal images of X. laevis tadpoles were obtained that visualized the major retinal layers. FD-OCT successfully detected the ablation of rod outer segments (OS) in degenerating tadpole eyes. Measurements from FD-OCT and histology showed a decrease in retinal thickness in transgenic mutant tadpoles relative to the wild-type control. The accumulation of phagosomes from dying rod OS was also visualized in the retinal pigment epithelium (RPE) in a degenerating tadpole retina.. This report demonstrates that FD-OCT is a viable technique for screening, diagnosing, and monitoring retinal degeneration in X. laevis tadpoles in vivo.

Topics: Animals; Animals, Genetically Modified; Caspase 9; Disease Models, Animal; Female; Fluorescent Antibody Technique, Indirect; Fourier Analysis; Larva; Male; Microscopy, Confocal; Phagosomes; Retinal Degeneration; Retinal Pigment Epithelium; Rhodopsin; Rod Cell Outer Segment; Tomography, Optical Coherence; Xenopus laevis

To identify a new mouse mutation developing early-onset dominant retinal degeneration, to determine the causative gene mutation, and to investigate the underlying mechanism.. Retinal phenotype was examined by indirect ophthalmoscopy, histology, transmission electron microscopy, immunohistochemistry, Western blot analysis, and electroretinography. Causative gene mutation was determined by genomewide linkage analysis and DNA sequencing. Structural modeling was used to predict the impact of the mutation on protein structure.. An ENU-mutagenized mouse line (R3), displaying attenuated retinal vessels and pigmented patches, was identified by fundus examination. Homozygous R3/R3 mice lost photoreceptors rapidly, leaving only a single row of photoreceptor nuclei at postnatal day 18. The a- and b-waves of ERG were flat in R3/R3 mice, whereas heterozygous R3/+ mice showed reduced amplitude of a- and b-waves. The R3/+ mice had a slower rate of photoreceptor cell loss than compound heterozygous R3/- mice with a null mutant allele. The R3 mutation was mapped and verified to be a rhodopsin point mutation, a c.553T>C for a p.C185R substitution. The side chain of Arg(185) impacted on the extracellular loop of the protein. Mutant rhodopsin-C185R protein accumulated in the photoreceptor inner segments, cellular bodies, or both.. Rhodopsin C185R mutation leads to severe retinal degeneration in R3 mutant mice. A dosage-dependent accumulation of misfolded mutant proteins likely triggers or stimulates the death of rod photoreceptors. The presence of a wild-type rhodopsin allele can delay the loss of photoreceptor cells in R3/+ mice.

Topics: Animals; Base Sequence; Blotting, Western; DNA Mutational Analysis; Electroretinography; Ethylnitrosourea; Genes, Dominant; Genome-Wide Association Study; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Molecular Sequence Data; Mutagenesis; Ophthalmoscopy; Phenotype; Photoreceptor Cells, Vertebrate; Point Mutation; Protein Structure, Secondary; Retinal Degeneration; Rhodopsin

Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death.. Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light.. Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times.. Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

Topics: Animals; Apoptosis; Cell Survival; Chromosomes, Artificial, Bacterial; Electrophysiology; Female; Fluorescent Antibody Technique, Indirect; G-Protein-Coupled Receptor Kinase 1; Gene Expression Regulation, Enzymologic; Genotype; Immunoblotting; In Situ Nick-End Labeling; Light; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Phosphorylation; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.

Topics: Animals; Brain; Calpain; Caspase 3; Cell Survival; Cells, Cultured; Coculture Techniques; Embryonic Stem Cells; Glial Fibrillary Acidic Protein; Humans; In Situ Nick-End Labeling; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred C3H; Microscopy, Fluorescence; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin

To characterize the influence of light and vitamin A on retinal degeneration in an animal model of retinitis pigmentosa caused by the rhodopsin P23H mutation.. Retinal degeneration was examined in transgenic Xenopus laevis expressing P23H rhodopsin, in which retinal degeneration is completely rescued by preventing light exposure. The sensitivity of this retinal degeneration to varying intensities, wavelengths, and durations of light exposure, and to vitamin A deprivation was characterized.. Green light was the most effective inducer of retinal degeneration in this model. Retinal degeneration was induced by prolonged exposure to green light and was prevented by filters that block short wavelengths. Reducing the duration of light exposure prevented retinal degeneration, even when the light intensity was proportionally increased. Vitamin A deprivation also induced retinal degeneration associated with defects in P23H rhodopsin biosynthesis. Vitamin A deprivation did not cause retinal degeneration in nontransgenic animals.. The mechanism of retinal degeneration in this animal model of RP involves the interaction of light with rhodopsin rather than with free chromophore or bleached rhodopsin. These results may explain the clinical benefits of vitamin A for patients with retinitis pigmentosa and may indicate that pharmacological chaperones are a viable approach to RP therapy. Results also suggest strategies for minimizing RD in patients through controlling light exposure duration or wavelengths.

Topics: Animals; Animals, Genetically Modified; Light; Male; Microscopy, Confocal; Mutation, Missense; Photometry; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Rhodopsin; Transgenes; Vitamin A; Vitamin A Deficiency; Xenopus laevis

Cone photoreceptors are responsible for color and central vision. In the late stage of retinitis pigmentosa and in geographic atrophy associated with age-related macular degeneration, cone degeneration eventually causes loss of central vision. In the present work, we investigated cone degeneration secondary to rod loss in the S334ter-3 transgenic rats carrying the rhodopsin mutation S334ter.. Recombinant human ciliary neurotrophic factor (CNTF) was delivered by intravitreal injection to the left eye of an animal, and vehicle to the right eye. Eyes were harvested 10 days after injection. Cone outer segments (COS), and cell bodies were identified by staining with peanut agglutinin and cone arrestin antibodies in whole-mount retinas. For long-term treatment with CNTF, CNTF secreting microdevices were implanted into the left eyes at postnatal day (PD) 20 and control devices into the right eyes. Cone ERG was recorded at PD 160 from implanted animals. Our results demonstrate that an early sign of cone degeneration is the loss of COS, which concentrated in many small areas throughout the retina and is progressive with age. Treatment with CNTF induces regeneration of COS and thus reverses the degeneration process in early stages of cone degeneration. Sustained delivery of CNTF prevents cones from degeneration and helps them to maintain COS and light-sensing function.. Loss of COS is an early sign of secondary cone degeneration whereas cell death occurs much later. At early stages, degenerating cones are capable of regenerating outer segments, indicating the reversal of the degenerative process. Sustained delivery of CNTF preserves cone cells and their function. Long-term treatment with CNTF starting at early stages of degeneration could be a viable strategy for preservation of central vision for patients with retinal degenerations.

Topics: Animals; Ciliary Neurotrophic Factor; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Models, Biological; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Recombinant Proteins; Regeneration; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin

We have recently developed transgenic X. laevis models of retinitis pigmentosa based on the rhodopsin P23H mutation in the context of rhodopsin cDNAs derived from several different species. The mutant rhodopsin in these animals is expressed at low levels, with levels of export from the endoplasmic reticulum to the outer segment that depend on the cDNA context. Retinal degeneration in these models demonstrates varying degrees of light dependence, with the highest light dependence coinciding with the highest ER export efficiency. Rescue of light dependent retinal degeneration by dark rearing is in turn dependent on the capacity of the mutant rhodopsin to bind chromophore. Our results indicate that rhodopsin chromophore can act in vivo as a pharmacological chaperone for P23H rhodopsin, and that light-dependent retinal degeneration caused by P23H rhodopsin is due to reduced chromophore binding.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum; Light; Models, Biological; Mutant Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65(-/-) mice lack 11-cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65(-/-) mice. Retina-RPE-choroids of Rpe65(-/-) mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65(-/-) mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at approximately 40-kDa as a major band in wild-type mice, whereas approximately 42-kDa S-opsin protein was detected in Rpe65(-/-) mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65(-/-) mice on SDS-PAGE was similar. In addition, approximately 25-kDa S-opsin polypeptide was notably detected in Rpe65(-/-) mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65(-/-) mice, but expression of M-opsin mRNA in Rpe65(-/-) mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65(-/-) mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65(-/-) mice.

Topics: Animals; Blotting, Western; Carrier Proteins; Choroid; cis-trans-Isomerases; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Glycosylation; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Protein Processing, Post-Translational; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Rod Opsins

Optokinetic testing is a non-invasive technique, widely used for visual functional evaluation in rodents. The modulatory influence of optokinetic stimulus parameters such as contrast level and grating speed on head-tracking response in normal and retinal degenerate (RD) mice (rd10) and rats (S334ter-line-3) was evaluated using a computer-based testing apparatus. In normal (non-RD) mice and rats, specific stripe width and grating speed was found to evoke maximum optokinetic head-tracking response. In line-3 RD rats, the contrast sensitivity loss was slow and remained close to the baseline (normal control) level until very late in the disease, whereas, in rd10 mice the progression of the contrast sensitivity loss was more rapid. Observed differences between rd10 mice and line-3 RD rats in the progression of contrast sensitivity loss may not be directly related to the degree of photoreceptor loss. In young RD mice, the modulatory influence of stimulus parameters on optokinetic head-tracking response was similar to normal control animals. During later stages, slower grating speed was required to evoke the maximum optokinetic response. Grating speed had lesser apparent influence on the response properties of line-3 RD rats. Discrepancies between the two RD models in the modulatory influence of optokinetic stimulus parameters can be the manifestation of fundamental species differences and/or differences in the degeneration pattern. This study highlights the importance of careful selection of appropriate stimulus parameters for testing optokinetic head-tracking response in RD animals.

Topics: Animals; Contrast Sensitivity; Head; Humans; Mice; Mice, Inbred C57BL; Motion Perception; Mutation; Nystagmus, Optokinetic; Photic Stimulation; Rats; Rats, Transgenic; Retinal Degeneration; Rhodopsin; Species Specificity

Q344ter is a naturally occurring rhodopsin mutation in humans that causes autosomal dominant retinal degeneration through mechanisms that are not fully understood, but are thought to involve an early termination that removed the trafficking signal, QVAPA, leading to its mislocalization in the rod photoreceptor cell. To better understand the disease mechanism(s), transgenic mice that express Q344ter were generated and crossed with rhodopsin knockout mice. Dark-reared Q344ter(rho+/-) mice exhibited retinal degeneration, demonstrating that rhodopsin mislocalization caused photoreceptor cell death. This degeneration is exacerbated by light-exposure and is correlated with the activation of transducin as well as other G-protein signaling pathways. We observed numerous sub-micrometer sized vesicles in the inter-photoreceptor space of Q344ter(rho+/-) and Q344ter(rho-/-) retinas, similar to that seen in another rhodopsin mutant, P347S. Whereas light microscopy failed to reveal outer segment structures in Q344ter(rho-/-) rods, shortened and disorganized rod outer segment structures were visible using electron microscopy. Thus, some Q344ter molecules trafficked to the outer segment and formed disc structures, albeit inefficiently, in the absence of full length wildtype rhodopsin. These findings helped to establish the in vivo role of the QVAPA domain as well as the pathways leading to Q344ter-induced retinal degeneration.

Topics: Animals; Blotting, Western; Catalysis; Cell Death; Disease Models, Animal; Mice; Mice, Transgenic; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Recent studies have demonstrated protective roles for autophagy in various neurodegenerative disorders, including the polyglutamine diseases; however, the role of autophagy in retinal degeneration has remained unclear. Accumulation of activated rhodopsin in some Drosophila mutants leads to retinal degeneration, and although it is known that activated rhodopsin is degraded in endosomal pathways in normal photoreceptor cells, the contribution of autophagy to rhodopsin regulation has remained elusive. This study reveals that activated rhodopsin is degraded by autophagy in collaboration with endosomal pathways to prevent retinal degeneration. Light-dependent retinal degeneration in the Drosophila visual system is caused by the knockdown or mutation of autophagy-essential components, such as autophagy-related protein 7 and 8 (atg-7/atg-8), or genes essential for PE (phosphatidylethanolamine) biogenesis and autophagosome formation, including Phosphatidylserine decarboxylase (Psd) and CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (Ept). The knockdown of atg-7/8 or Psd/Ept produced an increase in the amount of rhodopsin localized to Rab7-positive late endosomes. This rhodopsin accumulation, followed by retinal degeneration, was suppressed by overexpression of Rab7, which accelerated the endosomal degradation pathway. These results indicate a degree of cross talk between the autophagic and endosomal/lysosomal pathways. Importantly, a reduction in rhodopsin levels rescued Psd knockdown-induced retinal degeneration. Additionally, the Psd knockdown-induced retinal degeneration phenotype was enhanced by Ppt1 inactivation, which causes infantile neuronal ceroid lipofuscinosis, implying that autophagy plays a significant role in its pathogenesis. Collectively, the current data reveal that autophagy suppresses light-dependent retinal degeneration in collaboration with the endosomal degradation pathway and that rhodopsin is a key substrate for autophagic degradation in this context.

Topics: Animals; Animals, Genetically Modified; Autophagy; Disease Models, Animal; Drosophila; Drosophila Proteins; Endosomes; Gene Expression Regulation; Green Fluorescent Proteins; In Situ Nick-End Labeling; Larva; Light; Lysosomes; Membrane Proteins; Microscopy, Electron, Transmission; Microscopy, Immunoelectron; Mutation; Nerve Tissue Proteins; Photoreceptor Cells, Invertebrate; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Retinal Degeneration; Rhodopsin; RNA Interference; Statistics, Nonparametric; Thiolester Hydrolases; Time Factors

To determine whether docosahexaenoic acid can protect against hereditary retinal degenerations in transgenic mice expressing the V20G, P23H, and P27L (VPP) rhodopsin mutations.. Female transgenic mice expressing the VPP rhodopsin mutation, known to cause a retinal degeneration, were bred to male transgenic mice expressing the fat-1 gene, which can convert n6 to n3 polyunsaturated fatty acids (PUFA). Several weeks before breeding, the female mice were fed a standard diet containing 10% safflower oil (SFO), which is high in n6 and low in n3 PUFA (n6/n3=273). Offspring were genotyped and four groups identified: Fat1(+)/VPP(+), Fat1(-)/VPP(+), Fat1(+)/VPP(-), and Fat1(-)/VPP(-). Dams were maintained on the SFO diet during the nursing period and offspring were kept on the SFO diet after weaning. At 4, 16, and 28 weeks of age, retinal function was evaluated by electroretinography (ERG), photoreceptor cell loss was quantified by measuring outer nuclear layer thickness, and rhodopsin levels were determined. Fatty acid profiles were analyzed in whole retina, plasma, and liver at 4 and 28 weeks of age.. Expression of fat-1 in the absence of dietary n3 PUFA led to the generation of two groups of mice with distinctly different levels of n3 and n6 PUFA in the three tissues that were analyzed. Already at four weeks of age, the retinas of fat-1 positive animals had higher levels of n3 PUFA than their wild-type counterparts (23%-29% versus 6.4%-6.5%). In addition, by four weeks of age, there was a significant loss of rod photoreceptor cells in the VPP mice. Progression of retinal degeneration occurred with increasing age in VPP positive mice, with photoreceptor cell death occurring in both inferior and superior regions. Amplitudes of the a- and b-waves of the ERG were significantly reduced with age, with VPP positive mice showing the greatest change. Rhodopsin content was lower in the VPP transgenic mice. There were no significant differences in outer nuclear layer thickness or ERG amplitudes between Fat1(+)/VPP(+) and Fat1(-)/VPP(+), or between Fat1(+)/VPP(-)and Fat1(-)/VPP(-) mice at any of the three ages.. High levels of retinal docosahexaenoic acid do not protect mice expressing the VPP rhodopsin mutation from retinal degeneration.

Topics: Animals; Docosahexaenoic Acids; Electroretinography; Fatty Acids; Liver; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Rhodopsin

To determine whether photoreceptor degeneration in transgenic (Tg) rabbits carrying the Pro347Leu rhodopsin mutation alters the neural activity of the middle and inner retinal neurons.. Multifocal electroretinograms (mfERGs) were recorded from eight 12-week-old Tg rabbits both before and after intravitreal injection of the following: tetrodotoxin citrate (TTX), N-methyl-DL: -aspartic acid (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidine-dicarboxylic acid (PDA). Digital subtraction of the mfERGs recorded after the drugs were administered from those recorded before was used to extract the components that were eliminated by these drugs. Eight agematched, wild-type (WT) rabbits were studied with the same protocol.. There was no reduction in the amplitude of the cone photoreceptor response of the mfERGs in Tg rabbits. Both the first positive and the first negative waves of the ON-bipolar cell responses were significantly larger in the Tg than in the WT rabbits. Late negative waves of the ON-bipolar cell response were recorded only in the WT rabbits. The first negative wave of the inner retinal responses was larger in the Tg than in the Wt rabbits. The late positive waves were seen mainly in the WT rabbits.. The ON-bipolar cell and inner retinal responses were altered at the early stage of photoreceptor degeneration in Tg rabbits despite the preservation of the cone photoreceptor responses.

Topics: Aminobutyrates; Animals; Animals, Genetically Modified; Electroretinography; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; N-Methylaspartate; Photoreceptor Cells, Vertebrate; Pipecolic Acids; Point Mutation; Rabbits; Retinal Bipolar Cells; Retinal Degeneration; Rhodopsin; Sodium Channel Blockers; Synapses; Tetrodotoxin

To generate a transgenic (Tg) rabbit model of retinal degeneration and to characterize the pattern of degeneration by using histology and electrophysiology.. Rhodopsin Pro347Leu Tg rabbits were generated by BAC transgenesis. Tg rabbits were identified by Southern blot analysis, and the expression levels were measured by quantitative RT-PCR. Retinal histology was examined by light and electron microscopy and immunohistochemistry. Retinal function was assessed by full-field electroretinograms (ERGs).. Six lines of Tg rabbits were generated, and two lines with higher levels of expression showed rod-dominant progressive retinal degeneration. Retinal histology indicated a marked regional variation in the loss of photoreceptors with the central retina more severely affected than the peripheral retina. The characteristics of the ERGs of transgenic rabbits indicated that the rod components of the ERGs were reduced to only 5% by 48 weeks, whereas the cone components remained at 35% in the wild-type at the same time point. The retinal ultrastructure of Tg rabbits showed a large number of small vesicles that accumulated in the extracellular space of the photoreceptors.. To the best of the authors' knowledge, this is the first rabbit model of progressive retinal degeneration. Because rabbits have large eyes and are easy to handle and breed, they will provide a useful animal model for the study of the pathophysiology of and new treatments for retinal degeneration.

Topics: Animals; Animals, Genetically Modified; Blotting, Southern; Chromosomes, Artificial, Bacterial; Disease Models, Animal; Electroretinography; Fluorescein Angiography; In Situ Hybridization; Point Mutation; Rabbits; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Transgenes

Histone deacetylase 4 (HDAC4) shuttles between the nucleus and cytoplasm and serves as a nuclear co-repressor that regulates bone and muscle development. We report that HDAC4 regulates the survival of retinal neurons in the mouse in normal and pathological conditions. Reduction in HDAC4 expression during normal retinal development led to apoptosis of rod photoreceptors and bipolar (BP) interneurons, whereas overexpression reduced naturally occurring cell death of the BP cells. HDAC4 overexpression in a mouse model of retinal degeneration prolonged photoreceptor survival. The survival effect was due to the activity of HDAC4 in the cytoplasm and relied at least partly on the activity of hypoxia-inducible factor 1alpha (HIF1alpha). These data provide evidence that HDAC4 plays an important role in promoting the survival of retinal neurons.

Topics: Acetylation; Animals; Apoptosis; Cell Nucleus; Cell Survival; Cytoplasm; Electroporation; Histone Deacetylases; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mutation; Retina; Retinal Degeneration; Retinal Neurons; Retinal Rod Photoreceptor Cells; Rhodopsin; Transfection

To evaluate light-induced retinal damage in transgenic T17M rhodopsin mice as a novel model for bystander cone damage during retinal degeneration.. Mouse eyes were exposed to bright white light (15,000 lux, 2.5 minutes). After exposure, electroretinography was performed on mice dark adapted for 12 or more hours at 0 to 5 days to test photoreceptor response or for 0 to 12 hours to test response recovery. Retinal cryosections were examined by TUNEL staining and outer nuclear layer thickness measurements. Cone morphology was assessed by peanut agglutinin staining in retinal flatmounts and cryosections.. T17M retinal function and morphology changed rapidly after exposure to light. Scotopic and photopic electroretinogram responses declined progressively from 0.5 to 3 days. Scotopic response recovery peaked at 50% to 60% of the unilluminated response in 3 hours, indicating an early, rapid decline in scotopic signaling. Photopic responses were near normal or supernormal from 0 to 6 hours. Cell death peaked at 1 day, and outer nuclear layer thickness declined from 1 to 5 days. Disorganized cones were observed at 6 hours, intact and damaged cones were observed at 12 hours and 1 day, but only cone remnants were observed at 3 and 5 days. Light exposure had little to no effect on ERG responses in nontransgenic littermates and other retinal degeneration models.. The time course of light-induced T17M retinal damage is biphasic, with an initial decline in rod function within hours followed by bystander cone and rod deterioration within days. The rapid and synchronous induction of damage in this model is attractive for characterizing bystander effects in retinal degeneration.

Topics: Animals; Bystander Effect; Cell Death; Dark Adaptation; Disease Models, Animal; Electroretinography; In Situ Nick-End Labeling; Light; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutation; Polymerase Chain Reaction; Radiation Injuries, Experimental; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post-TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1-mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4-based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; ADP-Ribosylation Factors; Amino Acid Motifs; Amino Acid Sequence; Animals; Animals, Genetically Modified; Cilia; GTPase-Activating Proteins; Guanosine Triphosphate; Hydrolysis; I-kappa B Kinase; Intracellular Membranes; Molecular Sequence Data; Mutant Proteins; Organ Specificity; Protein Binding; Protein Sorting Signals; Protein Structure, Tertiary; Protein Transport; rab GTP-Binding Proteins; Retinal Degeneration; Rhodopsin; trans-Golgi Network; Xenopus; Xenopus Proteins

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.

Topics: Animals; Cell Death; Cell Survival; Drosophila melanogaster; Drosophila Proteins; Endocytosis; Endosomes; Humans; Lysosomes; Mutation; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin

AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light-damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model.. The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed ( approximately 15-17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry.. Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain.. Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods.

Topics: Animals; Dark Adaptation; Dexamethasone; Disease Models, Animal; Dog Diseases; Dogs; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique, Indirect; Glucocorticoids; Immunoblotting; Light; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Oligonucleotide Probes; Phosphorylation; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-fos; Radiation Injuries, Experimental; Retinal Degeneration; Rhodopsin; Transcription Factor AP-1; Triamcinolone Acetonide

Although the photoreceptors cell death is the main cause of some retinopathies diseases, the mechanisms involved in this process are poorly understood. The neuroprotective effects of interleukin-4 (IL-4) have been shown in several tissues, including retina. We demonstrate that treatment of rat retinal explants with IL-4 completely inhibited the thapsigargin-induced rod photoreceptor cell death after 24 hr in culture. We also showed that IL-4 receptor alpha subunit (IL-4Ralpha) is abundantly present in retina. Colocalization of IL-4Ralpha and rhodopsin indicate a direct effect of this cytokine in rod photoreceptor cells. Moreover, IL-4 increased the intracellular levels of cAMP in 7.4-fold, indicating that the neuroprotective effect of this cytokine was completely blocked by RpcAMP, an inhibitor of protein kinase (PKA). Our data demonstrate, for the first time, the neuroprotective effect of IL-4 through cAMP/PKA pathway in thapsigargin-induced photoreceptor cell death.

Topics: Animals; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Enzyme Inhibitors; Interleukin-4; Nerve Degeneration; Neuroprotective Agents; Organ Culture Techniques; Rats; Receptors, Interleukin-4; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction; Thapsigargin; Up-Regulation

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.

Topics: Acute Disease; Aging; Alcohol Oxidoreductases; Animals; Apoptosis; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; Caspases; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Chromatography, Liquid; Diterpenes; Ethanolamine; Humans; Light; Mass Spectrometry; Mice; Oxidation-Reduction; Rats; Retina; Retinal Degeneration; Retinal Diseases; Retinaldehyde; Retinyl Esters; Rhodopsin; Vitamin A

Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently, a novel treatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation and deliver retinal progenitor cells for cell replacement therapy. In this study, we engineered and analyzed a micro-fabricated polymer, poly(glycerol sebacate) (PGS) scaffold, whose useful properties include biocompatibility, elasticity, porosity, and a microtopology conducive to mouse retinal progenitor cell (mRPC) differentiation. In vitro proliferation assays revealed that PGS held up to 86,610 (+/-9993) mRPCs per square millimeter, which were retained through simulated transplantations. mRPCs adherent to PGS differentiated toward mature phenotypes as evidenced by changes in mRNA, protein levels, and enhanced sensitivity to glutamate. Transplanted composites demonstrated long-term mRPC survival and migrated cells exhibited mature marker expression in host retina. These results suggest that combining mRPCs with PGS scaffolds for subretinal transplantation is a practical strategy for advancing retinal tissue engineering as a restorative therapy.

Topics: Animals; Biocompatible Materials; Calcium; Cell Movement; Cell Proliferation; Decanoates; Glycerol; Humans; Materials Testing; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurotransmitter Agents; Polymers; Retina; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Stem Cells; Tissue Engineering; Tissue Scaffolds

A strong association between retinal degeneration and obesity has been shown in humans. However, the molecular basis of increased risk for retinal degeneration in obesity is unknown. Thus, an animal model with obesity and retinal degeneration would greatly aid the understanding of obesity-associated retinal degeneration. The retinal abnormalities in a novel rat model (WNIN-Ob) with spontaneously developed obesity are described.. Histologic and immunohistochemical examination were performed on retinal sections of 2- to 12-month-old WNIN-Ob rats, and findings were compared with those of lean littermate controls. RNA from retinas of 12-month-old WNIN-Ob and lean littermate rats was used for microarray and qRT-PCR analysis.. The WNIN-Ob rats developed severe obesity, with an onset at approximately 35 days. Evaluation of retinal morphology in 2- to 12-month-old WNIN-Ob and age-matched lean littermate controls revealed progressive retinal degeneration, with an onset between 4 to 6 months of age. Immunohistochemical analysis with anti-rhodopsin, anti-cone opsin, and PSD-95 antibodies further confirmed retinal degeneration, particularly rod cell loss and thinner outer plexiform layer, in the obese rat retina. Gene expression by microarray analysis and qRT-PCR established activation of stress response, tissue remodeling, impaired phototransduction, and photoreceptor degeneration in WNIN-Ob rat retina.. WNIN-Ob rats develop increased stress in retinal tissue and progressive retinal degeneration after the onset of severe obesity. The WNIN-Ob rat is the first rat model to develop retinal degeneration after the onset of obesity. This novel rat model may be a valuable tool for investigating retinal degeneration associated with obesity in humans.

Topics: Animals; Disease Models, Animal; Disks Large Homolog 4 Protein; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microscopy, Fluorescence; Obesity; Oligonucleotide Array Sequence Analysis; Opsins; Rats; Rats, Wistar; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

To determine the properties of the retina of a rhodopsin P347L transgenic (Tg) rabbit model of retinal degeneration by electroretinography (ERG).. Full-field ERGs were recorded in 12- to 48-week-old wild-type (WT) and Tg rabbits. The a-wave was analyzed by the a-wave fitting model of Hood and Birch. The stimulus-response function of the b-wave was analyzed by the Michaelis-Menten equation. Oscillatory potentials (OPs) were extracted by digital filtering after subtracting the a-wave. OPs were also recorded before and after an intravitreal injection of l-2 amino-4-phosphonobutyric acid (APB), cis-2,3 piperidine dicarboxylic acid (PDA), gamma-amino butyric acid (GABA), or tetrodotoxin citrate (TTX).. All the ERG components of Tg rabbits decreased progressively with age with the a-wave more affected than the b-wave, and the OPs were most preserved. Of interest, the summed OP amplitudes of the Tg rabbits were significantly larger than those of WT rabbits when they were 12 weeks of age. The changes in the amplitudes of the OPs after intravitreal injections of APB, PDA, or GABA in Tg rabbits did not differ significantly from those of WT rabbits. However, injection of TTX resulted in a significantly larger amplitude reduction of the OPs in Tg (65.3%) than in WT (28.6%) rabbits.. The significantly larger OPs in Tg rabbits resulted from alterations in the inner retinal neurons. The greater effect of TTX on the OP amplitudes in Tg rabbits suggests that the supernormal OPs in Tg rabbits may be related to secondary changes in the spiking neurons of the inner retina after photoreceptor degeneration.

Topics: Animals; Animals, Genetically Modified; Dark Adaptation; Electroretinography; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; GABA Agents; Light; Membrane Potentials; Mutation; Oscillometry; Photoreceptor Cells, Vertebrate; Rabbits; Retinal Degeneration; Rhodopsin; Tetrodotoxin

Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration.. The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry.. The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)).. Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

Topics: Animals; Calbindins; Cyclic GMP; Dark Adaptation; Electrophysiology; Fluorescent Antibody Technique, Indirect; Guanylate Cyclase; Hydrolysis; Light; Microspectrophotometry; Photic Stimulation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; S100 Calcium Binding Protein G; Vision, Ocular; Vitamin A Deficiency; Xenopus laevis

Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3.. Transgenic mice expressing mutant human ELOVL4 (TG2) were bred to mice expressing the fat-1 protein, which can convert n6 to n3 PUFA. Mice were maintained on an n3-deficient diet containing 10% safflower oil (SFO, enriched in n6 PUFA; n6/n3=273) so that four experimental groups were produced that differed only in levels of n3 PUFA and expression of the hELOVL4 transgene. These groups were identified by genotyping and named Fat1+/TG2+, Fat1(-)/TG2+, Fat1+/TG2(-), and Fat1(-)/TG2(-). All were continued on the SFO diet for 4 to 16 weeks such that those not expressing Fat1 would be deficient in n3 fatty acids. At both time points, animals were analyzed for retinal function by electroretinography (ERG), photoreceptor cell viability by outer nuclear layer (ONL) thickness measurements, fatty acid profiles in several tissues, and rhodopsin levels.. Mice expressing the fat-1 transgene had significantly higher levels of n3 PUFA, primarily DHA, in retina, liver, and plasma lipids at 4 and 16 weeks of age. Retinal DHA levels in fat-1 mice were twice those of controls. By 16 weeks of age, mice expressing the mutant hELOVL4 transgene had a significantly greater loss of photoreceptor cells, reduced ERG amplitudes, and lower rhodopsin levels than control mice. There was no effect of retinal fatty acids on the rate of degeneration of retinas expressing the ELOVL4 transgene.. We found no evidence that high levels of DHA in retinal membranes protected photoreceptor cells expressing mutant ELOVL4 from retinal degeneration. We conclude that DHA is not beneficial for the treatment of retinal degeneration in this animal model of human STGD3 macular dystrophy.

Topics: Animals; Caenorhabditis elegans Proteins; Docosahexaenoic Acids; Electroretinography; Eye Proteins; Fatty Acid Desaturases; Fatty Acids; Female; Gene Expression; Humans; Immunohistochemistry; Liver; Male; Membrane Proteins; Mice; Mice, Transgenic; Retina; Retinal Degeneration; Rhodopsin

This study tests the potential of light restriction to optimise retinal structure and function in adulthood, using the P23H-3 rhodopsin-mutant transgenic rat as a model. P23H-3 rats were reared in scotopic (5 lux) or mesopic (40-60 lux) cyclic (12 h/12 h light/dark) light. A further 2 groups were reared in one of these light conditions to P(postnatal day)30, and then were transferred to the other condition. Retinae were examined at P30-365. Rod and cone function were assessed by the dark-adapted flash electroretinogram. The rate of photoreceptor death was assessed with the TUNEL technique, and photoreceptor survival by the thickness of the outer nuclear layer (ONL). Photoreceptor structural changes were assessed by immunohistochemistry. Mesopic rearing severely reduced the number, function and outer segment (OS) length of photoreceptors. Light restriction in the adult (achieved by moving mesopic-reared animals to scotopic conditions at P30) slowed photoreceptor death, induced recovery of the ERG and of OS length in survivors, resulting in an adult retina that matched the scotopic-reared in function, photoreceptor survival (stability) and structure. Conversely, light exposure in the adult (achieved by moving scotopic-reared animals to mesopic conditions at P30) accelerated photoreceptor death, shortened OSs and reduced the ERG, resulting in a retina that was as damaged and dysfunctional as a mesopic-reared retina, and showed greater photoreceptor instability. Present observations suggest, that the stability and function of adult photoreceptors are determined by both early and adult ambient light experience. Light restriction in the adult was effective in inducing the self-repair of photoreceptors, and the recovery of their function and stability. Light restriction in the juvenile (before P30) improved early photoreceptor survival but made adult photoreceptors vulnerable to brighter light experienced in adulthood. For comparable human dystrophies, these results suggest that light restriction begun after retinal maturation may be effective in optimising the structure, function and stability of the adult retina.

Topics: Aging; Animals; Animals, Genetically Modified; Cell Survival; Electroretinography; In Situ Nick-End Labeling; Light; Male; Photoreceptor Cells, Vertebrate; Radiation Injuries, Experimental; Rats; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

Mutations in the rhodopsin gene that disrupt the encoded protein's folding properties are a major cause of autosomal dominant retinitis pigmentosa (ADRP). This disease is faithfully modeled in Drosophila where similar mutations in the ninaE gene, encoding rhodopsin-1 (Rh-1), cause ER stress and dominantly trigger age-related retinal degeneration. In addition, mutant flies bearing certain ninaE alleles have dramatically reduced Rh-1 protein levels, but the underlying mechanism for this reduction and significance of its contribution to the ADRP phenotype remains unclear. To address this question, we specifically analyzed the role of Drosophila genes homologous to the known yeast and animal regulators of the ER-associated degradation (ERAD) pathway, a process that reduces levels of misfolded proteins in the ER through proteasomal degradation. We found that loss-of-function of these putative ERAD factors resulted in increased levels of Rh-1 in ninaE mutant flies. Conversely, in an ER stress assay where mutant or wild-type Rh-1 were overexpressed in developing imaginal discs beyond the ER protein folding capacity of those cells, co-expression of certain ERAD factors was sufficient to reduce Rh-1 protein levels and to completely suppress ER stress reporter activation. Significantly, those ERAD factors that specifically reduced misfolded Rh-1 in the imaginal disc assay also delayed age-related retinal degeneration caused by an endogenous ninaE allele, indicating that ERAD acts as a protective mechanism against retinal degeneration in the Drosophila model for ADRP. These results suggest that manipulation of ERAD may serve as a powerful therapeutic strategy against a number of diseases associated with ER stress.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Line; Disease Models, Animal; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum; Eye Proteins; Green Fluorescent Proteins; Humans; Immunohistochemistry; Molecular Sequence Data; Mutation; Photoreceptor Cells, Invertebrate; Protein Folding; Retinal Degeneration; Rhodopsin; RNA Interference; Sequence Homology, Amino Acid; Signal Transduction

In several species, an acute injection of N-methyl-N-nitrosourea (MNU) induces a retinal degeneration characterized principally by a rapid loss of the outer nuclear layer, the other layers remaining structurally intact. It has, however, also been reported that down-regulation of melanopsin gene expression is associated with the degeneration and is detectable soon after injection. Melanopsin is expressed by a small subset of intrinsically photosensitive retinal ganglion cells and plays an important role in circadian behaviour photoentrainment. We injected MNU into Long Evans rats and investigated the ability of animals to entrain to three light/dark cycles of different light intensities (300, 15 and 1 lux). Control animals entrained their locomotor activity rhythms to the three cycles. In contrast, MNU-treated animals could only entrain properly to the 300 lux cycle. For the 15 lux cycle, their phase angle was much altered compared with control animals, and for the 1 lux cycle, MNU-injected animals were unable to photoentrain and exhibited an apparent free-run activity pattern with a period of 24.3 h. Subsequent to behavioural studies the animals were killed and rod, cone, melanopsin expression and melanopsin-expressing cells were quantified. Rod and cone loss was almost complete, melanopsin protein was reduced by 83% and melanopsin-expressing cells were reduced by 37%. Our study provides a comprehensive model of photoreceptor degeneration at the adult stage and a simple and versatile method to investigate the relation between retinal photoreceptors and the circadian system.

Topics: Animals; Circadian Rhythm; Disease Models, Animal; Gene Expression Regulation; Light; Light Signal Transduction; Lighting; Male; N-Methylaspartate; Opsins; Photic Stimulation; Photoreceptor Cells, Vertebrate; Rats; Rats, Long-Evans; Retinal Degeneration; Rhodopsin; Rod Opsins

To define the retinal pathology in an 88-year-old male affected with Goldmann-Favre syndrome with a 2 bp 5' A>C splice site mutation in the NR2E3 gene.. Retinal tissue from the macula and periphery was processed for immunohistochemistry. Perimacular retina was processed for transmission electron microscopy. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to rhodopsin, cone cytoplasm, and cone opsins. The affected donor eye was compared to a postmortem matched normal eye.. The retina was highly disorganized without laminar organization. The RPE was discontinuous in some perimacular regions. Large (>1 mm) spherical electrondense melanosomes were observed in the RPE and choroid by TEM. Rods were virtually absent in the affected retina. Cones were present in the macula, but were mostly absent from the retinal periphery. In addition, cone rosettes were observed in the perimacular area. Both red/green and blue cone opsins were distributed along the entire cellular expanse of the cone photoreceptors in the affected eye, but were restricted to the cone outer segments in the control retina.. The histological data obtained from the retina of an elderly male patient with Goldmann-Favre syndrome showed an absence of rods and abnormal distribution of red/green and blue cone opsins.

Topics: Aged; Aged, 80 and over; Arrestin; Fluorescent Antibody Technique, Indirect; Humans; Male; Night Blindness; Opsins; Orphan Nuclear Receptors; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Rhodopsin; Syndrome

To determine whether systemic minocycline can protect photoreceptors in experimental retinal detachment (RD).. Retinal detachment was induced in mice by subretinal injection of sodium hyaluronate, 1.4%. In 1 experiment, mice received daily injections of minocycline (group 1) or saline (group 2). In a second experiment, mice were treated with minocycline or saline beginning 24 hours prior, immediately after, or 24 hours after experimental RD. In both experiments, photoreceptor cell survival and apoptosis were assessed by immunohistochemistry with primary antibodies against photoreceptor cell markers, rod rhodopsin, and cone opsin, and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling.. Photoreceptor cell apoptosis was detected at day 1 after experimental RD, with apoptotic cells peaking in number at day 3 and dropping by day 7. Treatment with minocycline significantly reduced the number of apoptotic photoreceptor cells associated with RD when given 24 hours before or even 24 hours after RD.. Our data suggest that minocycline may be useful in the treatment of photoreceptor degeneration associated with RD, even when given up to 24 hours after RD.. Use of minocycline in patients with macula-off RD may prevent photoreceptor apoptosis and glial cell proliferation, improving final visual outcomes.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Caspase 3; Cell Survival; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Minocycline; Monocytes; Opsins; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Detachment; Rhodopsin

Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking KIF3A, membrane proteins involved in phototransduction did not traffic to the outer segments resulting in complete absence of a photopic electroretinogram and progressive cone degeneration. Rod photoreceptors lacking KIF3A degenerated rapidly between 2 and 4 weeks postnatally, but the phototransduction components including rhodopsin trafficked to the outer segments during the course of degeneration. Furthermore, KIF3A deletion did not affect synaptic anterograde trafficking. The results indicate that trafficking of membrane proteins to the outer segment is dependent on kinesin-II in cone, but not rod photoreceptors, even though rods and cones share similar structures, and closely related phototransduction polypeptides.

Topics: Animals; Kinesins; Membrane Proteins; Mice; Mice, Knockout; Mice, Transgenic; Microtubule-Associated Proteins; Protein Transport; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Pigments; Retinal Rod Photoreceptor Cells; Rhodopsin; Synapses; Time Factors

Several mutations in the N terminus of the G-protein-coupled receptor rhodopsin disrupt NXS/T consensus sequences for N-linked glycosylation (located at N2 and N15) and cause sector retinitis pigmentosa in which the inferior retina preferentially degenerates. Here we examined the role of rhodopsin glycosylation in biosynthesis, trafficking, and retinal degeneration (RD) using transgenic Xenopus laevis expressing glycosylation-defective human rhodopsin mutants. Although mutations T4K and T4N caused RD, N2S and T4V did not, demonstrating that glycosylation at N2 was not required for photoreceptor viability. In contrast, similar mutations eliminating glycosylation at N15 (N15S and T17M) caused rod death. Expression of T17M was more toxic than T4K to transgenic photoreceptors, further suggesting that glycosylation at N15 plays a more important physiological role than glycosylation at N2. Together, these results indicate that the structure of the rhodopsin N terminus must be maintained by an appropriate amino acid sequence surrounding N2 and may require a carbohydrate moiety at N15. The mutant rhodopsins were rendered less toxic in their dark inactive states, because RD was abolished or significantly reduced when transgenic tadpoles expressing T4K, T17M, and N2S/N15S were protected from light exposure. Regardless of their effect on rod viability, all of the mutants primarily localized to the outer segment and Golgi and showed little or no endoplasmic reticulum accumulation. Thus, glycosylation was not crucial for rhodopsin biosynthesis or trafficking. Interestingly, expression of similar bovine rhodopsin mutants did not cause rod cell death, possibly attributable to greater stability of bovine rhodopsin.

Topics: Amino Acids; Animals; Animals, Genetically Modified; Disease Models, Animal; Glycosylation; Green Fluorescent Proteins; Humans; Larva; Light; Mutation; Photoreceptor Cells, Vertebrate; Protein Folding; Protein Transport; Retinal Degeneration; Rhodopsin; Xenopus

We recently showed that subretinal CX3CR1-dependent microglial cell (MC) accumulation may lead to age-related macular degeneration. The fate of MC after engulfing retinal debris is poorly understood. Severe photoreceptor degeneration was observed 40days after exposure to bright light in CX3CR1-deficient but not control mice, and more MCs accumulated in the subretinal space of the former than the latter. To study the fate of subretinal MCs in CX3CR1 competent animals, we used a dystrophic rat model in which abundant subretinal MC accumulation is observed secondary to retinal degeneration. In dystrophic rats, MCs containing rhodopsin or rod outer segment (ROS) debris were found outside the outer retina at sites suggesting choroidal and ciliary egress. In conclusion, our data indicate that MC accumulation at injury sites is independent of CX3CR1 and precedes photoreceptor degeneration. The ectopic presence of rhodopsin-positive MCs suggests that CX3CR1 participates in MC egress from the outer retina.

Topics: Animals; Cell Movement; CX3C Chemokine Receptor 1; Gene Expression Regulation; Green Fluorescent Proteins; Light; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Microscopy, Electron, Transmission; Phagocytes; Phagocytosis; Photoreceptor Cells; Receptors, Chemokine; Retina; Retinal Degeneration; Rhodopsin; Statistics, Nonparametric; Time Factors

Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system treatment. Retinal stem cells (RSC) are a substrate for cell-replacement therapy, and previous studies have shown that RSCs from different developmental stages have distinct properties in proliferative capacity and differentiation potential. The tailbud stage is of special interest in retinogenesis, because RSCs commence differentiation after this period. However, no information about the characteristics of RSCs from the tailbud stage is available. In this study, the characteristics of cell cultures from the rat optic cup (referred to as optic-cup-derived RSCs; OC-RSCs) at embryonic day 12.5 (tailbud stage) were analyzed. OC-RSCs grew either as monolayers or as neurospheres in the presence of basic fibroblast growth factor and could be dissociated into a single cell suspension. Using the MTT assay, immunochemistry, cytogenetic analysis, and flow cytometry, we found that OC-RSCs were easily enriched to 92% by three passages, had a normal diploid karyotype, and exhibited no obvious differences in proliferative rate during eight passages (doubling time: 36 h). OC-RSCs produced retinal specific cells after the addition of serum to the medium, but the differentiation potential was affected by serum concentration. Preliminary results showed that transplanted OC-RSCs were incorporated into the degenerated retina of RCS rats and differentiated into rhodopsin-positive cells. Thus, OC-RSCs, after suitable enrichment, provide a population of stem cells with distinct growth and differentiation properties that make them suitable for research into RSC differentiation and transplantation.

Topics: Animals; Antigens, Differentiation; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cytogenetic Analysis; Disease Models, Animal; Flow Cytometry; Graft Survival; Immunohistochemistry; Rats; Rats, Long-Evans; Rats, Mutant Strains; Retina; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Stem Cells; Thy-1 Antigens

The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30-33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capill

Topics: Aging; Animals; Mutation; Neovascularization, Pathologic; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Degeneration; Retinal Pigment Epithelium; Retinal Vessels; Rhodopsin; Rod Cell Outer Segment

The Drosophila photoreceptor is a model system for genetic study of retinal degeneration. Many gene mutations cause fly photoreceptor degeneration, either because of excessive stimulation of the visual transduction (phototransduction) cascade, or through apoptotic pathways that in many cases involve a visual arrestin Arr2. Here we report a gene named tadr (for torn and diminished rhabdomeres), which, when mutated, leads to photoreceptor degeneration through a different mechanism. Degeneration in the tadr mutant is characterized by shrunk and disrupted rhabdomeres, the light sensory organelles of photoreceptor. The TADR protein interacted in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppressed the tadr mutation-caused degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 failed to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses revealed that, in the tadr mutant, the G(q) protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of G(q) level by introducing a hypomorphic allele of G(alphaq) gene greatly inhibited the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the G(q) signaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans.

Topics: Animals; Drosophila; Drosophila Proteins; Electroretinography; Genes, Insect; GTP-Binding Protein alpha Subunits, Gq-G11; Microscopy, Electron, Transmission; Mutation; Patch-Clamp Techniques; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Retinal prostheses are being developed to apply electrical stimulation to the retina in order to restore vision of individuals who suffer from diseases such as retinitis pigmentosa (RP) and aged related macular degeneration (AMD). Various electrical stimulus parameters have been extensively studied in both experimental and clinical settings. Both electrophysiological and psychophysical results have shown that outer retina disease exhibit higher stimulus threshold in one degenerate group versus the control group. Fewer studies have been conducted to investigate the change in threshold currents as a function of different degenerate stages. We propose to study the electrophysiological change in degenerate rat retinas by using an in vivo recording method. We recorded retinal-driven superior colliculus cells response in two control groups and four degenerate groups. Current pulses of seven different stimulus pulse durations were applied to the retinas to obtain strength duration curve per group. Preliminary results showed that for the postnatal (P) day 90 and 180 degenerate groups, threshold currents were not significantly different from the normal control group (P90 and P230). For P300 degenerate group, the threshold currents progressively increased. For P760 degenerate group, threshold currents were significantly elevated across all the stimulus pulse durations tested. Charge densities calculated for P760 degenerate group exceeded the safe limit of the stimulating electrode. Cell morphology in all control and degenerate groups is still under investigation for a correlation study.

Topics: Age Factors; Animals; Animals, Genetically Modified; Biomedical Engineering; Disease Models, Animal; Electric Stimulation Therapy; Electrophysiological Phenomena; Mutation; Rats; Retinal Degeneration; Rhodopsin; Sensory Thresholds; Superior Colliculi; Visual Pathways

To determine whether photoreceptor degeneration can stimulate Müller glia to transdifferentiate into neurons in adult mammalian retina, N-methyl-N-nitrosourea (MNU) was injected to induce complete loss of photoreceptors. Following MNU administration, Müller glia underwent reactive gliosis characterized by up-regulation of glial fibrillar acidic protein and nestin, and initiated proliferation through the cyclin D1 and D3 related pathways. Some Müller glia-derived cells were induced to express rhodopsin exclusively. These rhodopsin-positive cells exhibited synaptophysin around them, suggesting possible formation of synapses. After transplanted in to damaged retina, Müller glia migrated, grafted in host retina and produced rhodopsin. These results suggest that degeneration may promote preferential differentiation of Müller glia to photoreceptors and provide a potential therapeutic strategy for retinal degenerative diseases.

Topics: Animals; Cell Proliferation; Cyclin D; Cyclin D3; Cyclins; Glial Fibrillary Acidic Protein; Gliosis; Intermediate Filament Proteins; Methylnitrosourea; Mice; Nerve Regeneration; Nerve Tissue Proteins; Nestin; Neuroglia; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.

Topics: Albinism; Animals; Animals, Genetically Modified; Cell Death; Disease Models, Animal; Embryo, Nonmammalian; Eye Proteins; Gene Expression Regulation; Green Fluorescent Proteins; Homeodomain Proteins; Light; Microinjections; Neuroglia; Oligonucleotides; Paired Box Transcription Factors; PAX6 Transcription Factor; Proliferating Cell Nuclear Antigen; Regeneration; Repressor Proteins; Retinal Degeneration; Rhodopsin; Time Factors; Zebrafish; Zebrafish Proteins

Retinal degeneration slow (Rds) is a photoreceptor-specific tetraspanin glycoprotein essential for photoreceptor outer segment (OS) morphogenesis. Over 80 mutations in this protein are associated with several different retinal diseases. Rds forms a mixture of disulfide-linked homomeric dimers, octamers, and higher-order oligomers, with Cys150 playing a crucial role in its oligomerization. Rds also forms noncovalent homo- and hetero-tetramers with its nonglycosylated homologue, Rom-1. Here, we evaluated the subcellular site of Rds oligomerization and the pattern of Rds/Rom-1 complex assembly in several types of knockout mice, including rhodopsin (Rho-/-, lacking rod OS), Rom-1 (Rom-1-/-), neural retina leucine zipper (Nrl-/-, cone-dominant), and in comparison with wild-type (WT, rod-dominant) mice. Oligomerization and the pattern of complex assembly were also evaluated in OS-enriched vs OS-depleted preparations from WT and Rom-1-/- retinas. Velocity sedimentation under reducing- and nonreducing conditions and co-immunoprecipitation experiments showed the presence of Rds mainly as homo- and hetero-tetramers with Rom-1 in the photoreceptor inner segment (IS), while higher-order, disulfide-linked intermediate complexes and oligomers were exclusively present in the photoreceptor OS. Rom-1-independent oligomerization of Rds was observed in Rom-1-/- retinas. The pattern of Rds complexes in cones from Nrl-/- mice was comparable to that in rods from WT mice. On the basis of these findings, we propose that Rds traffics from the IS to the OS as homo- and hetero-tetramers, with subsequent disulfide-linked oligomerization occurring concomitant with OS disc morphogenesis (at either the base of OS or the tip of the connecting cilium). These results suggest that Rds mutations that interfere with tetramer formation can block Rds trafficking to the OS, leading to loss-of-function defects.

Topics: Animals; Basic-Leucine Zipper Transcription Factors; Eye Proteins; Gene Deletion; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Electron; Models, Animal; Protein Binding; Protein Subunits; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Subcellular Fractions; Tetraspanins

It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19.. Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression.. Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors.. RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Topics: Animals; Cell Transplantation; Cells, Cultured; Disease Models, Animal; Light; Microscopy, Confocal; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Protein Kinase C-alpha; Rats; Rats, Mutant Strains; Recoverin; Retinal Degeneration; Rhodopsin; Sensory Thresholds; Space Perception; Superior Colliculi; Transplantation, Heterologous

To study the distribution and differentiation of photoreceptor precursors in the ciliary epithelium in mice.. Proliferating cells in flat-mount specimens of the ciliary body and retina were studied by bromodeoxyuridine (BRDU; 150 mg/kg) labeling in young C57Bl mice. Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically to assess the distribution and differentiation of photoreceptor progenitors or precursors. Mice injected intraperitoneally with N-methyl-N-nitrosourea (MNU; 60 mg/kg) were also examined.. Part of the neuroblast layer composed of BrdU-positive retinal progenitor cells was identified within the ciliary epithelium of the pars plana in continuation of the layer of the peripheral retina during ocular development. In both the ciliary epithelium and the retina, the layer size decreased rapidly and disappeared mostly by postnatal day (P)9. Within the ciliary epithelium of the pars plana, numerous postmitotic rod and cone photoreceptor precursors were identified that rapidly differentiated morphologically and decreased in number with ocular development. Rod precursors were no longer seen in the pars plana at P12, whereas rare presumptive cone precursors persisted even at P120. An increase in the number of presumptive cone precursors (approximately 16-fold) was identified in the pars plana of adult mice with MNU-induced photoreceptor degeneration.. Rod and cone precursors were identified in the ciliary epithelium of the murine pars plana during ocular development but nearly disappeared after the completion of histogenesis. However, in response to retinal injury, an increased number of presumptive cone precursors was found even in the adult pars plana.

Topics: Animals; Bromodeoxyuridine; Cell Differentiation; Ciliary Body; Eye; Eye Proteins; Fluorescent Antibody Technique, Indirect; Homeodomain Proteins; Methylnitrosourea; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Paired Box Transcription Factors; PAX6 Transcription Factor; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Recoverin; Repressor Proteins; Retinal Degeneration; Rhodopsin; Stem Cells

The authors investigated photopic electroretinographic changes during degeneration in the Royal College of Surgeons (RCS) and transgenic P23H rhodopsin rat models, including the cellular origins of a large corneal-negative component that persists in the RCS rat.. Photopic and scotopic electroretinograms (ERGs) were recorded from dystrophic RCS (RCS-p(+)/Lav) rats (4-18 weeks old) and transgenic rhodopsin Pro23His line 1 (P23H) rats (4-30 weeks old). Age-matched congenic (RCS-rdy(+)p(+)/Lav) and Sprague-Dawley rats were used as controls. N-methyl-DL-aspartic acid (NMA), dopamine, and gamma-aminobutyric acid (GABA) were injected intravitreally, and optic nerve sectioning (ONS) was performed to suppress or remove inner retinal neuron activity. Retinal morphology for cone cell counts and immunohistochemistry for quantification of Kir4.1 channels were performed at various stages of degeneration.. As degeneration progressed, the photopic ERG of RCS dystrophic rats was distinctly different from that of P23H rats, primarily because of the growth of a corneal-negative response (RCS-NPR) after the b-wave in RCS rats. This response had a peak time similar to the photopic negative response (PhNR) in controls but with a more gradual recovery phase, and it was not affected by ONS. The PhNR in P23H rats declined linearly with the b-wave. NMA and GABA eliminated the RCS-NPR and uncovered a larger b-wave in RCS rats at late stages of degeneration, but NMA had little effect on the ERG in P23H rats. The NMA-sensitive negative response in RCS rats declined with age more slowly than did the NMA-isolated b-wave. The density of Kir4.1 channels at the endfeet of Müller cells and in the proximal retina increased significantly between 6 to 10 weeks and 14 weeks of age in the RCS rat retina but not in the P23H rat retina.. The photopic ERG of the dystrophic RCS rat retina becomes increasingly electronegative because of an aberrant negative response, originating from amacrine cell activity, which declines more slowly than the b-wave with degeneration. The absence of this response in the P23H rat indicates that the inner retinal cone pathway pathology is different in the two models. A relative increase in Kir4.1 channels on Müller cells of RCS retina may contribute to the enhanced negative ERG response in the RCS rat.

Topics: Amacrine Cells; Animals; Animals, Genetically Modified; Cell Count; Dopamine; Electroretinography; gamma-Aminobutyric Acid; Light; Microscopy, Confocal; N-Methylaspartate; Potassium Channels, Inwardly Rectifying; Rats; Rats, Long-Evans; Rats, Mutant Strains; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Signal Transduction

We propose two models of the human S-arrestin/rhodopsin complex in the inactive dark adapted rhodopsin and meta rhodopsin II form, obtained by homology modeling and knowledge based docking. First, a homology model for the human S-arrestin was built and validated by molecular dynamics, showing an average root mean square deviation difference from the pattern behavior of 0.76 A. Then, combining the human S-arrestin model and the modeled structure of the two human rhodopsin forms, we propose two models of interaction for the human S-arrestin/rhodopsin complex. The models involve two S-arrestin regions related to the N domain (residues 68-78; 170-182) and a third constituent of the C domain (248-253), with the rhodopsin C terminus (330-348). Of the 22 single point mutations related to retinitis pigmentosa and congenital night blindness located in the cytoplasmatic portion of rhodopsin or in S-arrestin, our models locate 16 in the interaction region and relate two others to possible dimer formation. Our calculations also predict that the light activated complex is more stable than the dark adapted rhodopsin and, therefore, of higher affinity to S-arrestin.

Topics: Arrestin; Computational Biology; Humans; Models, Molecular; Multiprotein Complexes; Point Mutation; Protein Binding; Retinal Degeneration; Rhodopsin

Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.

Topics: Amidohydrolases; Animals; Animals, Genetically Modified; Apoptosis; Arrestin; Ceramidases; Drosophila; Drosophila Proteins; Electroretinography; Embryo, Nonmammalian; Eye; Fat Body; Gene Expression Regulation, Developmental; Homeostasis; Membrane Potentials; Mutation; Photic Stimulation; Photoreceptor Cells, Invertebrate; Protein Binding; Retinal Degeneration; Rhodopsin; Sphingosine

Topics: Animals; Animals, Genetically Modified; Dark Adaptation; Electroretinography; Homozygote; Light; Phototherapy; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin

To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice.. RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30.. The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD.. RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Count; Dark Adaptation; In Situ Nick-End Labeling; Light; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Detachment; Retinal Vessels; Rhodopsin

Progression of retinal degeneration in a mouse model was studied in vivo with high-resolution spectral-domain optical coherence tomography (SD-OCT). Imaging in 3D with high depth resolution (<3 mum), SD-OCT resolved all the major layers of the retina of control C57BL/6J mice. Images of transgenic mice having a null mutation of the rhodopsin gene revealed the anatomical consequences of retinal degeneration: thinning of the outer retina, including the outer plexiform layer (OPL), outer nuclear layer (ONL), and inner and outer segments (IS/OS). We monitored the progression of retinal degeneration in rd1 mice (C3H/HeJ) by periodically imaging the same mice from the time the pups opened their eyes on P13 to P34. SD-OCT images showed that the outer retina (OPL, ONL, IS/OS) had already thinned by 73% (100 to 27 mum) at eye opening. The retina continued to degenerate, and by P20 the outer retina was not resolvable. The thickness of entire retina decreased from 228 mum (control) to 152 mum on P13 and to 98 mum by P34, a 57% reduction with the complete loss in the outer retina. In summary, we show that SD-OCT can monitor the progression of retinal degeneration in transgenic mice.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Disease Progression; Image Processing, Computer-Assisted; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Retina; Retinal Degeneration; Rhodopsin; Severity of Illness Index; Tomography, Optical Coherence

To describe and classify the morphologic changes in a naturally occurring dog model of early-onset cone-rod dystrophy (CRD) and to correlate these with earlier described clinical characteristics of the disease in dogs.. Purpose-bred Standard Wire-Haired Dachshunds (SWHDs) derived from a large pedigree of dogs with early-onset CRD were euthanatized at defined ages to characterize morphologic changes in the disease process. Specimens were examined by light microscopy, including morphometric studies, electron microscopy, and immunohistochemistry. Peanut agglutinin (PNA), protein kinase C (PKC), synaptophysin (Syn), rhodopsin (Rho)-63, glial fibrillary acidic protein (GFAP), and short-wavelength cone opsin (OS) were used for immunohistochemical characterization.. The photopic cone-system-derived ERG amplitudes were already significantly reduced or nonrecordable in CRD-affected dogs at 5 weeks, the earliest age studied. The outer retina was morphologically most severely affected initially, with a subsequent degeneration of the inner retina. Cone degeneration was more pronounced than rod degeneration in young CRD-affected dogs. There was a marked phenotypic variation based on morphologic findings in the affected dogs. At the earliest time point studied (5-8 weeks) cone photoreceptor and glial cell abnormalities were observed, in accordance with earlier studies based on electrophysiological and clinical findings in which day blindness and abnormal cone ERGs were observed in young affected SWHD puppies. Preliminary genetic studies have indicated an autosomal recessive mode of inheritance for the defect.. Through functional and structural characterization, early-onset cone abnormalities were found, consistent with a cone dysplasia at an age when rod structure was normal. Further studies are in progress to identify the gene(s) involved in this retinal disease process. The presently described natural animal model of primary cone dysplasia followed by rod degeneration may provide further insight into the human counterpart. Further studies are needed to ascertain an autosomal recessive mode of inheritance for CRD in the SWHD.

Topics: Animals; Disease Models, Animal; Dog Diseases; Dogs; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Male; Microscopy, Electron; Pedigree; Phenotype; Photoreceptor Cells, Vertebrate; Protein Kinase C; Retina; Retinal Degeneration; Rhodopsin; Rod Opsins; Synaptophysin

This study tests whether cones in the rhodopsin-mutant transgenic P23H-3 retina are damaged by ambient light and whether subsequent light restriction allows repair of damaged cones.. P23H-3 rats were raised in scotopic cyclic (12 hours of 5 lux, 12 hours of dark) ambient light. At postnatal day 90 to 130, some were transferred to photopic conditions (12 hours of 300 lux, 12 hours of dark) for 1 week and then returned to scotopic conditions for up to 5 weeks. Photoreceptor function was assessed by the dark-adapted flash-evoked electroretinogram, using a two-flash paradigm to isolate the cone response. Outer-segment structure was demonstrated by immunohistochemistry for cone and rod opsins and by electron microscopy.. Exposure for 1 week to photopic ambient light reduced the cone b-wave, the rod b-wave, and the rod a-wave by 40% to 60% and caused shortening and disorganization of cone and rod outer segments. Restoration of scotopic conditions for 2 to 5 weeks allowed partial recovery of the cone b-wave and the rod a- and b-waves, and regrowth of outer segments.. Modest increases in ambient light cause rapid and significantly reversible loss of cone and rod function in the P23H-3 retina. The reduction and recovery of cone function are associated with shortening and regrowth of outer segments. Because the P23H mutation affects a protein expressed specifically in rods, this study emphasizes the close dependence of cones on rod function. It also demonstrates the capacity of cones and rods to repair their structure and regain function.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Cell Death; Cell Survival; Dark Adaptation; Electroretinography; Fluorescent Antibody Technique, Indirect; Light; Mutation; Photic Stimulation; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Opsins

In previous studies of light-induced (LRD) and age-related (ageRD) retinal degeneration (RD) between the BALB/cByJ (BALB) and B6(Cg)-Tyr(c-2J)/J (B6a) albino mouse strains, RD-modifying quantitative trait loci (QTLs) were identified. After breeding BALB- and B6a-rd3/rd3 congenic strains and finding significant differences in RD, an F1 intercross to determine rd3 QTLs that influence this inherited RD was performed.. N10, F2 BALB- and B6a-rd3/rd3 strains were measured for retinal outer nuclear layer (ONL) thickness from 5 to 12 weeks of age. Since 10 weeks showed significant differences in the ONL, F2 progeny from an F1 intercross were measured for ONL thickness. F2 DNAs were genotyped for SNPs by the Center for Inherited Disease Research. Correlation of genotype with phenotype was made with Map Manager QTX.. One hundred forty-eight SNPs approximately 10 cM apart were typed in the F2 progeny and analyzed. Significant QTLs were identified on chromosomes (Chrs) 17, 8, 14, and 6 (B6a alleles protective) and two on Chr 5 (BALB alleles protective). Suggestive QTLs were found as well. For the strongest QTLs, follow-up SNPs were analyzed to narrow the critical intervals. Additional studies demonstrated that rd3 disease is exacerbated by light but not protected by the absence of rhodopsin regeneration.. QTLs were identified that modulate rd3-RD. These overlapped some QTLs from previous ageRD and LRD studies. The presence of some of the same QTLs in several studies suggests partial commonality in RD pathways. Identifying natural gene/alleles that modify RDs opens avenues of study that may lead to therapies for RD diseases.

Topics: Animals; Chromosomes, Mammalian; Eye; Genotype; Light; Mice; Mice, Congenic; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Polymorphism, Single Nucleotide; Quantitative Trait Loci; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Species Specificity

Genetically encoded optical neuromodulators create an opportunity for circuit-specific intervention in neurological diseases. One of the diseases most amenable to this approach is retinal degeneration, where the loss of photoreceptors leads to complete blindness. To restore photosensitivity, we genetically targeted a light-activated cation channel, channelrhodopsin-2, to second-order neurons, ON bipolar cells, of degenerated retinas in vivo in the Pde6b(rd1) (also known as rd1) mouse model. In the absence of 'classical' photoreceptors, we found that ON bipolar cells that were engineered to be photosensitive induced light-evoked spiking activity in ganglion cells. The rescue of light sensitivity was selective to the ON circuits that would naturally respond to increases in brightness. Despite degeneration of the outer retina, our intervention restored transient responses and center-surround organization of ganglion cells. The resulting signals were relayed to the visual cortex and were sufficient for the animals to successfully perform optomotor behavioral tasks.

Topics: Animals; Behavior, Animal; Disease Models, Animal; Electroporation; Evoked Potentials, Visual; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Light; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Patch-Clamp Techniques; Photic Stimulation; Piperazines; Quinoxalines; Retinal Bipolar Cells; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Time Factors; Vision, Ocular; Visual Pathways

EGCG, a major component of green tea, has a number of properties which includes it being a powerful antioxidant. The purpose of this investigation was to deduce whether inclusion of EGCG in the drinking water of albino rats attenuates the effect of a light insult (2200lx, for 24h) to the retina. TUNEL-positive cells were detected in the outer nuclear layer of the retina, indicating the efficacy of the light insult in inducing photoreceptor degeneration. Moreover, Ret-P1 and the mRNA for rhodopsin located at photoreceptors were also significantly reduced as well as the amplitude of both the a- and b-waves of the electroretinogram was also reduced showing that photoreceptors in particular are affected by light. An increase in protein/mRNA of GFAP located primarily to Müller cells caused by light shows that other retinal components are also influenced by the light insult. However, antigens associated with bipolar (alpha-PKC), ganglion (Thy-1) and amacrine (GABA) cells, in contrast, appeared unaffected. The light insult also caused a change in the content of various proteins (caspase-3, caspase-8, PARP, Bad, and Bcl-2) involved in apoptosis. A number of the changes to the retina caused by a light insult were significantly attenuated when EGCG was in the drinking water. The reduction of the a- and b-waves and photoreceptor specific mRNAs/protein caused by light were significantly less. In addition, EGCG attenuated the changes caused by light to certain apoptotic proteins (especially at after 2 days) but did not appear to significantly influence the light-induced up-regulation of GFAP protein/mRNA. It is concluded that orally administered EGCG blunts the detrimental effect of light to the retina of albino rats where the photoreceptors are primarily affected.

Topics: Administration, Oral; Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Catechin; Glial Fibrillary Acidic Protein; Light; Membrane Potentials; Neuroglia; Oxidative Stress; Photoreceptor Cells; Rats; Rats, Wistar; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger

In this study, the hypothesis that increased intraocular levels of iron cause oxidative damage to the retina was tested.. Adult C57BL/6 mice were given an intravitreous injection of saline or 0.10, 0.25, or 0.50 mM FeSO(4). Scotopic electroretinograms (ERGs) were performed 3, 7, and 14 days after injection, and photopic ERGs were performed on day 14. Hydroethidine was used to identify superoxide radicals and lipid peroxidation was visualized by staining for hydroxynonenal (HNE). Retinal cell death was evaluated by TUNEL and measurement of inner nuclear layer (INL) and outer nuclear layer (ONL) thickness. Levels of rhodopsin and cone-opsin mRNA were measured by quantitative real time RT-PCR. Cone density was assessed by peanut agglutinin staining and confocal microscopy.. Compared with retinas in saline-injected eyes, retinas from eyes injected with FeSO(4) showed greater fluorescence after intravenous injection of hydroethidine due to superoxide radicals in photoreceptors, greater photoreceptor staining for HNE, a marker of lipid peroxidation, and increased expression of Heme oxygenase 1, an indicator of oxidative stress. ERG b-wave amplitudes were reduced (photopic > scotopic) in FeSO(4)-injected eyes compared with those in saline-injected eyes. Numerous TUNEL-stained nuclei were seen along the outer border of the ONL, the location of cone cell nuclei, at 1 and 2 days after injection of FeSO(4). In FeSO(4)-injected eyes, the thickness of the ONL, but not the INL, was significantly reduced, and 17 days after injection, there were 3.8- and 2.6-fold reductions in the mRNAs for M-cone and S-cone opsin, respectively, whereas there was no significant difference in rhodopsin mRNA. Confocal microscopy of peanut agglutinin-stained sections showed dose-dependent FeSO(4)-induced cone drop out.. Increased intraocular levels of FeSO(4) cause oxidative damage to photoreceptors with greater damage to cones than rods. This finding suggests that the oxidative defense system of cones differs from that of rods and other retinal cells, and that cones are more susceptible to damage from the type of oxidative stress imposed by iron.

Topics: Animals; Apoptosis; Dose-Response Relationship, Drug; Electroretinography; Ferrous Compounds; Gene Expression; Heme Oxygenase-1; In Situ Nick-End Labeling; Injections; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Oxidative Stress; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Rod Opsins; Superoxides; Vitreous Body

The transcription factor neural retina leucine zipper (NRL) is required for rod photoreceptor differentiation during mammalian retinal development. NRL interacts with CRX, NR2E3, and other transcription factors and synergistically regulates the activity of photoreceptor-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. Here we report functional analyses of 17 amino acid variations and/or mutations of NRL. We show that 13 of these lead to changes in NRL phosphorylation. Six mutations at residues p.S50 (c.148T>A, c.148T>C, and c.149C>T) and p.P51 (c.151C>A, c.151C>T, and c.152C>T), identified in patients with autosomal dominant retinitis pigmentosa, result in a major NRL isoform that exhibits reduced phosphorylation but enhanced activation of the rhodopsin promoter. The truncated NRL mutant proteins-p.L75fs (c.224_225insC) and p.L160fs (c.459_477dup)-do not localize to the nucleus because of the absence of bZIP domain. The p.L160P (c.479T>C), p.L160fs, and p.R218fs (c.654delC) mutant proteins do not bind to the NRL-response element, as revealed by electrophoretic mobility shift assays. These three and p.S225N (c.674G>A) mutant show reduced transcriptional activity and may contribute to recessive disease. The p.P67S (c.199C>T) and p.L235F (c.703C>T) variations in NRL do not appear to directly cause retinitis pigmentosa, while p.E63K (c.187G>A), p.A76V (c.227C>T), p.G122E (c.365G>A), and p.H125Q (c.375C>G) are of uncertain significance. Our results support the notion that gain-of-function mutations in the NRL gene cause autosomal dominant retinitis pigmentosa while loss-of-function NRL mutations lead to autosomal recessive retinitis pigmentosa. We propose that differential phosphorylation of NRL fine-tunes its transcriptional regulatory activity, leading to a more precise control of gene expression.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Animals; Basic-Leucine Zipper Transcription Factors; Cell Line; Chlorocebus aethiops; COS Cells; DNA; DNA-Binding Proteins; Eye Proteins; Gene Expression Regulation; Genes, Dominant; Genes, Recessive; Humans; Molecular Sequence Data; Mutation; Phosphorylation; Promoter Regions, Genetic; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Homology, Amino Acid; Transcriptional Activation; Transfection

Topics: Animals; Apoptosis; Basic-Leucine Zipper Transcription Factors; Cell Differentiation; DNA-Binding Proteins; Eye Proteins; Genetic Therapy; Humans; Mice; Mice, Knockout; Mice, Mutant Strains; Mice, Transgenic; Photoreceptor Cells, Vertebrate; Retina; Retinal Bipolar Cells; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Synapses; Thioredoxins

To examine the long-term effects of acute photooxidative stress in the retina, retinal pigment epithelium (RPE), and choroid.. Albino rats injected with either the protective antioxidant phenyl-N-tert-butylnitrone (PBN) or saline 30 minutes before exposure to 5 klx white fluorescent light for 6 hours were kept for up to 3 months in 5 lux cyclic light. Electroretinograms were recorded, and the outer nuclear layer (ONL) and the choroidal thickness and area were measured after hematoxylin-eosin (H&E) staining. The expression of rod, cone, and RPE cell markers was detected by Western blotting, and apoptosis was analyzed by TUNEL staining. Oxidative stress was analyzed by immunohistochemistry against 4-hydroxynonenal (4-HNE)-modified proteins. Retinal and choroidal ultrastructures were observed by transmission electron microscopy (TEM). Choroidal circulation was analyzed by in vivo staining of the choroidal layer by trypan blue.. In the saline-injected animals, TUNEL- and 4-HNE-labeling in the ONL, RPE, and choroid were higher 24 hours and 7 days after light exposure, and ERG amplitude, ONL and choroidal thickness and area, and rhodopsin and RPE65 expression were lower 7 or more days after light exposure than in phenyl-N-tert-butylnitrone (PBN)-injected animals. In the saline-injected animals, the expression of mid-wavelength opsin and the presence of cone cells in the ONL and the choroidal circulation were preserved for 7 days after light exposure but started to decrease by 1 month and continued to decrease for 3 months after light exposure. An increase in TUNEL-positive cells was observed in the ONL at the inferior peripheral retina, just behind the iris, by 3 months after light exposure. Delayed loss of cone cells, remaining rod cells, and choroidal circulation were counteracted by PBN treatment.. Although cone cells are resistant to cell damage induced by acute photooxidative stress, progressive loss of cone cells continued for up to 3 months after light exposure. Impaired choroidal circulation is likely to be involved in the mechanism of delayed photoreceptor cell death after light exposure. Preserving choroidal circulation may provide a novel target for preserving the cone and the remaining rod cells in patients with retinal degeneration such as retinitis pigmentosa.

Topics: Aldehydes; Animals; Apoptosis; Blood Circulation; Blotting, Western; Carrier Proteins; Choroid; cis-trans-Isomerases; Cyclic N-Oxides; Electroretinography; Eye Proteins; Free Radical Scavengers; In Situ Nick-End Labeling; Light; Lipid Peroxidation; Neuroprotective Agents; Oxidative Stress; Photoreceptor Cells, Vertebrate; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin

Many genes from retinoid metabolism cause retinitis pigmentosa. Peropsin, an opsin-like protein with unknown function, is specifically expressed in apical retinal pigment epithelium microvilli. Since rhodopsin and RGR, another opsin-like protein, cause retinitis pigmentosa, we used D-HPLC to screen for the peropsin gene RRH in 331 patients (288 with retinitis pigmentosa and 82 with other retinal dystrophies). We found 13 nonpathogenic variants only, among which a c.730_731delATinsG that truncates the last two transmembrane-spanning fragments and the Lys284 required for retinol binding, but does not segregate with the disease phenotype. We conclude that RRH is not a frequent gene in retinitis pigmentosa.

Topics: Adult; Base Sequence; Case-Control Studies; DNA Mutational Analysis; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Pedigree; Polymorphism, Single Nucleotide; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Homology, Nucleic Acid

To describe a sensitivity to light-induced damage associated with expression of a T17M mutant human rhodopsin (hT17M) transgene in mice, with the goal of minimizing retinal injury during the subretinal delivery of rAAV-mediated gene therapy.. Mice were bred to express the hT17M rhodopsin transgene in a line that was hemizygous null for wild-type mouse rhodopsin (mrho(+/-)), and the eyes of transgenic mice and nontransgenic littermates were exposed for 2.5 minutes to unilateral illumination with fiber-optic light ranging from 5,000 to 10,000 lux. Funduscopic images were made with a handheld camera (Genesis; Kowa Company, Ltd., Tokyo, Japan). Full-field scotopic electroretinographic analysis (ERG) was performed to measure loss of retinal function. Morphometry in the light microscope was used to measure loss of rod photoreceptors. TUNEL staining and a nucleosome release assay were used to measure levels of apoptosis in retinal specimens.. mrho(+/-);hT17M mice exhibited a sensitivity to light-induced damage that caused severe loss of a- and b-wave ERG responses. hT17M transgenic mice on the mrho(+/+) background were equally sensitive to light-induced damage. Histologic analysis showed a concomitant loss of photoreceptors and TUNEL labeling of fragmented DNA in rod photoreceptor cells, demonstrating that the damage occurred via an apoptotic pathway. Nontransgenic littermate mice were not affected by this exposure to light. Mice expressing an hP23H mutant human rhodopsin transgene were minimally sensitive to light-induced damage at these intensities, in comparison to hT17M mice. Treating the hT17M mice with an equivalent regimen of exposure to red light was less damaging to the retina, as measured by ERG and histology.. Expression of a human hT17M mutant rhodopsin transgene in mice is associated with photoreceptor apoptosis in response to moderate exposure to light. This phenotype was not observed in nontransgenic littermates or in mice expressing an hP23H mutant human rhodopsin transgene. The results suggest that elimination of the glycosylation site at N15 is associated with increased sensitivity to light-induced damage.

Topics: Animals; Apoptosis; Dependovirus; Disease Models, Animal; Electroretinography; Enzyme-Linked Immunosorbent Assay; Female; Genes, Dominant; Genetic Therapy; In Situ Nick-End Labeling; Light; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Rhodopsin; Transgenes

Most transgenic animal models of retinal degeneration caused by rhodopsin mutations express the rhodopsin transgene on a wild-type (WT) genetic background. Previous studies have demonstrated that one mechanism of retinal degeneration is rhodopsin overexpression. To study the effect of C-terminal truncation of rhodopsin without the confounding factors of overexpression, several lines of transgenic mice were generated that expressed a C-terminal rhodopsin mutation on rhodopsin-knockout backgrounds.. Two lines of transgenic mice, expressing different levels of C-terminal truncated rhodopsin (S334ter) were mated with heterozygous rhodopsin-knockout (rho+/-) mice to express S334ter rhodopsin on a background with reduced endogenous rhodopsin expression. S334ter mice were mated to homozygous knockout (rho-/-) mice to examine the effect of S334ter rhodopsin on a null rhodopsin background. S334ter rhodopsin expression was estimated by Western blot. Retinal function was assessed by ERG and retinal degeneration by histopathology and morphometry. C-terminal rhodopsin sorting and trafficking was examined by fluorescence immunocytochemistry with detection by electron microscope.. Expression of S334ter truncated rhodopsin at low levels in the presence of decreased total rhodopsin in rods (S334ter, rho+/-) increased the rate of rod cell death in comparison to rho+/- littermates. In addition, S334ter rhodopsin prolonged the recovery time of the rod ERG to a light flash and diminished the a-wave amplitudes in comparison to their (rho+/-) littermates. Photoreceptors of S334ter mice on a homozygous rhodopsin-knockout background (S334ter+, rho-/-) had a fraction of mutant rhodopsin localized to the ciliary membranes.. Expression of S334ter rhodopsin without overexpression of total opsin in the rod photoreceptor decreased rod cell contribution to the ERG and compromised rod cell survival in adult mice. The increased cell death may be a consequence of C-terminal truncated rhodopsin mislocalization in membranes of the inner segment. Another possible pathologic mechanism is prolonged activation of phototransduction from the presence of mutant rhodopsin in the outer segment lacking the normal C-terminal binding sites for shutoff by arrestin and phosphorylation. These results suggest that rhodopsin lacking a C-terminal trafficking signal can be transported to the rod outer segment without cotransporting with full-length rhodopsin.

Topics: Animals; Blotting, Western; Cell Survival; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Genotype; Male; Mice; Mice, Knockout; Mice, Transgenic; Microscopy, Immunoelectron; Protein Transport; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Transgenes

The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo.. Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site.. Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33.. Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Topics: Aging; Animals; Animals, Newborn; Cyclic Nucleotide Phosphodiesterases, Type 6; Eye; Immunohistochemistry; Iontophoresis; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Oligonucleotides; Phosphoric Diester Hydrolases; Point Mutation; Retina; Retinal Degeneration; Rhodopsin; Staining and Labeling; Targeted Gene Repair

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Topics: Adaptation, Ocular; Animals; Aspartic Acid; Calcium; Cyclic GMP; Cysteine; Dark Adaptation; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Glycine; Guanylate Cyclase-Activating Proteins; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Physical Stimulation; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Tyrosine

To analyze the role of VEGF and its receptors, VEGFR2/Flk1 and VEGFR1/Flt1, on retinal progenitor cells (RPCs) in a murine model of inherited retinal degeneration (rd1 mice).. After proliferating RPCs in the retina of rd1 mice were labeled with bromodeoxyuridine (BrdU), expressions of VEGFR2/Flk1 and VEGFR1/Flt1 were immunohistochemically analyzed. To examine its effect on the proliferation of BrdU-positive RPCs in rd1 mice, VEGF was administered into retinal culture medium with or without blocking agents against VEGFR2/Flk1 or VEGFR1/Flt1 in vitro or injected into vitreous cavity in vivo.. BrdU-labeled RPCs in rd1 mice expressed VEGFR2/Flk1 but not VEGFR1/Flt1. These cells later expressed retinal neuronal markers such as Pax6 and rhodopsin. Exposure of the retinas from postnatal day (P) 9 rd1 mice to VEGF increased the number of proliferating RPCs by 61% in vitro. This effect was blocked by concomitant administration of VEGFR2/Flk1 kinase inhibitor. In vivo, a single intravitreal injection of VEGF in rd1 mice at P9 increased by 138% the number of RPCs and cells that developed from RPCs in the peripheral retina at P18.. VEGF stimulates the proliferation of RPCs through VEGFR2/Flk1 in rd1 mice. The observed proliferation of RPCs that have the potential to differentiate into retinal neurons may enhance the regeneration of the degenerating retina.

Topics: Animals; Animals, Newborn; Bromodeoxyuridine; Cell Count; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Eye Proteins; Fluorescent Antibody Technique, Indirect; Homeodomain Proteins; In Situ Nick-End Labeling; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Organ Culture Techniques; Paired Box Transcription Factors; PAX6 Transcription Factor; Repressor Proteins; Retina; Retinal Degeneration; Rhodopsin; Stem Cells; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas.. Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), supershift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling.. Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells.. The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa.

Topics: Animals; Blotting, Western; Cell Death; Cell Survival; DNA; Dog Diseases; Dogs; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Immunohistochemistry; Light; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-fos; Radiation Injuries, Experimental; Retinal Degeneration; Rhodopsin; Signal Transduction; Transcription Factor AP-1

Highly abundant short-chain alcohol dehydrogenases (RDHs) in the retina were assumed to be involved in the recycling of 11-cis-retinal chromophore in the visual cycle. Mutations in human RDH genes are associated with Fundus albipunctatus, a mild form of night blindness (RDH5) and an autosomal recessive, childhood-onset severe retinal dystrophy (RDH12). Rdh12 knockout mice were found to be susceptible to light-induced photoreceptor apoptosis, whereas Rdh5 and Rdh8 knockout mice displayed only delayed dark adaptation. However, each knockout mouse eventually regenerated normal levels of visual pigments, suggesting that RDHs compensate for each other in the visual cycle. Here, we established RDH double knockout (Rdh8(-/-)Rdh12(-/-)) and triple knockout (Rdh5(-/-)Rdh8(-/-)Rdh12(-/-)) mice generated on various genetic backgrounds including a rod alpha-transducin knockout to test cone function. RDH activity was severely reduced in Rdh8(-/-)Rdh12(-/-) retina extracts, whereas Rdh8(-/-) RDH activity was intermediate and Rdh12(-/-) RDH activity was reduced only slightly. Surprisingly, all multiple knockout mice produced sufficient amounts of the chromophore to regenerate rhodopsin and cone pigments in vivo. Three-month-old Rdh8(-/-)Rdh12(-/-) mice characteristically displayed a slowly progressing rod-cone dystrophy accompanied by accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic substance known to contribute to retinal degeneration. A2E accumulation and retinal degeneration were prevented by application of retinylamine, a potent retinoid cycle inhibitor. The results suggest that RDH8 and RDH12 are dispensable in support of the visual cycle but appear to be key components in clearance of free all-trans-retinal, thereby preventing A2E accumulation and photoreceptor cell death.

Topics: Alcohol Oxidoreductases; Animals; Dark Adaptation; Diterpenes; Mice; Mice, Knockout; Retina; Retinal Degeneration; Retinal Dehydrogenase; Retinoids; Rhodopsin

Rhodopsin mutations account for approximately 25% of human autosomal dominant retinal degenerations. However, the molecular mechanisms by which rhodopsin mutations cause photoreceptor cell death are unclear. Mutations in genes involved in the termination of rhodopsin signaling activity have been shown to cause degeneration by persistent activation of the phototransduction cascade. This study examined whether three disease-associated rhodopsin substitutions Pro347Ser, Lys296Glu, and the triple mutant Val20Gly, Pro23His, Pro27Leu (VPP) caused degeneration by persistent transducin-mediated signaling activity.. Transgenic mice expressing each of the rhodopsin mutants were crossed onto a transducin alpha-subunit null (Tr(alpha)(-/-)) background, and the rates of photoreceptor degeneration were compared with those of transgenic mice on a wild-type background.. Mice expressing VPP-substituted rhodopsin had the same severity of degeneration in the presence or absence of Tr(alpha). Unexpectedly, mice expressing Pro347Ser- or Lys296Glu-substituted rhodopsins exhibited faster degeneration on a Tr(alpha)(-/-) background. To test whether the absence of alpha-transducin contributed to degeneration by favoring the formation of stable rhodopsin/arrestin complexes, mutant Pro347Ser(+), Tr(alpha)(-/-) mice lacking arrestin (Arr(-/-)) were analyzed. Rhodopsin/arrestin complexes were found not to contribute to degeneration.. The authors hypothesized that the decay of metarhodopsin to apo-opsin and free all-trans-retinaldehyde is faster with Pro347Ser-substituted rhodopsin than it is with wild-type rhodopsin. Consistent with this, the lipofuscin fluorophores A2PE, A2E, and A2PE-H(2), which form from retinaldehyde, were elevated in Pro347Ser transgenic mice.

Topics: Animals; Arrestin; Genotype; Lipofuscin; Mice; Mice, Transgenic; Oligopeptides; Point Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Transducin; Vision, Ocular

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degeneration. This group of disorders essentially leads to blindness due to mutations in different genes. The genetic basis affected by sporadic and inherited autosomal dominant, autosomal recessive or X-linked mutations is complex. In humans, RP is in most cases associated with missense mutations in the rhodopsin gene (RHO). RHO plays an important role in phototransduction pathways. So far, few studies have described associations between chromosomal alterations and RP. In this study, we present a case report of a premature, 32-week-old male baby who suffered from retinopathy, facial dysmorphisms and other disorders. His chromosomes were analyzed by conventional and high-resolution chromosomal techniques. This analysis revealed structural aberrations on chromosomes 3 and 5 with an apparently balanced chromosomal translocation with karyotype 46,XY,t(3;5)(q25;q11.2). Remarkably, the 3q breakpoint on the long arm of chromosome 3 is located close to the physical RHO chromosomal gene location. In this study, we describe presumably for the first time a possible association between a 3q;5q chromosomal alteration and RP. We conclude that the new detected chromosomal translocation may lead either to loss or inactivation of the intragenic RHO gene or its respective gene regulatory region. As a consequence, the chromosomal aberration may be responsible for retinitis pigmentosa.

Topics: Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 5; Craniofacial Abnormalities; Humans; Infant, Newborn; Infant, Premature; Male; Retinal Degeneration; Rhodopsin; Translocation, Genetic

Ciliary neurotrophic factor (CNTF) provides morphologic preservation of rods in several animal models of retinitis pigmentosa (RP). However, CNTF may alter photoreceptor morphology and rod photoreceptor differentiation in vitro, as well as affecting normal retinal electrophysiology. In addition, the capacity of CNTF to support other cell types affected secondarily in RP (cones and ganglion cells) is unclear. The purposes of this study were to examine the effects of CNTF upon a canine model of RP, the rod-cone degeneration (rcd-1) dog. Archival tissue from a previous study assessing the capacity of CNTF to rescue photoreceptors in rcd-1 dogs was used. One eye was treated for 7 weeks before being explanted. The contralateral eye was untreated. A total of 23 rcd-1 dogs and seven control dogs (four untreated and three CNTF-treated) were used. Morphometric data describing outer and inner nuclear layer thickness, inner retinal thickness, cones and ganglion cells were collected at nine evenly spaced points along each retina and analysed using a mixed effects model. Immunohistochemistry was performed on a subset of 11 dogs for expression of rhodopsin, human cone arrestin (hCAR) and recoverin. CNTF protected the outer nuclear layer and increased inner retinal thickness in a dose-dependent manner (both were maximal at CNTF doses of 1-6 ng day-1). Significant cone loss or reduction of inner nuclear layer width in rcd-1 did not occur in this model, therefore we were unable to assess the protective effect of CNTF upon these parameters. CNTF did not afford significant ganglion cell protection. CNTF induced morphologic changes in rods and ganglion cells, as well as reducing expression of hCAR and rhodopsin, but not recoverin. The dose of CNTF which provided optimal outer nuclear layer protection also resulted in several other effects, including altered ganglion cell morphology, increased thickness of the entire retina, and reduced expression of some phototransduction proteins. These changes were more marked in rcd-1 retinas than in wild-type retinas. This implies that the consequences of CNTF treatment may be substantially influenced by the cellular context into which it is introduced.

Topics: Animals; Arrestin; Ciliary Neurotrophic Factor; Dogs; Dose-Response Relationship, Drug; Immunohistochemistry; Models, Animal; Recoverin; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin

Mutations in the C terminus of rhodopsin disrupt a rod outer segment localization signal, causing rhodopsin mislocalization and aggressive forms of retinitis pigmentosa (RP). Studies of cultured photoreceptors suggest that activated mislocalized rhodopsin can cause cell death via inappropriate G-protein-coupled signaling. To determine whether this pathway occurs in vivo, we developed a transgenic Xenopus laevis model of RP based on the class I rhodopsin mutation Q344Ter (Q350Ter in X. laevis). We used a second mutation, K296R, to block the ability of rhodopsin to bind chromophore and activate transducin. We compared the effects of expression of both mutants on X. laevis retinas alone and in combination. K296R did not significantly alter the cellular distribution of rhodopsin and did not induce retinal degeneration. Q350Ter caused rhodopsin mislocalization and induced an RP-like degeneration, including loss of rods and development of sprouts or neurites in some remaining rods, but did not affect the distribution of endogenous rhodopsin. The double mutant K296R/Q350Ter caused a similar degeneration and neurite outgrowth. In addition, we found no protective effects of dark rearing in these animals. Our results demonstrate that the degenerative effects of mislocalized rhodopsin are not mediated by the activated form of rhodopsin and therefore do not proceed via conventional G-protein-coupled signaling.

Topics: Animals; Animals, Genetically Modified; Chlorocebus aethiops; COS Cells; Female; Male; Neurites; Point Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis; Xenopus Proteins

In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration.

Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Blotting, Northern; Calcium; Calcium Signaling; Calnexin; Cell Survival; Chromatography, Affinity; DNA; Drosophila; Electrophoresis, Polyacrylamide Gel; Immunohistochemistry; Light; Microscopy, Electron; Molecular Chaperones; Molecular Sequence Data; Mutation; Patch-Clamp Techniques; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin

Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65(450Leu) or Rpe65(450Met)) can act as a modulator of light-damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild-type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c-Fos/AP-1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65(450Met) variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65(450Leu) variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c-Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c-Fos.

Topics: Age Factors; Animals; Carrier Proteins; cis-trans-Isomerases; Disease Models, Animal; Eye Proteins; Gene Expression Regulation, Developmental; Genetic Variation; Leucine; Methionine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Time Factors

Channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii has been shown to be a directly light-switched cation-selective ion channel, which employs 11-cis retinal as its chromophore. This is the same chromophore as the mammalian photoreceptor's visual pigment-rhodopsin. Previously, investigators demonstrated that ChR2 can be used to optically control neuronal firing by depolarizing the cell. In this issue of Neuron, Bi et al. apply viral-mediated gene transfer to deliver ChR2 to retinal ganglion cells (RGC) in a rodent model of inherited blindness. In this way, the authors have genetically engineered surviving retinal neurons to take on the lost photoreceptive function. The conversion of light-insensitive retinal interneurons into photosensitive cells introduces an entirely new direction for treatments of blinding retinal degeneration.

Topics: Animals; Disease Models, Animal; Genetic Therapy; Mice; Models, Biological; Photoreceptor Cells; Rats; Retina; Retinal Degeneration; Retinal Ganglion Cells; Rhodopsin; Vision, Ocular

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Dose-Response Relationship, Radiation; Evoked Potentials, Visual; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Green Fluorescent Proteins; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neurons; Patch-Clamp Techniques; Photic Stimulation; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Recovery of Function; Retina; Retinal Degeneration; Rhodopsin; Visual Pathways

The present experiments were undertaken to test recovery of function in the retina of the rhodopsin-mutant P23H-3 rat, in response to the management of ambient light. Observations were made in transgenic P23H-3 and non-degenerative Sprague-Dawley albino (SD) rats raised to young adulthood in scotopic cyclic light (12h 5 lx "daylight", 12h dark). The brightness of the day part of the cycle was increased to 300 lx (low end of daylight range) for 1 week, and then reduced to 5 lx for up to 5 weeks. Retinas were assessed for the rate of photoreceptor death (using the TUNEL technique), photoreceptor survival (thickness of the outer nuclear layer), and structure and function of surviving photoreceptors (outer segment (OS) length, electroretinogram (ERG)). Exposure of dim-raised rats to 300 lx for 1 week accelerated photoreceptor death, shortened the OSs of surviving photoreceptors, and reduced the ERG a-wave, more severely in the P23H-3 transgenics. Returning 300 lx-exposed animals to 5 lx conditions decelerated photoreceptor death and allowed regrowth of OSs and recovery of the a-wave. Recovery was substantial in both strains, OS length in the P23H-3 retina increasing from 17% to 90%, and a-wave amplitude from 33% to 45% of control values. Thinning of the ONL over the 6 week period studied was minimal. The P23H-3 retina thus shows significant recovery of function and outer segment structure in response to a reduction in ambient light. Restriction of ambient light may benefit comparable human forms of retinal degeneration in two ways, by reducing the rate of photoreceptor death and by inducing functional recovery in surviving photoreceptors.

Topics: Animals; Animals, Genetically Modified; Cell Death; Electroretinography; In Situ Nick-End Labeling; Photoreceptor Cells, Vertebrate; Phototherapy; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

To investigate the pathogenic mechanisms that underlie retinal degeneration induced by the rhodopsin mutation P23H in a Xenopus laevis model of RP.. Transgenic X. laevis were generated that expressed the rhodopsin mutants rhoP23H and rhoP23H/K29R (a variant incapable of transducin activation). Using quantitative dot blot assay, transgenic rhodopsin levels and the extent of retinal degeneration were determined. The contribution of rhodopsin signal transduction to cell death was assessed by comparison of rhoP23H and rhoP23H/K296R effects and by dark rearing of rhoP23H tadpoles. Intracellular localization and the oligomeric state of rhoP23H were determined by confocal immunofluorescence microscopy and Western blot analysis.. RhoP23H induced retinal degeneration in a dose-dependent manner whereas expression of a control rhodopsin did not, indicating that rod photoreceptor death was specific to the P23H mutation and was not caused by the overexpression of rhodopsin. Neither abolishment of rhoP23H photosensitivity and ability to activate transducin nor dark rearing rescued rod viability. RhoP23H was localized primarily to the endoplasmic reticulum (ER) of inner segments. Western blot analysis of transgenic retinas showed that rhoP23H was prone to form dimers and higher molecular weight oligomers. However, aggresomes were not observed in rhoP23H transgenic retinal sections, despite their being reported in cultured cells expressing rhoP23H.. These results support a role for rhoP23H misfolding and inner segment accumulation in rod death, possibly by ER overload or other cellular stress pathways rather than by altered rhodopsin signal transduction or aggresome formation.

Topics: Animals; Animals, Genetically Modified; Blotting, Western; Cell Death; Cell Line; Dark Adaptation; Disease Models, Animal; Dose-Response Relationship, Drug; Fluorescent Antibody Technique, Indirect; Kidney; Microscopy, Confocal; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transducin; Transfection; Transgenes; Xenopus laevis

Retinal neurodegeneration occurs in several inherited diseases. Some of the most severe disease alleles involve mutations at the C-terminus of rhodopsin, but in no case is the pathogenic mechanism leading to cell death well understood. We have examined a mouse model of recessive retinal degeneration caused by a knock-in of a human rhodopsin-EGFP fusion gene (hrhoG/hrhoG) at the rhodopsin locus. Whereas heterozygous mutant mice were indistinguishable from control mice, homozygous mutant mice had retinal degeneration. We hypothesized that degeneration might be due to aberrant rhodopsin signaling; however, inhibiting signaling by rearing mice in total darkness had no effect on the rate of degeneration. Using confocal and electron microscopy, we identified the fundamental defect as failed biogenesis of disk membranes, which is observed at the earliest stages of outer segment development. These results reveal that in addition to its role in transport and sorting of rhodopsin to disk membranes, rhodopsin is also essential for formation of disks.

Topics: Animals; Cell Membrane; Dark Adaptation; Electroretinography; Green Fluorescent Proteins; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Models, Animal; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Cell Outer Segment; Vision, Ocular

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo.

Topics: Alleles; Animals; Animals, Genetically Modified; Base Sequence; Dependovirus; Gene Silencing; Genetic Vectors; Mice; Models, Animal; Molecular Sequence Data; Mutation; Proline; Rats; Retinal Degeneration; Rhodopsin; RNA, Small Interfering

To determine the relationship between the presence of kinesin-2 and photoreceptor cell viability and opsin transport, by generating RHO-Cre transgenic mice and breeding them to mice with a floxed kinesin-2 motor gene.. Different lines of RHO-Cre transgenic mice were generated and characterized by transgene expression, histology, and electrophysiology. Mice from one line, showing uniform transgene expression, were crossed with Kif3a(flox)/Kif3a(flox) mice. The time courses of photoreceptor Cre expression, KIF3A loss, ectopic opsin accumulation, and photoreceptor cell death were determined by Western blot analysis and microscopy.. One of the RHO-Cre lines effected synchronous expression of Cre and thus uniform excision of Kif3a(flox) in rod photoreceptors across the retina. After the neonatal production of CRE and the initiation of KIF3A loss, ectopic accumulation of opsin was detected by postnatal day (P)7, and ensuing photoreceptor cell death was evident after P10 and almost complete by P28. Of importance, the photoreceptor cilium formed normally, and the disc membranes of the nascent outer segment remained normal until P10.. The RHO-Cre-8 mice provide an improved tool for studying gene ablation in rod photoreceptor cells. Regarding kinesin-2 function in photoreceptor cells, the relatively precise timing of events after CRE excision of Kif3a(flox) allows us to conclude that ectopic opsin is a primary cellular lesion of KIF3A loss, consistent with the hypothesis that opsin is a cargo of kinesin-2. Moreover, it demonstrates that KIF3A loss results in very rapid photoreceptor cell degeneration.

Topics: Animals; Blotting, Western; Electroretinography; Extracellular Matrix Proteins; Gene Expression Regulation, Enzymologic; Gene Silencing; Genotype; Immunoenzyme Techniques; Integrases; Kinesins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Fluorescence; Microtubule-Associated Proteins; Photoreceptor Cells, Vertebrate; Protein-Lysine 6-Oxidase; Retinal Degeneration; Rhodopsin

The effects of various light-induced stresses on the retina were examined in the retinal degenerative rat model.. Retinal morphology and electroretinograms (ERGs) were analyzed after application of light-induced stress of several intensities (650, 1300, 2500, or 5000 lux). For evaluation of rhodopsin (Rho) function, the kinetics of Rho regeneration and dephosphorylation were studied by spectrophotometric analysis and immunofluorescence labeling with antibodies specifically directed toward the phosphorylated residues (334)Ser and (338)Ser in the C terminus of Rho. Retinal cGMP concentration was determined by ELISA. Expression levels of neurotrophic factors (FGF2, brain-derived neurotrophic factor [BDNF], platelet-derived growth factor [PDGF], and ciliary neurotrophic factor [CNTF]) were evaluated quantitatively by RT-PCR.. Light intensity-dependent deterioration of ERG responses and thinning of the retinal outer nuclear layer were observed in wild-type and Royal College of Surgeons (RCS) rat retinas. Under dark adaptation after light-induced stress, the kinetics of Rho regeneration were not different between wild-type and RCS rat retinas. Rho dephosphorylation at (334)Ser and (338)Ser was extremely delayed in RCS rat retinas compared with wild-type without light-induced stress, but Rho dephosphorylation at those sites became slower in both RCS and wild-type rat retinas. In terms of expression of neurotrophic factors, almost no significant changes were observed between the animals after light-induced stress.. The present study indicates that light-induced stress causes intensity-dependent deterioration in retinal function and morphology in wild-type and RCS rat retinas. Disruption of the phototransduction cascade resulting from slower kinetics of Rho dephosphorylation appears to be involved in retinal degeneration.

Topics: Animals; Cyclic GMP; Dark Adaptation; Electroretinography; Enzyme-Linked Immunosorbent Assay; Light; Microscopy, Fluorescence; Nerve Growth Factors; Phosphorylation; Radiation Injuries, Experimental; Rats; Rats, Inbred BN; Rats, Mutant Strains; Rats, Sprague-Dawley; Regeneration; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger

This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression.. Retinas from rhodopsin knockout (Rho-/-) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with l-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density.. Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho-/- mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho-/- or with l-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1alpha DNA-binding and VEGF mRNA expression in Rho-/- retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho-/- mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas.. Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

Topics: Animals; Apyrase; Cyclic Nucleotide Phosphodiesterases, Type 6; Dark Adaptation; Diltiazem; Enzyme-Linked Immunosorbent Assay; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitroimidazoles; Oxygen Consumption; Phosphoric Diester Hydrolases; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Transcriptional Activation; Vascular Endothelial Growth Factor A

To investigate the peropsin gene (RRH), encoding a retinal pigment epithelium homolog of the rod-expressed opsin (rhodopsin), for the presence of pathogenic mutations causing retinitis pigmentosa (RP) or other retinal degenerations.. All seven exons composing the RRH open reading frame and the immediate intron sequences were analyzed by direct nucleotide sequencing of 613 patients with forms of retinal degeneration.. One patient with retinitis punctata albescens was a heterozygote with the missense change Cys98Tyr (TGT>TAT, c.293G>A). This change affects the homologous residue that is the target of the rhodopsin mutation Cys110Tyr, a reported cause of dominant RP. Unfortunately, none of the patient's relatives were available for a segregation analysis to determine if this change is unambiguously associated with disease. No definite pathogenic mutation was found in any of the other 612 patients who were evaluated.. The Cys98Tyr is a possible cause of retinitis punctata albescens, although this conclusion is tentative because the change was found in only one patient. Our results indicate that the peropsin gene is not a common cause of RP or some related retinal degenerations, at least in the set of patients we analyzed.

Topics: Cysteine; DNA Mutational Analysis; Heterozygote; Humans; Mutation, Missense; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tyrosine

Topics: Animals; Dextrans; Diabetic Retinopathy; Fluorescein; Humans; Mice; Mice, Transgenic; Perfusion; Photoreceptor Cells; Promoter Regions, Genetic; Retina; Retinal Degeneration; Rhodopsin; Vascular Endothelial Growth Factor A

Topics: Animals; Arrestin; Cattle; Cycloheximide; Green Fluorescent Proteins; Light; Lysine; Microscopy, Confocal; Neurons; Protein Transport; Proto-Oncogene Proteins c-myc; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Xenopus

To report the phenotype and characterization of a new, naturally occurring mouse model of hereditary retinal degeneration (rd12).. The retinal phenotype of rd12 mice were studied using serial indirect ophthalmoscopy, fundus photography, electroretinography (ERG), genetic analysis including linkage studies and gene identification, immunohistochemistry, and biochemical analysis.. Mice homozygous for the rd12 mutation showed small punctate white spots on fundus examination at 5 months of age. The retina in the rd12 homozygote had a normal appearance at the light microscopic level until 6 weeks of age when occasional voids appeared in the outer segments (OS) of the photoreceptor (PR) cells. The outer nuclear layer (ONL) appeared normal until 3 months of age though more obvious voids were detected in the OS. By 7 months of age, 6 to 8 layers of ONL remained in the mutant retina, and the OS were obviously shorter. The first sign of retinal degeneration was detected at the electron microscopic level around 3 weeks of age when occasional small lipid-like droplets were detected in the retinal pigment epithelium (RPE). By 3 months of age, much larger, lipid-like droplets accumulated in RPE cells accompanied by some OS degeneration. While the histology indicated a relatively slow retinal degeneration in the rd12 homozygous mutant mice, the rod ERG response was profoundly diminished even at 3 weeks of age. Genetic analysis showed that rd12 was an autosomal recessive mutation and mapped to mouse chromosome 3 closely linked to D3Mit19, a location known to be near the mouse Rpe65 gene. Sequence analysis showed that the mouse retinal degeneration is caused by a nonsense mutation in exon 3 of the Rpe65 gene, and the gene symbol for the rd12 mutation has been updated to Rpe65rd12 to reflect this. No RPE65 expression, 11-cis retinal, or rhodopsin could be detected in retinas from rd12 homozygotes, while retinyl esters were found to accumulate in the retinal pigment epithelium (RPE).. Mutations in the retinal pigment epithelium gene encoding RPE65 cause an early onset autosomal recessive form of human retinitis pigmentosa, known as Leber congenital amaurosis (LCA), which results in blindness or severely impaired vision in children. A naturally arising mouse Rpe65 mutation provides a good model for studying the pathology of human RPE65 mutations and the effects of retinyl ester accumulation.

Topics: Animals; Blindness; Carrier Proteins; cis-trans-Isomerases; Codon, Nonsense; Disease Models, Animal; Electroretinography; Exons; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Ophthalmoscopy; Phenotype; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Retinal Degeneration; Retinaldehyde; Rhodopsin

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft-host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft-host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.

Topics: Animals; Animals, Genetically Modified; Cell Differentiation; Cell Survival; Cells, Cultured; Disease Models, Animal; Glial Fibrillary Acidic Protein; Immunohistochemistry; Photoreceptor Cells; Rats; Retina; Retinal Degeneration; Rhodopsin; Stem Cell Transplantation; Stem Cells; Synapsins; Tomography, Optical Coherence

Genetic and environmental factors modify the severity of human neurodegenerations. Retinal degenerations caused by rhodopsin gene mutations show severity differences within and between families and even within regions of the same eye. Environmental light is thought to contribute to this variation. In the naturally occurring dog model of the human disorder, we found that modest light levels, as used in routine clinical practice, dramatically accelerated the neurodegeneration. Dynamics of acute retinal injury (consisting of abnormal intraretinal light scattering) were visualized in vivo in real time with high-resolution optical imaging. Long term consequences included fast or slow retinal degeneration or repair of injury depending on the dose of light exposure. These experiments provide a platform to study mechanisms of neuronal injury, repair, compensation, and degeneration. The data also argue for a gene-specific clinical trial of light reduction in human rhodopsin disease.

Topics: Animals; Base Sequence; Disease Models, Animal; DNA; Dogs; Humans; Light; Mutation; Nerve Regeneration; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transcription Factor AP-1

RPE65 is essential for the generation of 11-cis retinal. Rod photoreceptors in the RPE65-knockout (Rpe65(-/-)) mouse are known to degenerate slowly with age. This study was designed to examine cone photoreceptors and the expression of cone-specific genes in the Rpe65(-/-) mouse.. Gene expression changes were identified by microarray and confirmed by real-time RT-PCR. Cone photoreceptors were stained by peanut agglutinin (PNA) lectin in the flatmounted retina. The 9- or 11-cis retinal was supplied by intraperitoneal injections.. The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whereas the decrease in the middle-wavelength (MWL) cone opsin mRNA occurred relatively later in age. In contrast, the rhodopsin mRNA level did not show any significant change at all the ages analyzed. Consistent with the cone opsin changes, the cone transducin alpha-subunit mRNA decreased at both 4 and 8 weeks of age, whereas again the rod transducin alpha-subunit did not show any significant change. Rpe65(-/-) mice showed significant cone loss in both the central and ventral retina between 2 and 3 weeks of age. Administration of 9- or 11-cis retinal to Rpe65(-/-) mice 2 weeks of age increased cone density by twofold in these areas.. In the Rpe65(-/-) mouse, the expression of cone-specific genes is downregulated and is accompanied by cone degeneration at early ages. Early administration of 9- or 11-cis retinal can partially prevent cone loss, suggesting that the absence of 11-cis chromophore may be responsible for the early cone degeneration.

Topics: Animals; Carrier Proteins; Cell Count; cis-trans-Isomerases; Diterpenes; Down-Regulation; Eye Proteins; Gene Expression; Gene Expression Profiling; GTP-Binding Protein alpha Subunits; Heterotrimeric GTP-Binding Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Proteins; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinaldehyde; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Rod Opsins; Transducin

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld(null) flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

Topics: Animals; Drosophila melanogaster; Light; Membrane Proteins; Mutation; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phospholipase D; Phospholipid Transfer Proteins; Phospholipids; Photoreceptor Cells, Invertebrate; Retina; Retinal Degeneration; Rhodopsin; Type C Phospholipases; Vision, Ocular

To define the distribution of the red/green and blue opsins in cones from donor eyes from an affected member of a clinically well-characterized family with an autosomal dominant form of cone dystrophy.. Tissue was fixed and processed for immunohistochemistry. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to cone cytoplasm, rhodopsin, and cone opsins. The cone-associated matrix was also labeled with the lectin PNA. The affected donor eyes were compared to a postmortem matched normal eye.. Electroretinogram (ERG) testing three years prior to the affected member's death showed normal rod function, while the cone b-wave amplitude was reduced 40% below the lower limit of normal. Fundus exam showed only isolated drusen within the macula. Either a normal-appearing or only nonspecific macular findings were noted in the other affected family members who were examined. Immunofluorescence studies showed that blue cone opsin was restricted to the outer segments of blue cones in the affected retina. Red/green opsins were distributed along the entire plasma membrane of these cone types, from the tip of the outer segment to the synaptic base. Cone-associated matrix displayed a heterogeneous distribution. These patterns were observed both in the macula and in the periphery of the affected retina. Cone pedicles appeared larger than normal. In contrast, rhodopsin staining appeared normal.. The immunocytochemical data obtained suggest that the clinical manifestation of this dystrophy is associated with an abnormal distribution of cone red/green opsins. Additionally, changes in the cone pedicles could have contributed to the abnormal cone ERG in this patient.

Topics: Aged; Aged, 80 and over; Color Vision Defects; Electroretinography; Female; Genes, Dominant; Humans; Immunohistochemistry; Male; Pedigree; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Rod Opsins

Topics: Animals; Apoptosis; Awards and Prizes; Florida; History, 21st Century; Humans; Light; Ophthalmology; Radiation Injuries; Retina; Retinal Degeneration; Rhodopsin; Societies, Scientific; Vision Disorders

An unusual retinal vascular morphology in an enucleated eye from a patient with Leber congenital amaurosis (LCA) has been associated with a mutation in AIPL1. The AIPL1 protein is expressed in the pineal gland and retinal photoreceptors. In the retina, AIPL1 is expressed in both developing cone and rod photoreceptors, but it is restricted to rod photoreceptors in the adult human retina. Therefore, this study was conducted to determine the photoreceptor phenotype in this LCA patient to determine if photoreceptors were differentially affected.. Additional genetic screening was performed and the consequences of the H82Y amino acid substitution characterized in an in vitro assay of NUB1 modulation. The morphology of the photoreceptors was examined by light and electron microscopy. Immunohistochemistry and immunofluorescent confocal microscopy was performed using a range of retinal photoreceptor markers.. Transfection of the H82Y mutant AIPL1 in SK-N-SH cells revealed a normal subcellular localization and solubility but resulted in an increased ability of AIPL1 to redistribute GFP-NUB1 to the cytoplasm and resolve NUB1 fragment inclusion formation. Morphologically, the LCA retina appeared to be cone-dominant with a single layer of cone-like cells remaining in the central retina. Photoreceptor outer segments were absent and the surviving residual inner segments were severely shortened. Severe degeneration of the LCA retina was associated with upregulation of glial fibrillary acidic protein (GFAP). No signal was detected for AIPL1, rhodopsin, or L/M and S cone opsins in the LCA retina. Double labeling with peanut agglutinin (PNA) and wheat germ agglutinin (WGA) supported a cone-dominant phenotype for the surviving photoreceptors in the LCA retina, as did double labeling for cone arrestin, and rod and cone recoverin. The cone arrestin signal was restricted to the residual photoreceptor inner segments and was not detected in the cell bodies, axons, or axon terminals of the surviving photoreceptors. Recoverin immunoreactivity was most intense in the residual photoreceptor inner segments.. The phenotype in this patient suggests that although AIPL1 is required for the development of normal rod and cone photoreceptor function, it might only be essential for rod and not cone survival in the adult.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Blindness; Blotting, Western; Carrier Proteins; cis-trans-Isomerases; Eye Proteins; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Guanylate Cyclase; Homeodomain Proteins; Humans; Male; Microscopy, Confocal; Microscopy, Electron; Phenotype; Polymorphism, Single-Stranded Conformational; Receptors, Cell Surface; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Trans-Activators; Transfection; Up-Regulation

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin-EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.

Topics: Animals; Animals, Genetically Modified; Feasibility Studies; Genetic Therapy; Green Fluorescent Proteins; Mice; Photoreceptor Cells; Retina; Retinal Degeneration; Rhodopsin

In retinal degenerative diseases, rod photoreceptors typically deteriorate more rapidly than cone photoreceptors. In the Rpe65(-/-) mouse, a model for Leber's congenital amaurosis, cones degenerate much more rapidly than rods. In this model, the retinoid processing pathway in the retinal pigment epithelium is disrupted, and 11-cis retinal is not generated. This study was designed to investigate the feasibility of restoring functional cones with exogenous 11-cis retinal.. Rpe65(-/-)::Rho(-/-) mice were used to remove any interference of rods and compared with wild-type (wt) mice. Pups were injected intraperitoneally with 11-cis retinal, starting at postnatal day (P)10, and were maintained in complete darkness. At P25, cone function was assessed with photopic single-flash and flicker ERGs. Cone survival was determined immunohistochemically with cone-specific antibodies, and cone opsin levels were obtained by quantitative RT-PCR.. At P25, cone density and transcript levels of cone opsins were drastically reduced, but a minute cone electroretinogram was detected, indicating that the cones were functional. Confocal microscopy revealed that the cone opsins were mislocalized, suggesting that their transport to the outer segments was impaired. Intraperitoneal administrations of 11-cis retinal before P25 led to increased transport of cone opsins to the outer segments and preserved cones anatomically and functionally.. The results suggest that the ligand is required during cone opsin synthesis for successful opsin trafficking and that without 11-cis retinal, cones may degenerate because of opsin mislocalization. These results may have important consequences for the treatment of cone dystrophies.

Topics: Animals; Biological Transport; Carrier Proteins; Cell Survival; cis-trans-Isomerases; Electroretinography; Eye Proteins; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Photic Stimulation; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinaldehyde; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Rod Opsins

In animal models of retinitis pigmentosa, rod photoreceptor degeneration eventually leads to loss of cone photoreceptors. The purpose of this study was to characterize a transgenic model of rod degeneration in zebrafish.. Zebrafish transgenic for XOPS-mCFP, a membrane-targeted form of cyan fluorescent protein driven by the Xenopus rhodopsin promoter, were generated by plasmid injection. Immunohistochemistry was used to detect cell type, proliferation, and TUNEL markers in larval and adult retinas. Rod- and cone-specific transcripts were detected by RT-PCR. Visual responses in transgenic adults were measured by electroretinogram.. The XOPS promoter directed specific expression of mCFP in rods by 55 hours post fertilization (hpf). Rods in XOPS-mCFP heterozygotes began dying at 3.5 days post fertilization (dpf) and were almost completely absent by 5 dpf. A few rods were observed at the retinal margin, and numerous immature rods were observed in the outer nuclear layer (ONL) of transgenic adults. Apoptosis was increased in the ONL of larval and adult transgenic animals, and an elevation of rod precursor proliferation in adults was observed. ERG analysis confirmed that rod responses were absent in this line. Cone morphology and electrophysiology appeared normal in transgenic animals up to 7 months of age.. The XOPS-mCFP transgene causes selective degeneration of rods without secondary loss of cones in animals up to 7 months of age. This raises important questions about the significance of rod-cone interactions in zebrafish and their potential as a model of human inherited retinal degenerations.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Cell Survival; Disease Models, Animal; Electroretinography; Fluorescent Antibody Technique, Indirect; Green Fluorescent Proteins; In Situ Nick-End Labeling; Microscopy, Confocal; Plasmids; Recombinant Fusion Proteins; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Transgenes; Zebrafish

To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres.. rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections.. Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres.. A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

Topics: Animals; Antigens, Differentiation; Cell Count; Cell Proliferation; Cell Survival; Drug Carriers; Electroretinography; Fluorescent Antibody Technique, Indirect; Glial Cell Line-Derived Neurotrophic Factor; Glial Fibrillary Acidic Protein; Injections; Lactic Acid; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Microspheres; Photoreceptor Cells, Vertebrate; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Recombinant Proteins; Retinal Degeneration; Rhodopsin; Vitreous Body

1. Retinal dystrophies (RD) comprise a group of clinically and genetically heterogeneous retinal disorders, which typically result in the degeneration of photoreceptors followed by the impairment or loss of vision. Although age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are among the most common forms of RD, currently, there is no effective treatment for either disorder. 2. Recently, abnormal protein accumulation and aggregation due to protein misfolding and proteasome inhibition have been implicated in the pathogenesis of RD. In this paper we describe effects of several factors on protein aggregation and survival of photoreceptor cells. 3. Expression of rhodopsin carrying P23H mutation causes its accumulation in intracellular inclusion bodies in a perinuclear area of photoreceptor cells. beta- and gamma-synucleins and heat shock protein Hsp-70, but not alpha-synuclein, protect cultured ocular cells from mutant opsin accumulation. This effect might be explained by their chaperonic activity. 4. Knock-out of alpha- and gamma-synucleins does not affect gross retinal morphology, but induces tyrosine hydroxylase in the inner prexiform layer of the retina. Selegiline-a monoamine oxidase inhibitor used for the treatment of Parkinson's disease, reduces apoptosis and increases viability in cultured retinal pigment epithelium cells (APRE-19). 5. These results suggest that chaperones and selegiline may be considered promising candidates for the protection of ocular cells from the accumulation of misfolded and aggregated proteins.

Topics: Animals; Apoptosis; Cell Line; Cell Survival; Cytoprotection; Eye Proteins; gamma-Synuclein; Humans; Mice; Mice, Knockout; Molecular Chaperones; Neuroprotective Agents; Photoreceptor Cells; Protein Folding; Retina; Retinal Degeneration; Rhodopsin; Selegiline

Numerous mutations of rhodopsin lead to rod cell death and ultimately to complete blindness, yet little is known about the alterations in the physiology of the light sensors containing the aberrant protein, the rod photoreceptors.. Suction pipettes were used to record the light responses from single rod photoreceptors isolated from the retinas of transgenic pigs of various ages and at progressive stages of retinal degeneration.. We have observed changes in the photoresponse of transgenic porcine rods containing both wild type and mutant rhodospin. Our findings are consistent with the idea that substitutions at position proline 347 of rhodopsin interfere with the inactivation of R*. In addition the level of photoreceptor degeneration is positively correlated with an acceleration and desensitization of the photoresponse to dim flashes.. It appears that the phototransduction cascade, even when initiated by wild type rhodopsin molecules is altered in a way that is progressive with the level of retinal degeneration. A model consistent with our observations introduces the idea of a binding site for the carboxy terminus of rhodopsin on rhodopsin kinase.

Topics: Amino Acid Substitution; Animals; Animals, Genetically Modified; Electrophysiology; Immunohistochemistry; Kinetics; Leucine; Light; Mutation; Proline; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Swine; Vision, Ocular

Transgenic expression of ceramidase suppresses retinal degeneration in Drosophila arrestin and phospholipase C mutants. Here, we show that expression of ceramidase facilitates the dissolution of incompletely formed and inappropriately located elements of rhabdomeric membranes in ninaE(I17) mutants lacking the G protein receptor Rh1 in R1-R6 photoreceptor cells. Ceramidase expression facilitates the endocytic turnover of Rh1. Although ceramidase expression aids the removal of internalized rhodopsin, it does not affect the turnover of Rh1 in photoreceptors maintained in dark, where Rh1 is not activated and thus has a slower turnover and a long half-life. Therefore, the phenotypic consequence of ceramidase expression in photoreceptors is caused by facilitation of endocytosis. This study provides mechanistic insight into the sphingolipid biosynthetic pathway-mediated modulation of endocytosis and suppression of retinal degeneration.

Topics: Amidohydrolases; Animals; Animals, Genetically Modified; Cell Membrane; Ceramidases; Darkness; Drosophila melanogaster; Drosophila Proteins; Endocytosis; Eye Proteins; Half-Life; Ligands; Light; Mutation; Photoreceptor Cells, Invertebrate; Receptors, G-Protein-Coupled; Retinal Degeneration; Rhodopsin

Inherited retinal degenerations are a major cause of blindness for which there are currently no effective therapies. Significant progress concerning in vivo gene transfer has allowed retardation of degeneration or retinal functional improvement in different animal models. To date, there has been no evaluation of the impact of these treatments on higher visual function, a critical step for validating gene therapy treatment strategies. Here, we have used adeno-associated (AAV2)-mediated gene transfer of Prph2 in the Prph2(Rd2/Rd2) mouse model. We then assessed higher visual function by recording from central visually responsive neurons in the superior colliculus and improvements were correlated in individual animals with retinal function (ERG) and histological and biochemical changes. Although gene replacement therapy only partially restores photoreceptor morphology, it results in a 300% increase of the visual cycle protein rhodopsin, leading to retinal function improvement (250% increase of b-wave amplitude) and significantly higher central visual responses (166% increase at 24 cd/m(2)). These findings suggest that gene replacement therapy leading to even relatively modest structural improvement may result in improved central visual function.

Topics: Action Potentials; Animals; Disease Models, Animal; Electroretinography; Genetic Therapy; Genetic Vectors; Intermediate Filament Proteins; Membrane Glycoproteins; Membrane Potentials; Mice; Mice, Transgenic; Nerve Regeneration; Nerve Tissue Proteins; Peripherins; Photic Stimulation; Photoreceptor Cells; Recovery of Function; Retinal Degeneration; Rhodopsin; Superior Colliculi; Synaptic Transmission; Up-Regulation; Vision, Ocular; Visual Pathways

The Drosophila visual system has provided a model to study phototransduction and retinal degeneration. To identify new candidate proteins that contribute to these processes, we conducted a genome-wide screen for genes expressed predominately in the eye, using DNA microarrays. This screen appeared to be comprehensive as it led to the identification of all 22 eye-enriched genes previously shown to function in phototransduction or implicated in retinal degeneration. In addition, we identified 93 eye-enriched genes whose roles have not been previously defined. One of the eye-enriched genes encoded a member of a large family of transmembrane proteins, referred to as tetraspanins. We created a null mutation in the eye-enriched tetraspanin, Sunglasses (Sun), which resulted in light-induced retinal degeneration. We found that the Sun protein was distributed primarily in lysosomes, and functioned in a long-known but poorly understood phenomenon of light-induced degradation of rhodopsin. We propose that lysosomal tetraspanins in mammalian cells may also function in the downregulation of rhodopsin and other G-protein-coupled receptors, in response to intense or prolonged agonist stimulation.

Topics: Amino Acid Sequence; Animals; Drosophila melanogaster; Drosophila Proteins; Eye Proteins; Gene Expression Regulation; Lysosomes; Membrane Proteins; Microscopy, Electron, Transmission; Molecular Sequence Data; Mutation; Oligonucleotide Array Sequence Analysis; Retinal Degeneration; Rhodopsin

The primary purpose of this study was to evaluate the impact of caspase-3 ablation on photoreceptor degeneration in the rd-1 mouse. Concurrently, the role of caspase-3 in postnatal retinal development was evaluated. Caspase-3 is an important effector caspase that mediates many of the terminal proteolytic events of apoptosis. Its activation has been demonstrated in rodent models of photoreceptor degeneration and its ablation results in exencephaly and neonatal death.. Retinal morphometry was performed at the light microscopic level in caspase-3 mutant mice from PN0 through PN23, and in rd-1/caspase-3 double mutant mice at PN14, -16, and -18. This was supplemented by terminal dUTP transferase nick end labeling (TUNEL) and immunohistochemical staining for activated caspase-3, rhodopsin, factor VII-related antigen and proliferating cell nuclear antigen (PCNA).. Caspase-3-deficient animals display marginal microphthalmia, peripapillary retinal dysplasia, delayed regression of vitreal vasculature, and retarded apoptotic kinetics of the inner nuclear layer. Ablation of caspase-3 provided transient photoreceptor protection in rd-1, but TUNEL-positive rod death proceeded, despite the absence of caspase-3 activation.. In vivo, caspase-3 is not critical for rod photoreceptor development, nor does it play a significant role in mediating pathologic rod death. Peripapillary dysplastic lesions suggest that there is delayed fusion of the optic fissure, and inner nuclear layer abnormalities indicate a cell-specific dependency on the mitochondria-caspase axis during development. The temporal nature of apoptotic retardation in the absence of caspase-3 implies the presence of caspase-independent mechanisms of developmental and pathologic cell death.

Topics: Animals; Animals, Newborn; Antigens; Caspase 3; Caspases; Cell Death; Enzyme Activation; Factor VII; Female; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Mutant Strains; Photoreceptor Cells, Vertebrate; Proliferating Cell Nuclear Antigen; Retina; Retinal Degeneration; Rhodopsin

Nine neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) are caused by an expansion of a polyglutamine (polyQ) stretch in the respective proteins. Aggregation of expanded polyQ-containing proteins into the nucleus is a hallmark of these diseases. Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of these diseases. Using SCA7 and HD mouse models in which we recently described a retinal phenotype, we investigated whether altered gene expression underlies photoreceptor dysfunction. In both models, rhodopsin promoter activity was early and dramatically repressed, suggesting that downregulation of photoreceptor-specific genes plays a major role in polyQ-induced retinal dysfunction. Because the rhodopsin promoter drives mutant ataxin-7 expression in our SCA7 mice, we also assessed whether downregulation of mutant SCA7 transgene would reverse retinopathy progression and aggregate formation. Although residual expression of mutant ataxin-7 was found negligible from 9 weeks of age, SCA7 transgenic mice showed a progressive decline of photoreceptor activity leading to a complete loss of electroretinographic responses from 1 year of age. At this age, aggregates were cleared in only half of the photoreceptors, indicating that their formation is not fully reversible in this model. We demonstrate here that abolishing full-length mutant ataxin-7 expression did not reverse retinopathy progression in SCA7 mice, raising the possibility that some polyQ-induced pathological events might be irreversible.

Topics: Age Factors; Animals; Ataxin-7; Disease Models, Animal; Disease Progression; Down-Regulation; Electroretinography; Gene Expression Regulation, Developmental; Huntingtin Protein; Huntington Disease; Macromolecular Substances; Mice; Mice, Transgenic; Nerve Tissue Proteins; Nuclear Proteins; Peptides; Photoreceptor Cells, Vertebrate; Promoter Regions, Genetic; Recombinant Proteins; Retinal Degeneration; Rhodopsin; Spinocerebellar Ataxias; Transgenes; Trinucleotide Repeat Expansion

A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, we identified a dominant mutation in the ninaE gene (NinaE(pp100)) that encodes the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration was attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaE(pp100) mutant photoreceptor cells were strongly desensitized, which partially masked their constitutive activity. This desensitization primarily resulted from both the persistent binding of arrestin (ARR2) to the NINAE(pp100) mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE were shown to induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaE(pp100) represented the first rhodopsin mutation that stabilized this protein complex. Additionally, the NinaE(pp100) mutation led to elevated levels of G(q)alpha in the cytosol, which mediated a novel retinal degeneration pathway. Eliminating both G(q)alpha and arrestin completely rescued the NinaE(pp100)-dependent photoreceptor cell death, which indicated that the degeneration is entirely dependent on both G(q)alpha and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans.

Topics: Animals; Arrestins; Drosophila; Drosophila Proteins; Electroretinography; Eye Proteins; Female; Genes, Dominant; Genes, Recessive; GTP-Binding Protein alpha Subunits, Gq-G11; Macromolecular Substances; Male; Mutation; Patch-Clamp Techniques; Phosphoproteins; Photic Stimulation; Photoreceptor Cells, Invertebrate; Protein Transport; Retinal Degeneration; Rhodopsin; Signal Transduction

A subset of genetic mutations in photoreceptor-specific genes results in abnormally prolonged activation of transducin-mediated photosignaling in rod cells. In humans and animal models, these mutations cause visual dysfunctions ranging from a mild stationary night blindness to severe, early-onset retinal degeneration. There are mechanistic differences between mutations causing night blindness and those causing retinal degeneration. Here, we hypothesize that mutations causing continuous activation of the visual cascade as the result, for example, of the inability of the photoreceptor to regenerate rhodopsin, lead to retinal degeneration; those mutations that can terminate signaling, even if only partially and intermittently, slow the rate of degeneration sufficiently to give rise to stationary night blindness. Furthermore, we hypothesize that a prolonged decrease in intracellular calcium concentration resulting from persistent activation is responsible for triggering apoptotic rod-cell death.

Topics: Animals; Apoptosis; Calcium; Humans; Models, Biological; Mutation; Night Blindness; Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Opsins; Signal Transduction; Transducin

To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease.. Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium. After a survival time of 4 months, eye specimens were studied by light and electron microscopy as well as with immunohistochemical markers.. One eye developed endophthalmitis in the immediate postoperative period and was terminated. Laminated grafts with correct polarity were found in 13 of the remaining 19 eyes. In most cases, these grafts had well-developed organized photoreceptors with outer segments apposed to the host retinal pigment epithelium. The inner layers of the graft contained mostly Müller cells. Both eyes of the hosts had a reduction of photoreceptor cells in most of the retina, while inner layers remained relatively intact.. Full-thickness neuroretinal grafts can be transplanted to a large animal host with photoreceptor degeneration. The transplantation procedure is relatively atraumatic to both graft and host tissue, and the grafts survive well for at least 4 months. The graft and host retina does not seem to form extensive neuronal contacts, and future work must be directed at stimulating such activity without disrupting the retinal neuronal organization.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Calcium-Binding Proteins; Cell Polarity; Eye Proteins; Fluorescent Antibody Technique, Indirect; Graft Survival; Hippocalcin; Lipoproteins; Nerve Tissue Proteins; Neuroglia; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Protein Kinase C; Recoverin; Retina; Retinal Degeneration; Rhodopsin; Swine; Vimentin

To assess the structural changes in the retina caused by a functional blockade of rods and cones and to document the time course of their degeneration.. Double knockout mice were generated by cross-breeding CNGA3(-/-) mice with Rho(-/-) mice. Retinas of mutant and wild-type mice from 3 weeks up to 12 months of age were studied by confocal light and electron microscopy. The retinas were immunostained with cell-type-specific markers and with antibodies against synapse-associated proteins and transmitter receptors.. In 3-week-old CNGA3(-/-)Rho(-/-) mice, retinal layers showed normal structural organization, and photoreceptors established normal synaptic contacts. Until postnatal week (Pw)7, presynaptic markers and postsynaptic glutamate receptors were well expressed at the photoreceptor terminals. Photoreceptor degeneration started at Pw4, progressing to an almost complete loss by 3 months. Rod spherules showed an increase in the number of synaptic ribbons and postsynaptic elements during this early stage of degeneration, and horizontal cell processes grew into the outer nuclear layer. At later stages of retinal degeneration, the inner plexiform layer (IPL) was also affected. Rod bipolar cell axon terminals showed morphologic alterations, but the stratification pattern of cone bipolar cell axons and amacrine cell processes appeared unaffected. Transmitter receptors (GlyRalpha3, GABA(A) alpha2, GluR2/3) showed no obvious changes in the distribution and density of their synaptic clusters throughout the IPL at postnatal month 12.. The normal structural and synaptic organization of the mutant retina at Pw3 suggests that photoreceptor light responses are not essential for the development of the retinal circuitry. However, functional photoreceptors are necessary for the maintenance of rods and cones and their contacts in the OPL, because they degenerate almost completely by 3 months after birth. Degenerative changes can also be observed in the IPL; however, they appear to have a slower time course and by 12 months of age the IPL circuitry appears to be surprisingly intact.

Topics: Animals; Biomarkers; Cyclic AMP; Cyclic Nucleotide-Gated Cation Channels; Electroretinography; Ion Channels; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Nerve Endings; Nerve Tissue Proteins; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Rhodopsin; Synapses

The human rhodopsin gene is the locus for numerous alleles linked to the neurodegenerative disease retinitis pigmentosa. To facilitate the study of retinal degeneration and to test reagents designed to alter the structure and function of this gene, we have developed strains of mice whose native rhodopsin gene has been replaced with the corresponding human DNA modified to encode an enhanced GFP fusion at the C terminus of rhodopsin. The human rhodopsin-GFP fusion faithfully mimics the expression and distribution of wild-type rhodopsin in heterozygotes and serves as a sensitive reporter of rod-cell structure and integrity. In homozygotes, however, the gene induces progressive retinal degeneration bearing many of the hallmarks of recessive retinitis pigmentosa. When the gene is flanked by recognition sites for Cre recombinase, protein expression is reduced approximately 5-fold despite undiminished mRNA levels, suggesting translation inhibition. GFP-tagged human rhodopsin provides a sensitive method to monitor the development of normal and diseased retinas in dissected samples, and it offers a noninvasive means to observe the progress of retinal degeneration and the efficacy of gene-based therapies in whole animals.

Topics: Animals; Animals, Genetically Modified; Blastocyst; Cell Nucleus; Cell Survival; Disease Models, Animal; Gene Expression; Genetic Therapy; Green Fluorescent Proteins; Humans; Luminescent Proteins; Male; Mice; Recombinant Fusion Proteins; Retina; Retinal Degeneration; Rhodopsin; Stem Cells

The P23H transgenic rat was engineered to mimic a human form of retinal degeneration caused by a mutation in rhodopsin. We have tested whether the P23H transgene influences the vulnerability of photoreceptors to modest variations in ambient light, well within the physiological range. P23H-3 (P23H line 3) and control Sprague-Dawley (SD) rats were raised in cyclic light (12 h light, 12 h dark), with the light phase set at either 5 lx ('scotopic-reared') or 40-60 lx ('mesopic-reared'). Mesopic rearing reduced the length of outer segments (OSs) in both SD and P23H-3 strains, but the shortening was more marked in the P23H-3 strain. Mesopic rearing was associated with thinning of the ONL, again more prominently in the P23H-3. Correspondingly, mesopic rearing increased the rate of photoreceptor death (assessed by TUNEL labelling), the increase occurring during early postnatal life. Mesopic rearing upregulated FGF-2 (basic fibroblast growth factor) levels in photoreceptors and glial fibrillary acidic protein (GFAP) in Müller cells in both SD and P23H-3 strains; again the changes were more marked in the P23H-3. Finally, mesopic rearing decreased the amplitude of the a-wave of the ERG in both strains; again the effect was greater in the P23H-3 strain. The ERG decline induced in both strains by mesopic-rearing can be explained by a reduction of functional OS membrane, due to a combination of photoreceptor death and OS shortening. The P23H-3 transgene makes photoreceptors abnormally vulnerable to modest levels of ambient light, their vulnerability being evident in multiple ways. In humans suffering photoreceptor degeneration from comparable genetic causes, light restriction may preserve the number and the function of photoreceptors.

Topics: Animals; Animals, Genetically Modified; Cell Death; Dark Adaptation; Electroretinography; Fibroblast Growth Factor 2; Glial Fibrillary Acidic Protein; Light; Lighting; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin

Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments.

Topics: Animals; Apoptosis; Erythropoietin; Humans; Immunohistochemistry; Light; Mice; Mice, Transgenic; Mutation; Phosphoric Diester Hydrolases; Photoreceptor Cells; Protein Subunits; Recombinant Proteins; Retina; Retinal Degeneration; Rhodopsin

To use progenitor cells isolated from the neural retina for transplantation studies in mice with retinal degeneration.. Retinal progenitor cells from postnatal day 1 green fluorescent protein-transgenic mice were isolated and characterized. These cells can be expanded greatly in culture and express markers characteristic of neural progenitor cells and/or retinal development.. After they were grafted to the degenerating retina of mature mice, a subset of the retinal progenitor cells developed into mature neurons, including presumptive photoreceptors expressing recoverin, rhodopsin, or cone opsin. In rho-/- hosts, there was rescue of cells in the outer nuclear layer (ONL), along with widespread integration of donor cells into the inner retina, and recipient mice showed improved light-mediated behavior compared with control animals.. These findings have implications for the treatment of retinal degeneration, in which neuronal replacement and photoreceptor rescue are major therapeutic goals.

Topics: Animals; Animals, Newborn; Biomarkers; Calcium-Binding Proteins; Cell Differentiation; Cell Separation; Eye Proteins; Flow Cytometry; Green Fluorescent Proteins; Lipoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Multipotent Stem Cells; Photoreceptor Cells; Recoverin; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Rod Opsins; Vision, Ocular

Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE-/- retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE-/- mouse their processes become structured within two 'novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE-/- retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE-/- mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration.

Topics: Acetylcholinesterase; Age Factors; Animals; Animals, Newborn; Apoptosis; Calbindin 2; Calbindins; Chickens; Choline; Gene Expression Regulation, Developmental; Glutamate-Ammonia Ligase; Immunohistochemistry; In Situ Nick-End Labeling; Indoles; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Parvalbumins; Photoreceptor Cells; Radiometry; Retina; Retinal Degeneration; Rhodopsin; S100 Calcium Binding Protein G; Tritium

Continuous exposure to light, even at relatively low intensities, leads to retinal damage and blindness in wild-type animals. However, the molecular mechanisms underlying constant-light-induced blindness are poorly understood. It has been presumed that the visual impairment resulting from long-term, continuous exposure to ambient light is a secondary consequence of the effects of light on retinal morphology, but this has not been addressed.. To characterize the mechanism underlying light-induced blindness, we applied a molecular genetic approach using the fruit fly, Drosophila melanogaster. We found that the temporal loss of the photoresponse was paralleled by a gradual decline in the concentration of rhodopsin. The decline in rhodopsin and the visual response were suppressed by a C-terminal truncation of rhodopsin, by mutations in arrestin, and by elimination of a lysosomal protein, Sunglasses. Conversely, the visual impairment was greatly enhanced by mutation of the rhodopsin phosphatase, rdgC. Surprisingly, the mutations that suppressed light-induced blindness did not reduce the severity of the retinal degeneration resulting from constant light. Moreover, mutations known to suppress retinal degeneration did not ameliorate the light-induced blindness.. These data demonstrate that the constant light-induced blindness and retinal degeneration result from defects in distinct molecular pathways. Our results support a model in which visual impairment caused by continuous illumination occurs through an arrestin-dependent pathway that promotes degradation of rhodopsin.

Topics: Animals; Arrestin; Blindness; Calcium-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Electroretinography; Light; Microscopy, Electron, Transmission; Models, Biological; Mutation; Phosphoprotein Phosphatases; Photoperiod; Photoreceptor Cells, Invertebrate; Proteins; Retinal Degeneration; Rhodopsin

Mice lacking the transcription factor Nrl have no rod photoreceptors and an increased number of short-wavelength-sensitive cones. Missense mutations in NRL are associated with autosomal dominant retinitis pigmentosa; however, the phenotype associated with the loss of NRL function in humans has not been reported. We identified two siblings who carried two allelic mutations: a predicted null allele (L75fs) and a missense mutation (L160P) altering a highly conserved residue in the domain involved in DNA-binding-site recognition. In vitro luciferase reporter assays demonstrated that the NRL-L160P mutant had severely reduced transcriptional activity compared with the WT NRL protein, consistent with a severe loss of function. The affected patients had night blindness since early childhood, consistent with a severe reduction in rod function. Color vision was normal, suggesting the presence of all cone color types; nevertheless, a comparison of central visual fields evaluated with white-on-white and blue-on-yellow light stimuli was consistent with a relatively enhanced function of short-wavelength-sensitive cones in the macula. The fundi had signs of retinal degeneration (such as vascular attenuation) and clusters of large, clumped, pigment deposits in the peripheral fundus at the level of the retinal pigment epithelium (clumped pigmentary retinal degeneration). Our report presents an unusual clinical phenotype in humans with loss-of-function mutations in NRL.

Topics: Amino Acid Motifs; Basic-Leucine Zipper Transcription Factors; DNA Mutational Analysis; DNA-Binding Proteins; Eye Proteins; Female; Genes, Recessive; Humans; Male; Mutation; Pedigree; Promoter Regions, Genetic; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Pigments; Rhodopsin

In previous studies of subretinal transplantation in rabbits, the host photoreceptor layer seemed to prevent the bridging of neuronal fibers between the graft and the host retina. The current study was undertaken to determine whether the same phenomenon occurs in transplants to the subretinal space of the vascularized retina of rats. Bridging of fibers was examined in transplants to animals of different genetic backgrounds (normal versus dystrophic rats), of different ages, and after different survival times.. Sprague-Dawley (SD) rat retinal tissue from embryonic day (E)18 was subretinally grafted to adult (60-day-old) normal SD rats, to RCS rats (32 and 73 days old), and to adult (60-day-old) transgenic P23H rats. After various survival times (28-183 days), transplanted retinas were processed for routine histology and immunocytochemistry. Antibodies against calbindin, neuronal nitric oxide synthase (NOS), and protein kinase C (PKC) were used to identify specific retinal cell types and their processes.. The shape and position of the immunoreactive cell bodies indicated that the expected neuronal populations were labeled within the grafts and in the host retina. Labeled neuronal processes were also observed. In each case, NOS-, calbindin-, and PKC-immunolabeled fibers formed bridges between the graft and the host tissues. However, regardless of the extent of host photoreceptor cell loss, the age of the recipient, or the genetic background, bridging fibers were observed only in areas where the host photoreceptor layer was discontinuous or completely missing.. The present study demonstrates that the host photoreceptor layer plays a role in limiting graft-host anatomical integration.

Topics: Animals; Animals, Genetically Modified; Antibodies, Monoclonal; Biomarkers; Calbindins; Fetal Tissue Transplantation; Fluorescent Antibody Technique, Indirect; Graft Survival; Microscopy, Fluorescence; Nerve Fibers; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Protein Kinase C; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; S100 Calcium Binding Protein G; Synapses; Visual Pathways

To determine relative light-induced retinal damage susceptibility in transgenic rats expressing mutations in the N- or C-terminal region of rhodopsin.. Heterozygous transgenic rats, including P23H sublines 2 and 3 and S334ter sublines 4 and 9, were reared in dim cyclic light or in darkness before visible light exposure starting at various times of the day or night. Before exposure to light, some rats were given the synthetic antioxidant dimethylthiourea (DMTU). At various times after intense light treatment, rats were killed for determinations of rhodopsin and retinal DNA recovery, DNA fragmentation patterns, and Northern blot analysis of retinal heme oxygenase (HO)-1 and interphotoreceptor retinol binding protein (IRBP). Rod outer segments (ROSs) were isolated for Western blot analysis of rhodopsin using N- and C- terminal-specific monoclonal antibodies.. All rats incurred greater photoreceptor cell damage from exposure to light starting at 1 AM than from exposure at 5 PM. Among cyclic-light-reared rats, P23H line 3 animals were more susceptible to light-induced damage than P23H line 2 animals. S334ter rats exhibited retinal light damage profiles similar to those in normal rats. Dark-rearing potentiated retinal damage by light. However, dark-rearing alone prolonged photoreceptor cell life in P23H rats, but had no such effect in S334ter animals. DMTU pretreatment was effective in preventing or reducing light-induced retinal damage in all transgenic rats. S334ter rat ROSs contained the truncated form of rhodopsin. Intense light exposure resulted in DNA ladders typical of apoptotic cell death and the simultaneous induction of retinal HO-1 mRNA and reduced expression of IRBP.. Light-induced retinal damage in transgenic rats depends on the time of day of exposure to light, prior light-or dark-rearing environment, and the relative level of transgene expression. Retinal light damage leads to apoptotic visual cell loss and appears to result from oxidative stress. These results suggest that reduced environmental lighting and/or antioxidant treatment may delay retinal degenerations arising from rhodopsin mutations.

Topics: Animals; Animals, Genetically Modified; Blotting, Northern; Blotting, Western; Dark Adaptation; Disease Susceptibility; DNA; DNA Fragmentation; Eye Proteins; Female; Heme Oxygenase (Decyclizing); Light; Male; Mutation; Oxidative Stress; Radiation Injuries, Experimental; Rats; Retina; Retinal Degeneration; Retinol-Binding Proteins; Rhodopsin

To define the nature and estimate the molecular weight range of soluble endogenous retinal trophic activities on cone photoreceptor survival in two models of cone degeneration.. Diffusible factors from dissociated retinal cell cultures of 8-day normal-sighted (C57BL/6J) mice were tested for cone-survival-promoting activity by two approaches and by using two independent photoreceptor degeneration models. In the first approach, mouse retinal cells were cultured on semi-permeable membranes apposed to dissociated cultures of chick embryo retina. In the second approach, conditioned medium was collected from normal mouse retinal cultures and added to embryonic chicken retina cultures or to retinal explants obtained from 5-week retinal degeneration (rd1) mice. In some experiments, conditioned medium was heated or sequentially fractionated in dialysis tubing with molecular weight cutoffs of 8, 15, and 25 kDa. The number of chicken cones and viability were determined by using morphologic criteria, colorimetric assays, and labeling with antibodies raised against visinin. Mouse cones were counted by differential double immunolabeling with antibodies against rhodopsin (rods) and arrestin (rods and cones).. . Coculturing with normal mouse retinal cells delayed cone loss in dispersed embryonic chicken retina, by a maximum of 50% relative to the control. Conditioned medium derived from normal mouse retinas also significantly delayed cone loss in chicken cone cultures by a maximum of 1300%, compared with the control, and 40% in rd1 mouse retinal explant cultures. The survival activity in conditioned medium was destroyed by heat denaturation, and was partially retained by dialysis with a molecular weight cutoff of 25 kDa in both models.. These strategies have identified cone-survival-stimulating activities in normal mouse retina, capable of acting across species and enhancing both structural protection and viability. Such molecules may represent candidates for clinical treatment of inherited retinal degeneration.

Topics: Animals; Arrestin; Blotting, Western; Cell Survival; Chick Embryo; Coculture Techniques; Culture Media, Conditioned; Cytoprotection; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Microscopy, Fluorescence; Molecular Weight; Neuroprotective Agents; Rabbits; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin

To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK:. Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG).. The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained.. Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

Topics: Animals; c-Mer Tyrosine Kinase; Electroretinography; Immunoblotting; Mice; Mice, Inbred C57BL; Mice, Knockout; Phagocytosis; Phagosomes; Phenotype; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger

To determine whether ischemic preconditioning (IPC) upregulates certain retinal survival factors and to assess the protective effect of retinal IPC against light-induced photoreceptor degeneration.. Albino rats underwent IPC induced by raising the intraocular pressure in one eye to 120 mm Hg for 5 minutes. The fellow eye underwent sham treatment. Basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and Bcl-2 were measured after 6 and 48 hours, by the reverse transcription-polymerase chain reaction and immunoblot analysis. Other preconditioned rats received 48 hours of photic injury (2000 lux) 24 hours after IPC. The a- and b-wave amplitudes of the flash electroretinograms were measured 5 days later, followed by analysis of rhodopsin mRNA levels and histology. The influence of adenosine A1 receptor blockade was assessed.. bFGF, GFAP, and Bcl-2 were upregulated after IPC. BDNF was not upregulated. The marked reduction of the a- and b-wave amplitudes and the structural injury to the photoreceptors induced by the photic insult were significantly reduced by IPC. The protection afforded by IPC was not influenced by adenosine A1 antagonism.. IPC upregulates bFGF, GFAP, and Bcl-2 and protects photoreceptors against light-induced injury. These factors may be involved in the protective response.

Topics: Animals; Brain-Derived Neurotrophic Factor; Ciliary Neurotrophic Factor; Electroretinography; Female; Fibroblast Growth Factor 2; Glial Fibrillary Acidic Protein; Ischemic Preconditioning; Light; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-bcl-2; Radiation Injuries, Experimental; Rats; Rats, Wistar; Retinal Degeneration; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Up-Regulation

Topics: Amidohydrolases; Animals; Apoptosis; Arrestins; Cell Survival; Ceramidases; Ceramides; Clathrin; Drosophila; Endocytosis; Genes, Insect; Humans; Light; Models, Biological; Mutation; Necrosis; Phosphoproteins; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin; Vision, Ocular

Mutations in proteins of the Drosophila phototransduction cascade, a prototypic guanine nucleotide-binding protein-coupled receptor signaling system, lead to retinal degeneration and have been used as models to understand human degenerative disorders. Here, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration in Drosophila mutants. Targeted expression of Drosophila neutral ceramidase rescued retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppressed degeneration in these mutants. Both genetic backgrounds modulated the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlated with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration.

Topics: Acyltransferases; Amidohydrolases; Animals; Animals, Genetically Modified; Apoptosis; Arrestins; Ceramidases; Ceramides; Clathrin; Cloning, Molecular; Crosses, Genetic; Drosophila; Dynamins; Electroretinography; Endocytosis; Genes, Insect; Light; Mutation; Necrosis; Neutral Ceramidase; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoproteins; Photoreceptor Cells, Invertebrate; Receptors, Cell Surface; Retinal Degeneration; Rhodopsin; Serine C-Palmitoyltransferase; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Type C Phospholipases; Vision, Ocular

For most retinal degeneration disorders, no efficient treatment exists to preserve photoreceptors (PRs) and, consequently, to maintain vision. Gene transfer appears to be a promising approach to prevent PR loss. In order to design adequate vectors to target specific retinal cell types, we have analyzed the expression pattern of three different promoters (mouse phosphoglycerate kinase 1 (PGK), elongation factor-1 (EFS), rhodopsin (Rho)) in newborn and adult DBA/2 mice retinas using self-inactivating lentiviral vectors. At 7 days after intraocular injection and in optimal conditions, cell transduction was observed up to 1.5 mm from the injection site. PGK promoter expression was predominant in the retinal pigment epithelium (RPE), especially in adult mice, whereas the EFS promoter allowed a broad expression in the retina. Finally, as expected, the Rho promoter was specifically expressed in PRs. Differences in the cell types transduced and in transduction efficiency were observed between newborn and adult injected eyes emphasizing the importance of such basic studies for further gene therapy approaches as well as for understanding the transcriptional changes during retinal maturation. Thus, for future attempts to slow or rescue retinal degeneration by lentiviral delivery, PGK and EFS are more suitable to control the expression of a supporting secreted factor, PGK being mainly expressed in RPE and EFS in different cell types throughout the entire retina, whereas Rho should allow to specifically deliver the therapeutic gene to PRs.

Topics: Animals; Animals, Newborn; Gene Expression; Genetic Therapy; Genetic Vectors; Lentivirus; Mice; Mice, Inbred DBA; Microscopy, Fluorescence; Peptide Elongation Factor 1; Phosphoglycerate Kinase; Promoter Regions, Genetic; Retina; Retinal Degeneration; Rhodopsin; Virus Inactivation

Photoreceptor degeneration in retinitis pigmentosa (RP) runs an inevitable, gradually progressive course. A wide variety of growth factors of different origins have been shown to slow the rate of degeneration in some rodent models of RP. Recently, lens-derived neurotrophic factors have been shown to rescue degenerating ganglion cells in crush models of the optic nerve. Our objective was to evaluate the potential rescue effect of lensectomy and vitrectomy (L&V) on photoreceptor degeneration in a large-animal model, the rhodopsin P347L transgenic pig.. We operated on one eye of each of 49 3-week-old pigs--15 vitrectomies and 34 L&V, 6 of which received steroids. Retinal paraffin sections were prepared for all eyes, in addition to immunohistochemistry in four eyes, 8 weeks after L&V.. At eight weeks after L&V, operated eyes showed significantly more nuclei in the outer nuclear layer (ONL) than the unoperated fellow eyes. The better preservation of the ONL persisted but was less prominent by 20 weeks after surgery. Steroid treatment did not markedly reduce the better preservation of the ONL seen at 8, 10, and 12 weeks after surgery. The significant difference in cell count between operated and unoperated eyes in the L&V group at 8 weeks was due to the difference in the number of rods, not the cones.. Lensectomy and vitrectomy delay photoreceptor degeneration in rhodopsin P347L transgenic pigs. Lens-related rescue effect is a probable reason for the delayed degeneration.

Topics: Animals; Animals, Genetically Modified; Cell Count; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Lens, Crystalline; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Rhodopsin; Vitrectomy

There are no effective treatments for inherited retinal degenerations, which are prevalent causes of visual disability. Several proteins promote the survival of various types of neurons, and increasing expression of one or more of these survival factors is a promising strategy for a new treatment. Studies examining the effects of intravitreous injections of brain-derived neurotrophic factor (BDNF) in models of inherited retinal degenerations have suggested that BDNF has little survival-promoting activity for photoreceptors. In this study, we generated double transgenic mice with doxycycline-inducible expression of BDNF in the retina. In a model of primary rod photoreceptor degeneration, expression of BDNF resulted in significant delay in photoreceptor cell death and maintenance of retinal function assessed by electroretinogram recordings. Expression of BDNF also caused strong protection of photoreceptors from oxidative damage-induced cell death. These data suggest that continuous expression of BDNF, unlike intravitreous injections, results in morphologic and functional benefit in animal models of inherited retinal degeneration. Double transgenic mice with inducible expression of survival factors provide valuable tools for selection of survival factor candidates for gene therapy.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Death; Cell Survival; Disease Models, Animal; Doxycycline; Gene Expression Regulation; Gene Transfer Techniques; Hyperoxia; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Growth Factors; Oxygen; Photoreceptor Cells; Rats; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger

To test whether introduction of the Rpe65Leu(450) variant can overcome protection against light-induced photoreceptor apoptosis in mice without the activator protein (AP)-1 constituent c-Fos.. c-Fos-deficient mice (c-fos(-/-)) carrying the Leu(450) variant of RPE65 were compared with c-fos(-/-) mice with Rpe65Met(450). Expression of RPE65 was analyzed by Western blot analysis. Rhodopsin regeneration was determined by measuring rhodopsin after different times in darkness after bleaching. Susceptibility to light-induced damage was tested by exposure to white light and subsequent morphologic analysis. Activation of AP-1 and its complex composition was analyzed by electromobility shift assay (EMSA) and antibody interference. The contribution of AP-1 to apoptosis was tested by pharmacological inhibition of AP-1, using dexamethasone.. Compared with RPE65Met(450), introduction of the RPE65Leu(450) variant led to increased levels of RPE65 protein, accelerated rhodopsin regeneration, loss of protection against light-induced damage, and AP-1 responsiveness to toxic light doses, despite the absence of c-Fos. c-Fos was mainly replaced by Fra-2. Application of dexamethasone restored resistance to light-induced damage.. Increasing retinal photon catch capacity by introducing the Rpe65Leu(450) variant overcomes light damage resistance provided by c-fos deficiency. Thus, a variation of RPE65 at position 450 is a strong genetic modifier of susceptibility to light-induced damage in mice. Under conditions of high rhodopsin availability during exposure to light, Fra-2 and, to a minor degree, FosB substitute for c-Fos and enable light-induced AP-1 activity and thus photoreceptor apoptosis. Regardless of the AP-1 complex's composition, glucocorticoid receptor activation inhibits AP-1 and prevents apoptosis. Thus, not the absence of c-Fos per se, but rather impairment of AP-1 DNA binding is protective against light-induced damage. This impairment may result from the absence of c-Fos or glucocorticoid receptor-mediated transrepression.

Topics: Animals; Blotting, Western; Carrier Proteins; cis-trans-Isomerases; Dexamethasone; Disease Susceptibility; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Eye Proteins; Fos-Related Antigen-2; Glucocorticoids; Leucine; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Proteins; Proto-Oncogene Proteins c-fos; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Rhodopsin; Transcription Factor AP-1; Transcription Factors

Photoreceptor cells adapt to bright or continuous light, although the molecular mechanisms underlying this phenomenon are incompletely understood. Here, we report a mechanism of light adaptation in Drosophila, which is regulated by phosphoinositides (PIs). We found that light-dependent translocation of arrestin was defective in mutants that disrupt PI metabolism or trafficking. Arrestin bound to PIP(3) in vitro, and mutation of this site delayed arrestin shuttling and resulted in defects in the termination of the light response, which is normally accelerated by prior exposure to light. Disruption of the arrestin/PI interaction also suppressed retinal degeneration caused by excessive endocytosis of rhodopsin/arrestin complexes. These findings indicate that light-dependent trafficking of arrestin is regulated by direct interaction with PIs and is required for light adaptation. Since phospholipase C activity is required for activation of Drosophila phototransduction, these data point to a dual role of PIs in phototransduction.

Topics: Adaptation, Ocular; Animals; Animals, Genetically Modified; Arrestins; Cell Cycle Proteins; Drosophila; Electroretinography; Immunohistochemistry; Microscopy, Electron; Mutation; Phosphatidylinositols; Phosphoproteins; Photoreceptor Cells, Invertebrate; Protein Binding; Protein Conformation; Protein Transport; Radioligand Assay; Retinal Degeneration; Rhodopsin; Translocation, Genetic

Phenyl-N-tert-butylnitrone (PBN) protects rat retinas against light damage. Because the degenerative process involved in light damage and inherited retinal degeneration both lead to a common final cell death, apoptosis, we used transgenic rats with a P23H or S334ter rhodopsin mutation to test the effects of PBN on retinal degeneration and light damage and the susceptibility of the transgenic rats to light damage. In the first study, 3-week-old mutant and wild-type rats were given no drug, 0.25% PBN in drinking water, or 0.25% PBN in drinking water plus three daily intraperitoneal injections of PBN (100 mg/kg, i.p., every 8 hr). Electroretinograms were recorded at postnatal day 49, after which the rats were killed for morphometric analysis. There was no photoreceptor rescue by PBN in P23H or S334ter rats, as evidenced by equivalent loss of function and photoreceptor cells in the three treatment groups. In the second study, P23H, S334ter, and wild-type rats were exposed for 24 hr to 2700 lux light. The rats were untreated or treated with PBN (50 mg/kg per injection, every 6 hr, starting before exposure). ERGs were recorded before and 1 d after exposure. Animals were killed 6 d later for morphometric analysis. PBN protected wild-type and P23H but not S334ter retinas from light damage. S334ter retinas were relatively less susceptible to light damage than P23H and wild-type rats. The results suggest that the initiating event(s) that causes photoreceptor cell death in the mutated rats is different from that which occurs in light damage, although both ultimately undergo an apoptotic cell death.

Topics: Amino Acid Substitution; Animals; Animals, Genetically Modified; Cyclic N-Oxides; Disease Models, Animal; Electroretinography; Free Radical Scavengers; Genetic Predisposition to Disease; Light; Mutation; Nitrogen Oxides; Photic Stimulation; Photoperiod; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; Sensory Thresholds; Treatment Outcome

Many photoreceptor degenerations initially affect rods, secondarily leading to cone death. It has long been assumed that the surviving neural retina is largely resistant to this sensory deafferentation. New evidence from fast retinal degenerations reveals that subtle plasticities in neuronal form and connectivity emerge early in disease. By screening mature natural, transgenic, and knockout retinal degeneration models with computational molecular phenotyping, we have found an extended late phase of negative remodeling that radically changes retinal structure. Three major transformations emerge: 1) Müller cell hypertrophy and elaboration of a distal glial seal between retina and the choroid/retinal pigmented epithelium; 2) apparent neuronal migration along glial surfaces to ectopic sites; and 3) rewiring through evolution of complex neurite fascicles, new synaptic foci in the remnant inner nuclear layer, and new connections throughout the retina. Although some neurons die, survivors express molecular signatures characteristic of normal bipolar, amacrine, and ganglion cells. Remodeling in human and rodent retinas is independent of the initial molecular targets of retinal degenerations, including defects in the retinal pigmented epithelium, rhodopsin, or downstream phototransduction elements. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, it suggests that the neural retina may be more plastic than previously believed.

Topics: Aging; Amino Acids; Animals; Animals, Genetically Modified; Cell Death; Cell Movement; Disease Models, Animal; gamma-Aminobutyric Acid; Glutathione; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Indoles; Mice; Mice, Knockout; Microscopy, Electron; Mutation; Neuroglia; Neurons; Phenotype; Photoreceptor Cells; Pigment Epithelium of Eye; Rats; Retinal Degeneration; Rhodopsin; Synapses; Taurine; Tolonium Chloride

To determine whether subretinal Schwann cell transplantation can prolong the survival of photoreceptors in the rhodopsin knockout (rho(-/-)) mouse.. Schwann cells were prepared from postnatal day (PN) 5 to 7 mouse pups and grafted subretinally into the eyes of PN35 rho(-/-) mice. RT-PCR was performed on similarly prepared cells to determine growth factor production in vitro. Eyes were retrieved at PN70 for anatomic and statistical analysis. Control animals received grafts of fibroblasts or sham surgery.. RT-PCR demonstrated the presence of message for ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF) in the cultured Schwann cells. Schwann cell grafts produced a statistically significant rescue of photoreceptors in a restricted area of retina at PN70, but the effect was lost by PN140. Preserved inner segments could be identified, but outer segments were never present. Sham surgery also resulted in photoreceptor rescue but at a reduced level. Fibroblast grafts appeared to produce little or no rescue effect. Grafts of Schwann cells or fibroblasts and sham surgery induced a reactive Müller glial response.. Schwann cells can prolong photoreceptor survival in the rhodopsin knockout mouse until at least PN70.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Count; Cell Survival; Cell Transplantation; Ciliary Neurotrophic Factor; Fibroblasts; Glial Cell Line-Derived Neurotrophic Factor; Mice; Mice, Knockout; Nerve Growth Factors; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Schwann Cells; Sciatic Nerve; Transplantation, Isogeneic

This work examines the effects of cardiotrophin (CT)-1 on photoreceptor survival in transgenic rats that carry the rhodopsin mutation S334ter.. Recombinant CT-1 was injected intravitreally into eyes of heterozygous animals. Photoreceptor survival was analyzed by histology. Phosphorylation of signal transducer and activator of transcription1 (STAT1), STAT3, extracellular signal-regulated kinase (ERK), or Akt was assessed by immunoblot analysis. Localization of phosphorylated STAT3 was determined by immunocytochemistry.. Heterozygous S334ter rats experience rapid photoreceptor degeneration. By postnatal day (PD)20, the outer nuclear layer (ONL) retained only 1 to 2 rows of nuclei compared with 10 to 12 rows in wild-type animals. Repeated administration of CT-1 resulted in significant survival of photoreceptors. At PD20, a CT-1-treated eye (2 micro g/2 micro L every 3 days, starting at PD9) had six to seven rows of nuclei, and the vehicle-treated eyes had only one to two rows. At PD30, eyes treated every 3 days still had five to six rows of nuclei, in contrast to no rows to one row in vehicle-treated eyes. Eyes treated every 4 days retained three to four rows, whereas eyes treated every 5 days had two to three rows. There was a significant increase in phosphorylated STAT1 and -3 in the retina after CT-1 injection. The increase in phosphorylated STAT3 was colocalized with glutamine synthetase, a Müller cell marker, by immunocytochemistry.. These results indicate that CT-1 promotes photoreceptor survival and that Müller cells probably mediate this effect. They also suggest that sustained delivery of the protein is essential for long-term rescue of photoreceptors.

Topics: Animals; Animals, Genetically Modified; Cell Survival; Cytokines; Cytoprotection; DNA-Binding Proteins; Fluorescent Antibody Technique, Indirect; Immunoblotting; Immunoenzyme Techniques; Injections; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Photoreceptor Cells, Vertebrate; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Retinal Degeneration; Rhodopsin; STAT3 Transcription Factor; Trans-Activators; Vitreous Body

Topics: Animals; Hypoxanthine Phosphoribosyltransferase; IMP Dehydrogenase; In Situ Hybridization; Mice; Mice, Inbred Strains; Mice, Knockout; Mutation; Oligonucleotide Array Sequence Analysis; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Tissue Distribution

Topics: Carrier Proteins; Chromosome Mapping; Chromosomes, Human, Pair 17; Cohort Studies; Genes, Dominant; Genes, Recessive; Genetic Linkage; Genetic Testing; Humans; Molecular Biology; Mutation; Prevalence; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA-Binding Proteins; South Africa

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Apoptosis; Histidine; In Situ Nick-End Labeling; Mutation; Peptide Fragments; Photoreceptor Cells, Vertebrate; Proline; Rats; Retinal Degeneration; Rhodopsin; Serine

Phototransduction is initiated by the photoisomerization of rhodopsin (Rho) chromophore 11-cis-retinylidene to all-trans-retinylidene. Here, using Rho regenerated with retinal analogs with different ring sizes, which prevent isomerization around the C(11)=C(12) double bond, the activation mechanism of this G-protein-coupled receptor was investigated. We demonstrate that 11-cis-7-ring-Rho does not activate G-protein in vivo and in vitro, and that it does not isomerize along other double bonds, suggesting that it fits tightly into the binding site of opsin. In contrast, bleaching 11-cis-6-ring-Rho modestly activates phototransduction in vivo and at low pH in vitro. These results reveal that partial activation is caused by isomerization along other double bonds in more rigid 6-locked retinal isomers and protonation of key residues by lowering pH in 11-cis-6-ring-Rhos. Full activation is not achieved, because isomerization does not induce a complete set of conformational rearrangements of Rho. These results with 6- and 7-ring-constrained retinoids provide new insights into Rho activation and suggest a potential use of locked retinals, particularly 11-cis-7-ring-retinal, to inactivate opsin in some retinal degeneration diseases.

Topics: Animals; Hydrogen-Ion Concentration; Mice; Phosphorylation; Protein Conformation; Retinal Degeneration; Retinaldehyde; Rhodopsin; Spectroscopy, Fourier Transform Infrared

Previous studies have shown that the level of docosahexaenoic acid (22:6n-3, DHA) is lower in the rod outer segment (ROS) membranes of dogs and mice with inherited retinal degeneration than in ROS from appropriate controls. In the present study, we analyzed the ROS fatty composition of several lines of transgenic rats with P23H and S334ter rhodopsin mutations. Lines were chosen that have different rates of retinal degeneration.. At 21-22 days of age, animals were perfused and eyes fixed and sectioned for morphologic examination. Others were killed and retinas isolated for preparation of ROS by sucrose step-gradient centrifugation. Fatty acid composition of ROS phospholipids was determined by gas-liquid chromatography. Membrane purity was assessed by polyacrylamide gel electrophoresis.. Retinas of the slow degenerating lines were indistinguishable from controls, whereas there was a 15-20% and 50-60% loss of photoreceptor cell nuclei in intermediate and fast degenerating lines, respectively. Except for the slow P23H line, all mutant lines had lower levels of 22:6n-3 and total n-3 fatty acids in ROS phospholipids, compared to wild-type controls, and the level of 22:6n-3 was lowest in those lines with the fastest rate of degeneration. The relative levels of the other fatty acid families (saturated, monoenoic, and n-6) increased proportionately. The n-6/n-3 ratio increased in the more rapidly degenerating lines, but the phospholipid/protein ratios did not change. The low levels of 22:6n-3 in the ROS membranes were not compensated for by an increase in 22:5n-6, which always occurs in the retina of animals where 22:6n-3 levels are reduced by dietary manipulation.. Rats that express mutant rhodopsins have lower levels of 22:6n-3 in their ROS phospholipids than wild-type animals. We propose that photoreceptor-specific mutations provoke a metabolic stress in rod photoreceptor cells that generates an oxidant stress in these cells. The retina responds to this stress by reducing the level of substrate for lipid peroxidation (22:6n-3).

Topics: Animals; Animals, Genetically Modified; Cell Membrane; Chromatography, Gas; Docosahexaenoic Acids; Electrophoresis, Polyacrylamide Gel; Mutation; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Retinal dystrophy (Rdy) is an autosomal dominant photoreceptor dysplasia of Abyssinian cats and a model for autosomal dominant retinitis pigmentosa (ADRP) in man. We have pursued a candidate gene approach in the search for the causal mutation in Rdy. The genes RHO (encoding rhodopsin), ROM1 (encoding the structural retinal outer-membrane protein-1) and PDE6G (encoding the gamma subunit of the visual transduction protein cyclic guanosine monophosphate-phosphodiesterase) were polymerase chain reaction-amplified from normal feline genomic DNA. Leader, coding and 3' untranslated regions of each gene, and parts of introns were sequenced. Single-stranded conformation polymorphism (SSCP) analysis of Rdy-affected and normal cats was used to identify intragenic polymorphisms within ROM1 and PDE6G. DNA sequencing of all three genes in Rdy-affected cats was used to confirm results from SSCP. For both ROM1 and PDE6G polymorphisms identified by SSCP and sequencing showed disconcordance between the polymorphism and the disease phenotype within an Rdy disease pedigree. SSCP analysis of RHO performed across the 5' untranslated region, the entire coding sequence and the intron/exon boundaries in Rdy-affected and control cats failed to identify any intragenic polymorphisms that could be used for linkage analysis. DNA sequencing of these regions showed no differences between Rdy-affected and control cats. Mutations in ROM1 or in PDE6G are not causative of feline Rdy. The absence of potentially pathogenic polymorphisms in sequenced portions of the RHO gene makes it unlikely that a mutation in this gene is the cause of Rdy.

Topics: Animals; Base Sequence; Cat Diseases; Cats; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Eye Proteins; Female; Male; Membrane Proteins; Molecular Sequence Data; Phosphoric Diester Hydrolases; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA

To determine the fate of rhodopsin during endocytosis-mediated retinal degeneration.. Drosophila stocks were raised in complete darkness and shifted to light for 24 h prior to dissection and fixation of retinas. 1 microm frozen sections were cut on an ultracryomicrotome, then stained with antibodies specific for rhodopsin or arrestin. Localization of photoreceptor cell-specific proteins was determined by confocal microscopy.. Flies that are in the process of undergoing endocytosis-mediated retinal degeneration exhibit an apparent loss of rhabdomeric rhodopsin at early times during the degenerative process. Using different immunological agents, genetic backgrounds, and light treatments, we have found that the binding of arrestin to rhodopsin masked the C-terminal monoclonal antibody epitope and resulted in the loss of rhodopsin immunoreactivity. The loss of immunoreactive rhabdomeric rhodopsin only occurred when rhodopsin was depleted from the plasma membrane such that it was found within the rhabdomere at stoichiometric levels with arrestin.. When rhodopsin and arrestin are found at equal levels, binding of arrestin to rhodopsin results in the masking of the antibody epitope on the C-terminus of rhodopsin. Since masking can only occur after most of the rhodopsin has been depleted from the rhabdomere, it can be concluded that during endocytosis-induced retinal degeneration, much of the rhodopsin is localized to the cell body in small puncta. These data suggest that rhodopsin is at extremely high local concentrations in the cytoplasm. The data are discussed in the context of a model for photoreceptor cell apoptosis in retinal degenerative disorders.

Topics: Animals; Arrestins; Drosophila melanogaster; Endocytosis; Epitopes; Fluorescent Antibody Technique, Indirect; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoproteins; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin; Type C Phospholipases

The role of the carboxyl-terminal domain in rhodopsin transport was investigated using transgenic mice expressing a rhodopsin truncation mutant lacking the terminal 15 amino acids (S334ter). It was previously shown that S334ter translocates to the outer segment in the presence of endogenous rhodopsin. We now show that in the absence of endogenous rhodopsin S334ter mis-localizes to the plasma membrane and fails to reconstitute outer segment structures. Surprisingly, this mis-localization does not affect photoreceptor cell survival. These results provide further evidence on the important role of the COOH-terminal domain in rhodopsin trafficking and demonstrate an absolute requirement of this domain for correct vectorial transport of rhodopsin in rod photoreceptors.

Topics: Animals; Biological Transport; Blotting, Western; Immunohistochemistry; Mice; Mice, Transgenic; Microscopy, Confocal; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Terminator Regions, Genetic

Mutations in the photoreceptor transcription factor cone-rod homeobox (CRX) have been identified in patients with several forms of retinal degenerative disease. To investigate the mechanisms by which these mutations cause photoreceptor degeneration, CRX constructs representing eleven known mutations, as well as a set of C-terminal deletions, were generated and tested for their ability to activate a rhodopsin-luciferase reporter in a transient cell transfection assay. To further define functional domains, several Gal4dbd-Crx fusions were similarly tested using a Gal4 response element containing heterologous promoter. This analysis demonstrated that the C-terminal region, between amino acids 200 and 284, is essential for CRX-mediated transcriptional activation. Consistent with this, four mutants carrying C-terminal truncations demonstrated significantly reduced transcriptional activation. Confirming the importance of the homeodomain (HD), four of the five mutants carrying HD missense mutations displayed altered transactivating activity, either decreased (three) or increased (one). In vitro protein-DNA binding assays (EMSAs) with CRX-HD peptides representing the three HD mutants with decreased transactivating activity, indicated that the alteration was due to reduced, but not abolished, DNA binding to CRX targets. Taken together, these results support the hypothesis that CRX mutations involved in human photoreceptor degeneration act by impairing CRX-mediated transcriptional regulation of the photoreceptor genes. However, a clear relationship between the magnitude of biochemical abnormality and degree of disease severity was not observed, suggesting that other genetic and environmental modifiers may also contribute to the disease phenotype.

Topics: Amino Acid Sequence; Base Sequence; DNA; Eye Diseases, Hereditary; Gene Expression Regulation; Homeodomain Proteins; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Protein Binding; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Sequence Analysis, Protein; Structure-Activity Relationship; Trans-Activators

Topics: Animals; Animals, Genetically Modified; Awards and Prizes; Biological Transport; Cell Death; Cell Survival; Florida; GTP-Binding Proteins; Humans; Ophthalmology; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Societies, Scientific

To test whether high levels of cAMP promote apoptosis and shorten the life of retinal rod photoreceptors, the changes in cAMP levels during retinal degeneration were analyzed in two transgenic rat models that express rhodopsin P23H and S334ter mutations.. Dark- and light-adapted heterozygous P23H (lines 1 and 3; P23H-1 and -3), S334ter line 4 (S334ter-4), and Sprague-Dawley (control) rats were studied at 4 to 8 weeks by cAMP enzyme competitive immunoassay and by cAMP immunocytochemistry.. In control animals retinal cAMP content reached a steady state level at 30 days of age. Dark-adapted control retinas had up to 97% higher cAMP content than light-adapted retinas, and photoreceptor cells were the major source of this increase. Dark-adapted photoreceptors in all three lines of transgenic rats at advanced stages of retinal degeneration had cAMP content different from that of the control. In rats that express mutant rhodopsin, the number of photoreceptor cells was progressively reduced, because of retinal degeneration, but dark-adapted cAMP levels did not decline accordingly. P23H transgenic animals of both lines had higher levels of cAMP per photoreceptor cell count than control animals. This elevation was more pronounced as degeneration progressed. S334ter animals showed smaller cAMP elevation than P23H rats at a similar stage of retinal degeneration, but at a point when S334ter rats were undergoing rapid retinal degeneration, whereas in P23H rats retinal degeneration was slowing down.. All three lines of transgenic rats carrying rhodopsin mutations show an increase in dark-adapted photoreceptor cAMP levels. A complex relationship exists between cAMP levels and the rate of cell death in the retina. Although initially higher levels of cAMP may promote cell survival and slow down retinal degeneration, ultimately, elevated cAMP levels may become toxic and may contribute to retinal cell death.

Topics: Animals; Animals, Genetically Modified; Cell Count; Cyclic AMP; Dark Adaptation; Disease Progression; Female; Immunohistochemistry; Male; Mutation; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin

To define the molecular mechanism underlying light-induced oxidative damage to retinal photoreceptors.. Oxidative stress was induced in isolated rod photoreceptors by bright 470- to 490-nm light and monitored by measuring the conversion of dihydrorhodamine 123 to rhodamine, with fluorescence microscopy. The effect of the wavelength on oxidant generation was investigated by applying prebleaching stimuli of either 485- or 520-nm light before the bright 470- to 490-nm light. The role of internal messengers in photooxidative stress and membrane damage by bright 470- to 490-nm light was investigated by patch-clamp recording.. Constant illumination with bright 470- to 490-nm light caused a rapid increase in generation of oxidants, which peaked after approximately 60 seconds, and a decrease in membrane resistance, eventually producing irreversible membrane damage. The time course and extent of oxidant generation were not affected by the absence of intracellular guanosine triphosphate (GTP) or adenosine triphosphate (ATP), suggesting that oxidative stress and membrane damage induced by 470- to 490-nm light do not require coupling to a GTP-binding protein. Prebleaching exposure to 520-nm light suppressed oxidative stress and membrane damage by subsequent application of bright 470- to 490-nm light, and the extent of suppression increased with prebleaching duration.. Oxidative stress and damage induced in rods in response to 470- to 490-nm light require rhodopsin activation, but not visual transduction steps downstream of active rhodopsin. Prebleaching with 485- or 520-nm light has a different effect on the level of a transient rhodopsin intermediate required for lipid peroxidation by 470- to 490-nm light.

Topics: Animals; Electrophysiology; GTP-Binding Proteins; Light; Microscopy, Confocal; Oxidative Stress; Patch-Clamp Techniques; Radiation Injuries, Experimental; Rana pipiens; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodamines; Rhodopsin; Thiobarbituric Acid Reactive Substances; Vision, Ocular

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas-Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease-causing mutation in approximately one-third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone-rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone-rod homeobox gene, CRX. Based on this large survey, the prevalence of disease-causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation-specific studies.

Topics: Amino Acid Substitution; Animals; Arginine; Cysteine; Genetic Variation; Glutamine; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Leucine; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Optic Atrophies, Hereditary; Peripherins; Prevalence; Proline; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Trans-Activators; Tyrosine

Excessive light can cause retinal degeneration and may be an environmental cofactor accelerating retinal dystrophies and age-related diseases. In rodent models, the light damage susceptibility (LDS) of the retina is determined genetically. In two mouse strains, with different degrees of LDS, a Leu450Met variation in the pigment epithelial protein RPE65 was shown recently to cosegregate with low LDS. Because light damage is rhodopsin-mediated, and RPE65 is essential for the regeneration of rhodopsin in the visual cycle, we analyzed this variation regarding rhodopsin metabolism and LDS in four mouse strains. We found that, in contrast to previous assertions, LDS does not correlate with the maximal retinal content of rhodopsin present after dark adaptation. Instead, LDS correlated positively with the kinetics of rhodopsin regeneration, which determine rhodopsin availability during light exposure. Light damage occurred after absorption of a threshold dose of photons and thus fast regeneration, as observed in those two strains having Leu at position 450 of RPE65, was correlated with the occurrence of photoreceptor apoptosis after short exposure. In contrast, mice with the Leu450Met variation of Rpe65 regenerated rhodopsin with slow kinetics and showed an increased resistance to light-induced retinal degeneration. In these mice, RPE65 protein levels were reduced by a post-transcriptional mechanism. F(1) hybrid mice, carrying one normal and one variant Rpe65 gene, had intermediate levels of the corresponding protein and showed intermediate rhodopsin regeneration kinetics and an intermediate LDS. Thus, none of the two variants of Rpe65 had a dominant effect.

Topics: Amino Acid Substitution; Animals; Apoptosis; Carrier Proteins; cis-trans-Isomerases; Disease Models, Animal; Dose-Response Relationship, Radiation; Eye Proteins; Genetic Predisposition to Disease; Genetic Variation; Light; Mice; Mice, Inbred Strains; Photoreceptor Cells, Vertebrate; Proteins; Retina; Retinal Degeneration; Rhodopsin

To determine whether the volatile anesthetic halothane protects against light-induced photoreceptor degeneration in the rodent retina.. Albino mice and rats were anesthetized with halothane and exposed to high levels of white or blue light. Nonanesthetized animals served as controls. Retinal morphology was assessed by light microscopy, and apoptosis of photoreceptor cells was verified by detection of fragmented genomic DNA and in situ staining of apoptotic nuclei (TUNEL assay). Rhodopsin regeneration after bleaching was determined by measuring rhodopsin levels in retinas of mice or rats at different time points in darkness.. Halothane anesthesia reversibly inhibited metabolic rhodopsin regeneration and thus prevented rhodopsin from absorbing high numbers of photons during light exposure. Consequently, photoreceptors of mice and rats anesthetized with halothane were completely protected against degeneration induced by white light. In remarkable contrast, however, halothane anesthesia did not protect against blue-light-induced photoreceptor cell death.. After the initial bleach, halothane impeded photon absorption by rhodopsin by inhibiting metabolic rhodopsin regeneration. Apparently, the rhodopsin-mediated uptake of the critical number of photons to initiate white light-induced retinal degeneration was prevented. In contrast, halothane did not protect the retina against blue light. Blue light can efficiently restore functional rhodopsin from bleaching intermediates through a process termed photoreversal of bleaching. This process does not depend on the visual cycle via the pigment epithelium but nevertheless enables rhodopsin molecules to absorb the critical number of photons required to induce retinal degeneration.

Topics: Anesthesia, Inhalation; Anesthetics, Inhalation; Animals; Apoptosis; DNA; Halothane; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Regeneration; Retina; Retinal Degeneration; Rhodopsin

Acute white-light damage to rods depends on the amount of rhodopsin available for bleaching during light exposure. Bleached rhodopsin is metabolically regenerated through the visual cycle involving the pigment epithelium, or photochemically by deep blue light through photoreversal of bleaching. Because photoreversal is faster than metabolic regeneration of rhodopsin by several orders of magnitude, the photon catch capacity of the retina is significantly augmented during blue-light illumination, which may explain the greater susceptibility of the retina to blue light than to green light. However, blue light can also affect function of several blue-light-absorbing enzymes that may lead to the induction of retinal damage. Therefore, this study was conducted to test whether rhodopsin and its bleaching intermediates play a role in blue-light-induced retinal degeneration.. Eyes of anesthetized rats and mice that did or did not contain rhodopsin were exposed to green (550 +/- 10 nm) or deep blue (403 +/- 10 nm) light for up to 2 hours. Rats with nearly rhodopsinless retinas were obtained by bleaching rhodopsin in animals with inhibited metabolic rhodopsin regeneration-that is, under halothane anesthesia. In addition, Rpe65(-/-) mice that are completely without rhodopsin were used to test the susceptibility to blue-light damage of a rodent retina completely devoid of the visual pigment. Effects of illumination on photoreceptor morphology were assessed 24 hours or 10 days thereafter by morphologic and biochemical methods.. Exposure to blue light resulted in severe retinal damage and activation of the transcription factor AP-1 in rats. In contrast, green light had no effect. When rhodopsin was almost completely bleached by short-term green-light exposure while metabolic regeneration (but not photoreversal) was prevented by halothane anesthesia, blue-light exposure induced distinct lesions in rat retinas. When both metabolic rhodopsin regeneration and photoreversal of bleaching were almost completely inhibited, blue-light exposure caused only very moderate lesions. When mice without rhodopsin were exposed to blue light, no damage occurred, in contrast to wild-type control mice.. Short time exposure to blue light has deleterious effects on retinal morphology. Because damage was observed only in the presence of the visual pigment, blue-light-induced retinal degeneration is rhodopsin mediated. Absorption of blue light by other proteins is not sufficient to induce light damage. Photoreversal of bleaching, which occurs only in blue but not in green light, increases the photon-catch capacity of the retina and may thus account for the difference in the damage potential between blue and green light.

Topics: Animals; DNA; DNA Fragmentation; In Situ Nick-End Labeling; Light; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; Transcription Factor AP-1

In a previous study, both recoverin and heat shock cognate protein (hsc) 70 were recognized as autoantigens by sera from patients with cancer-associated retinopathy (CAR), and retinal dysfunction similar to CAR was inducible by intravitreous injection of anti-recoverin and anti-hsc 70 antibodies to Lewis rat. The purpose of the present study was to elucidate the effects of these antibodies on retinal photoreceptor cell functions, the contribution of caspase during the photoreceptor degeneration, and the roles of aberrant expression of recoverin in tumor cells.. As photoreceptor functions, rhodopsin phosphorylation using freshly prepared rod outer segments (ROS) and electroretinogram (ERG) were studied. Expression of recoverin in several kinds of tumors was examined by reverse transcription-polymerase chain reaction and Western blot analysis. The effects of recoverin on calcium-dependent protein phosphorylation were studied using the A549 lung adenocarcinoma cell line, which does not express recoverin.. Rhodopsin phosphorylation in bovine ROS was significantly promoted by the addition of anti-recoverin antibody. Similar effects on rhodopsin phosphorylation and ERG impairment were observed in rat eyes treated with anti-recoverin antibody. Co-injection of caspase inhibitors with anti-recoverin antibody inhibited ERG impairment and significantly suppressed the antibody-induced enhancement of rhodopsin phosphorylation. Aberrant expression of recoverin was found in 15 of 30 tumor tissues from patients with cancer without CAR. Profiles of calcium-dependent protein phosphorylation of cell lysate from A549 cells were modulated by the presence of purified recoverin.. These observations suggest that anti-recoverin antibody is incorporated into rod photoreceptor cells and modulates rhodopsin phosphorylation, which in turn produces activation of caspase-dependent apoptotic pathways. Regarding antibody generation in CAR, a high incidence of aberrant expression of recoverin in cancer tissues is important, as suggested previously.

Topics: Animals; Antigens, Neoplasm; Blotting, Western; Calcium-Binding Proteins; Caspase Inhibitors; Caspases; Cattle; Cell Death; DNA Primers; Electroretinography; Eye Proteins; Hippocalcin; Humans; Lipoproteins; Nerve Tissue Proteins; Paraneoplastic Syndromes; Phosphorylation; Photoreceptor Cells, Vertebrate; Rats; Rats, Inbred Lew; Recoverin; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation.. Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG).. Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT.. GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.

Topics: Animals; DNA Primers; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Gene Expression; Genotype; Immunoblotting; Male; Mice; Mice, Knockout; Mice, Transgenic; Microscopy, Immunoelectron; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Rhodopsin; Transgenes

The goal of these experiments was to evaluate the potential of the fibroblast growth factor family members FGF-5 and FGF-18 to rescue photoreceptors from cell death in retinal degenerative disease. Two strains of transgenic rats, expressing either a P23H or an S334ter rhodopsin mutation, were used as model systems. The neurotrophic growth factors were delivered by subretinal injection of adeno-associated virus vectors, driving expression of the genes with a constitutive CMV promoter. Morphological and functional analyses were performed to determine whether FGF-5 or FGF-18 overexpression could ameliorate cell death in the retina. Immunocytochemistry was used to determine the cellular sites of expression of the factors and to test for up-regulation of FGF receptors due to injection. Significant rescue from photoreceptor cell death was found after injections of vectors expressing either FGF-5 or FGF-18 in the animal models. Increased survival of photoreceptors did not produce a significant increase in electroretinographic responses, however, reflecting either trauma due to the surgery or a suppression of signaling due to expression of proteins. Three weeks after injections, both growth factors were localized to the inner and outer segments of photoreceptors, and the receptors FGFR1 and FGFR2 were also found to be up-regulated in these regions. No visible pathological changes were seen in the FGF-5- or FGF-18-treated eyes. These results indicate that the delivery of either FGF-5 or FGF-18 with adeno-associated virus protects photoreceptors from apoptosis in transgenic rat models of retinitis pigmentosa and that the rescue is probably mediated by conventional receptor tyrosine kinase pathways in photoreceptors.

Topics: Animals; Animals, Genetically Modified; Blotting, Western; Cell Death; Cell Line; Cell Survival; Cytomegalovirus; Dependovirus; Disease Models, Animal; Electroretinography; Fibroblast Growth Factor 5; Fibroblast Growth Factors; Genetic Vectors; Humans; Immunohistochemistry; Models, Genetic; Plasmids; Point Mutation; Promoter Regions, Genetic; Rats; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transfection; Up-Regulation

Canine generalized progressive retinal atrophies (gPRA) are a group of degenerative retinal diseases that are a major cause of hereditary blindness in a number of dog breeds. The expressed sequence tag (EST) approach was used to identify and characterize potential candidate genes from canine retinal cDNA libraries. Both conventional and subtractive canine retinal cDNA libraries were constructed and analyzed. Differential hybridization was performed to identify abundantly retinal expressed cDNA clones. Sequences of both random and abundantly expressed clones were analyzed using GCG software and searched against GenEMBL databases. For genes of interest isolated from the libraries, Northern blotting and RT-PCR were performed to determine mRNA expression of the genes. DNA sequences from 85 differentially expressed clones and 100 random cDNAs were obtained and analyzed. A higher percentage of abundantly retina-expressed clones showed homology to database sequences compared with random clones (72 versus 43%). Five retinal genes and 2 anonymous retinal ESTs were selected to analyze mRNA expression. The five known genes, namely HRG4/unc119, cGMP-PDEA, transducin 1A, opsin, and sFRP2 showed retina-specific expression. In anonymous ESTs, clone p81 revealed retina-specific expression, while p3 showed expression in each of 14 canine tissues. Transcripts of the canine secreted frizzled related protein 2 (sFRP2) gene showed surprisingly high abundance in the canine retina. The isolated retinal ESTs here will be useful resources for further investigation of canine retinal function and canine genome mapping.

Topics: Animals; Base Sequence; Cloning, Molecular; Databases, Factual; DNA Primers; DNA, Complementary; Dog Diseases; Dogs; Expressed Sequence Tags; Nucleic Acid Hybridization; Proteins; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger; Sequence Homology, Nucleic Acid; Tissue Distribution

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevis model of retinal degenerative disease.

Topics: Animals; Animals, Genetically Modified; Cell Death; Cell Membrane; Disease Models, Animal; Dogs; Genes, Reporter; Golgi Apparatus; Immunoblotting; Microscopy, Fluorescence; Protein Transport; rab GTP-Binding Proteins; Recombinant Fusion Proteins; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Xenopus laevis

Fibroblast growth factor-2 (FGF2) has neurotrophic effects in vitro and in vivo. It has been demonstrated to decrease photoreceptor cell death in rats exposed to constant light and in rats with an inherited defect in retinal pigmented epithelium (RPE) phagocytosis, but the effects of intravitreous injections of FGF2 in mice are equivocal. In this study, we used transgenic mice with increased expression of FGF2 in photoreceptors (rhodopsin promoter/FGF2 transgenics) to investigate the effects of sustained increased expression of FGF2 in mice with various types of photoreceptor degeneration, including rd mice that are homozygous for mutated phosphodiesterase beta subunit, Q344ter mice that undergo photoreceptor degeneration because of expression of mutated rhodopsin, and mice exposed to 75% oxygen for 1 or 2 weeks. At P21, the outer nuclear layer was markedly reduced in rd mice or Q344ter mice regardless of whether they inherited the rhodopsin promoter/FGF2 transgene. However, after 2 weeks of exposure to 75% oxygen, outer nuclear layer thickness was significantly reduced in littermate control mice compared to FGF2 transgenic mice (P = 0.0001). These data indicate that increased expression of FGF2 in photoreceptors protects them from hyperoxia-induced damage, but does not decrease cell death related to expression of mutated proteins involved in the phototransduction pathway. This suggests that FGF2 protects photoreceptors from oxidative damage, which may play a role in complex genetic diseases such as age-related macular degeneration.

Topics: Aging; Animals; Animals, Newborn; Cell Death; Fibroblast Growth Factor 2; Humans; Hyperoxia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Photoreceptor Cells, Vertebrate; Reference Values; Retinal Degeneration; Rhodopsin; RNA, Messenger

To evaluate the ocular toxicity of trypan blue (TB) injected into the vitreous cavity of rabbit eyes. TB is a dye that could be useful for staining epiretinal membranes during vitrectomy surgery.. Ten New Zealand White (NZW) rabbits underwent gas-compression vitrectomy. Rabbits were divided into three groups to receive injections of 0.1 ml basic salt solution, 0.1 ml of a 0.06% TB solution or 0.1 ml of a 0.2% TB solution. Ocular toxicity was assessed by slit-lamp biomicroscopy, ophthalmoscopy, electroretinography and histology.. Transient posterior capsule opacification was noted in all animals. No significant reductions in a-wave or b-wave amplitudes were found in any of the animals. Light and electron microscopic examination of the inferior retina in the 0.2% TB-treated eyes showed damaged photoreceptors and marked disorganization. Immunohistochemical staining for rhodopsin was strongly reduced in those sections and staining for proliferation with Ki-67 was positive. No histological abnormalities were found in the upper retina of the 0.2% TB-treated eyes or in any part of the retina of the 0.06% TB-treated or control eyes. No histological abnormalities were found in any of the anterior chamber angle specimens.. Although no signs of toxicity were found after the prolonged presence of TB at a concentration of 0.06% in the vitreous cavity of rabbit eyes, marked damage occurred in the lower retina of 0.2% TB-treated eyes. The short-term presence of TB at a concentration of 0.06% in the vitreous cavity is harmless to the rabbit eye but a higher concentration of TB could be unsafe.

Topics: Animals; Cataract; Coloring Agents; Electroretinography; Immunohistochemistry; Ki-67 Antigen; Lens Capsule, Crystalline; Ophthalmic Solutions; Rabbits; Retina; Retinal Degeneration; Rhodopsin; Trypan Blue; Vitrectomy; Vitreous Body

The E2F1 transcription factor controls cell proliferation and apoptosis. E2F1 activity is negatively regulated by the retinoblastoma (RB) protein. To study how inactivation of Rb and dysregulated E2F1 affects the developing retina, we analysed wild-type and Rb(-/-) embryonic retinas and retinal transplants and we established transgenic mice expressing human E2F1 in retinal photoreceptor cells under the regulation of the IRBP promoter (TgIRBPE2F1). A marked increase in cell proliferation and apoptosis was observed in the retinas of Rb(-/-) mice and TgIRBPE2F1 transgenic mice. In the transgenic mice, photoreceptor cells formed rosette-like arrangements at postnatal days 9 through 28. Complete loss of photoreceptors followed in the TgIRBPE2F1 mice but not in the Rb(-/-) retinal transplants. Both RB-deficient and E2F1-overexpressing photoreceptor cells expressed rhodopsin, a marker of terminal differentiation. Loss of p53 partially reduced the apoptosis and resulted in transient hyperplasia of multiple cell types in the TgIRBPE2F1 retinas at postnatal day 6. Our findings support the concept that cross-talk occurs between different retinal cell types and that multiple genetic pathways must become dysregulated for the full oncogenic transformation of neuronal retinal cells.

Topics: Animals; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; DNA Replication; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Eye Proteins; Female; Gene Expression Regulation; Genes, Synthetic; Humans; Hyperplasia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Photoreceptor Cells, Vertebrate; Promoter Regions, Genetic; Recombinant Fusion Proteins; Retina; Retinal Degeneration; Retinoblastoma Protein; Retinol-Binding Proteins; Rhodopsin; Transcription Factors; Transcription, Genetic; Transgenes

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. In the current work, we demonstrate that RDGC binds to calmodulin, and a mutation in an IQ motif that eliminates the calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. Our data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calmodulin; Catalytic Domain; Drosophila; Drosophila Proteins; Electroretinography; GTP-Binding Proteins; Humans; Molecular Sequence Data; Mutagenesis; Phosphoprotein Phosphatases; Photic Stimulation; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin

Topics: Animals; Animals, Genetically Modified; Mutation; Retinal Degeneration; Rhodopsin; Swine; Tomography

Mice without a functional c-Fos protein (c-fos-/- mice) do not exhibit light-induced apoptotic cell death of rods in contrast to their wild-type littermates (c-fos+/+ mice). To analyze the consequences of the absence of c-fos in the retina, we investigated whether the retinas of c-fos-/- mice have a reduced capacity to absorb and transduce light compared with c-fos+/+ mice.. Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts.. Although the configuration of the ERGs was comparable in both groups of mice, c-fos-/- mice showed a marked variability in all quantitative ERG-measures with lower mean amplitudes, longer latencies, and a 0.9-log-unit lower b-wave sensitivity on average. Morphometry showed that c-fos-/- mice have 23% fewer rods on average, whereas the number of cones was comparable among c-fos+/+ and c-fos-/- mice. Arrestin levels appeared slightly reduced in c-fos-/- mice when compared with c-fos+/+ mice, whereas Hsp70 levels were comparable in both genotypes. The kinetics of rhodopsin regeneration were similar, but c-fos-/- mice had a 25% lower rhodopsin content on average.. Compared with c-fos+/+ mice, retinal function in c-fos-/- mice is attenuated to a variable but marked degree, which may be, at least in part, related to the reduced number of rods and the reduced rhodopsin content. However, c-fos does not appear to be essential for the ability to absorb photons, nor for phototransduction or the function of second-order neurons. The resistance to light-induced apoptosis of photoreceptor cells in c-fos-/- mice may result from the acute deficit of c-fos in the apoptotic cascade rather than from developmental deficits affecting rod photoreceptor function.

Topics: Animals; Apoptosis; Arrestin; Blotting, Western; Dark Adaptation; Electroretinography; Gene Deletion; HSP70 Heat-Shock Proteins; Light; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Proto-Oncogene Proteins c-fos; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Rhodopsin; Vision, Ocular

Topics: Amino Acid Sequence; Animals; Base Sequence; DNA; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Molecular Sequence Data; Mutation; Phosphorylation; Retinal Degeneration; Rhodopsin; Transgenes

Light-induced apoptosis of photoreceptors represents an animal model for retinal degeneration. Major human diseases that affect vision, such as age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP), may be promoted by light. The receptor mediating light damage, however, has not yet been conclusively identified; candidate molecules include prostaglandin synthase, cytochrome oxidase, rhodopsin, and opsins of the cones and the retinal pigment epithelium (PE). We exposed to bright light two groups of genetically altered mice that lack the visual pigment rhodopsin (Rpe65-/- and Rho-/-). The gene Rpe65 is specifically expressed in the PE and essential for the re-isomerization of all-trans retinol in the visual cycle and thus for the regeneration of rhodopsin after bleaching. Rho-/- mice do not express the apoprotein opsin in photoreceptors, which, consequently, do not contain rhodopsin. We show that photoreceptors lacking rhodopsin in these mice are completely protected against light-induced apoptosis. The transcription factor AP-1, a central element in the apoptotic response to light, is not activated in the absence of rhodopsin, indicating that rhodopsin is essential for the generation or transduction of the intracellular death signal induced by light.

Topics: Animals; Apoptosis; Carrier Proteins; cis-trans-Isomerases; Eye Proteins; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Proteins; Retinal Degeneration; Rhodopsin; Transcription Factor AP-1

It is well established that the retina is damaged by intense visible light. Rhodopsin has been proposed to be involved in this process. We therefore undertook to examine whether rhodopsin isolated from light damaged animals is structurally altered at the molecular level.. Dark reared and dim cyclic light reared 8 week old Sprague-Dawley rats were exposed to intense visible light and sacrificed immediately or 24 h after exposure together with unexposed control animals reared under the same conditions. Rod outer segments were isolated by sucrose gradient ultracentrifugation, their membranes treated with urea, then washed with Tris buffer. The rhodopsin preparations were then reduced, pyridylethylated, delipidated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion trap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identification of glycosylation.. The rat rhodopsin protein was mapped with the exception of two single amino acid fragments. The reported sequence was confirmed with the exception of the controversial T/S320 residue, which was found to be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms of rhodopsin were found in the light damaged animals. Three sites of phosphorylation were confirmed with MS/MS (tandem mass spectral) data. Single or double phosphorylations were found among these three sites, in various combinations. Dark adaptation completely reversed the phosphorylation in all light damaged animals. Other posttranslational modifications were as previously reported.. Our results indicate that intense visible light exposure of rats does not lead to oxidative or other primary structural alterations in the rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous animals and that residue 320 in both normal and mutated rhodopsins is threonine, not serine.

Topics: Amino Acid Sequence; Animals; Light; Mass Spectrometry; Molecular Sequence Data; Peptide Fragments; Phosphorylation; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin

To determine whether the calcium channel blocker D-cis-diltiazem promotes photoreceptor survival in rats with the Pro23His rhodopsin mutation.. Heterozygous Pro23His rhodopsin line 1 rats (n = 11) were treated daily, according to a protocol applied successfully in rd mice, with D-cis-diltiazem hydrochloride increased incrementally from 21 to 54 mg/kg in a divided dose (8 AM and 8 PM) administered by intraperitoneal (i.p.) injection for 21 days, beginning on days of age 10 through 12. Saline-treated line 1 rats (n = 6) received i.p. injections of an equal volume of 0.9% saline. Analysis on day 35 of age included dark-adapted corneal electroretinogram (ERG) b- and a-waves recorded from threshold to 0.63 log candela-seconds [cd-s]/m2, saturated a-waves elicited with a 2.1 log cd-s/m2 flash, and morphometry of the outer nuclear layer (ONL) and rod outer segments (ROS).. ONL width and cell counts of diltiazem-treated and saline-treated animals at 35 days were reduced to 64%-68% of 15-day-old untreated P23H line 1 retinas. No photoreceptor rescue was found by measuring ONL width (P = 0.84), cell count (P = 0.42), or ROS length (P = 0.85). Functional assays by ERG b-wave threshold (P = 0.57), b-wave maximum amplitude (P = 0.46), and saturated a-wave amplitude (P = 0.59) also showed no rescue.. D-cis-Diltiazem did not rescue photoreceptors of Pro23His rhodopsin mutation line 1 rats treated according to the protocol used in rd mouse.

Topics: Animals; Calcium Channel Blockers; Cell Count; Cell Survival; Diltiazem; Electroretinography; Female; Histidine; Injections, Intraperitoneal; Male; Point Mutation; Proline; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

To determine the relative susceptibility of rats to retinal light damage at different times of the day or night.. Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure.. When dark-adapted, cyclic light-reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000-0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800-2000). The relative degree of light damage susceptibility persisted in cyclic light-reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light-reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light.. These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility.

Topics: Animals; Blotting, Northern; Circadian Rhythm; Dark Adaptation; DNA Damage; DNA Fragmentation; Electrophoresis, Agar Gel; Eye Proteins; Free Radical Scavengers; Gene Expression Profiling; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Light; Male; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Retinol-Binding Proteins; Rhodopsin; Thiourea

Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. We now demonstrate the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, we show that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. We propose that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders.

Topics: Animals; Apoptosis; Arrestin; Arrestins; Drosophila; Drosophila Proteins; Endocytosis; Eye Proteins; Light; Membrane Proteins; Mutation; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoproteins; Phosphorylation; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin; Type C Phospholipases

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. Here, we uncover a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability.

Topics: Animals; Apoptosis; Arrestins; Binding, Competitive; Calcium-Binding Proteins; Clathrin; Drosophila; Drosophila Proteins; Endocytosis; Gene Expression Regulation; Light; Mutation; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Photoreceptor Cells, Invertebrate; Retinal Degeneration; Rhodopsin

Several families have been described in which a variety of retinal dystrophies were apparently caused by a mutation in the peripherin/RDS gene. We present clinical and genetic findings in a new family affected with a retinal dystrophy with features of retinosis pigmentosa, pattern dystrophy and fundus flavimaculatus in which a mutation in the peripherin/RDS gene has been ruled out.. A screening in the rhodopsin, peripherin/RDS and ROM1 genes was done in the affected members of the family by PCR amplification and SSCP (single strand conformation polymorphism) analysis.. No mutation was found in any of the family members.. Mutations in other genes may be involved in retinal dystrophies.

Topics: Adult; Aged; Female; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Middle Aged; Mutation; Nerve Tissue Proteins; Nuclear Family; Pedigree; Peripherins; Pigment Epithelium of Eye; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Many retinal diseases, such as macular degeneration, affect both retinal pigment epithelium (RPE) and photoreceptors. Therefore, retinal repair may require transplantation of both tissues together as a cograft.. As recipients of retina-RPE cografts, 7- to 10-week-old albino Royal College of Surgeons rats that lose their photoreceptors because of a pigment epithelium defect were used. Freshly harvested intact sheets of RPE with neural retina from pigmented normal rat fetuses were gel embedded for protection and transplanted into the subretinal space.. After 6 to 7 weeks, with the support of the cografted RPE sheet, transplanted photoreceptors developed fully in organized parallel layers in the subretinal space. Immunohistochemistry for rhodopsin, rod alpha-transducin, and S-antigen and peanut agglutinin labeling for cone interphotoreceptor matrix domains suggested that the photoreceptors in the graft were capable of normal function.. Freshly harvested intact sheets of fetal RPE and retina, transplanted together into the subretinal space, can develop a normal morphology. Such transplants have the potential to benefit retinal diseases with dysfunctional RPE and photoreceptors.

Topics: Animals; Arrestin; Cell Survival; Cell Transplantation; Extracellular Matrix; Fetal Tissue Transplantation; Graft Survival; Immunohistochemistry; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Rats; Rats, Long-Evans; Rats, Mutant Strains; Retina; Retinal Degeneration; Rhodopsin; Transducin

The role of caspase-3 in photoreceptor degeneration was examined in a line of transgenic rats that carry a rhodopsin mutation S334ter. Photoreceptor degeneration in these animals is rapid. It is detected as early as postnatal day (PD) 8, and by PD 20, only one of the original 12 rows of nuclei remain in the outer nuclear layer. At PD 11 and 12, the number of photoreceptors dying per day reaches a peak of approximately 30% of the total photoreceptors in the retina. Coincident with this rapid degeneration is an increase in caspase-3-like activity as assessed by the cleavage of a fluorescent substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and an increase in activated caspase-3 as determined by Western blot analysis for its 12 kDa subunit. Intraocular injection of an irreversible caspase-3 inhibitor N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyk etone partially protected photoreceptors from degeneration. These findings indicate that a caspase-3-dependent mechanism is operative in photoreceptor death in the transgenic rats under investigation.

Topics: Animals; Animals, Genetically Modified; Caspase 3; Caspase Inhibitors; Caspases; Coumarins; Cysteine Proteinase Inhibitors; Heterozygote; Mutation; Oligopeptides; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin; Transgenes

Mice carrying a targeted disruption of the rhodopsin gene develop a severe degenerative retinopathy, failing to elaborate rod photoreceptor outer segments (ROS), having no recordable rod electroretinogram (ERG) and losing all of their rod cells over a period of approximately 12 weeks. Murine and human rhodopsins differ in their amino acid sequences. Whether, or to what extent, such variability might influence the ability of human rhodopsin to serve as an adequate structural and functional substitute for the endogenous protein in mouse rod cells bears direct relevance to exploiting the full utility of Rho-/-animals as a model of degenerative retinal disease in man. We crossed Rho-/-mice with mice expressing a wild-type human rhodopsin transgene at levels approximating to those of the endogenous protein. Immunohistological examination of retinal selections from such animals demonstrated ROS of normal number and length and temporal expression of rhodopsin similar to that observed in wild-type animals; that is, immunoreactivity to an anti-rhodopsin antibody became clearly evident by day 3 post-partum. Whereas Rho-/-mice never display a rod ERG response, and even lose cone responses by 12 weeks of age, rescued mice showed 75% normal maximum amplitudes and had ERG b-wave thresholds (based on a 50 microV criterion) within 0.1 log unit of normal wild-type at 20 weeks, and cone amplitudes remained normal at this age. These data demonstrate very substantial structural and functional rescue of the rod photoreceptors of Rho-/-mice and long-term preservation by the human rhodopsin transgene.

Topics: Animals; Genetic Therapy; Humans; Mice; Mice, Transgenic; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transgenes

To determine the effects of genetic background and light rearing conditions on intense-light-mediated retinal degeneration in young RCS rats.. Albino rats, homozygous or heterozygous for the rdy gene were bred and born in dim cyclic light. At P7 they were moved to a dark environment, and maintained there until exposure to intense visible (green) light at P18 or P25. Other rats remained in the dim cyclic light environment. At various times between P11 and P40 rats were killed for determinations of rhodopsin and photoreceptor cell DNA levels, western transblot analysis of retinal S-antigen (arrestin) and alpha-transducin, or northern slot blot analysis of their respective mRNA levels.. At P18, unexposed dark maintained homozygous RCS rats and their phenotypically normal heterozygous counterparts have nearly equivalent rhodopsin levels and photoreceptor cell DNA. Intense light exposure at this age, to 8 hours of continuous light or 3 hours of intermittent light, did not lead to a loss of either rhodopsin or retinal DNA when compared with their respective unexposed controls. At P25 rhodopsin levels were higher than at P18, while photoreceptor cell DNA was essentially the same as in the younger rats. However, intense light exposure at P25 resulted in substantial losses of rhodopsin and photorecptor cell DNA and the losses were greater in homozygous rats than in heterozygous animals. Light damage of P25 rats maintained in dim cyclic light was essentially the same as in dark maintained homozygous rats, but no damage was found in the heterozygous animals. By western analysis, alpha-transducin levels in the retina increased with time in darkness, while retinal S-antigen levels either remained the same or decreased during the period P15-P35. For rats in the cyclic light environment S-antigen expression was greater than alpha-transducin at all ages. Slot blot analysis of mRNAs for the two proteins generally followed the patterns seen by western analysis. S-antigen mRNA was expressed at an earlier age and at higher levels than alpha-transducin in both types of rats from both light rearing conditions. Peak expression of S-antigen most often occurred at P18 in both the heterozygous and homozygous rats.. The relative expressions of S-antigen and alpha-transducin in P18 and P25 rats correlates with their relative resistance to retinal light damage at P18 and their enhanced susceptibility at P25. Rats homozygous for the rdy gene also exhibit more damage than heterozygous animals when photoreceptor cell DNA is used to estimate the extent of retinal light damage.

Topics: Aging; Animals; Animals, Newborn; Arrestin; DNA; Heterozygote; Homozygote; Photoreceptor Cells, Vertebrate; Radiation Injuries, Experimental; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger; Transducin

The Tulp1 gene is a member of the tubby gene family with unknown function. Mutations in the human TULP1 gene cause autosomal recessive retinitis pigmentosa. To understand the pathogenic mechanism associated with TULP1 mutations and to explore the physiologic function of this protein, we examined tissue distribution of the Tulp1 protein in normal mice and the photoreceptor disease phenotype in Tulp1-ablated mice.. Tissue distribution of the Tulp1 protein in normal mice was examined by immunoblotting and immunocytochemistry. The disease phenotype in tulp1-/- mice was studied by light and electron microscopy, electroretinography (ERG), and immunocytochemistry. These results were compared with another mouse model of retinal degeneration carrying a rhodopsin mutation.. Tulp1 is found exclusively in photoreceptors, localizing predominantly in the inner segments. It is a soluble protein with an apparent molecular weight of approximately 70 kDa. Photoreceptor degeneration developed in tulp1-/- mice, with early involvement of both rods and cones. At the early stage of degeneration, rod and cone opsins, but not peripherin/RDS, exhibited prominent ectopic localization. Electron microscopy revealed massive accumulation of extracellular vesicles surrounding the distal inner segments.. The function of Tulp1 is required to maintain viability of rod and cone photoreceptors. Extracellular vesicular accumulation is not a common phenomenon associated with photoreceptor degeneration but appears to be a distinct ultrastructural feature shared by a small group of retinal disease models. The defect in tulp1-/- mice may be consistent with a loss of polarized transport of nascent opsin to the outer segments.

Topics: Animals; DNA Primers; Electroretinography; Extracellular Matrix; Eye Proteins; Immunoblotting; Immunoenzyme Techniques; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Rhodopsin

To determine whether constitutive signal flow arising from defective rhodopsin shut-off causes photoreceptor cell death in arrestin knockout mice.. The retinas of cyclic-light-reared, pigmented arrestin knockout mice and wild-type littermate control mice were examined histologically for photoreceptor cell loss from 100 days to 1 year of age. In separate experiments, to determine whether constant light would accelerate the degeneration in arrestin knockout mice, these animals and wild-type control mice were exposed for 1, 2, or 3 weeks to fluorescent light at an intensity of 115 to 150 fc. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal.. In arrestin knockout mice maintained in cyclic light, photoreceptor loss was evident at 100 days of age, and it became progressively more severe, with less than 50% of photoreceptors surviving at 1 year of age. The photoreceptor degeneration appeared to be caused by light, because when these mice were reared in the dark, the retinal structure was indistinguishable from normal. When exposed to constant light, the retinas of wild-type pigmented mice showed no light-induced damage, regardless of exposure duration. By contrast, the retinas of arrestin knockout mice showed rapid degeneration in constant light, with a loss of 30% of photoreceptors after 1 week of exposure and greater than 60% after 3 weeks of exposure.. The results indicate that constitutive signal flow due to arrestin knockout leads to photoreceptor degeneration. Excessive light accelerates the cell death process in pigmented arrestin knockout mice. Human patients with naturally occurring mutations that lead to nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form of stationary night blindness. The present findings suggest that such patients may be at greater risk of the damaging effects of light than those with other forms of retinal degeneration, and they provide an impetus to restrict excessive light exposure as a protective measure in patients with constitutive signal flow in phototransduction.

Topics: Animals; Arrestin; Dark Adaptation; Disease Susceptibility; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Night Blindness; Photoreceptor Cells, Vertebrate; Radiation Injuries, Experimental; Retinal Degeneration; Rhodopsin

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.

Topics: Adult; Alleles; Humans; Mutation; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin

To determine the effects of age and long-term light- or dark-rearing environments on acute, intense-light-mediated retinal degeneration.. Male albino rats were maintained in a dim cyclic light environment or in darkness for as long as 1 year. When aged 2, 4, 8, and 12 months, some rats were given the synthetic antioxidant dimethylthiourea (DMTU) by intraperitoneal injection and were exposed to intense visible light for as long as 24 hours. Uninjected control rats were exposed to light at the same time. Other rats were treated with light of lower intensity for various periods. Two weeks after intense-light treatment, photoreceptor cell degeneration was estimated by determining the level of rhodopsin and by measuring the content of photoreceptor cell DNA. Light-induced changes in retinal DNA were analyzed immediately after exposure by neutral gel electrophoresis and by 8-hydroxy-deoxyguanosine measurements. Expression of the antioxidative stress protein heme oxygenase-1 (HO-1) was determined by northern blot analysis of mRNA in retinal extracts.. At all ages, rats reared in cyclic dim-light conditions had lower rhodopsin levels than did rats reared in darkness; photoreceptor cell DNA levels were unaffected by the rearing environment. Senescent losses in rhodopsin and retinal DNA were significant after rats were 12 months old. Dim-light-reared rats exhibited an age-related increase in retinal light damage susceptibility, whereas dark-reared rats were equally susceptible to damage at all ages. In both types of rats, the mechanism of light-induced cell death involved an apoptotic process, visualized by the pattern of DNA fragments on electrophoretic gels. The process also induced the expression of HO-1 mRNA. Photoreceptor cell loss determined by biochemical measurement, DNA fragmentation, and HO-1 induction were dramatically reduced by the administration of DMTU.. The age-related increase in susceptibility to retinal light damage in rats is influenced by their long-term daily light history. Decreasing retinal irradiance by dark-rearing eliminates the age-related increase in light damage, suggesting a correlation between light environment and retinal gene expression associated with damage. In all rats, retinal light damage resulted in a pattern of DNA fragmentation consistent with apoptotic cell death and in an increased expression of HO-1 mRNA. Antioxidant treatment greatly reduced apoptosis and HO-1 expression. This indicates that light damage involves an oxidative process that may also trigger apoptosis in the retina. The rat aging model may provide useful insights into the role of light environment associated with retinal degeneration in an aging human population.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aging; Animals; Cell Death; Dark Adaptation; Deoxyguanosine; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Free Radical Scavengers; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Light; Male; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger; Thiourea

Phenotypic, genetic and molecular characterization of 69 index patients with retinitis pigmentosa (RP) and various inherited retinal diseases.. patients went through complete ocular examination and blood samples were drawn for mutational screening of three candidate genes: rhodopsin (RHO), peripherin/RDS, and ROM-1.. the most frequent type of RP among our population was the autosomal dominant (43.6%). Three RHO mutations were found among the RP patients. A RDS mutation was detected in three unrelated families segregating dominant macular dystrophy.. 18% of the autosomal dominant RP patients presented a RHO mutation; RDS R172W mutation was present in 25% of the dominant macular dystrophies.

Topics: Adult; Aged; Aged, 80 and over; DNA Mutational Analysis; Eye Proteins; Female; Fluorescein Angiography; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Membrane Proteins; Middle Aged; Nerve Tissue Proteins; Pedigree; Peripherins; Phenotype; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tetraspanins

The aim of this study was to establish a model for morphologic retinal reconstruction after destruction of photoreceptors.. Rat embryos were prelabeled by injection of bromodeoxyuridine (BrdU) into timed pregnant rats on 2 to 6 consecutive days. Pieces of fetal retinas (embryonic day [E] 17 to E22) were embedded in growth factor-reduced matrigel for protection and stored in medium on ice. With the use of a custom-mnade implantation tool, trimmed embedded pieces were placed into the subretinal space of albino rats whose photoreceptors had been damaged by continuous exposure to blue light for 3 to 4 days.. Donor cells were unequivocally identified by the BrdU label. Approximately 25% of transplants in the subretinal space developed parallel layers, with photoreceptor outer segments facing the host pigment epithelium. Transplants developed rosettes if host pigment epithelium had been damaged, if trauma to the donor tissue occurred during preparation or transplantation, and if the donor tissue was misplaced into the choroid or into the epiretinal space on top of the host retina. If the surgery was performed more than 4 weeks after the light damage, continued degeneration of the host retina caused secondary pigment epithelium damage, and transplants did not develop parallel layers of photoreceptor outer segments.. After transplantation to the subretinal space of a degenerated retina, gel-protected fetal retina can develop parallel layers and photoreceptor outer segments in contact with host pigment epithelium. Transplants can develop good fusion with the inner retina of a photoreceptor-deficient recipient.

Topics: Animals; Bromodeoxyuridine; DNA Replication; Female; Fetal Tissue Transplantation; Immunoenzyme Techniques; Light; Photoreceptor Cells; Pregnancy; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin

Gene transfer to retinal cells may provide a means to retard photoreceptor cell death and thus prevent blindness in diseases such as retinitis pigmentosa. We tested the possibility of interfering with apoptotic photoreceptor cell death in the rd mouse through subretinal delivery of a recombinant replication-defective adenovirus containing the human cDNA for bcl-2, Ad.2.5HRPbcl-2. Photoreceptor-specific transgene expression was accomplished through incorporation of the 2.5 kb human rhodopsin upstream fragment (HRP). Ad.2.5HRPbcl-2 was injected alone or in combination with Ad.CMVPDE beta. Ad.CMVPDE beta contains a cDNA encoding the beta subunit of cGMP phosphodiesterase (PDE beta). Recombinant viruses containing lacZ (driven either by the cytomegalovirus (CMV) promoter/enhancer or HRP) and of Ad.CMVPDE beta and vehicle alone were injected in contralateral eyes as control. Injection of Ad.2.5HRPbcl-2 in the rd mouse resulted in histologically detectable rescue lasting 6 weeks after birth. Extent of rescue was not as large as after delivery of wildtype PDE beta, the gene defective in the rd mouse. However, delivery of genes which prevent apoptotic cell death may have broad application to gene therapy of retinal degenerative diseases.

Topics: Adenoviridae; Animals; Cell Death; Fundus Oculi; Gene Expression; Gene Transfer Techniques; Genes, bcl-2; Genetic Therapy; Genetic Vectors; Mice; Mice, Mutant Strains; Photoreceptor Cells, Vertebrate; Promoter Regions, Genetic; Retinal Degeneration; Rhodopsin; Transgenes

Mutations of genes encoding various retina-specific proteins are known to cause a wide spectrum of inherited retinal dystrophies in different species. In the canine, several types of genetic retinal dystrophies have been described affecting primarily the photoreceptors and/or the retinal pigment epithelium. We are performing a systematic analysis of canine candidate genes for such diseases to identify the one mutated in the retinal dystrophy in Swedish Briard dogs.. We isolated and characterised the full length cDNA of canine retinal arrestin by the method of rapid amplification of cDNA ends (RACE).. The full length cDNA isolated by us is 1,575 base pairs (bp) long and contains a 1,218 bp-long open reading frame.. The homology of the canine arrestin protein is highest with the human analogue (88.9%) and lowest with mouse arrestin (85.3%). The most obvious sequence differences among the different arrestins are in the extreme carboxyl terminus. PCR-SSCP (single strand conformation polymorphism) analysis and direct sequencing of retinal cDNA didn't provide any evidence that mutations in the canine arrestin gene are responsible for the retinal dystrophy seen in the Swedish strain of Briard dogs. Similar data were obtained for the genes encoding rhodopsin and the beta-subunit of photoreceptor-specific phosphodiesterase by segregation analysis.

Topics: Amino Acid Sequence; Animals; Arrestins; Base Sequence; DNA Primers; DNA, Complementary; Dog Diseases; Dogs; Molecular Sequence Data; Polymerase Chain Reaction; Retinal Degeneration; Rhodopsin; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors are phosphorylated by kinases that mediate agonist-dependent receptor deactivation. Although many receptor kinases have been isolated, the corresponding phosphatases, necessary for restoring the ground state of the receptor, have not been identified. Drosophila RDGC (retinal degeneration C) is a phosphatase required for rhodopsin dephosphorylation in vivo. Loss of RDGC caused severe defects in the termination of the light response as well as extensive light-dependent retinal degeneration. These phenotypes resulted from the hyperphosphorylation of rhodopsin because expression of a truncated rhodopsin lacking the phosphorylation sites restored normal photoreceptor function. These results suggest the existence of a family of receptor phosphatases involved in the regulation of G protein-coupled signaling cascades.

Topics: Animals; Animals, Genetically Modified; Arrestin; Calcium-Binding Proteins; Darkness; Drosophila; Drosophila Proteins; Electroretinography; GTP-Binding Proteins; Light; Mutation; Phosphoprotein Phosphatases; Phosphorylation; Photoreceptor Cells, Invertebrate; Retina; Retinal Degeneration; Rhodopsin; Signal Transduction

Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.

Topics: Animals; Animals, Genetically Modified; Blotting, Southern; Disease Models, Animal; Electroretinography; Embryo Transfer; Gene Expression Regulation; Genetic Engineering; Microscopy, Electron; Molecular Sequence Data; Phenotype; Polymerase Chain Reaction; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Swine; Transgenes

Transgenic pigs with rhodopsin (Pro347Leu) mutation exhibited rod-cone degeneration. We compared the pathologic characteristics of the rod degeneration versus those of the cone cells.. The posterior and peripheral retinas of these transgenic pigs of age 4, 6, 8, 12, 24 and 33 weeks and normal pigs of age 4 and 8 weeks were studied by light and EM and morphometry.. The pathologic changes observed in the posterior and peripheral retinas of the transgenic pigs could be conveniently described in 3 phases: I) an initial phase of rapid and extensive degeneration of the rod cells in the first 6 weeks of age; II) an acute phase of cone cell degeneration involving approximately half of the population and lingering rod degeneration in the 6 to 12 weeks of age; and III) a partial cone recovery to be followed by a chronic degenerative phase of the remaining cones cells from 12 to 33 weeks of age.. Our study showed that the degenerative changes of rod cells could be differentiated from those of the cone cells. Cone and rod populations degenerated along different time schedule with different pathologic features. Hence, treatment for retinitis pigmentosa might vary with the different stages of the disease.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Photoreceptor Cells; Point Mutation; Retinal Degeneration; Rhodopsin; Swine

To screen for mutations in the rhodopsin, peripherin/RDS, and ROM1 genes in a family affected with retinitis punctata albescens. Because clinical heterogeneity was observed in this family, with some members affected with retinitis punctata albescens and one member affected with features typical of retinitis pigmentosa, we analyzed the apolipoprotein E gene to elucidate this unusual intrafamilial heterogeneity.. The coding sequences of these genes were analyzed with a combination of single-strand conformation polymorphism and direct sequence analysis. Haplotypes of the apolipoprotein E gene were analyzed by polymerase chain reaction and enzymatic digestion.. The Arg135Trp mutation in the rhodopsin gene was observed in all affected members of this family, but no mutation was detected in the peripherin/RDS or ROM1 genes. The e4 allele of the apolipoprotein E gene apparently cosegregated with the albescens phenotype in this family.. The albescent phenotype in retinal dystrophy appears to not be caused exclusively by a peripherin/RDS gene mutation, and we suggest that the apolipoprotein E gene may play a role in the albescent phenotype.

Topics: Adolescent; Adult; Amino Acid Sequence; Apolipoproteins E; Arginine; Base Sequence; DNA; Eye Proteins; Female; Fundus Oculi; Humans; Infant; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Membrane Proteins; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Pedigree; Peripherins; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Tetraspanins; Tryptophan

Mutations at various loci on the rhodopsin gene have been shown to cause autosomal dominant retinitis pigmentosa (ADRP). One of the most common is a point mutation (P23H) near the N-terminus of the protein. The authors have studied the effects of light deprivation on the rate of degeneration in pigmented transgenic mice expressing the P23H mutation as well as two additional mutations near the N-terminus of opsin (V20G, P27L).. Transgenic and normal littermates were reared in darkness or in cyclic light (approximately 7 foot-candle) for periods of 2, 4, or 6 months. Retinal structure and function were evaluated by electroretinography, retinal densitometry, light microscopy, and TUNEL labeling.. Retinas of normal animals, whether reared in darkness or in cyclic light, had no structural or functional abnormalities. The rate of photoreceptor degeneration in dark-reared transgenic mice was significantly slower than in transgenic mice raised under cyclic light conditions. Differences between the two groups of animals were evident in the retinal histology, the electroretinographically determined sensitivity to photic stimulation, and the rhodopsin levels in the retina. TUNEL labeling of retinal wholemounts showed that cyclic light-reared animals had a threefold higher incidence of photoreceptor cell death than their dark-reared counterparts; the density of apoptotic cells was greatest in the inferior retina, the region most severely affected in patients with the P23H mutation. In comparison, photoreceptor cell death was more uniformly distributed across the retina in dark-reared transgenic mice.. These findings suggest that light activation of rhodopsin contributes to the severity of the degenerative disease resulting from the P23H opsin mutation, and they raise the possibility that minimizing exposure to light may help to prolong useful vision of patients with this form of retinitis pigmentosa.

Topics: Animals; Apoptosis; Base Sequence; Cell Death; Dark Adaptation; DNA Primers; Electroretinography; Female; Gene Expression; Light; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Photoreceptor Cells; Point Mutation; Polymerase Chain Reaction; Retinal Degeneration; Rhodopsin; Sensory Deprivation

To test the hypothesis that overexpression of genes coding for the anti-apoptotic proteins Bcl-2 or Bcl-XL in photoreceptor cells may prevent or delay photoreceptor degenerations.. Transgenic mice were generated in which the bcl-2 or bcl-XL transgenes were expressed in photoreceptor cells under the transcriptional control of a rhodopsin gene promoter. Bcl-2 or bcl-XL transgenic mice were crossed separately to a mouse strain carrying the rd/rd mutation and to another mouse line carrying a dominant rhodopsin gene mutation; both genetic defects result in photoreceptor degeneration. Photoreceptor cell death in mice expressing one of the bcl transgenes and carrying either the rd mutation homozygously or the rhodopsin mutation heterozygously was examined by histologic and electroretinographic measurements. Bcl-2 and bcl-XL transgenic mice also were tested for possible resistance to light-induced photoreceptor damage under two different experimental conditions.. Bcl-2 or bcl-XL transgenes were expressed in photoreceptor cells of all lines of transgenic mice. In both the rd and the rhodopsin mutant mice, expression of either bcl-2 or bcl-XL transgenes did not prevent or measurably delay photoreceptor degeneration. Apoptosis-related nuclear DNA fragmentation, as assessed by in situ labeling with terminal deoxynucleotidyl transferase, was present in 13-day-old rd/rd mouse retinas with or without transgene expression. Twelve days after exposure to 2 hours of high-intensity light, bcl-2 transgenic mice retained approximately four rows of photoreceptor cells in the central retina as compared to none in littermate controls, whereas bcl-XL transgenic mice showed no increased resistance to light damage. Expression of the bcl-2 but not the bcl-XL transgene also was associated with a reduction in rhodopsin content.. Overexpression of bcl-2 or bcl-XL transgenes does not rescue photoreceptor cells from apoptosis caused by the two genetic mutations tested. Resistance to light damage seen in the bcl-2 transgenic mice is likely from a reduction in rhodopsin content rather than an anti-cell death activity of Bcl-2. Cell death pathways not regulated by Bcl-2 may be operative in photoreceptor degeneration.

Topics: Animals; Apoptosis; bcl-X Protein; Cell Death; Crosses, Genetic; DNA Damage; DNA Primers; Electroretinography; Female; Light; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Photoreceptor Cells; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger; Transgenes

To explore the pathogenic mechanism of dominant mutations affecting the carboxyl terminus of rhodopsin that cause retinitis pigmentosa, we generated five lines of transgenic mice carrying the proline-347 to serine (P347S) mutation. The severity of photoreceptor degeneration correlated with the levels of transgene expression in these lines. Visual function as measured by the electroretinogram was approximately normal at an early age when there was little histologic evidence of photoreceptor degeneration, but it deteriorated as photoreceptors degenerated. Immunocytochemical staining showed the mutant rhodopsin predominantly in the outer segments prior to histologically evident degeneration, a finding supported by quantitation of signal intensities in different regions of the photoreceptor cells by confocal microscopy. A distinct histopathologic abnormality was the accumulation of submicrometer-sized vesicles extracellularly near the junction between inner and outer segments. The extracellular vesicles were bound by a single membrane that apparently contained rhodopsin as revealed by ultrastructural immunocytochemical staining with anti-rhodopsin antibodies. The outer segments, although shortened, contained well-packed discs. Proliferation of the endoplasmic reticulum as reported in Drosophila expressing dominant rhodopsin mutations was not observed. The accumulation of rhodopsinladen vesicles likely represents aberrant transport of rhodopsin from the inner segments to the nascent disc membranes of the outer segments. It is possible that photoreceptor degeneration occurs because of a failure to renew outer segments at a normal rate, thereby leading to a progressive shortening of outer segments, or because of the loss of cellular contents to the extracellular space, or because of both.

Topics: Animals; Electroretinography; Genes, Dominant; Humans; Mice; Mice, Transgenic; Microscopy, Confocal; Microscopy, Electron; Microscopy, Electron, Scanning; Point Mutation; Proline; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Serine

The nature of the oligosaccharides of rhodopsin from normal rats and from the Royal College of Surgeons (RCS) rats was examined by chemical, enzymatic and chromatographic procedures. This report is the first description of the structures of oligosaccharides of rat rhodopsin. The major oligosaccharide isomer of rat rhodopsin was shown to have the same structure as that from cow, human and frog. No neutral galactosylated species were detected. Although the site of the dystrophy in the RCS rat has been shown to be located at the retinal pigment epithelium, the possibility was examined that alterations in the glycosylation of rhodopsin might also be present. No differences were observed in either the amounts or structures of the rhodopsin oligosaccharide chains from young or adult control rats and RCS rats, although some differences were observed in the relative distribution of some oligosaccharide isomers between the RCS and controls. In addition, no differences in the amino acid content or SDS-PAGE patterns were detected.

Topics: Amino Acid Sequence; Animals; Carbohydrate Sequence; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Oligosaccharides; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Rhodopsin

Mutations in the retinal degeneration slow (RDS)/peripherin gene have been shown to be associated with pattern dystrophy of the retina (PDR) and other retinal dystrophies. The aim of our study was to confirm or exclude the RDS locus and the rhodopsin (RHO) locus as the disease causing locus in a large Swiss family affected with pattern dystrophy of the retina.. A Swiss family with 14 members across 3 generations affected with PDR was examined. Eleven living family members were investigated using 6 markers surrounding the RDS and RHO loci.. Linkage to two possible candidate genes, the RDS gene on chromosome 6p and the rhodopsin gene on chromosome 3q, could be excluded.. The family provides evidence for genetic heterogeneity of PDR and is in agreement with heterogeneity in other retinal dystrophies. Further investigations are in progress to map the gene causing PDR in this family.

Topics: Adult; Aged; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 6; Female; Genetic Linkage; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Middle Aged; Nerve Tissue Proteins; Pedigree; Peripherins; Retinal Degeneration; Rhodopsin

Transgenic mice expressing the rhodopsin mutant Pro347Ser (Serine 6) display retinal degeneration through apoptosis that is characteristic of the disease retinitis pigmentosa. By 5 weeks after birth, these mice have lost approximately 35% of their photoreceptor cells. Retinas from these mice showed higher levels of cAMP compared to the levels in retinas of normal mice. Our studies provide evidence that elevated cAMP is common to the apoptotic process that occurs in retinitis pigmentosa. In addition, in vitro studies demonstrate no differences in the ability of the mutant and the wild-type rhodopsin to activate transducin, the rod cell G protein, to be phosphorylated by rhodopsin kinase or to bind arrestin. Mutants of rhodopsin, including Pro347Ser, are mistargeted to the rod inner segment, raising the possibility that rhodopsin triggers apoptosis through activation of signaling pathways not normally under its control.

Topics: Animals; Apoptosis; Cyclic AMP; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Mice, Transgenic; Mutation; Photoreceptor Cells; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Serine; Signal Transduction

Topics: Animals; Cyclic GMP; Humans; Ion Channel Gating; Ion Channels; Mice; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

In animal models for retinitis pigmentosa (RP), rod photoreceptors show abnormal distribution of rhodopsin prior to undergoing cell death. To elucidate the steps in degeneration of human photoreceptors, immunocytochemistry was performed on donor retinas from 15 RP patients and five normal subjects. Rhodopsin immunolabeling in the normal retinas was restricted to the rod outer segments. In the RP retinas, rhodopsin was present in shortened rod outer segments and in the surface membranes of the rod inner segments and somata. In regions of photoreceptor death, the surviving rods had sprouted rhodopsin-positive neurites that were closely associated with gliotic Müller cell processes and extended to the inner limiting membrane. Rods and cones in the RP maculas did not form neurites, but the axons of peripheral cones were abnormally elongated and branched. Double immunofluorescence labeling showed that the rod neurites bypassed the horizontal and rod bipolar cells that are normally postsynaptic to rod axons. To our knowledge, this is the first report of rod neurite sprouting in vivo. We were unable to find neurites on degenerate rods in old rds mice, an animal model for RP. The rod neurites in the human RP retinas resemble the long, branched processes formed by rods cultured on Müller cells or purified N-CAM. Neurite growth by surviving rods in the RP retinas may be a response to neurotrophic factor upregulation, loss of inhibitory factors, or changes in molecules associated with reactive Müller cells. Such changes in the retinal microenvironment may impede functional integration of transplanted photoreceptors. The contributions of the rhodopsin-positive rod neurites and abnormal cone axons to the functional abnormalities observed in RP are unknown.

Topics: Adult; Aged; Aged, 80 and over; Animals; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Mutant Strains; Microscopy, Electron; Middle Aged; Neurites; Reference Values; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Dominant mutations of the Drosophila ninaE-encoded rhodopsin are described that reduce the expression of wild-type rhodopsin and cause a slow, age-dependent form of retinal degeneration. A posttranslational event subsequent to the requirement for the ninaA-encoded cyclophilin is disrupted by the dominant mutations. Most of these dominant mutations are missense mutations that affect the physical properties of one of the seven transmembrane domains; another affects the cysteine involved in a disulfide linkage. The results indicate that misfolded or unstable mutant rhodopsin can interfere with maturation of wild-type rhodopsin, and that these cellular conditions may trigger retinal degeneration. In addition, these dominant rhodopsin mutations suppress the rapid degeneration seen in rdgC and norpA flies, indicating that high levels of rhodopsin are required.

Topics: Aging; Alleles; Amino Acid Sequence; Animals; Drosophila; Electroretinography; Gene Deletion; Gene Expression; Genes, Dominant; Genes, Insect; Genotype; Molecular Sequence Data; Mutation; Phenotype; Point Mutation; Protein Structure, Secondary; Retina; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.

Topics: Alleles; Amino Acid Sequence; Animals; Biological Transport; Cloning, Molecular; Drosophila; Electroretinography; Endoplasmic Reticulum; Ethyl Methanesulfonate; Genes, Dominant; Molecular Sequence Data; Mutagenesis; Photoreceptor Cells; Photoreceptor Cells, Invertebrate; Point Mutation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

To measure outer segment phagocytosis in cultures of Royal College of Surgeons (RCS) rat retinal pigment epithelium (RPE) that have been treated with carbachol. Carbachol treatment of RCS RPE results in an increase in the second messenger inositol triphosphate, which mimics that observed in normal RPE after interaction with rod outer segments (ROS).. Cultures of RCS RPE were phagocytically challenged with isolated rat ROS for 2 hours. Carbachol (1 mM) was added to some cultures to stimulate inositol triphosphate synthesis, and incubation continued for 30 minutes at 37 degrees C. Inositol triphosphate concentration was measured by radioreceptor assay. Bound and ingested outer segments were quantified by double immunofluorescent staining. Ingestion of outer segment membranes was confirmed by electron microscopy and immunogold staining.. Carbachol treatment was associated with a rapid and temporary increase in inositol triphosphate levels. Royal College of Surgeons rat RPE phagocytically challenged with outer segments and treated with carbachol showed significantly higher ingestion (34%) compared to untreated RCS RPE (9%) (P < 0.05).. Exposure of cultured RCS RPE to carbachol increases the intracellular concentration of inositol triphosphate and enhances phagocytosis of bound ROS. These results support the hypothesis that the phagocytic defect in RCS RPE is related to an abnormality in the generation of inositol triphosphate as a second messenger after outer segment recognition and binding.

Topics: Animals; Carbachol; Cells, Cultured; Fluorescent Antibody Technique; Immunohistochemistry; Inositol 1,4,5-Trisphosphate; Phagocytosis; Pigment Epithelium of Eye; Radioligand Assay; Rats; Rats, Mutant Strains; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

We identified genetic mutations and characterized their associated phenotypes in patients with retinitis pigmentosa.. Patients with retinitis pigmentosa were prospectively examined and screened for genetic mutations.. A 46-year-old man with retinitis pigmentosa was found to have a heterozygous mutation in the peripherin/RDS gene (arginine-46-stop). He had late onset of symptoms and demarcated peripheral retinal atrophy. All five first-degree relatives including his parents had no detectable mutations or retinitis pigmentosa. Genotypic data were consistent with reported family structure.. This study shows that new dominant mutations are a rare cause of isolated, or simplex, cases of retinitis pigmentosa. Identification of these mutations is helpful for genetic counseling.

Topics: Adult; Amino Acid Sequence; Arginine; Base Sequence; Codon; DNA; Electroretinography; Fundus Oculi; Genes, Dominant; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Middle Aged; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Neuropeptides; Pedigree; Peripherins; Polymerase Chain Reaction; Prospective Studies; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The retinotoxic drug diaminophenoxypentane (DAPP) was administered to rats to determine its long term effects on rhodopsin levels, retinal morphology and the retina's susceptibility to damage from visible light. In rats given 2 intraperitoneal injections of DAPP at doses of 65 mg/kg body wt, there was a dramatic and sustained loss of rhodopsin. One wk later visual pigment levels were 41% lower than in comparable dark maintained rats injected with saline. Rhodopsin levels in the DAPP treated rats remained lower than in control animals for the 13 wk period of the study. Morphologically, the ROS of rats 1-2 wks after DAPP treatment exhibited some disorganization and shortening; the RPE was unremarkable. Seven wks after DAPP treatment an occasional focal area of damage was seen in the RPE. Similarly, focal areas of degeneration were seen in the outer nuclear layer between the rows of photoreceptor cells. As determined by photoreceptor cell nuclear counts, the retinotoxic effect of DAPP persisted long after drug administration. In the treated rats the loss of visual cell nuclei was 11% at 7 wks; it was 22% 13 wks after DAPP treatment. Immediately after exposure to intense visible light, damage was seen in both the photoreceptor cells and RPE of DAPP treated rats. However, the effects of exposure in the treated rats were less dramatic than in retinas from rats without DAPP treatment. Following a 2 wk dark recovery period, the DAPP treated rats had a normal appearing retinal morphology and an intact RPE layer. The retinas of rats without DAPP treatment showed extensive visual cell and RPE loss.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aniline Compounds; Animals; Dark Adaptation; Injections, Intraperitoneal; Light; Longitudinal Studies; Male; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

To investigate the mechanism by which photoreceptors degenerate in transgenic mice carrying a mutant human rhodopsin gene (P23H).. The temporal and spatial pattern of the retinal degeneration caused by P23H rhodopsin was mapped using immunocytochemistry with rhodopsin-specific antibodies. The subcellular localizations of rhodopsin, transducin, and rod cGMP phosphodiesterase (PDE) were also determined, and rhodopsin localization was compared among P23H transgenic mice, rd mice, and Royal College of Surgeons (RCS) rats.. In transgenic mice that express P23H rhodopsin, photoreceptors are lost centrally by postnatal day 10. As the retina degenerates, rhodopsin accumulates in the outer nuclear layer and within the photoreceptor synaptic terminals. The P23H transgenic retinas also show an accumulation of transducin and PDE within the outer plexiform layer. In contrast, other types of hereditary retinal degenerations studied show a similar pattern of rhodopsin accumulation in the outer nuclear layer but not in the outer plexiform layer of the retina.. The pattern of degeneration in the P23H transgenic retina is consistent with a model in which the centrally located, first-born photoreceptors are the first to die. In contrast to other animal models for hereditary retinal degeneration (rd, RCS), a novel feature of the P23H degeneration is an accumulation of rhodopsin, transducin, and PDE within the outer plexiform layer of the retina. One hypothesis to explain this observation is that P23H rhodopsin is routed intracellularly through a pathway not used by normal rhodopsin. Nonmutant forms of the peripheral transducing proteins normally associated with disk membrane, such as transducin and PDE, may accompany the aberrantly routed rhodopsin.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Fluorescent Antibody Technique; Gene Expression; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Immunoelectron; Photoreceptor Cells; Retina; Retinal Degeneration; Rhodopsin; Transducin

The C57BL/6J-mivit/mivit mouse has a retinal degeneration in which photoreceptor cells are lost slowly at a rate of about one row per month beginning at 8 weeks. ROS are severely disrupted by 4 months, but inner segments maintain a normal length through 6 months. In addition to photoreceptor changes, the retinal pigment epithelium is unevenly pigmented. The present study utilized histological and biochemical techniques to assess the effects of dark-rearing on the progression of the retinal degeneration in the mivit/mivit mouse at ages 4, 6, 8, 12, 16, 20, 24, and 28 weeks. Results of systematic morphometric evaluation indicated that the rate of loss of photoreceptor cells did not differ significantly from the rate determined for mivit/mivit animals reared in a standard light cycle. Furthermore, retinal detachment from RPE, the displacement of darkly-staining cells into the subretinal space and the influx of macrophage-like cells in the area of the ROS were still present in mivit/mivit animals reared in darkness. ROS of mivit/mivit seemed to be preserved for a slightly longer period of time in the dark-rearing condition. Rhodopsin levels in 4-week dark-reared mivit/mivit mice were 0.32 +/- 0.04 nmol per retina which was comparable to mivit/mivit mice reared under standard lighting conditions. At 20 and 28 weeks, rhodopsin levels decreased in mivit/mivit retinas to a similar level regardless of their lighting history. The findings of the study suggest that light deprivation does not retard the degeneration mivit/mivit retina. Results are discussed in comparison with effects of dark-rearing on other models of retinal degeneration.

Topics: Animals; Cell Count; Choroid; Darkness; Mice; Mice, Inbred C57BL; Photoreceptor Cells; Pigment Epithelium of Eye; Retina; Retinal Degeneration; Rhodopsin; Sensory Deprivation; Time Factors

To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein.. Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels.. There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes.. There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Topics: Adolescent; Adult; Aged; Child; Codon; Dark Adaptation; Electroretinography; Female; Humans; Male; Middle Aged; Mutation; Phenotype; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA Splicing; Sensory Thresholds; Visual Field Tests; Visual Fields

Inflammatory mediators such as interferon-gamma (IFN-gamma) are thought to play a role in ocular disease. Although IFN-gamma was found in the vitreous of mice with experimentally induced autoimmune uveitis, intracameral injection of this cytokine did not induce intraocular inflammation in mice. Therefore, the authors created a transgenic mouse line using the rhodopsin promoter to direct the expression of IFN-gamma in the photoreceptor cells of the retina. These mice, designated rho gamma, enabled them to model intraocular inflammatory disease.. The authors fused a 2.1 kb 5' Hind III fragment from the murine rhodopsin gene to the IFN-gamma gene and introduced the DNA construct into fertilized zygotes. These were implanted into pseudopregnant C57BL/6 mice, and the resulting progeny were crossed back to balb/c mice. The transgene was identified by Southern blot hybridization. Eyes from the rho gamma mice were either fixed in zinc formalin and stained with hematoxylin and eosin or were frozen in OCT compound and processed for immunostaining using the indirect immunoperoxidase method with DAB as a chromogen.. The rho gamma transgenic mice developed intraocular disease, manifested as intraocular cellular infiltration, loss of photoreceptors, corneal clouding, cataract formation, and epithelial and microglial proliferation. Additionally, rho gamma mice exhibited antigenic changes, comprising GFAP expression on Müller cells, accumulation of neurofilament on photoreceptors, and expression of MHC class I and class II molecules on retinal cells.. IFN-gamma alters the antigenic properties of intraocular tissue and induces intraocular inflammation in mice. The results suggest a key position of IFN-gamma in the development of pathologic conditions related to intraocular inflammation and provide a useful animal model for the further study of inflammatory disorders, including autoimmune diseases.

Topics: Animals; Endophthalmitis; Eye; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Histocompatibility Antigens; Interferon-gamma; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Mice, Transgenic; Photoreceptor Cells; Retinal Degeneration; Rhodopsin

Retinitis pigmentosa (RP) is a group of inherited human diseases in which photoreceptor degeneration leads to visual loss and eventually to blindness. Although mutations in the rhodopsin, peripherin, and cGMP phosphodiesterase genes have been identified in some forms of RP, it remains to be determined whether these mutations lead to photoreceptor cell death through necrotic or apoptotic mechanisms. In this paper, we report a test of the hypothesis that photoreceptor cell death occurs by an apoptotic mechanism in three mouse models of RP: retinal degeneration slow (rds) caused by a peripherin mutation, retinal degeneration (rd) caused by a defect in cGMP phosphodiesterase, and transgenic mice carrying a rhodopsin Q344ter mutation responsible for autosomal dominant RP. Two complementary techniques were used to detect apoptosis-specific internucleosomal DNA fragmentation: agarose gel electrophoresis and in situ labeling of apoptotic cells by terminal dUTP nick end labeling. Both methods showed extensive apoptosis of photoreceptors in all three mouse models of retinal degeneration. We also show that apoptotic death occurs in the retina during normal development, suggesting that different mechanisms can cause photoreceptor death by activating an intrinsic death program in these cells. These findings raise the possibility that retinal degenerations may be slowed by interfering with the apoptotic mechanism itself.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Apoptosis; Disease Models, Animal; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Mutant Strains; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

A 49-year-old Japanese man had autosomal dominant retinitis pigmentosa with a point mutation in codon 17 of the rhodopsin gene, resulting in a threonine-to-methionine change, and retinal neovascularization in both eyes. Pigmentary degeneration mainly in the inferior area of the fundus, and severe loss in the upper portion of the visual field were observed. Moderately preserved rod and cone functions were demonstrated by electroretinograms. These findings differed from those of Japanese and white patients with autosomal dominant retinitis pigmentosa with a codon 347 mutation and were almost the same as those of white patients with the codon 17 mutation. Our study indicates that phenotypic similarities exist among patients with the same mutation, but of different racial backgrounds. The neovascularization in the right eye diminished over a two-year period in conjunction with the progression of retinal degeneration.

Topics: Adaptation, Ocular; Base Sequence; Codon; DNA; Electroretinography; Female; Fluorescein Angiography; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Neovascularization, Pathologic; Oligonucleotide Probes; Pedigree; Phenotype; Polymerase Chain Reaction; Retinal Degeneration; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin

Twenty five symptomatic individuals and six asymptomatic obligate gene carriers from four families with autosomal dominant retinitis pigmentosa (adRP) showing apparent incomplete penetrance have been studied. Symptomatic individuals from three families showed early onset of night blindness, non-recordable rod electroretinograms, and marked elevation of both rod and cone thresholds in all subjects tested. In the fourth family, there was more variation in the age of onset of night blindness and some symptomatic individuals showed well preserved rod and cone function in some retinal areas. All asymptomatic individuals tested had evidence of mild abnormalities of rod and cone function, indicating that these families show marked variation in expressivity rather than true non-penetrance of the adRP gene. No mutations of the rhodopsin or RDS genes were found in these families and the precise genetic mutation(s) remain to be identified.

Topics: Adolescent; Adult; Chromosomes, Human, Pair 7; Electrophysiology; Electroretinography; Female; Genetic Linkage; Heterozygote; Humans; Male; Middle Aged; Night Blindness; Pedigree; Photoreceptor Cells; Psychophysics; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Visual Fields

Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Pro347-->Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-specific RNA probe. Photoreceptors in the chimeric retina degenerated uniformly, independent of the genotype and similar to the photoreceptors in transgenic mice. However, the chimeric retinas showed varying proportions of normal and transgenic cells. The chimeric retina with a nearly even proportion of normal and transgenic photoreceptors displayed uniform but slower degeneration than that observed in a transgenic mouse of the same age. Our results demonstrate non-autonomy of gene action for the mutated rhodopsin gene and imply that cellular interactions between photoreceptors in the retina probably play a role in degeneration.

Topics: Amino Acid Sequence; Animals; Chimera; Crosses, Genetic; Female; Genotype; In Situ Hybridization; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mosaicism; Phenotype; Photoreceptor Cells; Point Mutation; Retinal Degeneration; Rhodopsin; RNA, Messenger

We have found four mutations in the human gene encoding the beta-subunit of rod cGMP phosphodiesterase (PDE beta) that cosegregate with autosomal recessive retinitis pigmentosa, a degenerative disease of photoreceptors. In one family two affected siblings both carry allelic nonsense mutations at codons 298 and 531. Affected individuals have abnormal rod and cone electroretinograms. PDE beta is the second member of the phototransduction cascade besides rhodopsin that is absent or altered as a cause of retinitis pigmentosa, suggesting that other members of this pathway may be defective in other forms of this disease.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Animals; Base Sequence; Codon; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; DNA Mutational Analysis; DNA, Single-Stranded; Dog Diseases; Dogs; Electroretinography; Eye Proteins; Female; Genes, Recessive; Humans; Male; Mice; Mice, Mutant Strains; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Pedigree; Phosphoric Diester Hydrolases; Photoreceptor Cells; Polymorphism, Genetic; Retinal Degeneration; Retinal Dysplasia; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Mutations in the retinal degeneration, retinal degeneration slow(/peripherin) and rhodopsin genes cause photoreceptor degeneration in humans and mice. Although the phenotypes arising from these mutations are different, suggesting different mechanisms of pathogenesis, we present evidence that apoptosis may be the final common pathway of the disease process linking genotype to phenotype. We observed internucleosomal cleavage of retinal DNA by gel electrophoresis and fragmented DNA at the single cell level by labeling the nicked DNA ends with biotinylated poly(dU). In retinal degeneration mice, DNA fragmentation occurred during the period of photoreceptor degeneration. In retinal degeneration slow mice and in transgenic mice expressing a mutant (Pro347Ser) rhodopsin gene, DNA fragmentation occurred after normal histogenetic cell death (also apoptosis) had ceased. Since DNA fragmentation by internucleosomal cleavage is a cardinal feature of apoptosis, our data suggest that all three of these genetic mutations lead to apoptosis.

Topics: Amino Acid Sequence; Animals; Apoptosis; DNA; DNA Damage; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Mutagenesis, Site-Directed; Photoreceptor Cells; Retina; Retinal Degeneration; Rhodopsin

Dietary deficiency in the retinoid precursors of the visual pigment chromophore 11-cis retinal eventually results in selective degeneration of the photoreceptor cells of the vertebrate retina. An early effect of retinoid deficiency is depletion of chromophore from the photoreceptor outer segments. Experiments were conducted to determine whether the rate of chromophore depletion was affected by the intensity of environmental light.. Rats were maintained on diets either containing or lacking retinoid precursors of 11-cis retinal for up to 30 weeks. Animals in both dietary groups were exposed to either bright (90 lux) or dim (5 lux) cyclic light for the duration of the experiment. At various time intervals retinal rhodopsin content and photoreceptor densities were determined in animals from each treatment group.. Bright light greatly accelerated the depletion of rhodopsin from the retina. Rhodopsin was almost completely depleted from the retinas of the retinoid-deficient animals raised under bright light for 25 weeks, whereas the dim-light-reared animals fed the retinoid-deficient diet still had significant amounts of retinal rhodopsin even after 30 weeks. Bright light alone moderately depressed retinal rhodopsin levels in animals fed the diet containing a vitamin A precursor of 11-cis retinal. Among rats fed the latter diet, retinal rhodopsin content in the animals kept in bright cyclic light was maintained throughout the experiment at about 70% of the amount of rhodopsin in rats housed in dim cyclic light. The light-related rhodopsin depletion in the retinoid-deprived rats was accompanied by photoreceptor cell death. After 30 weeks of treatment, photoreceptor cell densities remained similar in all treatment groups except the retinoid-deprived group housed under bright cyclic light. In the latter group, photoreceptor cell densities in the central retinas were reduced by an average of more than 50% after 30 weeks. Retinoid deficiency and bright light exposure alone each resulted in a reduction in rod outer segment size. An even greater reduction in outer segment size was observed in vitamin A-deprived animals housed under bright cyclic light.. These findings indicate that light accelerates the depletion of retinoids from the retina and the accompanying photoreceptor cell degeneration.

Topics: Animals; Cell Count; Cell Death; Dark Adaptation; Diet; Disease Models, Animal; Light; Male; Photoreceptor Cells; Rats; Rats, Inbred F344; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Vitamin A; Vitamin A Deficiency

The C57BL/6J-vit/vit mouse is a newly described model of retinal degeneration in which photoreceptor cells die over the course of a year and the retinal pigment epithelium is unevenly pigmented. The present study utilized histological and biochemical techniques to assess the progression of the retinal degeneration in the vit/vit mouse ages 2 weeks to 8 months. Results of systematic morphometric evaluation indicated that the inner nuclear and plexiform layers of the retina are similar in thickness to age-matched C57BL/6J controls, but the outer plexiform layer is significantly thinner by 4 months. Rows of photoreceptor cells are lost at a rate of about one per month beginning at 2 months of age. By 8 months, the photoreceptor cell nuclei have diminished to only two to three rows. Inner segments of the vit/vit retina are similar in length to controls. Outer segments separate from the RPE during the first 2 months, they seem to be elongated at 2-3 months, but become severely disrupted past 4 months. Beginning at about 5 months, numerous darkly-staining cells resembling photoreceptor cell nuclei are observed in the area of the inner and outer segments and the subretinal space. Spectrophotometric analysis of rhodopsin indicated similar levels in vit/vit and controls at 6 weeks but a 50% reduction by 22 weeks. At 46 weeks, the level of rhodopsin in the mutant animal was less than 0.1 nmol per retina. The loss of rhodopsin in the vit/vit retinas correlated strongly with the decreasing number of rows of photoreceptor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Disease Models, Animal; Mice; Mice, Inbred C57BL; Photoreceptor Cells; Retina; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Visual pigment in normal miniature poodles and those with progressive rod-cone degeneration (prcd), a late-onset autosomal recessive photoreceptor degeneration, has been studied using imaging fundus reflectometry (IFR). The stage to which the disease had advanced in the animals with prcd was assessed with electroretinography (ERG). Measurements were carried out on seven affected, two heterozygous and three homozygous normal animals. The IFR measurements showed that the in situ difference spectrum of visual pigment measured in the central retina of the normal poodle is typical of vertebrate rhodopsin, with a maximum at about 510 nm. Rhodopsin regeneration following extensive bleaches continues for up to 70 min. In poodles with prcd, rhodopsin is spectrally normal and regenerates at normal rates. In young affected animals under 1 year of age, the final levels of rhodopsin could already be substantially reduced. Serial measurements of visual pigment in these dogs showed differences in the degree and spatial pattern of pigment loss and rate of progression between animals. The extent of visual pigment loss also differed among the older (greater than 4.5 years) affected animals: while in one animal no pigment could be detected, in another a central band of retina was relatively spared, and significant levels of visual pigment were measured within it. Pigment levels measured within the central 25 degrees of the retinas of poodles heterozygous for prcd were lower than those in normal animals, even though their ERGs were within the normal range.

Topics: Animals; Dark Adaptation; Dogs; Electroretinography; Fundus Oculi; Light; Photoreceptor Cells; Retina; Retinal Degeneration; Rhodopsin; Sensory Thresholds

Scotopic visual thresholds and time courses for dark adaptation were determined in eight patients with Sorsby's fundus dystrophy. Rhodopsin regeneration also was recorded in two. All patients had poor night vision and a visible yellow deposit at the level of Bruch's membrane that was confluent in the posterior pole. In retinal regions with the yellow deposit, scotopic thresholds were elevated, the rod-cone break was delayed or indistinct, the time courses for the rod portion of the dark adaptation curve was prolonged, and rhodopsin regeneration was slow in the one patient in whom measurements were made. In regions of ophthalmoscopically normal retina, dark adaptation was affected minimally, and in one patient, rhodopsin was regenerated at a normal rate. It was hypothesized that the abnormal dark adaptation and rhodopsin kinetics might be caused by reduced metabolic exchange across a thickened Bruch's membrane.

Topics: Adult; Bruch Membrane; Dark Adaptation; Fundus Oculi; Humans; Kinetics; Middle Aged; Night Blindness; Retinal Degeneration; Rhodopsin; Sensory Thresholds; Vision Disorders

To assess the protective effect of ascorbic acid in retinal light damage of rats, we have determined the uptake and retinal tissue distributions of its L- and D- stereoisomers following interperitoneal or intraocular injections. The effects of intense-intermittent light exposure and darkness on tissue ascorbate were compared by measuring its levels in retina and retinal pigment epithelial tissues at various times after administration. The protective effects of the two forms of ascorbate against retinal light damage were also compared by measuring rhodopsin levels 2 weeks after intense light exposure. After interperitoneal injection, both forms of ascorbic acid were higher in the retinal pigment epithelial-choroid-scleral complex (eye cup) than in the retina. Over a 2 hr post-injection period, L-ascorbate in the eye cup was 2 to 4 fold higher than normal (10-11 nmol); D-ascorbate levels were between 15 and 30 nmol. During the same period retinal L-ascorbate was just above normal (12-14 nmol), whereas less than 5 nmol of D-ascorbate was present. When ascorbate was given by the intraocular route the opposite effect was found. During the 2 hr post-injection period retinal L-ascorbate levels were 2 to 5 fold higher than normal; D-ascorbate was between 25 and 50 nmol/retina. Within 1 hr post-injection, L-ascorbate in the eye cup was near normal and D-ascorbate levels were 10 nmol or less. In uninjected rats perfused with normal saline, the endogenous L-ascorbate was distributed 55% in the retina with 9% and 36%, respectively, in the RPE-choroid and sclera. Ten-thirty min after interperitoneal peritoneal injection about 40% of the L-ascorbate was present in the retina with 17% and 44% in the RPE-choroid and sclera. Total ascorbate (L + D) levels in the same tissues of D- injected rats were similar to those found for rats given L-ascorbate. Following 7 hrs of darkness, tissue ascorbate levels in the injected rats decreased to approximately the same levels present in uninjected animals. For rats exposed to intense light average retinal ascorbate levels decreased further, while RPE-choroid and scleral levels were largely unchanged from the dark control levels. About 50% of the tissue ascorbate was present in the retina 10-30 min after intraocular injection. The RPE-choroid contained between 10 and 14% of the ascorbate, with 35-40% present in the sclera. Retinal ascorbate levels remained high in the injected eyes following 2.5 hrs of darkness, but decreased as a res

Topics: Animals; Ascorbic Acid; Chromatography, High Pressure Liquid; Dark Adaptation; Injections; Injections, Intraperitoneal; Light; Male; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Rhodopsin; Stereoisomerism; Time Factors; Tissue Distribution

Phosphorylation of rhodopsin is not detectable in vitro in the retina of the rd mouse. We investigated the enzymatic system responsible for this abnormality by measuring the levels of rhodopsin kinase and protein phosphatase 2A in normal (rd/+) and diseased (rd/rd) mouse retinas of several ages. For each enzyme, we developed micro assays that were suitable for measuring enzyme activity in one-half mouse retina. Our results indicate that rhodopsin kinase activity is identical in rd/+ and rd/rd retinas until post-natal day 11, and it decreases thereafter in the rd/rd retina, correlating with the loss of rod photoreceptors that occurs in this tissue. Protein phosphatase 2A has a constant level of activity in rd/+ retinas from ages 5 to 32 days but it is higher than normal in rd/rd retinas from post-natal days 5 to 10. It then decreases to levels that are comparable to those in rd/+ retina. Although the rd/rd extract contains the elevated protein phosphatase 2A activity, when rd/rd and rd/+ retinal extracts are each subjected to gel filtration, the elution profiles of protein phosphatase 2A activity appear to be quantitatively identical. This apparent loss of rd/rd phosphatase activity suggests a difference in the regulatory behavior of the enzyme in the normal and degenerative retinas. Thus, the failure to detect in vitro phosphorylation of rhodopsin in the rd/rd retina seems to result from the elevated level of protein phosphatase 2A activity which could more rapidly remove the phosphate from phosphorylated rhodopsin. Since protein phosphatase 2A is a ubiquitous enzyme with broad specificity, an elevation in its activity also could affect other protein phosphorylations.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Chromatography, Gel; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 2; Retina; Retinal Degeneration; Rhodopsin

Dissociated photoreceptor cells from a transgenic strain of mice, containing a bovine promoter lac Z gene construct, were transplanted to the dystrophic retinas of a strain of mice (C3H, rd/rd) without obvious photoreceptors. The transgenic photoreceptor cells expressed beta-galactosidase and were distinguishable from the host retinal cells by light and electron microscopy after the 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemical reaction. These results showed that transplanted transgenic photoreceptor cells survived at least 1 month in the host retina and had a primitive outer segment and a well-developed synaptic terminal.

Topics: Animals; beta-Galactosidase; Cell Survival; Disease Models, Animal; Galactosides; Gene Expression Regulation, Enzymologic; Genetic Markers; Histocytochemistry; Indoles; Lac Operon; Mice; Mice, Inbred C3H; Mice, Transgenic; Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Mutations in the rhodopsin gene are associated with as many as one quarter of all cases of autosomal dominant retinitis pigmentosa (RP). A number of different rhodopsin mutations have been reported but only the proline to histidine mutation in codon 23 (Pro-23-His) has been well characterized clinically. One recent report described a "sectoral" distribution of the retinal degeneration associated with this mutation, while another reported only that pigment was present in all four quadrants in 13 of 17 patients. This asymmetric distribution of pigmentation and visual field loss may prove to be an important clinical sign of a type of RP with a relatively good visual prognosis. The authors present a family with Pro-23-His rhodopsin-associated RP in which all six affected individuals had a regional distribution of the retinal degeneration in which the inferior hemisphere of the retina was most severely affected.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Base Sequence; Codon; DNA; Female; Histidine; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Proline; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Progressive degeneration of retinal photoreceptor cells occurs in albino rats when exposed to continuous lighting. Three proteins involved in the phototransduction cascade were immunodetected in these cells after various durations of continuous illumination. We found that S-antigen (arrestin) and rhodopsin immunoreactivities persisted for 1-2 months during the degenerative process, whilst immunoreactivity of the alpha subunit of transducin totally disappeared between day 2 and day 4 of continuous light exposure. This suggests that continuous illumination could impair alpha-transducin synthesis, a possible causal factor of photoreceptor damage.

Topics: Animals; Antibodies, Monoclonal; Antigens; Arrestin; Autoantigens; Blotting, Western; Dark Adaptation; Disease Models, Animal; Eye Proteins; Fluorescent Antibody Technique; Immunoenzyme Techniques; Light; Male; Membrane Proteins; Phosphodiesterase Inhibitors; Photoreceptor Cells; Rats; Rats, Inbred Lew; Retinal Degeneration; Rhodopsin; Transducin

We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and measuring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and +/+). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80-100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in +/+ retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.

Topics: Animals; Antibodies; Chromatography, High Pressure Liquid; Circadian Rhythm; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Motor Activity; Ocular Physiological Phenomena; Photic Stimulation; Photoreceptor Cells; Retina; Retinal Degeneration; Retinaldehyde; Rhodopsin

A recent study demonstrated the amelioration of light-induced photoreceptor degeneration by flunarizine and suggested that the protective effect achieved by the drug may be due to the inhibition of inositol 1,4,5-trisphosphate-induced calcium release from intracellular stores or the inhibition of calcium entry through putative voltage-sensitive calcium channels. In the current study, we investigated the effect of nimodipine, a specific voltage-sensitive calcium channel blocker, in light-induced retinal degeneration to further define the factors involved in the retinal degenerative process. Morphological, morphometric, and biochemical results demonstrated that nimodipine was ineffective in ameliorating light-induced retinal degeneration, and implied that calcium entry through voltage-sensitive channels is unlikely to be involved in this disease process, and inositol 1,4,5-trisphosphate-induced calcium release from intracellular stores may play a dominant role in light-induced photoreceptor cell damage.

Topics: Animals; Light; Male; Nimodipine; Rats; Rats, Inbred Lew; Retina; Retinal Degeneration; Rhodopsin

Although free radical formation and lipid peroxidation have been implicated in photoreceptor degeneration following continuous light exposure, recent evidence led us to hypothesize that excessive stimulation of the photoreceptor cells in prolonged light exposure may cause intracellular calcium overload and consequent photoreceptor cell injury. To test this hypothesis, we studied the effects of flunarizine hydrochloride, a calcium overload blocker that inhibits the inositol 1,4,5-triphosphate-induced release of intracellular stores of calcium, in an established rat model of light-induced retinal degeneration. Light and electron microscopic examination of the flunarizine-treated retinas revealed remarkable preservation of the retinal pigment epithelium, rod inner and outer segments, nuclei, and synapses of the photoreceptor cells at all phases of the recovery period. This observation was further supported by morphometric evaluation of the outer nuclear layer thickness, which revealed a greater preservation of the photoreceptor nuclei in the drug-treated animals at 6 and 14 days after exposure. In addition, the rhodopsin levels in the flunarizine-treated retinas were also significantly higher than in the controls in all phases of recovery. The ability of flunarizine to ameliorate light-induced retinal degeneration in the rat supports our hypothesis that elevated intracellular calcium may indeed play a role in light-induced photoreceptor degeneration.

Topics: Animals; Calcium; Disease Models, Animal; Flunarizine; Light; Photoreceptor Cells; Rats; Rats, Inbred Lew; Retina; Retinal Degeneration; Rhodopsin

Retinas of 9-10-day-old rd and control mice were incubated for 2 hr with [14C]leucine along with either tritiated palmitic acid or galactose to investigate the acylation or glycosylation, respectively, of rhodopsin. Although other laboratories have reported that phosphorylation of rhodopsin is not detectable in rd retinas, the two post-translational modifications of rhodopsin investigated in the present work are detectable. The rod outer segments (ROS) were separated from the retinal debris containing the rough endoplasmic reticulum (RER) of photoreceptor cells by vortexing and then by linear sucrose gradients. The rhodopsin from the RER was purified by affinity chromatography and gel electrophoresis. In the acylation studies, the mean ratio of palmitate to leucine in the rd mouse was nearly twice that of controls (11.73 +/- 2.84 v. 6.81 +/- 1.04). Possible explanations for the disparity between the two groups could include: (1) a diminished internal pool size of the fatty acid; or (2) acylation of amino acids such as serine or threonine which normally are not acylated in rhodopsin. Treatment of purified rhodopsin with 1 M hydroxylamine released similar amounts of palmitate from the rd mice and controls. Hence, the higher ratio of palmitate to leucine in rd mice is apparently due to a diminished internal pool size. In the glycosylation studies, the ratio of galactose to leucine was very similar between rd mice and controls, 1.7 +/- 0.43 v. 2.47 +/- 0.74. Protein content and specific activity were determined for the crude ROS preparations and for the remaining retinal debris. Although the amount of ROS protein differed significantly between the two groups, the specific activities did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acylation; Animals; Eye Proteins; Glycosylation; Mice; Mice, Inbred Strains; Palmitic Acid; Palmitic Acids; Retina; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Development and loss of photoreceptor cells in mice, afflicted by the rds (retinal degeneration slow) gene, was analyzed by measuring the ocular visual pigment content as rhodopsin (spectroscopy) and opsin (immunoassay). With regard to the postnatal age, where opsin was just detectable, and to the initial rate of opsin synthesis, the mutants did not strongly deviate from the normal animals. The final maximal visual pigment level was, however, about half of normal for the heterozygous mutants and about 3% of normal for the homozygous mutants, both in the pigmented and in the albino strain. In the pigmented normal or heterozygous mutant the (rhod)opsin levels remain stable up to at least 1 year of age. For the corresponding albino animals this was only observed up to 9 months of age. Thereafter the level declines. In the homozygous mutants, maximal opsin levels were observed at about 3 weeks postnatal. Subsequently, this level gradually declined to about 40% in the pigmented and about 15% in the albino mutant. The results indicate that the rds gene does not directly affect the biosynthetic pathways of opsin. The physiological effect of the rds gene is aggravated by photodamage for which the albino animal is particularly susceptible.

Topics: Aging; Animals; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Eye; Eye Proteins; Heterozygote; Homozygote; Immunoblotting; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Mutant Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Rod Opsins; Spectrophotometry

Topics: Animals; Cats; Cyclic GMP; Phosphorylation; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Photoreceptor-mediated mechanisms were studied in patients with a recently identified retinopathy typified by night blindness, cystoid maculopathy, and similar scotopic and photopic electroretinograms (ERGs). Dark-adapted spectral sensitivity functions were only partly explained as composites of rod and cone curves shifted to lower sensitivities; there was unusually high sensitivity from 400-460 nm. A rod mechanism, reduced in sensitivity by at least 3 log units, was detectable with dark adaptometry. No measurable rhodopsin was found with fundus reflectometry. Light-adapted spectral sensitivities were subnormal for wavelengths greater than 500 nm but supernormal from 420-460 nm. On a yellow adapting field, the supernormal spectrum approximated that of the short-wavelength-sensitive (SWS) cone system. With spectral ERGs, two mechanisms were demonstrated. Dark- and light-adapted ERGs to green, orange-yellow, and red stimuli had similar waveforms and coincident intensity-response functions on a photopic intensity axis. ERGs to blue and blue-green stimuli were similar, and intensity-response functions coincided on a SWS cone intensity axis. Patients varied in the degree to which rod and midspectral cone function were decreased and SWS cone function was increased.

Topics: Adolescent; Adult; Child; Dark Adaptation; Electroretinography; Female; Fundus Oculi; Humans; Macular Degeneration; Male; Middle Aged; Night Blindness; Photoreceptor Cells; Psychophysics; Retinal Degeneration; Rhodopsin; Sensory Thresholds; Visual Acuity; Visual Field Tests

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.

Topics: Aging; Animals; Chromosome Mapping; Drosophila; Electrophysiology; Light; Microscopy, Electron; Mutation; Phenotype; Photoreceptor Cells; Retinal Degeneration; Retinal Pigments; Rhodopsin; Type C Phospholipases

In both of the early onset systems, the rd mouse and the rcdl Irish Setter, early elevation of cyclic GMP may be the ultimate cause of accelerated photoreceptor degeneration. This would be consistent with the data utilizing in vitro systems in which retinal samples, in culture, undergo degeneration in response to constant exposure to high levels of this nucleotide. However, the ultimate cause of the elevated cyclic GMP in the rd mouse or in the rcdl Irish Setter still remains a mystery. It appears that all of the necessary proteins of the visual cascade are produced, although they are lost at different rates. The phosphodiesterase appears to be reduced faster than other proteins. This may, in turn, account for the elevation in cyclic GMP levels. The cause of this enhanced disappearance could reside in the phosphodiesterase protein itself, or in other more distal components. The alteration in rhodopsin reaction to the specific rhod-4 antisera suggests that this protein is not properly oriented in the disc membrane. Although this may or may not alter the visual cascade, it does suggest that these membranes are not identical to those of the normal dog retina. Future studies should focus on the individual functional activities of each component, on their structures, and on their proper assembly within the disc.

Topics: Amino Acid Sequence; Animals; Autoradiography; Blotting, Western; Disease Models, Animal; Dogs; Eye Proteins; Molecular Sequence Data; Phosphoric Diester Hydrolases; Retina; Retinal Degeneration; Retinol-Binding Proteins; Rhodopsin; Rod Cell Outer Segment

Topics: 5'-Nucleotidase; Animals; Antigens; Arrestin; DNA; Electrophoresis, Polyacrylamide Gel; Eye Proteins; GTP-Binding Proteins; Light; Lipid Metabolism; Peroxides; Photoreceptor Cells; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Sodium-Potassium-Exchanging ATPase; Superoxide Dismutase; Transducin

Abyssinian cats with different stages of a slowly progressive autosomal recessively-inherited retinal degeneration were studied with imaging fundus reflectometry (IFR) and electroretinography (ERG). Maps of the visual pigment distribution were made in an area of retina extending from the posterior pole to the midperiphery. Rhodopsin levels in the midperipheral retina of a 6-month-old affected cat (stage of suspected disease) were reduced about 20% relative to the mean normal value. The same cat, tested at 2.5 yr of age (now moderately advanced stage), showed a 60% reduction. A 3-yr-old affected cat (also moderately advanced) had a reduction in rhodopsin of about 60%. There was no measurable rhodopsin in a 7-yr-old affected cat (advanced stage). Rhodopsin regeneration kinetics at the different stages of disease were found to be similar to those of normal cats. The rod ERG b-wave threshold in the 6-month-old cat was elevated by 0.26 log units; at 2.5 yr of age, the threshold was elevated by 0.48 log units. A 0.34 log units threshold elevation was found in the 3-yr-old cat. There was no detectable ERG in the 7-yr-old cat. The relationship between the rod ERG threshold elevations and the rhodopsin levels was close to that expected if the dysfunction was caused by decreased quantal absorption.

Topics: Animals; Cats; Electroretinography; Female; Fundus Oculi; Male; Photoreceptor Cells; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

Topics: Amino Acids; Animals; Antioxidants; Disease Models, Animal; Disease Susceptibility; Glutathione; Light; Lipid Metabolism; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Retina; Retinal Degeneration; Rhodopsin

We used reflection retinal densitometric, psychophysical, and electroretinographic techniques to study the scotopic retinal function of a mother and her three daughters who had clinical evidence of a sectoral type of retinitis pigmentosa. Retinal regions with, and those without, ophthalmoscopic signs of degeneration were investigated. During dark adaptation, the time courses of rhodopsin regeneration and recovery of scotopic sensitivity were similar to normal as was the relation of rhodopsin to scotopic threshold. In dark-adapted eyes, threshold increases were not proportional to rhodopsin loss. The results of psychophysical tests of background adaptation and temporal summation, and analysis of the relation of electroretinographic a- to b-wave amplitudes, led to the conclusion that abnormalities of photoreceptor cell function central to the rhodopsin-bearing outer segments accounted for the increased thresholds.

Topics: Adult; Dark Adaptation; Densitometry; Electroretinography; Female; Humans; Ophthalmoscopy; Photoreceptor Cells; Regeneration; Retina; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Visual Field Tests

On the basis of the amino acid sequence of bovine rhodopsin, a series of peptides from the C-terminus (Rhod-4 and Rhod-1) and external loops (Rhod-10) were synthesized. Rabbit antisera to these peptides recognize the rhodopsin molecule in whole retina from 8-week-old normal and affected rcdl (rod/cone-dysplasic) Irish setters (8- and 4-weeks-old). When the rhodopsin content was equalized by using a solid-phase radioimmunoassay, the reaction with anti-peptide antisera to the C-terminal octapeptide (residues 341-348) is severely decreased in the rcdl-dog retinas. The results of mixing experiments suggest that this is not due to proteolytic clipping of the rhodopsin C-terminus from the affected dogs. Treatment of retinas with 1.0 mM-NaF, a phosphatase inhibitor, or pretreatment with alkaline and acid phosphatases does alter the reaction of the rhodopsin with anti-rhodopsin antisera. This suggests that the decreased reaction of the affected rhodopsin with the anti-peptide antisera may partially result from differences in intrinsic rhodopsin phosphorylation. However, since the reaction of rcdl retinas cannot be restored to that of the normals, these results suggest that the rhodopsin molecule from the rcdl dogs may be structurally altered in other ways.

Topics: Alkaline Phosphatase; Animals; Carboxypeptidases; Dog Diseases; Dogs; Immunoelectrophoresis; Peptides; Phosphorylation; Photoreceptor Cells; Radioimmunoassay; Retinal Degeneration; Retinal Pigments; Rhodopsin

Topics: Animals; Ascorbic Acid; Biomechanical Phenomena; Docosahexaenoic Acids; Light; Lipids; Male; Photoreceptor Cells; Rats; Rats, Inbred Strains; Reference Values; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

Cats on a taurine-deficient diet were studied with imaging fundus reflectometry and full-field electroretinography. The pattern of rhodopsin loss and the natural history of the disease were determined from maps of the rhodopsin distribution in the central and nasal retina of cats with different degrees of severity of the retinopathy. Rhodopsin loss is first detectable in a focal region of the central retina. Subsequently, there are decreases in rhodopsin in the paracentral and nasal midperipheral retina. The horizontal streak of high rhodopsin levels is preferentially reduced in this retinopathy. The b-wave amplitude of the rod-dominated ERG is markedly reduced in cats with only mildly decreased levels of rhodopsin in the peripheral retina. In an affected cat with moderate rhodopsin loss in the central retina but minimal loss nasally, a light-microscopic study of the retina showed that there was disorganization and shortening of rod outer segments and loss of rod photoreceptor cells in the areas of reduced rhodopsin levels.

Topics: Animals; Cats; Dark Adaptation; Electroretinography; Female; Fundus Oculi; Male; Photoreceptor Cells; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Taurine

Psychophysical, reflectometric, and electrophysiologic studies were done on four members of a dominant pedigree with progressive cone dystrophy. The two youngest individuals were asymptomatic at the initial examination, and none of the subjects complained of problems associated with night vision. Nevertheless, absent or grossly reduced cone-mediated electroretinographic (ERG) responses showed the widespread loss of cone function, and moderate elevations (less than 1 log unit) in absolute threshold together with reductions in rhodopsin levels in the mid-peripheral retina provided evidence of impairment of the rod system. The progressive nature of the disease was apparent from the case histories and the changes in visual performance that occurred on re-test after a 5-year interval. Moreover, the results of increment threshold measurements at several retinal loci suggested that peripheral cones may be affected earlier and more severely than those in the central retina.

Topics: Adolescent; Adult; Color Perception; Dark Adaptation; Electrooculography; Electroretinography; Female; Genes, Dominant; Humans; Male; Pedigree; Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Sensory Thresholds

The developmental pattern of rhodopsin phosphorylation in degenerative (rdle homozygote) and normal (rd/+ heterozygote) mouse retina has been investigated. The results indicate that rhodopsin levels are comparable in the 2 retinas up to about 10 days of age but that rhodopsin phosphorylation is not. The phosphorylation of rhodopsin is substantially reduced in the degenerative retina during development. This abnormality may be an expression of the rd lesion. The rhodopsin kinase/phosphatase system, the G protein, and the visual pigment are all involved in the modulation of cGMP-phosphodiesterase activity in normal retinas. A defect in any of these components could account for the reduced level of cGMP-phosphodiesterase activity in rd retinas, resulting in cGMP accumulation and subsequent photoreceptor degeneration.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Female; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Phosphorylation; Photoreceptor Cells; Rabbits; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Rod Cell Outer Segment

During inherited retinal dystrophy in Irish Setter dogs, decreased activity of cGMP phosphodiesterase (PDE) results in high cGMP levels and retinal degeneration (1-3). This defect could be in PDE itself, or in its interactions with other proteins of the rod outer segment. We report herein that when retinas from 8-week-old dogs were phosphorylated with gamma-32P-ATP, and separated on SDS-PAGE, phosphorylation of rd dog rhodopsin was reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Since rd-mediated inhibition was prevented by 1 mM NaF, the results suggest that the cause of reduced rd phosphorylation is increased phosphatase activity. Together, these results demonstrate that decreased phosphorylation of rhodopsin due to increased phosphatase activity is a fundamental biochemical change which may partially account for the degenerative process and loss of visual acuity during inherited retinal dystrophy.

Topics: Animals; Dog Diseases; Dogs; Phosphoprotein Phosphatases; Phosphorylation; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

Cyclic GMP metabolism has been investigated in the retinas of mice that are heterozygous for a 'photoreceptor dystrophy' gene and have a lowered concentration of cGMP in their photoreceptor cells. The concentration of rhodopsin, retinal morphology and guanylate cyclase kinetics were normal. Cyclic GMP phosphodiesterase had a lowered affinity for cGMP. In accord with previous observations, chelation of exogenous calcium had no effect on cGMP levels in light-adapted retinas but increased them in dark-adapted tissue. The difference between cGMP concentrations in heterozygous and normal retinas in the dark was then eliminated. It was concluded that a modulator of cGMP phosphodiesterase activity is most likely to be causing the lowered steady-state level of cGMP in heterozygous retinas and that calcium is not involved.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium; Cyclic GMP; Dark Adaptation; Guanylate Cyclase; Heterozygote; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Rhodopsin

Retinas obtained from 7-day-old rd mice show less reaction with antirhodopsin antisera than retinas from normal mice of the same age. Likewise, antisera prepared against synthetic peptides, which corresponds to the carboxyl terminus of rhodopsin, also react less with rd retinas from 7-day-old mice. In contrast, Western blots of denatured rhodopsin from rd vs. normal retinas of the same age indicate no change in the total quantity of this protein. These results demonstrate that in the 7-day-old rd mouse retina, rhodopsin is not altered in quantity; rather, it is less accessible to reaction with anti-rhodopsin antisera. Furthermore, these results suggest that the site of altered accessibility is on the carboxyl terminus of rhodopsin.

Topics: Age Factors; Animals; Calmodulin; Cyclic GMP; Immunologic Techniques; Mice; Mice, Mutant Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Rod Cell Outer Segment

Topics: Animals; Extracellular Matrix; Eye Proteins; Molecular Weight; Photoreceptor Cells; Pigment Epithelium of Eye; Rats; Rats, Mutant Strains; Retinal Degeneration; Retinol-Binding Proteins; Rhodopsin; Vitamin A

Topics: Age Factors; Animals; Dark Adaptation; Docosahexaenoic Acids; Eye Proteins; Fatty Acids, Unsaturated; Female; Lipids; Male; Periodicity; Phospholipids; Photoreceptor Cells; Rats; Rats, Inbred Strains; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment; Rod Opsins

Topics: Aging; Animals; Carbon Radioisotopes; Dogs; Glycine; Mice; Radioisotope Dilution Technique; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Species Specificity

Adaptation to steady background lights has been investigated in pcd/pcd mice, a mutant strain with retinal degeneration. The hyperbolic stimulus/response functions of the scotopic b-wave of the electroretinogram show progressive changes. For the dark-adapted eyes the decrease in log sensitivity between ages 1 and 12 months is related linearly to the decreasing rhodopsin content. The observed decline in amplitude of maximum responses from dark-adapted eyes begins only after age 5 months and is accompanied by gradual prolongation of the time to the peak of half-maximum b-wave responses. At all ages, the sensitivity of response is changed little by increments of steady red background lights; the greatest slope of log sensitivity vs. log background plots is about +0.2. (In normal mice the slope is about +0.9.) The pcd/pcd b-wave results do not fit the normal empirical relation that is thought to reflect neural processing in the distal retina. Thus the present results suggest that neural processing is abnormal in pcd/pcd retinas.

Topics: Adaptation, Physiological; Age Factors; Animals; Dark Adaptation; Electroretinography; Female; Light; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

Topics: Aging; Animals; Antigens; Autoantibodies; Autoimmune Diseases; Cattle; Dose-Response Relationship, Immunologic; Female; Lymphocyte Activation; Male; Rats; Rats, Inbred Strains; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

A correlation was noted between alterations in activity of cGMP phosphodiesterase /PDE/ and in content of rhodopsin /R/ from rats of the Campbell strain with hereditary retina degeneration as well as from control animals of the Wistar strain. Dynamics of alterations in the PDE/R ratio was similar to both in impaired and healthy animals within the first 35 days of life. These data suggest that the decrease of the PDE activity in retina of the Campbell rats occurred due to destruction and removing of outer layer of photoreceptor cell segments, i.e. it was of the secondary nature rather than of primary pathogenetic importance. After 1.5 month of the life retina aqueous extract activated PDE of cyclic nucleotides in impaired rats, but did not cause inhibition as it was observed in healthy animals. The phenomenon appears to be due to various localization of PDE inhibitors and activators in retina as well as to destruction of the layer of rod outer segments in retina of impaired rats.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Phosphoric Diester Hydrolases; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Species Specificity

Topics: Aging; Alcohol Oxidoreductases; Animals; Cytochrome P450 Family 2; Darkness; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Rhodopsin; Vitamin A

In mice homozygous for the newly reported rds (retinal degeneration slow) gene, a progressive loss of visual cells occurs throughout life. Histogenesis of the retina proceeds normally with the exception of the receptor layer which remains rudimentary. Electron microscopic observations of the retina at different stages of development have revealed that the outer segments of the visual cells in this mutant fail to develop and that the receptor layer consists of inner segments only. In the absorption spectra of the retinal extract from the mutant the characteristic rhodopsin peak is lacking.

Topics: Animals; Mice; Mice, Inbred BALB C; Mice, Neurologic Mutants; Microscopy, Electron; Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Rodent Diseases

Topics: Humans; Ophthalmoscopes; Ophthalmoscopy; Retinal Degeneration; Retinal Pigments; Rhodopsin; Television

Topics: Absorption; Animals; Color; Electroretinography; Light; Rats; Retinal Degeneration; Rhodopsin; Time Factors

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Enzyme Inhibitors; Nerve Tissue Proteins; Phosphodiesterase Inhibitors; Photoreceptor Cells; Rats; Retinal Degeneration; Rhodopsin

Resynthesis of rhodopsin both in the retina and the eye cup was studied in albino rats (Campbell line) and in rats with pigmented eyes (Hunter line) with hereditary retinal dystrophy. Wistar rats and those of MSU line were used as controls, respectively. The rate of resynthesis of rhodopsin after its bleaching in the retina of dystrophic animals appeared to be much less than that in the normal ones, and decreased during the disease. When clear morphological changes were seen, only 50% of the previously bleached pigment was capable of regeneration during 2 hours of dark adaptation, the time being quite adequate for complete regeneration of rhodopsin in normal animals. It was found that in Campbell and Hunter rats breakdown and resynthesis of rhodopsin takes place not only in the retina but also in the layer of outer segment debris of photoreceptors located between the pigment epithelium cells and the retina.

Topics: Animals; Rats; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

1. Rhodopsin concentration has been measured by the method of densitometry in retinae of rats with inherited retinal dystrophy (RCS) raised in darkness and compared with that of normal rats similarly reared. 2. In both RCS and normal rats the fraction of rhodopsin bleached is always directly proportional to the photon content of the light, I.t, where I is the light intensity in effective quanta (500 nm) cm-2 sec-1 and t is the duration of the bleaching exposure in seconds. 3. Rhodopsin photosensitivity for bleaching is slightly higher in RCS rats than in normals (2.3 (10)-16 cm2 chromophore-1 compared with 1.3 (10)-16 cm2 chromophore-1). 4. Rhodopsin regeneration in the dark in both RCS and normal rats cannot be described by the kinetics of a simple monomolecular chemical reaction. 5. Following 5 min bleaches, the regeneration rate becomes slower as the preceding bleach is made stronger. Regeneration in the dark is significantly faster in the RCS rats than in the normal ones. 6. In normal rats, after a full bleach, rhodopsin regenerates back to the dark-adapted level within 3--4 hr. In RCS rats rhodopsin regenerates to reach a plateau level, below the previous dark-adapted level, that lasts for several hours. 7. The faction of total rhodopsin that can regenerate gradually declines with age until in 70 days old RCS rats no rhodopsin regeneration can be measured by the ensitometer. However, total rhodopsin density (fully bleached-dark-adapted) is still close to normal.

Topics: Age Factors; Animals; Dark Adaptation; Kinetics; Rats; Regeneration; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

1. Electroretinogram (e.r.g.) responses recorded from dark-reared rats with inherited retinal dystrophy (RCS) showed progressive decline in b-wave ampliture and prolongation of the time to the peak of the b-wave with age when compared with records obtained from dark-reared normal albino rats. 2. Dark-adaptation was followed in RCS and normal rats by recording the light intensity needed to evoke a criterion e.r.g. response at different time intervals after bleaching and 90% of the rhodopsin. 3. In normal rats, dark-adaptation was governed by two mechanisms. The first 25--35 min of recovery was determined by cones. The second branch, determined by the recovery of rods, lasted for about 3 hr and proceeded along an exponential time course with time constant of 41.4 +/- 2.4 min (S.E. of mean). 4. In RCS rats, the time course of the dark-adaptation after a 90% bleach depended on age. In 25--30 day old rats the recovery curve had at least three breaks separating three different mechanisms. Rats, 35--40 days old, exhibited double exponential recovery curves, while 45--70 day old rats recovered along a single exponential curve similar in time course to the cone branch of dark-adaptation found in normal rats. 5. Action spectra obtained from RCS rats at different time intervals of the recovery curve showed that in young rats, 25--30 days old, small e.r.g. responses recorded before bleaching and at the end of the recovery period were determined by rhodopsin while those recorded during the first part of the recovery from 90% bleach were determined by a combination of rods and cones. In RCS rats of advanced age (45--70 days old), rhodopsin was the major contributor to the e.r.g. responses recorded either before bleaching or at the end of the recovery period. 6. The gradual deterioration with age of the e.r.g. in RCS rats cannot be explained by either the decrease in quantum catch due to the decrease in rhodopsin content or by the linear relationship between log e.r.g. threshold and pigment concentration. 7. Using estimates of rhodopsin density within surviving rods obtained from retinal densitometry, it was shown that in RCS rats where more than 30% of normal levels of rhodopsin was located within the functioning rods, the log intensity needed for a criterion e.r.g. response measured at the end of the recovery period from a 90% bleach was linearly related to the fraction of 'functional' rhodopsin. 8. No simple relationship between log e.r.g. threshold and rhodopsin con

Topics: Age Factors; Animals; Dark Adaptation; Densitometry; Electroretinography; Kinetics; Photoreceptor Cells; Rats; Retinal Degeneration; Rhodopsin

Topics: Animals; In Vitro Techniques; Rats; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

Topics: Animals; In Vitro Techniques; Photolysis; Rats; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Temperature

Electroretinographic changes as well as rhodopsin content in the isolated retina and the eye cup in Hunter rats were studied during inherited retinal degeneration. The concentration of rhodopsin in the eye cup increases for the first 45 days after birth and then decreases slowly; the visual pigment concentration in the retina reaches its maximum by the 25th day and drops abruptly after the 35th day. The amplitude of the electroretinographic b-wave evoked by near-threshold and saturating stimuli decreases evenly till 35th day. After this day the threshold drastically increases and a-wave disappears. Thus, the 35th day is crucial in the degeneration process. The possible role played by abnormal rhodopsin metabolism in the changes observed is discussed.

Topics: Animals; Electroretinography; Rats; Rats, Inbred Strains; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin

Topics: Age Factors; Animals; Calcium; Cyclic GMP; DNA; Magnesium; Mice; Photoreceptor Cells; Rats; Retina; Retinal Degeneration; Rhodopsin

Topics: Age Factors; Animals; Cholesterol; Darkness; Fatty Acids; Lipids; Phospholipids; Phosphorus; Rats; Retina; Retinal Degeneration; Rhodopsin; Sphingomyelins

Topics: Animals; Autoimmune Diseases; Blindness; Disease Models, Animal; Haplorhini; Macaca mulatta; Retinal Degeneration; Retinal Pigments; Rhodopsin

Of 107 consecutive patients with genetically-determined retinitis pigmentosa, 23 were provisionally diagnosed as having inherited the disease in an X-linked fashion. 42 affected males and 61 females were examined, and from the data obtained the following conclusions were drawn: (1) X-linked retinitis pigmentosa exists and is distinct from choroideremia. (2) In contrast to the results of previous surveys, X-linked retinitis pigmentosa is a common form of this disease and over 20 per cent. of retinitis pigmentosa is probably transmitted in an X-linked manner. (3) (a) In contradistinction to the findings of previous investigators, most if not all adult heterozygous females have detectable degenerative changes in the ocular fundus. (b) The ocular changes in heterozygous females are most easily detected by fundus examination, visual field testing, dark adaptation measurements, and estimation of retinal rhodopsin concentration. The single most frequent abnormality is peripheral retinal pigment epithelial atrophy, which is found in all adult heterozygous females. (c) The pattern of retinal dysfunction in heterozygous females, and in particular preservation of the ocular electrical responses, suggests that the disease in women is qualitatively different from that in men and in other genetic forms of retinitis pigmentosa. There is some evidience that the disease in heterozygous women is patchy. (d) Degeneration in heterozygous females is usually symmetrical, but great variation was found in the severity of degeneration amongst heterozygotes of similar ages. No non-genetic influences were found to account for this. No evidence came to light by which the importance of X-chromosome inactivation could be assessed in determining the phenotype of heterozygous women. (4) No evidience is available to determine the number of X-linked genes transmitting the disease.

Topics: Adult; Child; Dark Adaptation; Electrooculography; Female; Fluorescein Angiography; Genetic Linkage; Genetic Variation; Heterozygote; Humans; Male; Pedigree; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sex Chromosomes; Sex Factors; Visual Fields

Topics: Age Factors; Albinism; Animals; Cell Membrane; Darkness; Eye; Optic Nerve; Photoreceptor Cells; Pigmentation; Rats; Retina; Retinal Degeneration; Rhodopsin; Sensory Deprivation; Time Factors

Topics: Age Factors; Animals; Darkness; Diet; Light; Pigment Epithelium of Eye; Rats; Retina; Retinal Degeneration; Retinal Pigments; Rhodopsin; Time Factors; Vitamin A; Vitamin A Deficiency

Topics: Animals; Mice; Retinal Degeneration; Retinal Pigments; Rhodopsin; Vertebrates