11-cis-retinal and Disease-Models--Animal

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

In industrialized countries, age-related macular degeneration (AMD) is the leading cause of blindness in elderly people. Hallmarks of the non-neovascular (dry) form of AMD are the formation of drusen and geographic atrophy, whereas the exudative (wet) form of the disease is characterized by invading blood vessels. In retinal angiomatous proliferation (RAP), a special form of wet AMD, intraretinal vessels grow from the deep plexus into the subretinal space. Little is known about the mechanisms leading to intraretinal neovascularization, but age-related changes such as reduction of choroidal blood flow, accumulation of drusen, and thickening of the Bruch's membrane may lead to reduced oxygen availability in photoreceptors. Such a chronic hypoxic situation may induce several cellular response pathways including the stabilization of hypoxia-inducible factors (HIFs) and the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Here, we discuss the potential contribution of hypoxia and HIFs in RAP disease pathology and in some mouse models for subretinal neovascularization.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Disease Models, Animal; Forecasting; Genes, Synthetic; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Mice, Transgenic; Mitochondrial Proteins; Neoplasm Proteins; Neovascularization, Pathologic; Promoter Regions, Genetic; Receptors, LDL; Retinal Drusen; Retinal Vessels; Rhodopsin; Vascular Endothelial Growth Factor A; Wet Macular Degeneration

Mutations in rhodopsin are one of the most common causes of retinitis pigmentosa (RP). Misfolding of rhodopsin can result in disruptions in cellular protein homeostasis, or proteostasis. There is currently no available treatment for RP. In this review, we discuss the different approaches currently being investigated for treatment of rhodopsin RP, focusing on the potential of manipulation of the proteostasis network as a therapeutic approach to combat retinal degeneration.

Topics: Animals; Disease Models, Animal; Genetic Predisposition to Disease; Humans; Molecular Targeted Therapy; Mutation; Proteostasis Deficiencies; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

The first autosomal dominant mutation identified to cause retinitis pigmentosa in the North American population was the substitution of proline to histidine at position 23 of the rhodopsin gene (P23H RHO). Many biochemical studies have demonstrated that P23H mutation induces rhodopsin (RHO) misfolding leading to endoplasmic reticulum stress. Herein, we review current thinking of this topic.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum Stress; Genes, Dominant; Humans; Proteostasis Deficiencies; Retinitis Pigmentosa; Rhodopsin

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

Gene therapy for dominantly inherited genetic disease is more difficult than gene-based therapy for recessive disorders, which can be treated with gene supplementation. Treatment of dominant disease may require gene supplementation partnered with suppression of the expression of the mutant gene either at the DNA level, by gene repair, or at the RNA level by RNA interference or transcriptional repression. In this review, we examine some of the gene delivery approaches used to treat animal models of autosomal dominant retinitis pigmentosa, focusing on those models associated with mutations in the gene for rhodopsin. We conclude that combinatorial approaches have the greatest promise for success.

Topics: Adenoviridae; Animals; Disease Models, Animal; Gene Expression; Gene Transfer Techniques; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Mice; Mutation; Retinitis Pigmentosa; Rhodopsin; RNA Interference; Transcription, Genetic

Topics: Animals; c-Mer Tyrosine Kinase; Carrier Proteins; cis-trans-Isomerases; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Drug Design; Eye Proteins; Genetic Therapy; Humans; Intercellular Signaling Peptides and Proteins; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Targeted Therapy; Mutation; Nerve Tissue Proteins; Nifedipine; Peripherins; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Retinitis Pigmentosa; Rhodopsin; Vitamin A

Topics: Adolescent; Adult; Animals; Carrier Proteins; cis-trans-Isomerases; Disease Models, Animal; Eye Proteins; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Leber Congenital Amaurosis; Macaca fascicularis; Nerve Growth Factors; Rats; Retinitis Pigmentosa; Rhodopsin; Serpins; Simian Immunodeficiency Virus; Young Adult

The existence of constitutive activity for G protein-coupled receptors (GPCRs) was first described in 1980s. In 1991, the first naturally occurring constitutively active mutations in GPCRs that cause diseases were reported in rhodopsin. Since then, numerous constitutively active mutations that cause human diseases were reported in several additional receptors. More recently, loss of constitutive activity was postulated to also cause diseases. Animal models expressing some of these mutants confirmed the roles of these mutations in the pathogenesis of the diseases. Detailed functional studies of these naturally occurring mutations, combined with homology modeling using rhodopsin crystal structure as the template, lead to important insights into the mechanism of activation in the absence of crystal structure of GPCRs in active state. Search for inverse agonists on these receptors will be critical for correcting the diseases cause by activating mutations in GPCRs. Theoretically, these inverse agonists are better therapeutics than neutral antagonists in treating genetic diseases caused by constitutively activating mutations in GPCRs.

Topics: Animals; Disease Models, Animal; Drug Inverse Agonism; Humans; Mutation; Receptors, G-Protein-Coupled; Rhodopsin; Sequence Homology, Amino Acid; Structural Homology, Protein

Retinitis pigmentosa (RP) is an inherited retinal degeneration characterized by nyctalopia, ring scotoma, and bone-spicule pigmentation of the retina. So far, no effective therapy has been found for RP. As a possible molecular etiology of RP, retina-specific gene deficits are most likely involved, but little has been identified in terms of intracellular mechanisms leading to retinal photoreceptor cell death at post-translational levels. In order to find an effective therapy for RP, we must look for underlying common mechanisms that are responsible for the development of RP, instead of designing a specific therapy for each of the RP types with different causes. Therefore, in the present study, several animal models with different causes of RP were studied, including (1)Royal College of Surgeons (RCS) rats with a deficit of retinal pigment epithelium (RPE) function caused by rhodopsin mutation; (2) P23H rats, (3) S334ter rats, (4) photo stress rats, (5) retinal degeneration (rd) mice with a deficit of phosphodiesterase(PDE) function; and (6) cancer-associated retinopathy (CAR) model rats with a deficit of recoverin-dependent photoreceptor adaptation function. In each of these models, the following assessments were made in order to elucidate common pathological mechanisms among the models: (1) retinal function assessed by electroretinogram (ERG), (2) retinal morphology, (3) retinoid analysis, (4) rhodopsin regeneration, (5) rhodopsin phosphorylation and dephosphorylation, and (6) cytosolic cGMP levels. We found that unregulated photoreceptor adaptation processes caused by an imbalance of rhodopsin phosphorylation and dephosphorylation caused retinal dysfunction leading to photoreceptor cell death. As possible candidate drugs for normalizing these retinal dysfunctions and stopping further retinal degeneration, nilvadipine, a Ca channel blocker, retinoid derivatives, and anthocyanine were chosen and tested to determine their effect on the above animal models with retinal degeneration. Nilvadipine showed beneficial effects against retinal degeneration in all models tested, but retinoid derivatives and anthocyanine showed these beneficial effects in only some models. Thus our present data allowed us to test the effectiveness of nilvadipine in the treatment of human RP patients.

Topics: Animals; Calcium Channel Blockers; Dark Adaptation; Disease Models, Animal; Humans; Mice; Mutation; Nifedipine; Phosphorylation; Rats; Retinitis Pigmentosa; Rhodopsin

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

Mutations in the rhodopsin gene are the most common cause of retinitis pigmentosa (RP) among human patients. The nature of the rhodopsin mutations has critical implications for the design of strategies for gene therapy. Nearly all rhodopsin mutations are dominant. Although dominance does not arise because of haploinsufficiency, it is unclear whether it is caused by gain-of-function or dominant-negative mutations. Current strategies for gene therapy have been devised to deal with toxic, gain-of-function mutations. However, analysis of results of transgenic and targeted expression of various rhodopsin genes in mice suggests that dominance may arise as a result of dominant-negative mutations. This has important consequences for gene therapy. The effects of dominant-negative mutations can be alleviated, in principle, by supplementation with additional wild-type rhodopsin. If added wild-type rhodopsin could slow retinal degeneration in human patients, as it does in mice, it would represent a valuable new strategy for gene therapy of RP caused by dominant rhodopsin mutations.

Topics: Animals; Cell Death; Disease Models, Animal; Genes, Dominant; Genetic Therapy; Humans; Mice; Mice, Transgenic; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Disease Models, Animal; Genes, Dominant; Genetic Therapy; Mice; Mice, Transgenic; Mutation; Night Blindness; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin

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; Animals; Awards and Prizes; Base Sequence; Chromosome Aberrations; Chromosome Disorders; Disease Models, Animal; Electroretinography; Humans; Mice; Mice, Transgenic; Molecular Sequence Data; Mutation; Ophthalmology; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Societies, Medical; United States

Recent biochemical findings in the human disease, retinitis pigmentosa, and related retinal degenerative diseases in animals were reviewed and discussed. While the biochemical etiology of the human disease is not known, there are indications that retinal degeneration in the rd mouse and the Irish Setter dog are related to a deficiency in cGMP phosphodiesterase and the accumulation of cGMP in the photoreceptor outer segments. The biochemical basis of retinal degeneration in the Royal College of Surgeons (RCS) rat does not seem to be related to a defect in the metabolism of cGMP, but there are suggestions that a defect in retinoid metabolism may be involved. The possibility that the defect in RCS rats may involve receptors on the membranes of the cells of the retinal pigment epithelium or phagocytic markers on those of the rod outer segment disks was discussed.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium; Disease Models, Animal; Dogs; Eye Proteins; Guanylate Cyclase; Mice; Nucleotides, Cyclic; Rats; Retinitis Pigmentosa; Rhodopsin; Taurine; Vitamin A

Topics: Adenylyl Cyclases; Animals; Anura; Calcium; Cattle; Cell Membrane; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Enzyme Activation; GTP Phosphohydrolases; Phosphoric Diester Hydrolases; Photic Stimulation; Photoreceptor Cells; Retinal Diseases; Rhodopsin; Vision, Ocular

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Topics: Animals; Disease Models, Animal; Dogs; Electroretinography; Humans; Mice; Mice, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering

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

Gene therapy for autosomal dominant retinitis pigmentosa (adRP) is challenged by the dominant inheritance of the mutant genes, which would seemingly require a combination of mutant suppression and wild-type replacement of the appropriate gene. We explore the possibility that delivery of a nanoparticle (NP)-mediated full-length mouse genomic rhodopsin (gRho) or human genomic rhodopsin (gRHO) locus can overcome the dominant negative effects of the mutant rhodopsin in the clinically relevant P23H

Topics: Animals; Disease Models, Animal; Genomics; Humans; Mice; MicroRNAs; Mutation; Nanoparticles; Retinitis Pigmentosa; Rhodopsin

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

Rhodopsin is a G-protein-coupled receptor that is specifically and abundantly expressed in rod photoreceptors. Over 150 rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). The most common mutation in the United States is the conversion of proline to histidine at position 23 (P23H) in the N-terminal domain of rhodopsin. We previously found that P23H rhodopsin was misfolded, ubiquitinylated, and rapidly degraded. Here, we investigated the role of lysine residues on P23H rhodopsin ubiquitinylation and turnover. We transfected HEK293 cells with a P23H human rhodopsin construct where all 11 lysine residues were mutated to arginine (K-null P23H). We found that the K-null P23H rhodopsin was significantly less ubiquitylated than intact P23H rhodopsin. We found that K-null P23H protein turnover was significantly slower compared to P23H rhodopsin through cycloheximide chase analysis. Finally, we also generated a wild-type rhodopsin construct where all lysines were converted to arginine and found significantly reduced ubiquitylation. Our findings identify ubiquitinylation of lysine residues as an important posttranslational modification involved in P23H rhodopsin protein degradation.

Topics: Animals; Disease Models, Animal; HEK293 Cells; Humans; Lysine; Mutation; Proteolysis; Rhodopsin; Ubiquitination

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

The balanced homeostasis of the G protein-coupled receptor (GPCR), rhodopsin (Rho), is required for vision. Misfolding mutations in Rho cause photoreceptor death, leading to retinitis pigmentosa (RP) and consequently blindness. With no cure currently available, the development of efficient therapy for RP is an urgent need. Pharmacological supplementation with molecular chaperones, including flavonoids, improves stability, folding, and membrane targeting of the RP Rho mutants in vitro. Thus, we hypothesized that flavonoids by binding to P23H Rho and enhancing its conformational stability could mitigate detrimental effects of this mutation on retinal health. In this work, we evaluated the pharmacological potential of two model flavonoids, quercetin and myricetin, by using in silico, in vitro, and in vivo models of P23H Rho. Our computational analysis showed that quercetin could interact within the orthosteric binding pocket of P23H Rho and shift the conformation of its N-terminal loop toward the wild type (WT)-like state. Quercetin added to the NIH-3T3 cells stably expressing P23H Rho increased the stability of this receptor and improved its function. Systemic administration of quercetin to P23H Rho knock-in mice substantially improved retinal morphology and function, which was associated with an increase in levels of Rho and cone opsins. In addition, treatment with quercetin resulted in downregulation of the UPR signaling and oxidative stress-related markers. This study unravels the pharmacological potential of quercetin to slow down the progression of photoreceptor death in Rho-related RP and highlights its prospective as a lead compound to develop a novel therapeutic remedy to counter RP pathology.

Topics: Animals; Disease Models, Animal; Mice; Mutation; Prospective Studies; Quercetin; Retina; 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

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

Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.

Topics: Animals; Disease Models, Animal; Homeostasis; Mice; Molecular Chaperones; NIH 3T3 Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Michael Robichaux is first author on ' Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa', published in DMM. Michael conducted the research described in this article while a postdoctoral fellow in Ted Wensel's lab at Baylor College of Medicine, Houston, TX, USA. He is now an assistant professor at West Virginia University, Morgantown, WV, USA, investigating the intricate subcellular processes in photoreceptor neurons that enable vision.

Topics: Animals; Disease Models, Animal; Humans; Male; Mice; 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

In Parkinson's disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics.

Topics: Animals; Biomechanical Phenomena; Corpus Striatum; Disease Models, Animal; Glutamic Acid; Light; Locomotion; Mice; Mice, Transgenic; Midbrain Reticular Formation; Neurons; Optogenetics; Oxidopamine; Parkinson Disease; Rhodopsin

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

Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.

Topics: Alleles; Animals; Cell Line; CRISPR-Cas Systems; Dependovirus; Disease Models, Animal; Electroretinography; Gene Editing; Genetic Therapy; Humans; INDEL Mutation; Mice; Mice, Transgenic; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retina; Retinitis Pigmentosa; Rhodopsin

Mechanisms contributing to the progression of autosomal dominant retinitis pigmentosa (adRP) due to the P23H rhodopsin mutation are complex and diverse. Previous studies showed that mechanisms like endoplasmic reticulum (ER) stress, pyroptosis, and oxidative stress were involved in the pathogenesis of the disease. However, the roles and relationships of different mechanisms are not precisely known. In this study, we aimed to evaluate certain mechanisms and find novel genes involved in P23H-related adRP.. Total RNA extracted at postnatal day (PN) 14, PN21, and PN35 was used for RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses were conducted for RNA-seq data. Additionally, data from the clustered regularly interspaced short palindromic repeats (CRISPR) screening library and the RNA-seq data of several mechanisms were used for generating custom gene sets for gene set enrichment analysis (GSEA). Next, we obtained the intersection of the aforementioned gene sets and our RNA-seq data to identify candidate genes, which were verified using real-time quantitative PCR (qPCR).. Functional enrichment analyses were consistent with disease phenotypes. All time points observed pyroptosis. In the results of GSEA, ER stress, pyroptosis, and oxidative stress were observed at PN14. ER stress and pyroptosis were shown on PN35. A total of 22 candidate genes were identified. The expression levels of selected genes verified by qPCR were concordant with the RNA-seq data.. In our study, we conclude that pyroptosis and ER stress might play a central role in RP progression. We also identified differentially expressed gene clusters related to ER stress and pyroptosis, which deserve further study. These findings provide a novel perspective for the investigation of P23H-related adRP.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum Stress; Mice; Retinitis Pigmentosa; Rhodopsin; RNA-Seq

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.

Topics: Animals; Dexamethasone; Disease Models, Animal; Evaluation Studies as Topic; Inflammation; Mice; Mice, Inbred C57BL; Retina; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Tight Junctions; Zonula Occludens-1 Protein

Gene-expression programs modulated by transcription factors (TFs) mediate key developmental events. Here, we show that the synthetic transcriptional repressor (TR; ZF6-DB), designed to treat Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP), does not perturb murine retinal development, while maintaining its ability to block Rho expression transcriptionally. To express ZF6-DB into the developing retina, we pursued two approaches, (i) the retinal delivery (somatic expression) of ZF6-DB by Adeno-associated virus (AAV) vector (AAV-ZF6-DB) gene transfer during retinogenesis and (ii) the generation of a transgenic mouse (germ-line transmission, TR-ZF6-DB). Somatic and transgenic expression of ZF6-DB during retinogenesis does not affect retinal function of wild-type mice. The P347S mouse model of RHO-adRP, subretinally injected with AAV-ZF6-DB, or crossed with TR-ZF6-DB or shows retinal morphological and functional recovery. We propose the use of developmental transitions as an effective mode to challenge the safety of retinal gene therapies operating at genome, transcriptional, and transcript levels.

Topics: Animals; Dependovirus; Disease Models, Animal; Female; Gene Expression; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; Transcription Factors; Transcriptome; Zinc Fingers

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

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

Topics: Amino Acid Substitution; Animals; Antirheumatic Agents; Disease Models, Animal; Endothelium, Vascular; Gene Expression; Gene Knock-In Techniques; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinases; Leukemia Inhibitory Factor; Mice; Mice, Transgenic; Microglia; Mutation; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; STAT Transcription Factors; Transgenes; Tumor Necrosis Factor-alpha

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

Rod-cone degenerations, for example, retinitis pigmentosa are leading causes of blindness worldwide. Despite slow disease progression in humans, vision loss is inevitable; therefore, development of vision restoration strategies is crucial. Among others, promising approaches include optogenetics and prosthetic implants, which aim to bypass lost photoreceptors (PRs). Naturally, the efficacy of these therapeutic strategies will depend on inner retinal structural and functional preservation. The present study shows that in photoinducible I307N rhodopsin mice (Translational Vision Research Model 4 [Tvrm4]), a 12k lux light exposure eliminates PRs in the central retina in 1 week, but interneurons and their synapses are maintained for as long as 9 weeks postinduction. Despite bipolar cell dendritic retraction and moderate loss of horizontal cells, the survival rate of various cell types is very high. Significant preservation of conventional synapses and gap junctions in the inner plexiform layer is also observed. We found the number of synaptic ribbons to gradually decline and their ultrastructure to become transiently abnormal, although based on our findings intrinsic retinal architecture is maintained despite complete loss of PRs. Unlike common rodent models of PR degeneration, where the disease phenotype often interferes with retinal development, in Tvrm4 mice, the degenerative process can be induced after retinal development is complete. This time course more closely mimics the timing of disease onset in affected patients. Stability of the inner retina found in these mutants 2 months after PR degeneration suggests moderate, stereotyped remodeling in the early stages of the human disease and represents a promising finding for prompt approaches of vision restoration.

Topics: Animals; Disease Models, Animal; Mice; Mice, Mutant Strains; Mutation; Photoreceptor Cells; Retina; Retinitis Pigmentosa; 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

USH2A variants are the most common cause of Usher syndrome type 2, characterized by congenital sensorineural hearing loss and retinitis pigmentosa (RP), and also contribute to autosomal recessive non-syndromic RP. Several treatment strategies are under development; however, sensitive clinical trial endpoint metrics to determine therapeutic efficacy have not been identified. In the present study, we have performed longitudinal retrospective examination of the retinal and auditory symptoms in (i) 56 biallelic molecularly confirmed USH2A patients and (ii) ush2a mutant zebrafish to identify metrics for the evaluation of future clinical trials and rapid preclinical screening studies. The patient cohort showed a statistically significant correlation between age and both rate of constriction for the ellipsoid zone length and hyperautofluorescent outer retinal ring area. Visual acuity and pure tone audiograms are not suitable outcome measures. Retinal examination of the novel ush2au507 zebrafish mutant revealed a slowly progressive degeneration of predominantly rods, accompanied by rhodopsin and blue cone opsin mislocalization from 6 to 12 months of age with lysosome-like structures observed in the photoreceptors. This was further evaluated in the ush2armc zebrafish model, which revealed similar changes in photopigment mislocalization with elevated autophagy levels at 6 days post fertilization, indicating a more severe genotype-phenotype correlation and providing evidence of new insights into the pathophysiology underlying USH2A-retinal disease.

Topics: Adolescent; Adult; Aged; Animals; Autophagy; Disease Models, Animal; Electroretinography; Extracellular Matrix Proteins; Female; Genetic Association Studies; Genotype; Hearing Loss, Sensorineural; Humans; Male; Middle Aged; Mutation; Opsins; Retina; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Usher Syndromes; Visual Acuity; Young Adult; Zebrafish

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

Bombina variegata 8 (Bv8), also known as prokineticin-2 (PK-2), is a potent pro-angiogenic factor. However, its role in retinal neovascularization (RNV) remains unknown. In this study, we explored the role of Bv8 in the pathogenesis of RNV. We found that the expression of Bv8 was significantly increased in two different models of retinal neovascularization: the oxygen-induced retinopathy (OIR) mouse model and the rhodopsin promoter (rho)/VEGF transgenic mouse model. Neutralization of Bv8 by intravitreal injections of its antibody, not only inhibited retinal and subretinal neovascularization but also decreased the mRNA and protein levels of several pro-angiogenic factors. Our in vitro assay showed that recombinant human Bv8 (RhBv8) protein promoted human retinal microvascular endothelial cells (HRECs) tube-formation, cell proliferation and vascular endothelial growth factor receptor 1 (VEGFR1) and receptor 2 (VEGFR2) expression. Our findings suggest that Bv8 could be used as a novel target for the treatment of RNV-related ocular diseases.

Topics: Amphibian Proteins; Animals; Animals, Newborn; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuropeptides; Oxygen; Promoter Regions, Genetic; Retinal Neovascularization; Retinal Vessels; Rhodopsin; RNA; Vascular Endothelial Growth Factor A

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.

Topics: Animals; Disease Models, Animal; Electroretinography; Endoplasmic Reticulum; Gene Knock-In Techniques; HSP40 Heat-Shock Proteins; Mice; Mice, Knockout; Molecular Chaperones; Mutation; Photoreceptor Cells, Vertebrate; Rats; Retina; Retinitis Pigmentosa; Rhodopsin; Transfection

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

Corynetoxins, members of the tunicamycin group of antibiotics, produce a severe and frequently fatal neurological disorder in ruminant livestock, and guinea pigs are a useful model to study the pathology and pathogenesis of this disease. The aim of this study was to determine whether tunicamycin produced ocular damage in this species, which could have pharmacotherapeutic and diagnostic value. Four 8-week-old guinea pigs were treated with tunicamycin, and two control animals were given the drug vehicle only. Guinea pigs were injected subcutaneously with 400 μg/kg of tunicamycin, in dimethyl sulphoxide, and killed 48 h post-injection. The eyes were then examined by light microscopy. Immunohistochemistry for rhodopsin was also performed. The principal pathological finding was marked retinal photoreceptor damage, which was characterised by disruption and disorganisation of rods, sometimes progressing to necrosis and separation of the outer segment. The cytoplasm of some rods was focally distended by accumulated, proteinaceous material. Rhodopsin immunopositivity in injured rods was markedly diminished and associated with shrinkage and shortening of the injured rod's outer segment. Ocular pathology, in the form of reproducible and extensive retinal photoreceptor damage, was found in guinea pigs given tunicamycin, extending the range of species found to be susceptible to this toxic injury. The guinea pig could prove to be a good animal model to test potential therapeutic interventions, and as brain lesions are often minimal and liver pathology non-specific in intoxicated ruminants, any spontaneously arising ophthalmic injury found in these species could be diagnostically useful.

Topics: Animals; Disease Models, Animal; Guinea Pigs; Photoreceptor Cells; Photoreceptor Cells, Vertebrate; Rhodopsin; Tunicamycin

Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the

Topics: Animals; Cone-Rod Dystrophies; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; HEK293 Cells; Homeodomain Proteins; Humans; Mice, Transgenic; Ophthalmoscopy; Retinal Cone Photoreceptor Cells; Rhodopsin; Rod Opsins; Tomography, Optical Coherence; Trans-Activators; Vision, Ocular

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

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

Progressive rod-cone degeneration (PRCD) is a small protein localized to photoreceptor outer segment (OS) disc membranes. Several mutations in PRCD are linked to retinitis pigmentosa (RP) in canines and humans, and while recent studies have established that PRCD is required for high fidelity disc morphogenesis, its precise role in this process remains a mystery. To better understand the part which PRCD plays in disease progression as well as its contribution to photoreceptor OS disc morphogenesis, we generated a Prcd-KO animal model using CRISPR/Cas9. Loss of PRCD from the retina results in reduced visual function accompanied by slow rod photoreceptor degeneration. We observed a significant decrease in rhodopsin levels in Prcd-KO retina prior to photoreceptor degeneration. Furthermore, ultrastructural analysis demonstrates that rod photoreceptors lacking PRCD display disoriented and dysmorphic OS disc membranes. Strikingly, atomic force microscopy reveals that many disc membranes in Prcd-KO rod photoreceptor neurons are irregular, containing fewer rhodopsin molecules and decreased rhodopsin packing density compared to wild-type discs. This study strongly suggests an important role for PRCD in regulation of rhodopsin incorporation and packaging density into disc membranes, a process which, when dysregulated, likely gives rise to the visual defects observed in patients with PRCD-associated RP.

Topics: Animals; Disease Models, Animal; Eye Proteins; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Atomic Force; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Mutations in the gene rhodopsin are one of the major causes of autosomal dominant retinitis pigmentosa (adRP). Mutant forms of Rhodopsin frequently accumulate in the endoplasmic reticulum (ER), cause ER stress, and trigger photoreceptor cell degeneration. Here, we performed a genome-wide screen to identify suppressors of retinal degeneration in a Drosophila model of adRP, carrying a point mutation in the major rhodopsin, Rh1 (Rh1G69D). We identified two novel E3 ubiquitin ligases SORDD1 and SORDD2 that effectively suppressed Rh1G69D-induced photoreceptor dysfunction and retinal degeneration. SORDD1/2 promoted the ubiquitination and degradation of Rh1G69D through VCP (valosin containing protein) and independent of processes reliant on the HRD1 (HMG-CoA reductase degradation protein 1)/HRD3 complex. We further demonstrate that SORDD1/2 and HRD1 function in parallel and in a redundant fashion to maintain rhodopsin homeostasis and integrity of photoreceptor cells. These findings identify a new ER-associated protein degradation (ERAD) pathway and suggest that facilitating SORDD1/2 function may be a therapeutic strategy to treat adRP.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Electroretinography; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Gene Knockout Techniques; Humans; Larva; Microscopy, Electron, Scanning; Point Mutation; Proteasome Endopeptidase Complex; Proteolysis; Retina; Retinitis Pigmentosa; Rhodopsin; Ubiquitin-Protein Ligases; Ubiquitination; Valosin Containing Protein

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Choroideremia; Disease Models, Animal; Female; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Mitochondria; Photoreceptor Cells, Vertebrate; Rhodopsin; Rod Cell Outer Segment

Retinopathy of prematurity (ROP) is a severe complication of premature infants, leading to vision loss when untreated. Presently, the molecular mechanisms underlying ROP are still far from being clearly understood. This study sought to investigate whether thyroid hormone (TH) signaling contributes to the neuropathology of ROP using the mouse model of ROP to evaluate longitudinal photoreceptor function.. Animals were exposed to hyperoxia from P7 to P12 to induce retinopathy, thereafter the animals were returned to room air (normoxia). The thyroid-activating enzyme type 2 deiodinases (Dio2) knockout (KO) mice and the littermate controls that were exposed to hyperoxia or maintained in room air and were then analyzed. The retinal function was evaluated using electroretinograms (ERGs) at three and seven weeks followed by histologic assessments with neuronal markers to detect cellular changes in the retina. Rhodopsin protein levels were measured to validate the results obtained from the immunofluorescence analyses.. In the ROP group, the photoreceptor ERG responses are considerably lower both in the control and the Dio2 KO animals at P23 compared to the non-ROP group. In agreement with the ERG responses, loss of Dio2 results in mislocalized cone nuclei, and abnormal rod bipolar cell dendrites extending into the outer nuclear layer. The retinal function is compromised in the adult Dio2 KO animals, although the cellular changes are less severe. Despite the reduction in scotopic a-wave amplitudes, rhodopsin levels are similar in the adult mice, across all genotypes irrespective of exposure to hyperoxia.. Using the mouse model of ROP, we show that loss of Dio2 exacerbates the effects of hyperoxia-induced retinal deficits that persist in the adults. Our data suggest that aberrant Dio2/TH signaling is an important factor in the pathophysiology of the visual dysfunction observed in the oxygen-induced retinopathy model of ROP.

Topics: Animals; Animals, Newborn; Blotting, Western; Disease Models, Animal; Electroretinography; Enzyme Activators; Hyperoxia; Immunohistochemistry; Iodide Peroxidase; Iodothyronine Deiodinase Type II; Mice; Mice, Knockout; Mice, Transgenic; Oxygen; Photoreceptor Cells, Vertebrate; Retinopathy of Prematurity; Rhodopsin; Thyroid Gland

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

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

Ligand-driven modulation of the mitochondrial translocator protein 18 kDa (TSPO) was recently described to dampen the neuroinflammatory response of microglia in a retinal light damage model resulting in protective effects on photoreceptors. We characterized the effects of the TSPO ligand XBD173 in the postischemic retina focusing on changes in the response pattern of the major glial cell types of the retina-microglia and Müller cells.. Retinal ischemia was induced by increasing the intraocular pressure for 60 min followed by reperfusion of the tissue in mice. On retinal cell types enriched via immunomagnetic separation expression analysis of TSPO, its ligand diazepam-binding inhibitor (DBI) and markers of glial activation were performed at transcript and protein level using RNA sequencing, qRT-PCR, lipid chromatography-mass spectrometry, and immunofluorescent labeling. Data on cell morphology and numbers were assessed in retinal slice and flatmount preparations. The retinal functional integrity was determined by electroretinogram recordings.. We demonstrate that TSPO is expressed by Müller cells, microglia, vascular cells, retinal pigment epithelium (RPE) of the healthy and postischemic retina, but only at low levels in retinal neurons. While an alleviated neurodegeneration upon XBD173 treatment was found in postischemic retinae as compared to vehicle controls, this neuroprotective effect of XBD173 is mediated putatively by its action on retinal glia. After transient ischemia, TSPO as a marker of activation was upregulated to similar levels in microglia as compared to their counterparts in healthy retinae irrespective of the treatment regimen. However, less microglia were found in XBD173-treated postischemic retinae at 3 days post-surgery (dps) which displayed a more ramified morphology than in retinae of vehicle-treated mice indicating a dampened microglia activation. Müller cells, the major retinal macroglia, show upregulation of the typical gliosis marker GFAP. Importantly, glutamine synthetase was more stably expressed in Müller glia of XBD173-treated postischemic retinae and homeostatic functions such as cellular volume regulation typically diminished in gliotic Müller cells remained functional.. In sum, our data imply that beneficial effects of XBD173 treatment on the postischemic survival of inner retinal neurons were primarily mediated by stabilizing neurosupportive functions of glial cells.

Topics: Animals; Antigens, Differentiation; Arginase; Carrier Proteins; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Glutamate-Ammonia Ligase; Ischemia; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neuroglia; Purines; Receptors, GABA; Retina; Retinal Diseases; Retinal Neurons; Rhodopsin; RNA, Messenger

The rhodopsin mutation P23H is responsible for a significant portion of autosomal-dominant retinitis pigmentosa, a disorder characterized by rod photoreceptor death. The mechanisms of toxicity remain unclear; previous studies implicate destabilization of P23H rhodopsin during light exposure, causing decreased endoplasmic reticulum (ER) exit and ER stress responses. Here, we probed phototransduction in Xenopus laevis rods expressing bovine P23H rhodopsin, in which retinal degeneration is inducible by light exposure, in order to examine early physiological changes that occur during retinal degeneration.. We recorded single-cell and whole-retina responses to light stimuli using electrophysiology. Moreover, we monitored morphologic changes in rods after different periods of light exposure.. Initially, P23H rods had almost normal photoresponses, but following a brief light exposure varying from 4 to 32 photoisomerizations per disc, photoresponses became irreversibly prolonged. In intact retinas, rods began to shed OS fragments after a rod-saturating exposure of 12 minutes, corresponding to approximately 10 to 100 times more photoisomerizations.. Our results indicate that in P23H rods light-induced degeneration occurs in at least two stages, the first involving impairment of phototransduction and the second involving initiation of morphologic changes.

Topics: Animals; Animals, Genetically Modified; Dark Adaptation; Disease Models, Animal; Electrophysiological Phenomena; Electroretinography; Female; Male; Microscopy, Confocal; Photic Stimulation; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Vision, Ocular; Xenopus laevis

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

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Electroretinography; Gene Knock-In Techniques; Glycosylation; Mice; Mice, Inbred C57BL; Mutation, Missense; Protein Structure, Tertiary; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Sequence Alignment

To determine the relationship between the amplitudes of the electrically evoked potentials (EEPs) and the number of optic nerve axons at a late stage of retinal degeneration in rhodopsin P347L transgenic (Tg) rabbits, a model of retinitis pigmentosa.. Six eyes of six wild-type (WT) (43.8 ± 7.5 months of age) and six eyes of six Tg (40.3 ± 2.6 months of age) rabbits were studied. The EEPs were elicited by 1 to 5 mA of transcorneal electrical stimulation. The first positive wave, the P1 component, was analyzed. After euthanasia, the number of axons in the optic nerve was counted.. The threshold current to elicit a P1 was significantly higher in Tg rabbits than WT rabbits. The amplitude of P1 elicited by 5 mA in Tg rabbits was about 24% of that in WT rabbits (P < 0.01). The number of axons in the optic nerve of Tg rabbits was reduced to about 59% of that of WT rabbits (P < 0.01). The correlation between the axon number and the amplitude of the P1 in Tg and WT rabbits was not significant. The mean ratio of the P1 amplitude/axon in Tg rabbits was decreased to 53% of that in WT rabbits (P < 0.05).. The degree of reduction in the EEP in Tg rabbits is more severe than the reduction in the number of optic nerve axons. The use of transcorneal electrical stimulation to determine the suitable candidates for prosthesis at the end-stage of retinitis pigmentosa may underestimate the condition of the optic nerves.

Topics: Animals; Animals, Genetically Modified; Axons; Cell Count; Disease Models, Animal; Electric Stimulation; Evoked Potentials, Visual; Optic Nerve; Photoreceptor Cells, Vertebrate; Rabbits; Retinitis Pigmentosa; Rhodopsin

The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit.. Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts.. Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group.. Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.

Topics: Animals; Animals, Genetically Modified; cis-trans-Isomerases; Disease Models, Animal; Electroretinography; Filtration; Genotyping Techniques; Immunohistochemistry; Light; Mice; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Phototherapy; Polymorphism, Single Nucleotide; Radio Waves; Retina; Retinitis Pigmentosa; Rhodopsin

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

Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaE

Topics: Animals; Carboxypeptidases; Disease Models, Animal; Drosophila melanogaster; Mutation; Photoreceptor Cells, Invertebrate; Retinoids; Rhodopsin

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

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the

Topics: Animals; Carboxylic Acids; Dark Adaptation; Disease Models, Animal; Lipofuscin; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Pyrimidines; Pyrroles; Retina; Retinoids; Retinol-Binding Proteins, Plasma; Rhodopsin; Stargardt Disease

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

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

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

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease, in which the death of mutant rod photoreceptors leads secondarily to the non-cell autonomous death of cone photoreceptors. Gene therapy is a promising treatment strategy. Unfortunately, current methods of gene delivery treat only a fraction of diseased cells, yielding retinas that are a mosaic of treated and untreated rods, as well as cones. In this study, we created two RP mouse models to test whether dying, untreated rods negatively impact treated, rescued rods. In one model, treated and untreated rods were segregated. In the second model, treated and untreated rods were diffusely intermixed, and their ratio was controlled to achieve low-, medium-, or high-efficiency rescue. Analysis of these mosaic retinas demonstrated that rescued rods (and cones) survive, even when they are greatly outnumbered by dying photoreceptors. On the other hand, the rescued photoreceptors did exhibit long-term defects in their outer segments (OSs), which were less severe when more photoreceptors were treated. In summary, our study suggests that even low-efficiency gene therapy may achieve stable survival of rescued photoreceptors in RP patients, albeit with OS dysgenesis.

Topics: Animals; Cell Death; Disease Models, Animal; Genetic Therapy; Mice; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; 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

Increasing evidence suggests that nerve growth factor (NGF) exerts protective effects against retinal degeneration in animal models of retinitis pigmentosa (RP). This study aims at investigating the effects of intravitreal injection of recombinant human NGF (rhNGF) on retinal photoreceptors apoptosis in an animal model of RP, the Royal College of Surgeons (RCS) rats.. Thirty-six RCS rats were treated with intravitreal injection of rhNGF or murine NGF (mNGF) or vehicle at 20 postnatal days (pd) and sacrificed at 40 pd. The eyes were enucleated and evaluated by histology, flow cytometric analysis for rhodopsin expression, Western blot for TrkA and activated (phosphorylated) TrkA (pTrkA) levels, and TUNEL assay for apoptosis' detection.. RCS rats showed a significant retinal degeneration associated with cell apoptosis at 40 pd when compared to wild-type animals. Histology showed that rhNGF intravitreal treatment significantly increased retinal thickness when compared to untreated eyes. Photoreceptors' number evaluated by flow cytometry was significantly increased in both intravitreal rhNGF- and mNGF-treated groups when compared to untreated eyes. This protective effect was associated with an increase in TrkA and activated pTrkA levels and an inhibition of apoptosis. Intravitreal NGF injection was well tolerated and did not show clinical and histological signs of adverse effects.. Intravitreal rhNGF injection proved safe and effective in favoring retinal cell survival in RCS rats. This is the first report showing that the novel rhNGF already proved safe in a phase I study exerts a biologic effect similar to the well-characterized mNGF-induced retinal protection. These results may trigger further studies to investigate rhNGF administration for the treatment of progressive degenerative retinal disorders such as retinitis pigmentosa.

Topics: Animals; Apoptosis; Blotting, Western; Cell Survival; Disease Models, Animal; Flow Cytometry; Humans; In Situ Nick-End Labeling; Intravitreal Injections; Male; Mice; Nerve Growth Factor; Photoreceptor Cells, Vertebrate; Rats; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin

Secondary generalized seizure (sGS) is a major source of disability in temporal lobe epilepsy (TLE) with unclear cellular/circuit mechanisms. Here we found that clinical TLE patients with sGS showed reduced volume specifically in the subiculum compared with those without sGS. Further, using optogenetics and extracellular electrophysiological recording in mouse models, we found that photoactivation of subicular GABAergic neurons retarded sGS acquisition by inhibiting the firing of pyramidal neurons. Once sGS had been stably acquired, photoactivation of GABAergic neurons aggravated sGS expression via depolarized GABAergic signaling. Subicular parvalbumin, but not somatostatin subtype GABAergic, neurons were easily depolarized in sGS expression. Finally, photostimulation of subicular pyramidal neurons genetically targeted with proton pump Arch, rather than chloride pump NpHR3.0, alleviated sGS expression. These results demonstrated that depolarized GABAergic signaling in subicular microcircuit mediates sGS in TLE. This may be of therapeutic interest in understanding the pathological neuronal circuitry underlying sGS. VIDEO ABSTRACT.

Topics: Adolescent; Adult; Animals; Case-Control Studies; Disease Models, Animal; Electroencephalography; Epilepsy, Temporal Lobe; GABAergic Neurons; gamma-Aminobutyric Acid; Hippocampus; Humans; Magnetic Resonance Imaging; Male; Mice; Mice, Transgenic; Middle Aged; Nerve Net; Optogenetics; Organ Size; Parvalbumins; Pyramidal Cells; Rhodopsin; Seizures; Somatostatin; Young Adult

Diabetic retinopathy is a common complication of diabetes mellitus. Diabetic patients experience functional deficits in dark adaptation, contrast sensitivity, and color perception before microvascular pathologies become apparent. Herein, we evaluated early changes in neural retinal function and in retinoid metabolism in the eye in diabetes. Streptozotocin-induced diabetic rats showed decreased a- and b-wave amplitudes of scotopic and photopic electroretinography responses 4 months after diabetes induction compared to nondiabetic controls. Although Western blot analysis revealed no difference in opsin expression, rhodopsin content was decreased in diabetic retinas, as shown by a difference in absorbance. Consistently, levels of 11-cis-retinal, the chromophore for visual pigments, were significantly lower in diabetic retinas compared to those in controls, suggesting a retinoid deficiency. Among visual cycle proteins, interphotoreceptor retinoid-binding protein and stimulated by retinoic acid 6 protein showed significantly lower levels in diabetic rats than those in nondiabetic controls. Similarly, serum levels of retinol-binding protein 4 and retinoids were significantly lower in diabetic rats. Overall, these results suggest that retinoid metabolism in the eye is impaired in type 1 diabetes, which leads to deficient generation of visual pigments and neural retinal dysfunction in early diabetes.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Disease Models, Animal; Male; Photoreceptor Cells, Vertebrate; Rats, Wistar; Retina; Retinaldehyde; Retinol-Binding Proteins, Plasma; Rhodopsin; Visual Pathways

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Disease Models, Animal; Female; Gene Editing; Genes, Dominant; Genes, Recessive; Green Fluorescent Proteins; Humans; Male; Phenotype; Point Mutation; Retinitis Pigmentosa; Rhodopsin; RNA, Guide, Kinetoplastida; Sequence Deletion; Xenopus laevis; Xenopus Proteins

Retinal ischemia is common in eye disorders, like diabetic retinopathy or retinal vascular occlusion. The goal of this study was to evaluate the potential protective effects of an intravitreally injected vascular endothelial growth factor (VEGF) inhibitor (ranibizumab) on retinal cells in an ischemia animal model via immunohistochemistry (IF) and quantitative real-time PCR (PCR). A positive binding of ranibizumab to rat VEGF-A was confirmed via dot blot. One eye underwent ischemia and a subgroup received ranibizumab. A significant VEGF increase was detected in aqueous humor of ischemic eyes (p = 0.032), whereas VEGF levels were low in ranibizumab eyes (p = 0.99). Ischemic retinas showed a significantly lower retinal ganglion cell number (RGC; IF Brn-3a: p<0.001, IF RBPMS: p<0.001; PCR: p = 0.002). The ranibizumab group displayed fewer RGCs (IF Brn-3a: 0.3, IF RBPMS: p<0.001; PCR: p = 0.007), but more than the ischemia group (IF Brn-3a: p = 0.04, IF RBPMS: p = 0.03). Photoreceptor area was decreased after ischemia (IF: p = 0.049; PCR: p = 0.511), while the ranibizumab group (IF: p = 0.947; PCR: p = 0.122) was comparable to controls. In the ischemia (p<0.001) and ranibizumab group (p<0.001) a decrease of ChAT+ amacrine cells was found, which was less prominent in the ranibizumab group. VEGF-receptor 2 (VEGF-R2; IF: p<0.001; PCR: p = 0.021) and macroglia (GFAP; IF: p<0.001; PCR: p<0.001) activation was present in ischemic retinas. The activation was weaker in ranibizumab retinas (VEGF-R2: IF: p = 0.1; PCR: p = 0.03; GFAP: IF: p = 0.1; PCR: p = 0.015). An increase in the number of total (IF: p = 0.003; PCR: p = 0.023) and activated microglia (IF: p<0.001; PCR: p = 0.009) was detected after ischemia. These levels were higher in the ranibizumab group (Iba1: IF: p<0.001; PCR: p = 0.018; CD68: IF: p<0.001; PCR: p = 0.004). Our findings demonstrate that photoreceptors and RGCs are protected by ranibizumab treatment. Only amacrine cells cannot be rescued. They seem to be particularly sensitive to ischemic damage and need maybe an earlier intervention.

Topics: Amacrine Cells; Animals; Aqueous Humor; Calcium-Binding Proteins; Cell Count; Cholinergic Neurons; Disease Models, Animal; Glial Fibrillary Acidic Protein; Humans; Ischemia; Mice; Microfilament Proteins; Microglia; Photoreceptor Cells, Vertebrate; Protective Agents; Protein Binding; Ranibizumab; Rats; Reperfusion Injury; Retina; Retinal Ganglion Cells; Rhodopsin; RNA, Messenger; Synapses; Vascular Endothelial Growth Factor A

Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause X-linked RP (XLRP), an untreatable, inherited retinal dystrophy that leads to premature blindness. RPGR localises to the photoreceptor connecting cilium where its function remains unknown. Here we show, using murine and human induced pluripotent stem cell models, that RPGR interacts with and activates the actin-severing protein gelsolin, and that gelsolin regulates actin disassembly in the connecting cilium, thus facilitating rhodopsin transport to photoreceptor outer segments. Disease-causing RPGR mutations perturb this RPGR-gelsolin interaction, compromising gelsolin activation. Both RPGR and Gelsolin knockout mice show abnormalities of actin polymerisation and mislocalisation of rhodopsin in photoreceptors. These findings reveal a clinically-significant role for RPGR in the activation of gelsolin, without which abnormalities in actin polymerisation in the photoreceptor connecting cilia cause rhodopsin mislocalisation and eventual retinal degeneration in XLRP.Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause retinal dystrophy, but how this arises at a molecular level is unclear. Here, the authors show in induced pluripotent stem cells and mouse knockouts that RPGR mediates actin dynamics in photoreceptors via the actin-severing protein, gelsolin.

Topics: Actins; Animals; Carrier Proteins; Cilia; Disease Models, Animal; Eye Proteins; Gelsolin; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Protein Transport; Retinitis Pigmentosa; Rhodopsin

In some diseases the TrkC.T1 isoform is upregulated in glia, associated with glial TNF-α production and neuronal death. What remains unknown are the activating signals in glia, and how paracrine signals may be selective for a targeted neuron while sparing other proximate neurons. We studied these questions in the retina, where Müller glia contacts photoreceptors on one side and retinal ganglion cells on the other. In a mutant Rhodopsin mouse model of retinitis pigmentosa (RP) causing progressive photoreceptor death-but sparing retinal ganglion cells-TrkC.T1 and NT-3 ligand are upregulated in Müller glia. TrkC.T1 activity generates p-Erk, which causes increased TNF-α. These sequential events take place predominantly in Müller fibers contacting stressed photoreceptors, and culminate in selective death. Each event and photoreceptor death can be prevented by reduction of TrkC.T1 expression, by pharmacological antagonism of TrkC or by pharmacological inhibition Erk. Unmasking the sequence of non-cell autologous events and mechanisms causing selective neuronal death may help rationalize therapies.

Topics: Animals; Cell Death; Disease Models, Animal; Ependymoglial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation; HEK293 Cells; Heterozygote; Humans; Mice; Mice, Knockout; Mutation; Nerve Growth Factors; Neuroglia; Neurotrophin 3; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-akt; Rats; Receptor, trkC; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

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

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

Compelling evidence suggests that transplantation of neural stem cells (NSCs) from multiple sources ameliorates motor deficits after stroke. However, it is currently unknown to what extent the electrophysiological activity of grafted NSC progeny participates in the improvement of motor deficits and whether excitatory phenotypes of the grafted cells are beneficial or deleterious to sensorimotor performances. To address this question, we used optogenetic tools to drive the excitatory outputs of the grafted NSCs and assess the impact on local circuitry and sensorimotor performance. We genetically engineered NSCs to express the Channelrhodopsin-2 (ChR2), a light-gated cation channel that evokes neuronal depolarization and initiation of action potentials with precise temporal control to light stimulation. To test the function of these cells in a stroke model, rats were subjected to an ischemic stroke and grafted with ChR2-NSCs. The grafted NSCs identified with a human-specific nuclear marker survived in the peri-infarct tissue and coexpressed the ChR2 transgene with the neuronal markers TuJ1 and NeuN. Gene expression analysis in stimulated versus vehicle-treated animals showed a differential upregulation of transcripts involved in neurotransmission, neuronal differentiation, regeneration, axonal guidance, and synaptic plasticity. Interestingly, genes involved in the inflammatory response were significantly downregulated. Behavioral analysis demonstrated that chronic optogenetic stimulation of the ChR2-NSCs enhanced forelimb use on the stroke-affected side and motor activity in an open field test. Together these data suggest that excitatory stimulation of grafted NSCs elicits beneficial effects in experimental stroke model through cell replacement and non-cell replacement, anti-inflammatory/neurotrophic effects.

Topics: Animals; Cell Separation; Disease Models, Animal; Down-Regulation; Gene Expression Profiling; Human Embryonic Stem Cells; Humans; Inflammation; Male; Neostriatum; Neural Stem Cells; Oligonucleotide Array Sequence Analysis; Optogenetics; Rats, Sprague-Dawley; Rhodopsin; Stroke; Synaptic Transmission; Transduction, Genetic; 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

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications.

Topics: Animals; Disease Models, Animal; DNA; DNA Methylation; DNA, Complementary; Gene Expression; Gene Silencing; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Introns; Mice; Mice, Inbred C57BL; Mice, Knockout; Nanoparticles; Plasmids; Promoter Regions, Genetic; Retinitis Pigmentosa; Rhodopsin; Transgenes

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

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

Most retinitis pigmentosa (RP) mutations arise in rod photoreceptor genes, leading to diminished peripheral and nighttime vision. Using a pig model of autosomal-dominant RP, we show glucose becomes sequestered in the retinal pigment epithelium (RPE) and, thus, is not transported to photoreceptors. The resulting starvation for glucose metabolites impairs synthesis of cone visual pigment-rich outer segments (OSs), and then their mitochondrial-rich inner segments dissociate. Loss of these functional structures diminishes cone-dependent high-resolution central vision, which is utilized for most daily tasks. By transplanting wild-type rods, to restore glucose transport, or directly replacing glucose in the subretinal space, to bypass its retention in the RPE, we can regenerate cone functional structures, reactivating the dormant cells. Beyond providing metabolic building blocks for cone functional structures, we show glucose induces thioredoxin-interacting protein (Txnip) to regulate Akt signaling, thereby shunting metabolites toward aerobic glucose metabolism and regenerating cone OS synthesis.

Topics: Animals; Disease Models, Animal; Fatty Acids; Glucose; Green Fluorescent Proteins; MicroRNAs; Retinal Cone Photoreceptor Cells; Retinal Photoreceptor Cell Inner Segment; Retinal Photoreceptor Cell Outer Segment; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering; Sus scrofa; Thioredoxins

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

Rett syndrome (RTT) is a neurodevelopmental disorder affecting multiple functions, including the norepinephrine (NE) system. In the CNS, NE is produced mostly by neurons in the locus coeruleus (LC), where defects in intrinsic neuronal properties, NE biosynthetic enzymes, neuronal CO2 sensitivity, and synaptic currents have been reported in mouse models of RTT. LC neurons in methyl-CpG-binding protein 2 gene (Mecp2) null mice show a high rate of spontaneous firing, although whether such hyperexcitability might increase or decrease the NE release from synapses is unknown. To activate the NEergic axonal terminals selectively, we generated an optogenetic mouse model of RTT in which NEergic neuronal excitability can be manipulated with light. Using commercially available mouse breeders, we produced a new strain of double-transgenic mice with Mecp2 knockout and channelrhodopsin (ChR) knockin in catecholaminergic neurons. Several RTT-like phenotypes were found in the tyrosine hydroxylase (TH)-ChR-Mecp2(-/Y) mice, including hypoactivity, low body weight, hindlimb clasping, and breathing disorders. In brain slices, optostimulation produced depolarization and an increase in the firing rate of LC neurons from TH-ChR control mice. In TH-ChR control mice, optostimulation of presynaptic NEergic neurons augmented the firing rate of hypoglossal neurons (HNs), which was blocked by the α-adrenoceptor antagonist phentolamine. Such optostimulation of NEergic terminals had almost no effect on HNs from two or three TH-ChR-Mecp2(-/Y) mice, indicating that excessive excitation of presynaptic neurons does not benefit NEergic modulation in mice with Mecp2 disruption. These results also demonstrate the feasibility of generating double-transgenic mice for studies of RTT with commercially available mice, which are inexpensive, labor/time efficient, and promising for cell-specific stimulation. © 2016 Wiley Periodicals, Inc.

Topics: Action Potentials; Adrenergic alpha-Antagonists; Animals; Disease Models, Animal; Dopamine beta-Hydroxylase; Female; Locus Coeruleus; Luminescent Proteins; Male; Methyl-CpG-Binding Protein 2; Mice; Mice, Transgenic; Neurons; Norepinephrine; Optogenetics; Phentolamine; Rett Syndrome; Rhodopsin; Tyrosine 3-Monooxygenase

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

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

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

Rhodopsin, a G-protein coupled receptor, most abundant protein in retinal rod photoreceptors, is glycosylated at asparagines-2 and 15 on its N-terminus. To understand the role of rhodopsin's glycosylation in vivo, we generated and characterized a transgenic mouse model that expresses a non-glycosylated form of rhodopsin. We show that lack of glycosylation triggers a dominant form of progressive retinal degeneration. Electron microscopic examination of retinas at postnatal day 17 revealed the presence of vacuolar structures that distorted rod photoreceptor outer segments and became more prominent with age. Expression of non-glycosylated rhodopsin alone showed that it is unstable and is regulated via ubiquitin-mediated proteasomal degradation at the base of outer segments. We observed similar vacuolization in outer segments of transgenic mice expressing human rhodopsin with a T17M mutation (hT17M), suggesting that the mechanism responsible for the degenerative process in mice expressing the non-glycosylated rhodopsin and the RHO(hT17M) mice is likely the cause of phenotype observed in retinitis pigmentosa patients carrying T17M mutation.

Topics: Animals; Disease Models, Animal; Gene Expression; Glycosylation; Humans; Mice; Mice, Transgenic; Microscopy, Electron; Mutation, Missense; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Ubiquitination

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

Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as introns may help promote more endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time points when cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools.

Topics: Animals; Blotting, Western; Disease Models, Animal; DNA; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Humans; Introns; Mice, Knockout; Microscopy, Confocal; Microscopy, Electron, Transmission; Nanoparticles; Phenotype; Reproducibility of Results; Retina; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding.. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses.. Two stabilization assays of P23H mutant opsin were developed and validated, one based on β-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin.. Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Drug Evaluation, Preclinical; Immunoblotting; Mice; Mutant Proteins; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.

Topics: Animals; Animals, Newborn; Cell Survival; Disease Models, Animal; Gene Expression; Mice; Nerve Growth Factor; Photoreceptor Cells, Vertebrate; Primary Cell Culture; Rats; Recombinant Proteins; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin

Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3' UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.

Topics: Amino Acid Sequence; Animals; Base Sequence; Color Vision; Disease Models, Animal; Electroretinography; Gene Dosage; Hemizygote; Molecular Sequence Data; Mutation; Phenotype; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Tomography, Optical Coherence; Transgenes

To investigate the process of retinal degeneration by analyzing the functional and morphological findings in transgenic rabbits with a Pro347Leu rhodopsin mutation.. Wild-type (WT) and transgenic (Tg) rabbits at ages 4, 8 and 12 months were used. We conducted functional evaluation by recording the changes in the pupil response to red and blue light stimulation and the amplitude of the electroretinography (ERG). Morphologically, rod and cone distribution was examined using light and electron microscopy. Immunostaining for the identification of retinal ganglion cells (RGCs) was also confirmed by injecting a TUJ-1 monoclonal antibody.. Pupil constriction for infrared pupillography and the a- and b-waves for ERG in Tg rabbits decreased with increasing age; the differences were compared to the age-matched WT rabbits. The subnormal ERG in the Tg rabbits, especially the a-wave decrease and pupil constriction with a long latency time, was induced only during exposure to blue light stimulation at 12 months. Light and electron microscopic findings showed a progressive loss of photoreceptor cells over time manifesting by 8 months in the peripheral retina. Moreover, pyknotic nuclei of the outer nuclear layer in the center of the visual streak were observed. At 12 months, there was disappearance of the rods and ballooning degeneration of the cones. Some remaining RGCs had large cell bodies with long branching dendrites.. The changes in the pupil light response and amplitude of the ERG could be used to predict the state of retinal degeneration in the Tg rabbit.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Light; Photoreceptor Cells, Vertebrate; Point Mutation; Pupil; Rabbits; Reflex, Pupillary; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin; Tubulin

Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.

Topics: Alleles; Animals; Arrhythmias, Cardiac; Caenorhabditis elegans; Calcium Channels, L-Type; Disease Models, Animal; Electrophysiological Phenomena; Gene Expression; Kymography; Light; Microscopy, Video; Muscle Contraction; Mutation; Optogenetics; Pharyngeal Muscles; Rhodopsin

To preserve photoreceptor cell structure and function in a rodent model of retinitis pigmentosa with P23H rhodopsin by selective inhibition of the mutant rhodopsin allele using a second generation antisense oligonucleotide (ASO).. Wild-type mice and rats were treated with ASO by intravitreal (IVT) injection and rhodopsin mRNA and protein expression were measured. Transgenic rats expressing the murine P23H rhodopsin gene (P23H transgenic rat Line 1) were administered either a mouse-specific P23H ASO or a control ASO. The contralateral eye was injected with PBS and used as a comparator control. Electroretinography (ERG) measurements and analyses of the retinal outer nuclear layer were conducted and correlated with rhodopsin mRNA levels.. Rhodopsin mRNA and protein expression was reduced after a single ASO injection in wild-type mice with a rhodopsin-specific ASO. Transgenic rat eyes that express a murine P23H rhodopsin gene injected with a murine P23H ASO had a 181 ± 39% better maximum amplitude response (scotopic a-wave) as compared with contralateral PBS-injected eyes; the response in control ASO eyes was not significantly different from comparator contralateral eyes. Morphometric analysis of the outer nuclear layer showed a significantly thicker nuclear layer in eyes injected with murine P23H ASO (18%) versus contralateral PBS-injected eyes.. Allele-specific ASO-mediated knockdown of mutant P23H rhodopsin expression slowed the rate of photoreceptor degeneration and preserved the function of photoreceptor cells in eyes of the P23H rhodopsin transgenic rat. Our data indicate that ASO treatment is a potentially effective therapy for the treatment of retinitis pigmentosa.

Topics: Alleles; Animals; Blotting, Western; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Macular Degeneration; Male; Mice; Oligonucleotides, Antisense; Rats; Rats, Transgenic; Real-Time Polymerase Chain Reaction; Rhodopsin; RNA, Messenger

Retinal prosthetic implants are the only approved treatment for retinitis pigmentosa, a disease of the eye that causes blindness through gradual degeneration of photoreceptors. An array of microelectrodes triggered by input from a camera stimulates surviving retinal neurons, with each electrode acting as a pixel. Unintended stimulation of retinal ganglion cell axons causes patients to see large oblong shapes of light, rather than focal spots, making it difficult to perceive forms. To address this problem, we performed calcium imaging in isolated retinas and mapped the patterns of cells activated by different electrical stimulation protocols. We found that pulse durations two orders of magnitude longer than those typically used in existing implants stimulated inner retinal neurons while avoiding activation of ganglion cell axons, thus confining retinal responses to the site of the electrode. Multielectrode stimulation with 25-ms pulses can pattern letters on the retina corresponding to a Snellen acuity of 20/312. We validated our findings in a patient with an implanted epiretinal prosthesis by demonstrating that 25-ms pulses evoke focal spots of light.

Topics: Animals; Biosensing Techniques; Disease Models, Animal; Electric Stimulation; Evoked Potentials, Visual; Female; Genetic Predisposition to Disease; Humans; In Vitro Techniques; Microelectrodes; Miniaturization; Mutation; Phenotype; Phosphenes; Prosthesis Design; Rats, Long-Evans; Retina; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin; Synaptic Potentials; Synaptic Transmission; Time Factors; Transfection; Visual Pathways; Visual Perception; Visual Prosthesis

We previously reported a transgenic Xenopus laevis model of retinitis pigmentosa in which tadpoles express the bovine form of P23H rhodopsin (bP23H) in rod photoreceptors. In this model, retinal degeneration was dependent on light exposure. Here, we investigated ultrastructural changes that occurred in the rod photoreceptors of these retinas when exposed to light.. Tadpoles expressing bP23H in rods were transferred from constant darkness to a 12-hour light:12-hour dark (12L:12D) regimen. For comparison, transgenic tadpoles expressing an inducible form of caspase 9 (iCasp9) were reared in a 12L:12D regimen, and retinal degeneration was induced by administration of the drug AP20187. Tadpoles were euthanized at various time points, and eyes were processed for confocal light and transmission electron microscopy.. We observed defects in outer and inner segments of rods expressing bP23H that were aggravated by light exposure. Rod outer segments exhibited vesiculations throughout and were rapidly phagocytosed by the retinal pigment epithelium. In rod inner segments, we observed autophagic compartments adjacent to the endoplasmic reticulum and extensive vesiculation at later time points. These defects were not found in rods expressing iCasp9, which completely degenerated within 36 hours after drug administration.. Our results indicate that ultrastructural defects in outer and inner segment membranes of bP23H expressing rods differ from those observed in drug-induced apoptosis. We suggest that light-induced retinal degeneration caused by P23H rhodopsin occurs via cell death with autophagy, which may represent an attempt to eliminate the mutant rhodopsin and/or damaged cellular compartments from the secretory pathway.

Topics: Animals; Animals, Genetically Modified; Autophagy; Caspase 9; Disease Models, Animal; Light; Photoperiod; Radiation Injuries, Experimental; Retinal Photoreceptor Cell Inner Segment; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Tacrolimus; Xenopus laevis

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 gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

Topics: Animals; ATP-Binding Cassette Transporters; Dependovirus; Disease Models, Animal; Gene Transfer Techniques; Genetic Vectors; HEK293 Cells; Humans; Injections; Lipofuscin; Macular Degeneration; Melanosomes; Mice; Mice, Inbred C57BL; Myosin VIIa; Myosins; Phenotype; Photoreceptor Cells, Vertebrate; Retina; Retinal Pigment Epithelium; Rhodopsin; Stargardt Disease; Sus scrofa; Trans-Splicing; Transduction, Genetic; Usher Syndromes

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

The P23H mutation in rhodopsin (Rho(P23H)) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the Rho(P23H) rhodopsin transgene (GHL(+)). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for GHL(+), GHL(+)/Chop(-/-) and GHL(+)/Ask1(-/-) animals between 4-28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of Chop and Ask1 did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of GHL(+)/Chop(-/-) and GHL(+)/Ask1(-/-), a region that was severely degenerated in GHL(+) mice. Our results show that in the presence of the Rho(P23H) transgene, the rate of decline in retinal sensitivity is similar in Chop or Ask1 ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in GHL(+) mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression.

Topics: Animals; Apoptosis; Disease Models, Animal; Electrophysiology; Electroretinography; Genotype; MAP Kinase Kinase Kinase 5; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Photophobia; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transcription Factor CHOP; Vision, Ocular

Functional studies have detected deficits in retinal signaling in asymptomatic children from families with inherited autosomal dominant retinitis pigmentosa (RP). Whether retinal abnormalities are present earlier during gestation or shortly after birth in a subset of children with autosomal dominant RP is unknown and no appropriate animal RP model possessing visual function at birth has been available to examine this possibility. In a recently developed transgenic P23H (TgP23H) rhodopsin swine model of RP, we tracked changes in pre- and early postnatal retinal morphology, as well as early postnatal retinal function.. Domestic swine inseminated with semen from a TgP23H miniswine founder produced TgP23H hybrid and wild type (Wt) littermates. Outer retinal morphology was assessed at light and electron microscopic levels between embryonic (E) and postnatal (P) day E85 to P3. Retinal function was evaluated using the full field electroretinogram at P3.. Embryonic TgP23H rod photoreceptors are malformed and their rhodopsin expression pattern is abnormal. Consistent with morphological abnormalities, rod-driven function is absent at P3. In contrast, TgP23H and Wt cone photoreceptor morphology (E85-P3) and cone-driven retinal function (P3) are similar.. Prenatal expression of mutant rhodopsin alters the normal morphological and functional development of rod photoreceptors in TgP23H swine embryos. Despite this significant change, cone photoreceptors are unaffected. Human infants with similarly aggressive RP might never have rod vision, although cone vision would be unaffected. Such aggressive forms of RP in preverbal children would require early intervention to delay or prevent functional blindness.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; DNA; DNA Mutational Analysis; Electroretinography; Genotype; Humans; Immunohistochemistry; Microscopy, Electron, Transmission; Mutation; Polymerase Chain Reaction; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Swine, Miniature

Human and swine retinas have morphological and functional similarities. In the absence of primate models, the swine is an attractive model to study retinal function and disease, with its cone-rich visual streak, our ability to manipulate their genome, and the differences in susceptibility of rod and cone photoreceptors to disease. We characterized the normal development of cone function and its subsequent decline in a P23H rhodopsin transgenic (TgP23H) miniswine model of autosomal dominant RP.. Semen from TgP23H miniswine 53-1 inseminated domestic swine and produced TgP23H and Wt hybrid littermates. Retinal function was evaluated using ERGs between postnatal days (P) 14 and 120. Retinal ganglion cell (RGC) responses were recorded to full-field stimuli at several intensities. Retinal morphology was assessed using light and electron microscopy.. Scotopic retinal function matures in Wt pigs up to P60, but never develops in TgP23H pigs. Wt and TgP23H photopic vision matures similarly up to P30 and diverges at P60 where TgP23H cone vision declines. There are fewer TgP23H RGCs with visually evoked responses at all ages and their response to light is compromised. Photoreceptor morphological changes mirror these functional changes.. Lack of early scotopic function in TgP23H swine suggests it as a model of an aggressive form of RP. In this mammalian model of RP, normal cone function develops independent of rod function. Therefore, its retina represents a system in which therapies to rescue cones can be developed to prolong photopic visual function in RP patients.

Topics: Animals; Animals, Genetically Modified; Cell Count; Disease Models, Animal; Electroretinography; Microscopy, Electron, Transmission; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Swine, Miniature

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

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

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

Visual sensory impairments are common in Mental Deficiency (MD) and Autism Spectrum Disorder (ASD). These defects are linked to cerebral dysfunction in the visual cortical area characterized by the deregulation of axon growth/guidance and dendrite spine immaturity of neurons. However, visual perception had not been addressed, although the retina is part of the central nervous system with a common embryonic origin. Therefore, we investigated retinal perception, the first event of vision, in a murine model of MD with autistic features. We document that retinal function is altered in Fmr1 KO mice, a model of human Fragile X Syndrome. Indeed, In Fmr1 KO mice had a lower retinal function characterized by a decreased photoreceptors neuron response, due to a 40% decrease in Rhodopsin content and to Rod Outer Segment destabilization. In addition, we observed an alteration of the visual signal transmission between photoreceptors and the inner retina which could be attributed to deregulations of pre- and post- synaptic proteins resulting in retinal neurons synaptic destabilization and to retinal neurons immaturity. Thus, for the first time, we demonstrated that retinal perception is altered in a murine model of MD with autistic features and that there are strong similarities between cerebral and retinal cellular and molecular defects. Our results suggest that both visual perception and integration must be taken into account in assessing visual sensory impairments in MD and ASD.

Topics: Animals; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Male; Mice; Mice, Knockout; Phenotype; Retina; Rhodopsin; Visual Perception

For sensitive detection of rare gene repair events in terminally differentiated photoreceptors, we generated a knockin mouse model by replacing one mouse rhodopsin allele with a form of the human rhodopsin gene that causes a severe, early-onset form of retinitis pigmentosa. The human gene contains a premature stop codon at position 344 (Q344X), cDNA encoding the enhanced green fluorescent protein (EGFP) at its 3' end, and a modified 5' untranslated region to reduce translation rate so that the mutant protein does not induce retinal degeneration. Mutations that eliminate the stop codon express a human rhodopsin-EGFP fusion protein (hRho-GFP), which can be readily detected by fluorescence microscopy. Spontaneous mutations were observed at a frequency of about one per retina; in every case, they gave rise to single fluorescent rod cells, indicating that each mutation occurred during or after the last mitotic division. Additionally, the number of fluorescent rods did not increase with age, suggesting that the rhodopsin gene in mature rod cells is less sensitive to mutation than it is in developing rods. Thus, there is a brief developmental window, coinciding with the transcriptional activation of the rhodopsin locus, in which somatic mutations of the rhodopsin gene abruptly begin to appear.

Topics: Aging; Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation, Developmental; Gene Knock-In Techniques; Green Fluorescent Proteins; Humans; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Mutation; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transcriptional Activation

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

Astrocytes provide neuroprotective effects against degeneration of dopaminergic (DA) neurons and play a fundamental role in DA differentiation of neural stem cells. Here we show that light illumination of astrocytes expressing engineered channelrhodopsin variant (ChETA) can remarkably enhance the release of basic fibroblast growth factor (bFGF) and significantly promote the DA differentiation of human embryonic stem cells (hESCs) in vitro. Light activation of transplanted astrocytes in the substantia nigra (SN) also upregulates bFGF levels in vivo and promotes the regenerative effects of co-transplanted stem cells. Importantly, upregulation of bFGF levels, by specific light activation of endogenous astrocytes in the SN, enhances the DA differentiation of transplanted stem cells and promotes brain repair in a mouse model of Parkinson's disease (PD). Our study indicates that astrocyte-derived bFGF is required for regulation of DA differentiation of the stem cells and may provide a strategy targeting astrocytes for treatment of PD.

Topics: Animals; Astrocytes; Cell Differentiation; Cell- and Tissue-Based Therapy; Cells, Cultured; Disease Models, Animal; Dopaminergic Neurons; Embryonic Stem Cells; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Light; Mice; Neural Stem Cells; Neurogenesis; Parkinson Disease; Protein Engineering; Recombinant Proteins; Rhodopsin; Stem Cell Transplantation; Substantia Nigra

The P23H-1 transgenic rat carries a mutated mouse opsin gene, in addition to endogenous opsin genes, and undergoes progressive photoreceptor loss that is generally characteristic of human autosomal dominant retinitis pigmentosa (RP). Here, we examined morphological changes correlated with visual function that is comparable to clinical application in the pigmented P23H-1 rat retina as photoreceptor degeneration progressed. We found that rod function was compromised as early as postnatal day 28 and was a good indicator for tracking retinal degeneration. Cone function was normal and did not change until the thickness of the photoreceptor layer was reduced by 75%. Similar to the threshold versus intensity curves used to evaluate vision of RP patients, light-adaptation curves showed that cone thresholds depended on the number of remaining functioning cones, but not on its length of outer segments (OS). By 1 year of age, both rod and cone functions were significantly compromised. Correlating with early abnormal rod function, rods and related secondary neurons also underwent progressive degeneration, including shortening of inner and OS of photoreceptors, loss of rod bipolar and horizontal cell dendrites, thickening of the outer Müller cell processes, and reduced density of pre- and postsynaptic markers. Similar early morphological modifications were also observed in cones and their related secondary neurons. However, cone function was maintained at nearly normal level for a long period. The dramatic loss of rods at late stage of degeneration may contribute to the dysfunction of cones. Attention has to be focused on preserving cone function and identifying factors that damage cones when therapeutic regimes are applied to treat retinal degeneration. As such, these findings provide a foundation for future studies involving treatments to counter photoreceptor loss.

Topics: Adaptation, Ocular; Age Factors; Animals; Disease Models, Animal; Electroretinography; Eye Proteins; Gene Expression Regulation; Humans; Nerve Tissue Proteins; Neuroglia; Neurons; Neurotransmitter Agents; Rats; Rats, Long-Evans; Rats, Transgenic; Receptors, Glutamate; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Fields

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

Rhodopsin has been used as a prototype system to investigate G protein-coupled receptor (GPCR) internalization and endocytic sorting mechanisms. Failure of rhodopsin recycling upon light activation results in various degenerative retinal diseases. Accumulation of internalized rhodopsin in late endosomes and the impairment of its lysosomal degradation are associated with unregulated cell death that occurs in dystrophies. However, the molecular basis of rhodopsin accumulation remains elusive. We found that the novel norpA(P24) suppressor, diehard4, is responsible for the inability of endo-lysosomal rhodopsin trafficking and retinal degeneration in Drosophila models of retinal dystrophies. We found that diehard4 encodes Osiris 21. Loss of its function suppresses retinal degeneration in norpA(P24), rdgC(306), and trp(1), but not in rdgB(2), suggesting a common cause of photoreceptor death. In addition, the loss of Osiris 21 function shifts the membrane balance between late endosomes and lysosomes as evidenced by smaller late endosomes and the proliferation of lysosomal compartments, thus facilitating the degradation of endocytosed rhodopsin. Our results demonstrate the existence of negative regulation in vesicular traffic between endosomes and lysosomes. We anticipate that the identification of additional components and an in-depth description of this specific molecular machinery will aid in therapeutic interventions of various retinal dystrophies and GPCR-related human diseases.

Topics: Animals; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Endocytosis; Endosomes; Humans; Lysosomes; Membrane Proteins; Phospholipase C beta; Photoreceptor Cells, Invertebrate; Retinal Dystrophies; Rhodopsin; Vesicular Transport Proteins

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

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.

Topics: Animals; Apoptosis Inducing Factor; Calpain; Cell Death; Cell Nucleus; Disease Models, Animal; Electroretinography; Gene Expression Regulation; In Situ Nick-End Labeling; Intravitreal Injections; Mitochondria; Mitochondrial Proteins; Mutation; Ophthalmic Solutions; Peptides; Photoreceptor Cells, Vertebrate; Protein Transport; Rats; Rats, Transgenic; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

A rhodopsin P347L transgenic (Tg) rabbit, a model of retinitis pigmentosa, has been generated in our laboratory. The purpose of this study was to determine the properties of focal areas of the retina in this rabbit model during the course of retinal degeneration. To accomplish this, we recorded focal ERGs from wild-type (WT) and Tg rabbits at ages 3, 6, and 12 months. A 15° stimulus spot was used to elicit the focal ERGs from the center of the visual streak and from four surrounding areas. We found that the amplitudes of the focal cone ERG b-waves and oscillatory potentials (OPs) of the Tg rabbits in the five areas decreased progressively with increasing age and became almost non-recordable at 12 months. There were no significant regional differences in the b-waves of Tg rabbits recorded from the 5 areas. The amplitudes of the OPs were better preserved than the b-waves and the OPs/b-wave ratio was higher than that in WT rabbits at every recording area. The summed OPs amplitudes, which most likely originate from the amacrine and/or ganglion cells, recorded from the area superior to the optic disc was significantly larger than that from other areas at 3- and 6-months-old. This indicated that the inner retinal neurons were not altered equally after photoreceptor degeneration in this rabbit model.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Rabbits; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis Pigmentosa (RP) is the leading cause of inherited blindness in the developed world, affecting approximately 1 in 3000 individuals. Although there is currently no cure for RP, the genetic pathology has been well established. In this study, we developed a novel mouse model of RP (huRhoP347S) expressing a pathogenic human rhodopsin gene with a Pro347Ser (P347S) mutation on a rhodopsin knockout background. These mice undergo severe retinal degeneration at 1 month of age. In contrast to prior studies, this model was administered a gene therapy treatment at 19 days postnata. We evaluated several self-complementary adeno-associated virus (AAV) serotypes for photoreceptor tropism, including scAAV2/2, scAAV2/5, scAAV2/6.2 and scAAV2/9, and found that scAAV2/9 transduced photoreceptors with greater efficiency and expression than other vectors. We engineered an scAAV2/9 vector to contain a microRNA sequence specifically targeting the human rhodopsin gene and demonstrated its ability to silence rhodopsin by 60.2±8.2% in vitro. In addition, we constructed an scAAV2/9 vector to contain a replacement 'codon-modified' rhodopsin transgene (RhoR2) that was resistant to degradation by the microRNA. We found that delivery of the RhoR2 by scAAV2/9 is capable of restoring vision to rhodopsin knockout mice, and rescuing our novel transgenic huRhoP347S mouse model of dominant RP. Average a-wave responses of RhoR2-injected eyes were 1.8-fold higher than those of control-injected eyes. We found that delivery of the microRNA and replacement rhodopsin in a 1:2 ratio produced an average electroretinography (ERG) a-wave response of 17.4±2.9 compared to 6.5±2.8 μV for eyes injected with negative control virus.

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Silencing; Genetic Therapy; Mice; MicroRNAs; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Transgenes

Retinal detachment (RD) is a leading cause of blindness, and although final surgical re-attachment rate has greatly improved, visual outcome in many macula-off detachments is disappointing, mainly because of photoreceptor cell death. We previously showed that lutein is anti-apoptotic in rodent models of ischemia/reperfusion injury. The objective of this study is to investigate lutein as a possible pharmacological adjunct to surgery.. Subretinal injections of 1.4 % sodium hyaluronate were used to induce RD in Sprague-Dawley rats until their retinae were approximately 70 % detached. Daily injections of corn oil (control group) or 0.5 mg/kg lutein in corn oil (treatment group) were given intraperitoneally starting 4 h after RD induction. Animals were euthanized 3 days and 30 days after RD and their retinae were analyzed for photoreceptor apoptosis and cell survival at the outer nuclear layer (ONL) using TUNEL staining and cell counting on retinal sections. Glial fibrillary acidic protein (GFAP) and rhodopsin (RHO) expression were evaluated with immunohistochemistry. Western blotting was done with antibodies against cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 to delineate lutein's mechanism of action in the apoptotic cascade. To seek a possible therapeutic time window, the same set of experiments was repeated with treatment commencing 36 h after RD.. When lutein was given 4 h after RD, there were significantly fewer TUNEL-positive cells in ONL 3 days after RD when compared with the vehicle group. Cell counting showed that there were significantly more nuclei in ONL in lutein-treated retinae by day 30. Treatment groups also showed significantly reduced GFAP immunoreactivity and preserved RHO expression. At day 3 after RD, Western blotting showed reduced expression of cleaved caspase-3 and cleaved caspase-8 in the treatment group. No difference was found for cleaved caspase-9. When lutein was given 36 h after RD similar results were observed.. Our results suggest that lutein is a potent neuroprotective agent that can salvage photoreceptors in rats with RD, with a therapeutic window of at least 36 h. The use of lutein in patients with RD may serve as an adjunct to surgery to improve visual outcomes.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Caspase 8; Cell Count; Cell Survival; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Injections, Intraperitoneal; Lutein; Male; Neuroprotective Agents; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Detachment; Rhodopsin

Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6α(nmf363) mouse model of this disease, defects in the α-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6α levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6α, to transduce Pde6α(nmf363) retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6α deficiency in humans, in addition to PDE6β deficiency, is also likely to be treatable by gene therapy.

Topics: Animals; Cyclic Nucleotide Phosphodiesterases, Type 6; Dependovirus; Disease Models, Animal; Electroretinography; Genetic Therapy; Genetic Vectors; Immunoblotting; Mice; Mice, Inbred C57BL; Mice, Transgenic; Promoter Regions, Genetic; Retina; Retinitis Pigmentosa; Rhodopsin; Transduction, Genetic

We have recently described the surviving cones and Müller-glia process remodeling in retinitis pigmentosa (RP) and shown that rod degeneration triggers the reorganization of the cone mosaic into an orderly array of rings. Within these rings, remodeled Müller-glia processes envelope cones. Here, we report the spatiotemporal pattern of healthy rods, their relationship with dying rods and the way that rod death stimulates the modification of cone spatial-distribution patterns and Müller-glia processes in the S334ter-line-3 rat, a transgenic model expressing a rhodopsin mutation that causes RP. The spatial patterns of rods, cones, microglial and Müller cells were labeled by immunocytochemistry with cell-type-specific markers at various stages of deveopment in rat whole-mount retinas. Spatial patterns of dying cells were examined by TUNEL staining. The S334ter rod mosaic began to develop small holes around postnatal day 10. These hot-spots of cell death progressively increased in size, leaving larger rod-less holes behind. The holes were temporarily occupied by active microglial cells, before being replaced by remodeled Müller-cell processes. Our data suggest that the hot spots of rod death create holes in the rod mosaic early in retinal degeneration and that the resulting pattern triggers the modification of the spatial-distribution patterns of cones and glia cells.

Topics: Animals; Cell Death; Disease Models, Animal; Disease Progression; Microglia; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Time Factors

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

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; Dependovirus; Disease Models, Animal; Electroretinography; Gene Transfer Techniques; Genes, Dominant; Genetic Therapy; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; 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

The Pro23His (P23H) rhodopsin (RHO) mutation underlies the most common form of human autosomal dominant retinitis pigmentosa (adRP). The objective of this investigation was to establish a transgenic miniature swine model of RP using the human P23H RHO gene.. Somatic cell nuclear transfer (SCNT) was used to create transgenic miniature pigs that expressed the human P23H RHO mutation. From these experiments, six transgenic founders were identified whose retinal function was studied with full-field electroretinography (ffERG) from 3 months through 2 years. Progeny from one founder were generated and genotyped to determine transgene inheritance pattern. Retinal mRNA was isolated, and the ratio of P23H to wild-type pig RHO was measured.. A single transgene integration site was observed for five of the six founders. All founders had abnormal scotopic and photopic ffERGs after 3 months. The severity of the ffERG phenotype was grouped into moderately and severely affected groups. Offspring of one founder inherited the transgene as an autosomal dominant mutation. mRNA analyses demonstrated that approximately 80% of total RHO was mutant P23H.. Expression of the human RHO P23H transgene in the retina creates a miniature swine model with an inheritance pattern and retinal function that mimics adRP. This large-animal model can serve as a novel tool for the study of the pathogenesis and therapeutic intervention in the most common form of adRP.

Topics: Animals; Animals, Genetically Modified; Blotting, Southern; Cell Line; Disease Models, Animal; Electroretinography; Female; Follow-Up Studies; Gene Expression Regulation; Genotype; Humans; In Situ Hybridization, Fluorescence; Male; Mutation; Nuclear Transfer Techniques; Retina; Retinitis Pigmentosa; Rhodopsin; RNA; Swine; Swine, Miniature

Rhodopsin is the G protein-coupled receptor in charge of initiating signal transduction in rod photoreceptor cells upon the arrival of the photon. D190N (Rho(D190n)), a missense mutation in rhodopsin, causes autosomal-dominant retinitis pigmentosa (adRP) in humans. Affected patients present hyperfluorescent retinal rings and progressive rod photoreceptor degeneration. Studies in humans cannot reveal the molecular processes causing the earliest stages of the condition, thus necessitating the creation of an appropriate animal model. A knock-in mouse model with the D190N mutation was engineered to study the pathogenesis of the disease. Electrophysiological and histological findings in the mouse were similar to those observed in human patients, and the hyperfluorescence pattern was analogous to that seen in humans, confirming that the D190N mouse is an accurate model for the study of adRP.

Topics: Animals; Disease Models, Animal; Gene Order; Gene Targeting; Genes, Dominant; Humans; Mice; Mice, Transgenic; Mutation; Protein Transport; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The a-wave of the photopic electroretinogram (ERG) of macaque monkeys is made up of the electrical activities of cone photoreceptors and post-photoreceptoral neurons. However, it is not known whether the contributions of these two components change in retinas with inherited photoreceptor degeneration. The purpose of this study was to determine the contributions of cones and post-photoreceptoral neurons to the a-wave of the photopic ERGs in rhodopsin Pro347Leu transgenic (Tg) rabbits.. Ten Tg and 10 wild-type (WT) New Zealand White rabbits were studied at 4 and 12 months of age. The a-waves of the photopic ERGs were elicited by xenon flashes of different stimulus strengths before and after the activities of post-photoreceptoral neurons were blocked by intravitreal injections of a combination of 0.2 to 0.4 mM of 6-cyano-7-nitrouinoxaline-2,3(1H,4H)-dione, disodium (CNQX) and 2 to 4 mM of (±)-2-amino-4-phosphonobutyric acid.. The percentage contribution of the cone photoreceptors to the photopic ERG a-waves increased with increasing stimulus strength, and the percentage ranged from 54% to 75% in 4-month-old WT rabbits. In contrast, the percentage contribution of the cone photoreceptors in 4-month-old Tg rabbits ranged from 32% to 51% (P < 0.05). The mean percentage contribution of cone photoreceptors became still smaller at 11% to 48% in 12-month-old Tg rabbits.. These results suggest that the relative contribution of cone photoreceptors to the photopic ERG a-wave is smaller in retinas with inherited photoreceptor degeneration. This indicates that the a-waves of the photopic ERGs in patients with retinitis pigmentosa must consider this lower contribution from the cone photoreceptors.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Excitatory Amino Acid Antagonists; Glutamic Acid; Interneurons; Intravitreal Injections; Rabbits; Retinal Cone Photoreceptor Cells; Rhodopsin

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

Chronic stress in the endoplasmic reticulum (ER) underlies many degenerative and metabolic diseases involving apoptosis of vital cells. A well-established example is autosomal dominant retinitis pigmentosa (ADRP), an age-related retinal degenerative disease caused by mutant rhodopsins. Similar mutant alleles of Drosophila Rhodopsin-1 also impose stress on the ER and cause age-related retinal degeneration in that organism. Well-characterized signalling responses to ER stress, referred to as the unfolded protein response (UPR), induce various ER quality control genes that can suppress such retinal degeneration. However, how cells activate cell death programs after chronic ER stress remains poorly understood. Here, we report the identification of a signalling pathway mediated by cdk5 and mekk1 required for ER-stress-induced apoptosis. Inactivation of these genes specifically suppressed apoptosis, without affecting other protective branches of the UPR. CDK5 phosphorylates MEKK1, and together, they activate the JNK pathway for apoptosis. Moreover, disruption of this pathway can delay the course of age-related retinal degeneration in a Drosophila model of ADRP. These findings establish a previously unrecognized branch of ER-stress response signalling involved in degenerative diseases.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Line; Cells, Cultured; Chromosome Aberrations; Cyclin-Dependent Kinase 5; Disease Models, Animal; Drosophila; Drosophila Proteins; Endoplasmic Reticulum Stress; Genes, Dominant; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Molecular Sequence Data; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Time Factors

In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.

Topics: Adolescent; Adult; Aged, 80 and over; Animals; Biomarkers; Cadherins; Ciliary Body; Disease Models, Animal; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Male; Mice; Mice, Inbred C57BL; Middle Aged; Nerve Tissue Proteins; Nestin; Paired Box Transcription Factors; PAX6 Transcription Factor; Pigment Epithelium of Eye; Real-Time Polymerase Chain Reaction; Repressor Proteins; Retinal Detachment; Retinal Neurons; Rhodopsin; SOXB1 Transcription Factors; Stem Cells; Vitreoretinopathy, Proliferative; Vitreous Body

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

To determine whether transcorneal electrical stimulation (TES) has neuroprotective effects on the photoreceptors, and whether it slows the rate of decrease of the electroretinogram (ERG) in rhodopsin P347L transgenic (Tg) rabbits.. Six-week-old Tg rabbits received TES through a contact lens electrode on the left eye weekly for 6 weeks. The right eyes received sham stimulation on the same days. Electroretinograms (ERGs) were recorded before and at 12 weeks after the TES. After the last ERG recordings, the animals were euthanized for morphologic analysis of the retinas. Immunohistochemical (IHC) analysis was performed to detect the immunostaining by peanut agglutinin (PNA) and rhodopsin antibodies in the retinas.. The a- and b-wave amplitudes of the photopic ERGs and the b-wave amplitudes of the scotopic ERGs at higher stimulus intensities were significantly larger in the TES eyes than in the sham stimulated eyes (P<0.05, respectively). Morphologic analyses showed that the mean thickness of the outer nuclear layer (ONL) in the visual streak at 12 weeks was significantly thicker in TES eyes than in sham-stimulated eyes (P<0.05). IHC showed that the immunostaining by PNA and rhodopsin antibody in the TES-treated retinas was stronger than that in the sham-stimulated retinas.. TES promotes the survival of photoreceptors and preserves the ERGs in Tg rabbits. Although further investigations are necessary before using TES on patients, these findings indicate that TES should be considered for therapeutic treatment for RP patients with a P347L mutation of rhodopsin.

Topics: Animals; Animals, Genetically Modified; Cornea; Disease Models, Animal; Electric Stimulation; Electroretinography; Immunohistochemistry; Photoreceptor Cells, Vertebrate; Rabbits; Retinal Diseases; Rhodopsin

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

Innate mechanisms of inter-organ protection underlie the phenomenon of remote ischaemic preconditioning (RPc) in which episode(s) of ischaemia and reperfusion in tissues remote from the heart reduce myocardial ischaemia/reperfusion injury. The uncertainty surrounding the mechanism(s) underlying RPc centres on whether humoral factor(s) produced during ischaemia/reperfusion of remote tissue and released into the systemic circulation mediate RPc, or whether a neural signal is required. While these two hypotheses may not be incompatible, one approach to clarify the potential role of a neural pathway requires targeted disruption or activation of discrete central nervous substrate(s).. Using a rat model of myocardial ischaemia/reperfusion injury in combination with viral gene transfer, pharmaco-, and optogenetics, we tested the hypothesis that RPc cardioprotection depends on the activity of vagal pre-ganglionic neurones and consequently an intact parasympathetic drive. For cell-specific silencing or activation, neurones of the brainstem dorsal motor nucleus of the vagus nerve (DVMN) were targeted using viral vectors to express a Drosophila allatostatin receptor (AlstR) or light-sensitive fast channelrhodopsin variant (ChIEF), respectively. RPc cardioprotection, elicited by ischaemia/reperfusion of the limbs, was abolished when DVMN neurones transduced to express AlstR were silenced by selective ligand allatostatin or in conditions of systemic muscarinic receptor blockade with atropine. In the absence of remote ischaemia/reperfusion, optogenetic activation of DVMN neurones transduced to express ChIEF reduced infarct size, mimicking the effect of RPc.. These data indicate a crucial dependence of RPc cardioprotection against ischaemia/reperfusion injury upon the activity of a distinct population of vagal pre-ganglionic neurones.

Topics: Action Potentials; Adenoviridae; Animals; Atropine; Autonomic Fibers, Preganglionic; Brain Stem; Constriction; Disease Models, Animal; Drosophila Proteins; Genetic Vectors; Heart; Hindlimb; Ischemic Preconditioning, Myocardial; Lentivirus; Male; Muscarinic Antagonists; Muscle, Skeletal; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neural Pathways; Neuropeptides; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Recombinant Fusion Proteins; Rhodopsin; Time Factors; Transduction, Genetic; Vagus Nerve

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

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

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

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

To determine the effect of light/dark cycles on the cones of 11-cis retinal-treated RPE65/rhodopsin double knockout (Rpe65(-/-)Rho(-/-)) mice. Studies have shown that cones degenerate in chromophore-deficient mouse models for Leber Congenital Amaurosis (LCA), but exogenous supplementation of the native 11-cis retinal chromophore can inhibit this degeneration, suggesting that 11-cis retinal could be used as a therapeutic agent for preserving functional cones in patients with LCA. However, these treated mice were maintained in the dark.. 11-cis Retinal was introduced into Rpe65(-/-)Rho(-/-) mice at postnatal day 10 as a single subcutaneous injection mixed with a basement membrane matrix. The mice were maintained in either normal light/dark cycles or constant dark conditions. Fluorescence microscopy was used to assess retinal morphology. Cone cell survival was determined by counting cone opsin-containing cells on flat-mounted P30 retinas. Cross-sections of P21 mouse retina were used to assess cone cell integrity by visualizing opsin localization. Cone function was determined by electroretinography (ERG).. Previous studies have shown that 11-cis retinal-treated mice lacking RPE65 and raised in constant dark have higher cone photoreceptor cell number, improved cone opsin localization, and enhanced cone ERG signals when compared with untreated mice. However, in this study the authors show that 11-cis retinal-treated Rpe65(-/-)Rho(-/-) mice raised in cyclic light did not show the improvements seen with the dark-reared mice.. Thus, 11-cis retinal by itself, as well as other agents that form photosensitive pigments, will not be good therapeutic candidates for preserving cones in LCA.

Topics: Animals; Carrier Proteins; Cell Count; Cell Survival; cis-trans-Isomerases; Dark Adaptation; Disease Models, Animal; Electroretinography; Eye Proteins; Gene Knockout Techniques; Leber Congenital Amaurosis; Light; Mice; Mice, Knockout; Microscopy, Fluorescence; Opsins; Retinal Cone Photoreceptor Cells; Retinaldehyde; Rhodopsin

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a 'repression-replacement' strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations.

Topics: Animals; Disease Models, Animal; Down-Regulation; Gene Knockdown Techniques; Genetic Therapy; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ophthalmoscopy; Repressor Proteins; Retinitis Pigmentosa; Rhodopsin; Transcription, Genetic

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

Retinitis pigmentosa (RP) is a type of inherited retinal degenerative disease, which leads to blindness. The primary pathological event of this disease is the death of rods because of genetic mutations. The S334ter-line-3 rat is a transgenic model developed to express a rhodopsin mutation similar to that found in RP. In this study, the rod's death triggered are organization of the cone mosaic into an orderly array of rings. Four observations were relevant to understand this reorganization. First, rods died in hot spots, which progressively increased as circular waves, leaving rod-less zones behind. Second, rings of cones formed around these zones. Third, remodeled Müller glia processes enveloped cones and filled the center of their rings. Zonula occludens-1 located between the photoreceptor inner segments and the apical processes of Müller cells showed in the rings. Fourth, these rings were formed before the onset of cone cell deaths and were maintained until late stages of RP. From these observations,we hypothesize that cone-Müller-cell interactions mediate and maintain the rings. A test of this hypothesis can be performed by injecting DL-a-aminoadipic acid (AAA), which is known to disrupt Müller cell metabolism. A single intravitreal injection of AAA at P50 disrupted the rings of cones 3 days after the injection. These findings indicate that the rearrangement of cones in rings is modulated by Müller cells in RP. Thus, if the relationship between photoreceptors and Müller glia is better understood, the latter could potentially be manipulated for effective neuroprotection or the restoration of normal cone arrays.

Topics: 2-Aminoadipic Acid; Age Factors; Animals; Animals, Newborn; Cell Communication; Cell Death; Cell Movement; Disease Models, Animal; Excitatory Amino Acid Antagonists; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; In Situ Nick-End Labeling; Intravitreal Injections; Membrane Proteins; Mice; Microscopy, Confocal; Mutation; Neuroglia; Opsins; Phosphoproteins; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Zonula Occludens-1 Protein

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

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

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

To develop a robust ex vivo model for evaluating cone survival in end-stage retinitis pigmentosa (RP) and apply this to quantify the effects of putative neuroprotective compounds.. Rhodopsin knockout mice were crossed with OPN1-GFP reporter mice so that GFP-positive cones could be identified against the background of a rod-specific degeneration. Retinal explants were harvested from 10-week-old mice and maintained in organotypic culture. Ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), or vascular endothelial growth factor 165b (VEGF(165b)) was administered daily to treatment groups at three doses (200 ng/mL, 100 ng/mL, or 50 ng/mL; n = 5 explants per group). Fluorescence microscopy was performed on days 1, 3, 5, 7, 9, and 12 to document the number of GFP-expressing cones.. Cone survival could be assessed reliably and reproducibly in this model, and cone degeneration was significantly greater in the absence of rods, in keeping with clinical observations of RP. Daily administration of 200 ng/mL CNTF led to significantly increased cone survival compared with sham-treated controls. The effect was dose dependent; 100 ng/mL CNTF reduced cone loss but to a lesser extent, and 200 ng/mL GDNF showed significant protection against cone loss at later time points (day 9-12) but was much less effective than CNTF at all doses. VEGF(165b) showed no neuroprotective effect in this model at any dose.. This model allows precise quantification of the neuroprotective effects of various compounds on cone survival and may therefore provide a robust method of screening neuroprotective compounds before application in vivo.

Topics: Animals; Cell Survival; Ciliary Neurotrophic Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Glial Cell Line-Derived Neurotrophic Factor; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Nerve Growth Factors; Neuroprotective Agents; Organ Culture Techniques; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vascular Endothelial Growth Factor A

To engineer a knockin mouse model that can be used to monitor the effects of treatments on degradation and mislocalization of proline-to-histidine change at codon 23 (P23H) rhodopsin, a common cause of autosomal dominant retinitis pigmentosa (ADRP). The goal was to introduce a gene that expressed rhodopsin at low levels to avoid rapid retinal degeneration, and with a readily visible tag to make it easy to distinguish from wild type rhodopsin.. One copy of the endogenous mouse rhodopsin gene was replaced with a mutant human rhodopsin gene that encodes P23H-rhodopsin fused to enhanced green fluorescent protein (GFP) at its C terminus. The gene includes a LoxP site in the sequence corresponding to the 5'-untranslated region, which greatly reduces translation efficiency. Characterized are the resulting heterozygous and homozygous P23H-hRho-GFP mouse lines for mRNA and protein expression, P23H-rhodopsin localization in rod cells, effects on visual function, and retinal degeneration.. The retinas of heterozygous P23H-hRho-GFP mice are morphologically and functionally very similar to those of wild type mice, and they display little cell death over time. P23H-hRho-GFP mice transcribe the knockin gene as efficiently as the endogenous mouse allele, but they contain much less of the protein product than do knockin mice expressing nonmutant hRho-GFP, indicating that substantial degradation of P23H-rRho-GFP occurs in mouse rod cells. The remaining P23H-hRho-GFP mislocalizes to the inner segment and outer nuclear layer, with only approximately 20% in rod outer segments.. P23H-hRho-GFP mice provide a valuable tool for evaluating the efficacy of potential therapies for ADRP that influence the levels or localization of P23H-rhodopsin.

Topics: Animals; Blotting, Northern; Codon; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Gene Knock-In Techniques; Genotyping Techniques; Green Fluorescent Proteins; Histidine; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Mutagenesis, Site-Directed; Mutation; Proline; Recombinant Fusion Proteins; Retinal Photoreceptor Cell Inner Segment; Retinal Photoreceptor Cell Outer Segment; Retinitis Pigmentosa; Rhodopsin

Rho GTPases such as RAS-related C3 botulinum substrate 1 (RAC1) and cell division cycle 42 homolog (S. cerevisiae; CDC42) have been linked to cellular processes including movement, development, and apoptosis. Recently, RAC1 has been shown to be a pro-apoptotic factor in the retina during light-induced photoreceptor degeneration. Here, we analyzed the role of CDC42 in the degenerating retina.. Photoreceptor degeneration was studied in a mouse model for autosomal dominant retinitis pigmentosa (VPP) with or without a rod-specific knockdown of Cdc42, as well as in wild-type and Cdc42 knockdown mice after light exposure. Gene and protein expression were analyzed by real-time PCR, western blotting, and immunofluorescence. Retinal morphology and function were assessed by light microscopy and electroretinography, respectively.. CDC42 accumulated in the perinuclear region of terminal deoxynucleotidyl transferase dUTP nick end labeling-negative photoreceptors during retinal degeneration induced by excessive light exposure and in the rd1, rd10, and VPP mouse models of retinitis pigmentosa. The knockdown of Cdc42 did not affect retinal morphology or function in the adult mice and did not influence photoreceptor apoptosis or molecular signaling during induced and inherited retinal degeneration.. Retinal degeneration induces the accumulation of CDC42 in the perinuclear region of photoreceptors. In contrast to RAC1, however, lack of CDC42 does not affect the progression of degeneration. CDC42 is also dispensable for normal morphology and function of adult rod photoreceptor cells. RECEIVED: May 25, 2011 ACCEPTED: November 10, 2011.

Topics: Animals; Apoptosis; Blotting, Western; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Electroretinography; Fluorescent Antibody Technique; Gene Expression; Gene Knockdown Techniques; GTPase-Activating Proteins; In Situ Nick-End Labeling; Light; Mice; Mice, Knockout; Real-Time Polymerase Chain Reaction; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; 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 establish mouse models for RPGR-associated diseases by generating and characterizing an Rpgr mutation (in-frame deletion of exon 4) in two different genetic backgrounds (BL/6 and BALB/c).. Gene targeting in embryonic stem (ES) cells was performed to introduce a in-frame deletion of exon 4 in the Rpgr gene (Rpgr(DeltaEx4)). Subsequently, the mutation was introduced in two different inbred mouse strains by successive breeding. Mutant and wild-type mice of both strains were characterized by electroretinography (ERG) and histology at five time points (1, 3, 6, 9, and 12 months). RPGR transcript amounts were assessed by quantitative RT-PCR. A variety of photoreceptor proteins, including RPGR-ORF15, RPGRIP, PDE6delta/PrBPdelta, rhodopsin, and cone opsin, were localized on retinal sections by immunohistochemistry.. Mislocalization of rhodopsin and cone opsin was an early pathologic event in mutant mice of both lines. In contrast, RPGR-ORF15 as well as RPGRIP1 and PDE6delta/PrBPdelta showed similar localizations in mutant and wild-type animals. Functional and histologic studies revealed a mild rod-dominated phenotype in mutant male mice on the BL/6 background, whereas a cone-dominated phenotype was observed for the same mutation in the BALB/c background.. Both Rpgr mutant mouse lines developed retinal disease with a striking effect of the genetic background. Cone-specific modifiers might influence the retinal phenotype in the BALB/c strain. The two lines provide models to study RPGR function in rods and cones, respectively.

Topics: Animals; Carrier Proteins; Cyclic Nucleotide Phosphodiesterases, Type 6; Cytoskeletal Proteins; Disease Models, Animal; Electroretinography; Embryonic Stem Cells; Exons; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Genotype; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Opsins; Proteins; Retinal Cone Photoreceptor Cells; Retinal Diseases; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Bone spicule pigments (BSP) are a hallmark of retinitis pigmentosa (RP). In this study, we examined the process of BSP formation in the rhodopsin knockout (rho (-/-)) mouse, a murine model for human RP.. In rho (-/-) mice from 2 to 16 months of age, representing the range from early to late stages of degeneration, retinal sections and whole mounts were examined morphologically by light and electron microscopy. The results were compared to scanning laser ophthalmoscopy of BSP degeneration in human RP.. After the loss of all photoreceptor cells in rho-/- mice, the outer retina successively degenerated, leading to approximation and finally a direct contact of inner retinal vessels and the retinal pigment epithelium (RPE). We could show that it was the event of proximity of retinal vessel and RPE that triggered migration of RPE cells along the contacting vessels towards the inner retina. Ultrastructurally, these mislocalized RPE cells partially sealed the vessels by tight junction linkage and deposited extracellular matrix perivascularly. Also, the vascular endothelium developed fenestrations similar to the RPE-choroid interface. In whole mounts, the pigmented cell clusters outlining retinal capillaries correlated well with BSPs in human RP. The structure of the inner retina remained well preserved, even in late stages.. The Rho (-/-) mouse is the first animal model that depicts all major pathological changes, even in the late stages of RP. Using the rho (-/-) mouse model we were able to analyze the complete dynamic process of BSP formation. Therefore we conclude that: (1) In rho (-/-) retinas, BSPs only form in areas devoid of photoreceptors; (2) Direct contact between inner retinal vessels and RPE appears to be a major trigger for migration of RPE cells; (3) The distribution of the RPE cells in BSPs reflects the vascular network at the time of formation. The similarity of the disease process between mouse and human and the possibility to study all consecutive steps of the course of the disease makes the rho (-/-) mouse valuable for further insights in the dynamics of BSP formation in human RP.

Topics: Animals; Cell Movement; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Scanning; Nerve Tissue Proteins; Photoreceptor Cells, Vertebrate; Platelet Endothelial Cell Adhesion Molecule-1; Retinal Pigment Epithelium; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

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

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

Topics: Amino Acid Substitution; Animals; Apoptosis; Base Sequence; Dependovirus; Disease Models, Animal; Electroretinography; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; HeLa Cells; Humans; Mice; Models, Biological; Multiprotein Complexes; Mutation, Missense; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Recombinant Proteins; Retina; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Stress, Physiological; Transcription Factor CHOP; Transfection; Unfolded Protein Response; Vision, Ocular

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

No effective treatment currently exists for prion diseases and therefore the development of experimental non-human primate models of prion neurotoxicity, to better understand the underlying mechanism and to test new treatments relevant to humans, represents an urgent medical need. However, the establishment of such models is challenging due to animal welfare and cost considerations. We describe here the use of Microcebus murinus retina, in primary cultures and in vivo, as a new experimental primate model to rapidly examine the effects in the central nervous system of PrP(106-126), a neurotoxic fragment of the human prion protein. We demonstrate that PrP(106-126) triggered rod photoreceptor cell loss by apoptosis and a change in morphology of microglial cells in mixed neuronal-glial cultures of retinal cells. In addition, 2days after intravitreal injection of PrP(106-126), retinas showed a significant increase in the number of apoptotic nuclei, mainly in the ganglion cell layer.

Topics: Analysis of Variance; Animals; Cell Death; Cells, Cultured; Cheirogaleidae; Disease Models, Animal; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Microscopy, Electron, Transmission; Neurons; Neurotoxicity Syndromes; Parvalbumins; Peptide Fragments; Prions; Retina; Rhodopsin; Vimentin

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

TM601 is a synthetic polypeptide with sequence derived from the venom of the scorpion Leiurus quinquestriatus that has anti-neoplastic activity. It has recently been demonstrated to bind annexin A2 on cultured tumor and vascular endothelial cells and to suppress blood vessel growth on chick chorioallantoic membrane. In this study, we investigated the effects of TM601 in models of ocular neovascularization (NV). When administered by intraocular injection, intravenous injections, or periocular injections, TM601 significantly suppressed the development of choroidal NV at rupture sites in Bruch's membrane. Treatment of established choroidal NV with TM601 caused apoptosis of endothelial cells and regression of the NV. TM601 suppressed ischemia-induced and vascular endothelial growth factor-induced retinal NV and reduced excess vascular permeability induced by vascular endothelial growth factor. Immunostaining with an antibody directed against TM601 showed that after intraocular or periocular injection, TM601 selectively bound to choroidal or retinal NV and co-localized with annexin A2, which is undetectable in normal retinal and choroidal vessels, but is upregulated in endothelial cells participating in choroidal or retinal NV. Intraocular injection of plasminogen or tissue plasminogen activator, which like TM601 bind to annexin A2, also suppressed retinal NV. This study supports the hypothesis that annexin A2 is an important target for treatment of neovascular diseases and suggests that TM601, through its interaction with annexin A2, causes suppression and regression of ocular NV and reduces vascular leakage and thus may provide a new treatment for blinding diseases such as neovascular age-related macular degeneration and diabetic retinopathy.

Topics: Angiogenesis Inhibitors; Animals; Animals, Newborn; Annexin A2; Apoptosis; Bruch Membrane; Capillary Permeability; Choroidal Neovascularization; Disease Models, Animal; Endothelial Cells; Female; Fibrinolysin; Humans; Infant, Newborn; Injections, Intravenous; Mice; Mice, Inbred C57BL; Mice, Transgenic; Promoter Regions, Genetic; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Rhodopsin; Scorpion Venoms; Tissue Plasminogen Activator; Vascular Endothelial Growth Factor A

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

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (Rh(P23H)). Unlike the wild-type Rh, mutant Rh(P23H) exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded Rh(P23H) and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that Rh(P23H) is a substrate of the ER-associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1(P37H) (the equivalent of mammalian Rh(P23H)) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1(P37H) toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1(P37H) and further activated the Ire1/Xbp1 ER stress pathway in the Rh1(P37H) retina. Despite this, Rh1(P37H) flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1(P37H) retina. Pharmacological treatment of Rh1(P37H) flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration.

Topics: Adenosine Triphosphatases; Animals; Cell Cycle Proteins; Disease Models, Animal; Down-Regulation; Drosophila; Drosophila Proteins; Endoplasmic Reticulum; Female; Gene Silencing; Humans; Male; Mutation, Missense; Protein Folding; Retina; Retinitis Pigmentosa; Rhodopsin; Valosin Containing Protein

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.

Topics: Aging; Animals; Animals, Genetically Modified; Apoptosis; Axons; Cell Count; Cytoskeletal Proteins; Disease Models, Animal; Female; Fluorescent Antibody Technique, Indirect; Mutation; Photoreceptor Cells, Vertebrate; Rats; Retinal Dystrophies; Retinal Ganglion Cells; Rhodopsin; Stilbamidines; Transcription Factor Brn-3A

Lutein has been the focus of recent study as a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate, with the use of a mouse endotoxin-induced uveitis (EIU) model, the neuroprotective effects of lutein against retinal neural damage caused by inflammation.. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Each animal was given a subcutaneous injection of lutein or vehicle three times: concurrently with and 3 hours before and after the LPS injection. Analysis was carried out 24 hours after EIU induction. Levels of rhodopsin protein and STAT3 activation were analyzed by immunoblotting. Lengths of the outer segments of the photoreceptor cells were measured. Dark-adapted full-field electroretinograms were recorded. Oxidative stress in the retina was analyzed by dihydroethidium and fluorescent probe. Expression of glial fibrillary acidic protein (GFAP) was shown immunohistochemically.. The EIU-induced decrease in rhodopsin expression followed by shortening of the outer segments and reduction in a-wave amplitude were prevented by lutein treatment. Levels of STAT3 activation, downstream of inflammatory cytokine signals, and reactive oxygen species (ROS), which are both upregulated during EIU, were reduced by lutein. Pathologic change of Müller glial cells, represented by GFAP expression, was also prevented by lutein.. The present data revealed that the antioxidant lutein was neuroprotective during EIU, suggesting a potential approach for suppressing retinal neural damage during inflammation.

Topics: Animals; Antioxidants; Disease Models, Animal; Electroretinography; Escherichia coli; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; In Situ Nick-End Labeling; Lipopolysaccharides; Lutein; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neuroglia; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species; Retinitis; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; STAT3 Transcription Factor; Uveitis, Posterior

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

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

In several disease states, abnormal growth of blood vessels is associated with local neuronal degeneration. This is particularly true in ocular diseases such as retinal angiomatous proliferation (RAP) and macular telangiectasia (MacTel), in which, despite the absence of large-scale leakage or hemorrhage, abnormal neovascularization (NV) is associated with local neuronal dysfunction. We describe here a retinal phenotype in mice with dysfunctional receptors for VLDL (Vldlr-/- mice) that closely resembles human retinal diseases in which abnormal intra- and subretinal NV is associated with photoreceptor cell death. Such cell death was evidenced by decreased cone and, to a lesser extent, rod opsin expression and abnormal electroretinograms. Cell death in the region of intraretinal vascular abnormalities was associated with an increased presence of markers associated with oxidative stress. Oral antioxidant supplementation protected against photoreceptor degeneration and preserved retinal function, despite the continued presence of abnormal intra- and subretinal vessels. What we believe to be novel, Müller cell-based, virally mediated delivery of neurotrophic compounds specifically to sites of NV was also neuroprotective. These observations demonstrate that neuronal loss secondary to NV can be prevented by the use of simple antioxidant dietary measures or cell-based delivery of neurotrophic factors, even when the underlying vascular phenotype is not altered.

Topics: Angiogenesis Inhibitors; Animals; Antioxidants; Aptamers, Nucleotide; Disease Models, Animal; Electroretinography; Gene Expression; Gene Expression Profiling; Gene Transfer Techniques; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Growth Factors; Opsins; Oxidative Stress; Receptors, LDL; Retina; Retinal Cone Photoreceptor Cells; Retinal Neovascularization; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vascular Endothelial Growth Factor A

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

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light-induced, oxygen-dependent, free radical-mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light-induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide-induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light-induced oxidative injury.

Topics: Animals; Animals, Newborn; Apoptosis; bcl-X Protein; Caspase 3; Cell Survival; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation; Heme Oxygenase-1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Janus Kinase 2; Light; Mice; Mice, Inbred C57BL; Neurons; Proto-Oncogene Proteins c-fos; Receptors, Erythropoietin; Retinal Diseases; Retinal Pigment Epithelium; Rhodopsin; Thioredoxins

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

This study was designed to determine if low power laser therapy can achieve amelioration of vasoproliferation yet preserve useful vision in the treated area in a transgenic mouse model of retinal neovascularisation. The mice were anaesthetised and the pupils dilated for ERG and fundus fluorescein angiography on postnatal day 32. The left eyes were treated with approximately 85 laser spots (532 nm, 50 ms, 300 microm diameter) at a power level of 20 mW at the cornea. The eyes were examined using ERG and fluorescein angiography, one, four and six weeks later. Flat mounts of FITC-dextran infused retinas, retinal histology and PEDF immunohistochemistry was studied one or six weeks after laser treatment. In untreated eyes the expected course of retinal neovascularisation in this model was observed. However, retinal neovascularisation in the laser treated eye was significantly reduced. The laser parameters chosen produced only mild lesions which took 10-20 s to become visible. ERG responses were comparable between the treated and untreated eyes, and histology showed only partial loss of photoreceptors in the treated eyes. PEDF intensity corresponded inversely with the extent of neovascularisation. Low power panretinal photocoagulation can inhibit retinal neovascularisation and yet preserve partial visual function in this transgenic mouse model of retinal neovascularisation.

Topics: Animals; Disease Models, Animal; Electroretinography; Eye Proteins; Fluorescein Angiography; Immunohistochemistry; Laser Coagulation; Low-Level Light Therapy; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Growth Factors; Photoreceptor Cells, Vertebrate; Promoter Regions, Genetic; Retinal Neovascularization; Retinal Vessels; Rhodopsin; Serpins; Time Factors; Vascular Endothelial Growth Factor A; Vision Disorders; Vision, Ocular

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 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

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

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

We studied the potential of systemically administered aminoglycosides as a therapy for retinal degeneration resulting from premature termination codon (PTC) mutations. Aminoglycosides were systemically delivered to two rodent models of retinal degeneration: a transgenic rat model of dominant disease caused by a PTC in rhodopsin (S334ter); and a mouse model of recessive disease (rd12) caused by a PTC in the retinoid isomerase Rpe65. Initial luciferase reporter assays were undertaken to measure the efficiency of gentamicin-induced read-through in vitro. These experiments indicated that gentamicin treatment induced on average a 5.3% extra read-through of the S334ter PTC in vitro, but did not affect the rd12 PTC. Beginning at postnatal day 5, animals received daily subcutaneous injections of gentamicin or geneticin at a range of doses. The effect of the treatment on retinal degeneration was examined by histopathology and electroretinography (ERG). Systemic treatment with aminoglycoside significantly increased the number of surviving photoreceptors in the S334ter rat model over several weeks of treatment, but was not effective in slowing the retinal degeneration in the rd12 mouse model. Similarly, ERG recordings indicated better preservation of retinal function in the treated S334ter rats, but no difference was observed in the rd12 mice. Daily subcutaneous injection of 12.5mug/g gentamicin was the only regimen that inhibited retinal degeneration without apparent adverse systemic side effects. Reduced effectiveness beyond postnatal day 50 correlated with reduced ocular penetration of drug as seen in gentamicin-Texas red (GTTR) conjugation experiments. We conclude that, in the rat model, an approximately 5% reduction of abnormal truncated protein is sufficient to enhance photoreceptor survival. Such a change in truncated protein is consistent with beneficial effects seen when aminoglycosides has been used in other, non-ocular animal models. In the rd12 mouse, lack of efficacy was seen despite this particular PTC being theoretically more sensitive to aminoglycoside modification. We conclude that aminoglycoside read-through of PTCs in vitro and in vivo cannot be predicted just from genomic context. Because there is considerable genetic heterogeneity amongst retinal degenerations, pharmacologic therapies that are not gene-specific have significant appeal. Our findings suggest that if adverse issues such as systemic toxicity and limited ocular penetration can be ove

Topics: Aminoglycosides; Animals; Carrier Proteins; cis-trans-Isomerases; Codon, Nonsense; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Electroretinography; Eye Proteins; Fluorescent Dyes; Gentamicins; Mice; Mice, Mutant Strains; Rats; Rats, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; Xanthenes

Perturbed microRNA (miR) expression is a feature of, and may play a fundamental role in, certain disease states such as different forms of cancer. Retinitis pigmentosa (RP) a group of inherited retinal degenerations is characterised by a progressive loss of photoreceptor cells and consequent visual handicap. We have previously reported an altered pan-retinal expression of miR-96, -183, -1 and -133 in a P347S-Rhodopsin transgenic mouse model of RP. As many different mutations in Rhodopsin and other genes such as RDS/Peripherin can lead to RP, it was of interest to explore whether the characterized retinal miR expression signature was observed in three other mouse models of RP linked to rhodopsin and rds/peripherin. Therefore, pan-retinal expression of miR-96, -182, -183, -1, -133 and -142 was analysed using quantitative real-time RT-PCR. A common signature of altered miR expression was found; expression of miR-96, -182 and -183 decreased by 14.1-53.2%, while expression of miR-1, -133 and -142 was up-regulated by 186.1-538.5%. Significantly, the detected pan-retinal miR signature was mirrored by similar miR expression profiles in FACS-isolated rod photoreceptors from these mice. In an attempt to understand the function of these miRs, corresponding target genes were predicted using computational means. Many 'enriched' targets (with binding sites for at least two of the above miRs) were found to be regulatory molecules and members of intracellular signalling circuits. However, further studies are required to highlight which of the large number of in silico predicted targets are actually controlled by these miRs.

Topics: Animals; Disease Models, Animal; Gene Expression; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Knockout; MicroRNAs; Nerve Tissue Proteins; Peripherins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.

Topics: Animals; Aspartic Acid; Calcium; Carrier Proteins; cis-trans-Isomerases; Dark Adaptation; Disease Models, Animal; Dose-Response Relationship, Radiation; Eye Proteins; Glycine; Kinetics; Light Signal Transduction; Membrane Potentials; Mice; Mice, Transgenic; Mutation; Night Blindness; Opsins; Photic Stimulation; Retinal Rod Photoreceptor Cells; Retinaldehyde; Rhodopsin; Spectrum Analysis; Time Factors

Paralysis is a major consequence of spinal cord injury (SCI). After cervical SCI, respiratory deficits can result through interruption of descending presynaptic inputs to respiratory motor neurons in the spinal cord. Expression of channelrhodopsin-2 (ChR2) and photostimulation in neurons affects neuronal excitability and produces action potentials without any kind of presynaptic inputs. We hypothesized that after transducing spinal neurons in and around the phrenic motor pool to express ChR2, photostimulation would restore respiratory motor function in cervical SCI adult animals. Here we show that light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory diaphragmatic motor activity. Furthermore, robust rhythmic activity persisted long after photostimulation had ceased. This recovery was accomplished through a form of respiratory plasticity and spinal adaptation which is NMDA receptor dependent. These data suggest a novel, minimally invasive therapeutic avenue to exercise denervated circuitry and/or restore motor function after SCI.

Topics: Animals; Anterior Horn Cells; Diaphragm; Disease Models, Animal; Female; Neural Pathways; Neuronal Plasticity; Paralysis; Periodicity; Phototherapy; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Respiration; Respiratory Insufficiency; Rhodopsin; Spinal Cord; Spinal Cord Injuries; Treatment Outcome

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

Mutational heterogeneity in genes causative of dominantly inherited disorders represents a significant barrier for development of therapies directed towards correction of the primary genetic defect. To circumvent the mutational heterogeneity present in rhodopsin- (RHO-) linked autosomal dominant Retinitis Pigmentosa (adRP), a strategy involving suppression and replacement of RHO has been adopted. RNA interference- (RNAi-) mediated suppression of RHO has been explored as has the generation of an RNAi-resistant replacement gene using the degeneracy of the genetic code. Additionally, the functional equivalence of codon-modified replacement genes has been demonstrated in a transgenic animal (RHO-M). Suppression and replacement, while exemplified by adRP, may also be relevant to many other dominantly inherited diseases with the hallmark of mutational heterogeneity.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Electroretinography; Gene Expression; Genetic Therapy; Mice; Mice, Transgenic; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; RNA, Small Interfering

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

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

Topics: Amino Acid Substitution; Animals; Base Sequence; Disease Models, Animal; Histidine; In Vitro Techniques; Membrane Proteins; Mice; Mice, Transgenic; Molecular Sequence Data; Nucleic Acid Conformation; Perilipin-2; Rhodopsin; RNA, Catalytic; Substrate Specificity

Neural ectopic rewiring in retinal degeneration such as retinitis pigmentosa (RP) may form functional synapses between cones and rod bipolar cells that cause atypical signal processing. In this study, the multifocal electroretinograms (mfERGs) of a large animal model of RP, the rhodopsin P347L transgenic (Tg) pig, were measured to examine the sources and nature of altered signal processing.. mfERG responses from a 6-week-old Tg pig were recorded before and after sequential application of tetrodotoxin (TTX), N-methyl-D-aspartate (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidinedicarboylic acid (PDA), to identify contributions to the retinal signal from inner retinal neurons, the ON-pathway, the OFF-pathway, and photoreceptors. The mfERG response contributions from different retinal components of in the Tg eyes were estimated and compared with control data from eyes of age-matched wild-type (WT) pigs.. There was a prominent difference in the estimates of the inner retinal response and ON-bipolar cell pathway contribution between the Tg and WT mfERG responses. In particular, the early components of the inner retinal contribution were obviously altered in the Tg mfERG. The inner retinal components at approximately 24 and 40 ms appeared to be inverted. Differences in the estimates of OFF-bipolar cell pathway contributions were minimal. There was no change in cone cell responses in the Tg mfERG.. In Tg retinas, ectopic synapses formed between cones and rod bipolar cells probably altered signal processing of the ON-bipolar cell pathway. In response to the altered visual signal input from the outer retina, signal processing in inner retinal neurons was also modified.

Topics: Aminobutyrates; Animals; Animals, Genetically Modified; Computers, Handheld; Disease Models, Animal; Electroretinography; Mutation; N-Methylaspartate; Photoreceptor Cells, Vertebrate; Pipecolic Acids; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Synapses; Synaptic Transmission; Tetrodotoxin; Vision, Ocular

Rhodopsin transgenes carrying mutations that cause autosomal dominant retinitis pigmentosa in humans have been used to study rod photoreceptor degeneration in various model organisms including Xenopus laevis. To date, the only transgenes shown to cause rod photoreceptor degeneration in Xenopus laevis have been either mammalian rhodopsins or chimeric versions of rhodopsin based mainly on Xenopus laevis rhodopsin sequences but with a mammalian C-terminus. Since the C-terminal sequence of rhodopsin is highly conserved in mammals and divergent in Xenopus laevis, and mammalian and epitope-tagged rhodopsins may have unexpected properties as transgenes, we decided to test whether a Xenopus laevis rhodopsin transgene carrying only the P23H mutation could also cause rod photoreceptor degeneration. Xenopus laevis tadpoles expressing these transgenes indeed had shortened outer segments and, in severely affected animals, the loss of rod photoreceptors but not the loss of cone photoreceptors. RT-PCR analyses showed that less than 10% of mutant transgenic rhodopsin relative to wild-type endogenous rhodopsin mRNA was sufficient to produce severe rod photoreceptor degeneration. As observed in other animal models as well as humans carrying this particular rhodopsin mutation, the rod photoreceptor degeneration was most severe in the ventral retina and was modified by light. Thus, the rod photoreceptor degeneration produced in Xenopus laevis by the P23H mutation in an otherwise untagged Xenopus laevis rhodopsin is generally similar to that seen with mammalian rhodopsins and epitope-tagged versions of Xenopus laevis rhodopsin, though some differences remain to be explained.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Gene Expression; Genotype; Humans; In Situ Hybridization; Light; Molecular Sequence Data; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Sequence Alignment; Species Specificity; Transgenes; Xenopus laevis

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

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

The study of some 4-substituted-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, designed as hA(3) adenosine receptor antagonists, is reported. The new compounds bear on the four-position different acylamino, sulfonylamino, benzylureido and benzyloxy moieties, which have also been combined with a para-methoxy group on the 2-phenyl ring or with a nitro residue at the six-position. Many derivatives show high hA(3) adenosine receptor affinities and selectivities both versus hA(1) and hA(2A) receptors. The observed structure-affinity relationships of this class of antagonists have been exhaustively rationalized using the recently published ligand-based homology modeling (LBHM) approach. The selected 4-bismethanesulfonylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (13), which shows high hA(3) affinity (K(i)=5.5nM) and selectivity versus hA(1), hA(2A) (both selectivity ratios>1800) and hA(2B) (cAMP assay, IC(50)>10,000nM) receptors, was tested in an in vitro rat model of cerebral ischemia, proving to be effective in preventing the failure of synaptic activity, induced by oxygen and glucose deprivation in the hippocampus, and in delaying the occurrence of anoxic depolarization.

Topics: Adenosine A3 Receptor Antagonists; Animals; Binding, Competitive; Brain Ischemia; Cattle; Cell Membrane; Cerebral Cortex; Disease Models, Animal; Humans; Hydrogen Bonding; Ligands; Models, Molecular; Protein Binding; Quinoxalines; Rats; Receptor, Adenosine A3; Rhodopsin; Structural Homology, Protein; Structure-Activity Relationship; Triazoles; Xanthines

Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse.. For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections.. Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected.. Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.

Topics: Adenoviridae; Animals; Bone Marrow Cells; Cell Survival; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Gene Silencing; Genetic Vectors; Green Fluorescent Proteins; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Transfection

The purpose of this study was to explore neuroretinal transplantation in a large animal model of severe retinitis pigmentosa and to establish graft development, long-term survival, graft-host integration, and effects on the host retina.. Rhodopsin transgenic pigs, aged 6 months, received in one eye a fetal full-thickness neuroretinal sheet in the subretinal space by means of vitrectomy and retinotomy. Six months postoperatively, eyes were studied in the light microscope and with immunohistochemical markers. Full-field electroretinography (ERG) was performed at 4 and 6 months.. Laminated grafts with well-organized photoreceptors, rod bipolar cells, and Müller cells were found in five of six eyes. Neuronal connections between graft and host retina were not seen. In the five eyes containing a graft, the number of surviving rods in the host retina was significantly higher compared with unoperated eyes. The ERG did not reveal any significant difference in b-wave amplitude between operated and control eyes, but the cone-derived response in operated eyes increased significantly from 4 to 6 months while the rod response in control eyes decreased significantly.. Fetal full-thickness neuroretina can be transplanted safely to an eye with severe retinal degeneration. In their major part, the transplants develop a normal laminated morphology and survive for at least 6 months. Graft and host retinal neurons do not form connections. Retinal function in the host is reduced initially by the surgical trauma, but the presence of a well-laminated graft counteracts this effect and rescues rods from degeneration.

Topics: Animals; Animals, Genetically Modified; Biomarkers; Dark Adaptation; Disease Models, Animal; Electroretinography; Female; Fetal Tissue Transplantation; Fluorescent Antibody Technique, Indirect; Follow-Up Studies; Glial Fibrillary Acidic Protein; Graft Survival; Neuroglia; Pregnancy; Protein Kinase C; Retina; Retinal Bipolar Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Transducin; Transplantation, Homologous; Vitrectomy

To characterize molecular and cellular changes induced by sustained expression of ciliary neurotrophic factor (CNTF) in the rds mutant mouse retina.. Recombinant adeno-associated virus (rAAV) expressing CNTF was injected subretinally, for transduction of peripherin/rds(+/)(-) transgenic mice that carry the P216L mutation found in human retinitis pigmentosa. Characterization of retinal neurons and glia was performed by immunocytochemistry with cell-type-specific markers. Activation of signaling molecules was examined by Western blot and immunostaining. Alterations of gene transcription profiles were studied by microarray analyses.. CNTF viral transduction maintained rhodopsin expression in surviving rod photoreceptors, but greatly reduced both S- and M-opsin normally expressed in cones. In addition, CNTF treatment resulted in increased numbers and dispersion of Müller glia and Chx10-positive bipolar cells within the inner nuclear layer. Persistent CNTF signaling also caused enhanced phosphorylation of STAT1, STAT3, and p42/44 ERK, as well as their levels of expression. Moreover, altered transcription profiles were detected for a large number of genes. Among these, Crx and Nrl involved in photoreceptor differentiation and several genes involved in phototransduction were suppressed.. Despite the rescue from cell death, continuous exposure to CNTF changed photoreceptor cell profiles, especially resulting in the loss of cone immunoreactivity. In addition, the Müller glia and bipolar cells became disorganized, and the number of cells expressing Müller and bipolar cell markers increased. Constitutive CNTF production resulted in sustained activation of cytokine signal transduction and altered the expression of a large number of genes. Therefore, stringent regulation of CNTF may be necessary for its therapeutic application in preventing retinal degeneration.

Topics: Animals; Blotting, Western; Ciliary Neurotrophic Factor; Dependovirus; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Genetic Vectors; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroglia; Oligonucleotide Array Sequence Analysis; Phosphorylation; Photoreceptor Cells, Vertebrate; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; STAT1 Transcription Factor; STAT3 Transcription Factor; Transfection

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

Retinitis pigmentosa (RP) is a heterogeneous group of diseases in which one of a wide variety of mutations selectively causes rod photoreceptor cell death. After rods die, cone photoreceptors gradually die resulting in blindness. Antioxidants reduce cone cell death in rd1/rd1 mice indicating that cones die from oxidative damage in that model of rapidly progressive RP. In this study, we sought to determine if this observation could be generalized to models of other types of RP, rd10/rd10 mice, a model of more slowly progressive recessive RP, and Q344ter mice, a model of rapidly progressive dominant RP. Compared to appropriate vehicle-treated controls, rd10/rd10 and Q344ter mice treated between P18 and P35 with a mixture of antioxidants previously found to be effective in rd1/rd1 mice showed significantly greater cone survival. Antioxidant-treated rd10/rd10 mice showed preservation of cone function as shown by a significant increase in photopic ERG b-wave amplitudes, and surprisingly showed temporary preservation of scotopic a-wave amplitudes, prolonged rod survival, and slowed depletion of rhodopsin mRNA. These data suggest that oxidative damage contributes to cone cell death regardless of the disease causing mutation that leads to the demise of rods, and that in more slowly progressive rod degenerations, oxidative damage may also contribute to rod cell death. Protection from oxidative damage may be a broadly applicable treatment strategy in RP.

Topics: alpha-Tocopherol; Animals; Antioxidants; Ascorbic Acid; Cell Death; Codon, Nonsense; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Drug Administration Schedule; Electroretinography; Exons; Heterozygote; Homozygote; Injections, Intraperitoneal; Kinetics; Mice; Mice, Mutant Strains; Mutation, Missense; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Thioctic Acid

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 elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic approximately 27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

Topics: Animals; Animals, Genetically Modified; Cattle; Cell Line, Transformed; Darkness; Disease Models, Animal; Gene Expression Regulation; Histidine; Humans; Mice; Microscopy, Electron, Scanning; Mutation; Peptide Fragments; Proline; Retinal Rod Photoreceptor Cells; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Transfection; Xenopus laevis

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

Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.

Topics: Activating Transcription Factor 6; Animals; Animals, Genetically Modified; Apoptosis; Cell Line; Cell Survival; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum; Endoribonucleases; Humans; Kinetics; Membrane Proteins; Mice; Mutation; Protein Folding; Protein Serine-Threonine Kinases; Proteins; Rats; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a protein responsible for isomerization of all-trans-retinaldehyde to its photoactive 11-cis-retinaldehyde and is essential for the visual cycle. RPE65 mutations can cause severe, early onset retinal diseases such as Leber congenital amaurosis (LCA). A naturally occurring rodent model of LCA with a recessive nonsense Rpe65 mutation, the rd12 mouse, displays a profoundly diminished rod electroretinogram (ERG), an absence of 11-cis-retinaldehyde and rhodopsin, an overaccumulation of retinyl esters in retinal pigmented epithelial (RPE) cells, and photoreceptor degeneration. rd12 mice were injected subretinally at postnatal day 14 with rAAV5-CBA-hRPE65 vector. RPE65 expression was found over large areas of RPE soon after treatment. This led to improved rhodopsin levels with ERG signals restored to near normal. Retinyl ester levels were maintained at near normal, and fundus and retinal morphology remained normal. All parameters of restored retinal health remained stable for at least 7 months. The Morris water maze behavioral test was modified to test rod function under very dim light; rd12 mice treated in one eye performed similar to normally sighted C57BL/6J mice, while untreated rd12 mice performed very poorly, demonstrating that gene therapy can restore normal vision-dependent behavior in a congenitally blind animal.

Topics: Animals; Behavior, Animal; Carrier Proteins; cis-trans-Isomerases; Dependovirus; Disease Models, Animal; Esters; Eye Proteins; Genetic Therapy; Genetic Vectors; Mice; Mice, Inbred C57BL; Optic Atrophy, Hereditary, Leber; Retina; Rhodopsin; Vision, Ocular

Platelet-derived growth factor-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured fibroblasts with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with 20 microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.

Topics: 3T3 Cells; Animals; Aptamers, Nucleotide; Cell Proliferation; Disease Models, Animal; Epiretinal Membrane; Eye; Injections; Mice; Mice, Inbred C57BL; Mice, Transgenic; Proto-Oncogene Proteins c-sis; Rabbits; Retina; Retinal Detachment; Retinal Diseases; 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

One eye of rd12 mice received a sub-retinal injection of a vector carrying normal human RPE65 cDNA at post-natal day 18, and at 6- and 13-months of age. Electroretinograms (ERGs) and visual-evoked potentials (VEPs) were recorded to luminance, and to spatially and temporally modulated stimuli to assess the consequences of delayed treatment on visual pathway function. Early treatment resulted in better overall retinal rescue and better rescue of cone-mediated function. VEPs to low temporal frequency luminance modulation were well preserved at all but the oldest treatment age and corresponded to predictions based on the amount of retinal rescue. In contrast, VEPs to high frequency spatially and temporally modulated stimuli were impaired even at the earliest age. These results provide further support that early treatment in human LCA will have the most hope for optimal visual performance.

Topics: Animals; Blindness; Dark Adaptation; Disease Models, Animal; Electroretinography; Evoked Potentials, Visual; Genetic Therapy; Mice; Mice, Mutant Strains; Retina; Rhodopsin; Time Factors; Vision Tests; Visual Acuity; Visual Cortex

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

Autosomal dominant Stargardt-like (STGD3) disease results from mutations in the ELOVL4 gene (elongation of very-long-chain fatty acids). This study was undertaken to characterize a mouse model with a targeted deletion of Elovl4 and to explore the role of this gene in retinal/macular degeneration.. A construct targeted to exon 2 of the Elovl4 gene was used to suppress expression of the gene. Elovl4 homozygous pups were nonviable and were not available for study. Hence, the analysis was performed on heterozygous Elovl4(+/-) mice 16 to 22 month of age and littermate wild-type (WT) control mice of the same age. Characterization included examining gene message and protein levels, electroretinogram (ERG), retinal morphology and ultrastructure, and plasma and retinal fatty acid composition.. Although the level of Elovl4 mRNA was reduced in Elovl4(+/-) retinas, only minimal morphologic abnormalities were found, and the retinal (ERG) function was essentially normal in Elovl4(+/-) retinas compared with the WT control retinas. Systemic fatty acid profiles of Elovl4(+/-) mice were unremarkable, although the concentration of several fatty acids was significantly lower in Elovl4(+/-) retinas, particularly the monounsaturated fatty acids.. The detailed characterization of this animal model provides the first in vivo evidence that Elovl4 haploinsufficiency is not the underlying key disease mechanism in STGD3. The results are consistent with a dominant negative mechanism for the deletion mutation. The Elovl4 knockout mouse is one of three complementary animal models that will help elucidate the disease mechanism.

Topics: Animals; Blotting, Western; Disease Models, Animal; Electroretinography; Eye Proteins; Fatty Acids; Female; Gene Deletion; Gene Expression Regulation; Genotype; Haplotypes; Macular Degeneration; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Retina; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger

To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR).. Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy.. Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest.. Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease.

Topics: Animals; Capillary Permeability; Diabetic Retinopathy; Disease Models, Animal; Fluorescein Angiography; Immunoenzyme Techniques; Mice; Mice, Transgenic; Microscopy, Confocal; Nerve Fibers; Retina; Retinal Ganglion Cells; Retinal Neovascularization; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A

Over 100 rhodopsin mutation alleles have been associated with autosomal dominant retinitis pigmentosa (ADRP). These mutations appear to cause photoreceptor cell death through diverse molecular mechanisms. We show that K296E, a rhodopsin mutation associated with ADRP, forms a stable complex with arrestin that is toxic to mouse rod photoreceptors. This cell death pathway appears to be conserved from flies to mammals. A genetics approach to eliminate arrestin unmasked the constitutive activity of K296E and caused photoreceptor cell death through a transducin-dependent mechanism that is similar to light damage. Expressing K296E in the arrestin/transducin double knock-out background prevented transducin signaling and led to substantially improved retinal morphology but did not fully prevent cell death caused by K296E. The adverse effect of K296E in the arrestin/transducin knock-out background can be mimicked by constant exposure to low light. Furthermore, we found that arrestin binding causes K296E to mislocalize to the wrong cellular compartment. Accumulation of stable rhodopsin/arrestin complex in the inner segment may be an important mechanism for triggering the cell death pathway in the mammalian photoreceptor cell.

Topics: Animals; Arrestin; Cell Compartmentation; Cell Death; Chromosome Disorders; Disease Models, Animal; Female; Genes, Dominant; Humans; Light; Macromolecular Substances; Male; Mice; Mice, Knockout; Mice, Transgenic; Mutation; Protein Binding; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transducin

To investigate the retinal neural damage that occurs during inflammation and the therapeutic effects of the angiotensin II type 1 receptor (AT1R) blocker, telmisartan, using a model of endotoxin-induced uveitis (EIU).. The localization of AT1R and AT2R was shown by immunohistochemistry. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Animals were treated with telmisartan for 2 days and were evaluated 24 hours later. Expression levels of angiotensin II, STAT3 activation induced by inflammatory cytokines, and retinal proteins essential for neural activities (e.g., synaptophysin, rhodopsin) were analyzed by immunoblot. An AT2R antagonist was administered to evaluate the contribution of AT2R signaling in this therapy. Dark-adapted full-field electroretinography (ERG) was also performed.. AT1R and AT2R were expressed in presynaptic terminals in most of the retinal neurons. AT1R was also expressed in Müller glial cells. During inflammation, angiotensin II expression was elevated, STAT3 was activated, and synaptophysin and rhodopsin expression were reduced. The expression of glial fibrillary acidic protein (GFAP), downstream of STAT3 activation, was induced in Müller glial cells. However, treatment with telmisartan successfully avoided all these changes. An AT2R antagonist lowered synaptophysin expression despite the treatment. STAT3 activity was negatively correlated with rhodopsin expression. Furthermore, ERG responses, which were mostly prevented by telmisartan, were disturbed during inflammation.. Retinal protein expression and visual function are both disturbed by inflammation. Treatment with the AT1R blocker telmisartan efficiently prevented these signs of retinal neural damage through the reduction of local angiotensin II expression, the blockade of AT1R, and the relative upregulation of AT2R function.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Benzoates; Dark Adaptation; Disease Models, Animal; Electroretinography; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Immunoblotting; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neuroglia; Neuroprotective Agents; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Retina; Rhodopsin; STAT3 Transcription Factor; Synaptophysin; Telmisartan; Uveitis

Topics: Animals; Disease Models, Animal; Humans; Mice; Mice, Transgenic; Microscopy, Confocal; Retina; Retinal Neovascularization; Rhodopsin; Time Factors; Vascular Endothelial Growth Factor A

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

rhodopsin mutations result in autosomal dominant retinitis pigmentosa (ADRP), the most frequent being Proline-23 substitution by histidine (RhoP23H). Although cellular and rodent animal models have been developed, the pathogenic mechanisms leading to RhoP23H-induced cell death are still poorly understood. For this, we have used a Drosophila model by introducing a mutation in the fly rhodopsin-1 gene (Rh1P37H) that corresponds to human RhoP23H. Rh1P37H transgenic flies show dominant photoreceptor degeneration that mimics age-, light-dependent and progressive ADRP. Moreover, we clarify the pathogenic mechanism of Rh1P37H mutation that acts as an antimorph. First, we show the dual-localization of mutant Rhodopsin since most of Rh1P37H accumulates in endoplasmic reticulum. Second, expression of mutant, mislocalized, Rhodopsin leads to cytotoxicity, via the activation of two stress-specific mitogen-activated protein kinases (MAPKs), p38 and JNK, which are known to control stress-induced apoptosis. In Rh1P37H flies, visual loss and degeneration are indeed accompanied by apoptotic features and prevented by expression of p35 apoptosis inhibitor. Finally, we show for the first time that properly localized, mutant, Rhodopsin is active. Thus, the development of a fly model that faithfully reproduces the human disease sheds light onto the molecular defects causing ADRP thereby making it possible to devise potential therapeutic approaches.

Topics: Amino Acid Substitution; Animals; Apoptosis; Base Sequence; Cell Death; Diptera; Disease Models, Animal; Humans; Molecular Sequence Data; Mutation, Missense; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

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

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

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

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

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3-4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R-/-) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/-/R-/- or G+/+/R-/-) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light-sensitive pigment in the retinas of G+/+/R-/- mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light-sensitive visual pigment.

Topics: Animals; Blotting, Northern; Blotting, Western; Disease Models, Animal; Electroretinography; Immunohistochemistry; Mice; Mice, Knockout; Mice, Transgenic; Microscopy, Electron; Nerve Degeneration; Night Blindness; Point Mutation; Retina; Rhodopsin; Rod Opsins; Spectrophotometry; Transgenes

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

Topics: Alleles; Amino Acid Sequence; Animals; Chromatography, Liquid; Cytoplasm; Detergents; Disease Models, Animal; Dogs; Electrophoresis, Polyacrylamide Gel; Glycosylation; Immunoblotting; Immunohistochemistry; Ligands; Light; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Mutation; Peptides; Protein Structure, Tertiary; Proteins; Receptors, G-Protein-Coupled; Retina; Retinitis Pigmentosa; Retinoids; rho GTP-Binding Proteins; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Time Factors; Ultraviolet Rays

Nine neurodegenerative diseases, including spinocerebellar ataxia type 7 (SCA7), are caused by the expansion of polyglutamine stretches in the respective disease-causing proteins. A hallmark of these diseases is the aggregation of expanded polyglutamine-containing proteins in nuclear inclusions that also accumulate molecular chaperones and components of the ubiquitin-proteasome system. Manipulation of HSP70 and HSP40 chaperone levels has been shown to suppress aggregates in cellular models, prevent neuronal death in Drosophila, and improve to some extent neurological symptoms in mouse models. An important issue in mammals is the relative expression levels of toxic and putative rescuing proteins. Furthermore, overexpression of both HSP70 and its co-factor HSP40/HDJ2 has never been investigated in mice. We decided to address this question in a SCA7 transgenic mouse model that progressively develops retinopathy, similar to SCA7 patients. To co-express HSP70 and HDJ2 with the polyglutamine protein, in the same cell type, at comparable levels and with the same time course, we generated transgenic mice that express the heat shock proteins specifically in rod photoreceptors. While co-expression of HSP70 with its co-factor HDJ2 efficiently suppressed mutant ataxin-7 aggregation in transfected cells, they did not prevent either neuronal toxicity or aggregate formation in SCA7 mice. Furthermore, nuclear inclusions in SCA7 mice were composed of a cleaved mutant ataxin-7 fragment, whereas they contained the full-length protein in transfected cells. We propose that differences in the aggregation process might account for the different effects of chaperone overexpression in cellular and animal models of polyglutamine diseases.

Topics: Animals; Ataxin-7; Cell Line; Cell Nucleus; Disease Models, Animal; DNA, Complementary; Electroretinography; Heat-Shock Proteins; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Immunoblotting; Immunohistochemistry; Mice; Mice, Transgenic; Microscopy; Models, Genetic; Molecular Chaperones; Mutation; Nerve Tissue Proteins; Neurons; Peptides; Phenotype; Plasmids; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Retina; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Time Factors; Transfection; Ubiquitin

Bardet-Biedl syndrome (BBS) is a heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, hypogenitalism, and an increased incidence of diabetes and hypertension. No information is available regarding the specific function of BBS2. We show that mice lacking Bbs2 gene expression have major components of the human phenotype, including obesity and retinopathy. In addition, these mice have phenotypes associated with cilia dysfunction, including retinopathy, renal cysts, male infertility, and a deficit in olfaction. With the exception of male infertility, these phenotypes are not caused by a complete absence of cilia. We demonstrate that BBS2 retinopathy involves normal retina development followed by apoptotic death of photoreceptors, the primary ciliated cells of the retina. Photoreceptor cell death is preceded by mislocalization of rhodopsin, indicating a defect in transport. We also demonstrate that Bbs2(-/-) mice and a second BBS mouse model, Bbs4(-/-), have a defect in social function. The evaluation of Bbs2(-/-) mice indicates additional phenotypes that should be evaluated in human patients, including deficits in social interaction and infertility.

Topics: Animals; Apoptosis; Bardet-Biedl Syndrome; Cilia; Disease Models, Animal; Gene Targeting; Humans; Kidney Diseases, Cystic; Male; Mice; Mice, Knockout; Obesity; Phenotype; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Sensation Disorders; Social Dominance; Spermatogenesis

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

Three different mutations of rhodopsin are known to cause autosomal dominant congenital night blindness in humans. Although the mutations have been studied for 10 years, the molecular mechanism of the disease is still a subject of controversy. We show here, using a transgenic Xenopus laevis model, that the photoreceptor cell desensitization that is a hallmark of the disease results from persistent signaling by constitutively active mutant opsins.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Electrophysiology; Green Fluorescent Proteins; Humans; Luminescent Proteins; Membrane Potentials; Microscopy, Fluorescence; Mutation; Night Blindness; Phenotype; Photic Stimulation; Photoreceptor Cells; Protein Conformation; Retinaldehyde; Rhodopsin; Xenopus laevis

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

Combretastatin A-4 (CA-4) is a naturally occurring agent that binds tubulin and causes necrosis and shrinkage of tumors by damaging their blood vessels. In this study the effect of a CA-4 prodrug, combretastatin A-4-phosphate (CA-4-P), was tested in two models of ocular neovascularization.. The effect of CA-4-P was quantitatively assessed in transgenic mice with overexpression of vascular endothelial growth factor in the retina (rho/VEGF mice) and mice with choroidal neovascularization (CNV) due to laser-induced rupture of Bruch's membrane.. In rho/VEGF mice, daily intraperitoneal injections of 4.0 mg/kg CA-4-P starting at postnatal day (P)7, the time of onset of transgene expression, resulted in a significant reduction in the number of neovascular lesions and total area of neovascularization per retina at P21, compared with vehicle-injected mice. In mice with laser-induced rupture of Bruch's membrane, daily intraperitoneal injections of 75 or 100 mg/kg CA-4-P resulted in a significant reduction in the area of CNV at rupture sites compared with vehicle-injected mice. In mice with established CNV, daily intraperitoneal injections of 100 mg/kg CA-4-P for 1 week resulted in a significant reduction in CNV area at rupture sites compared with the baseline area before treatment or the area of CNV in vehicle-treated mice.. These data indicate that CA-4-P suppresses the development of VEGF-induced neovascularization in the retina and both blocks development and promotes regression of CNV. Therefore, CA-4-P shows potential for both prevention and treatment of ocular neovascularization.

Topics: Animals; Antineoplastic Agents, Phytogenic; Choroidal Neovascularization; Disease Models, Animal; Endothelial Growth Factors; Female; Intercellular Signaling Peptides and Proteins; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Transgenic; Prodrugs; Remission Induction; Rhodopsin; Stilbenes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Sorsby fundus dystrophy (SFD) is a rare, late-onset macular dystrophy caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. The known mutations introduce potentially unpaired cysteine residues in the C terminus of the protein and result in the formation of higher-molecular-weight protein complexes of as yet unknown composition and functional consequences in the pathologic course of SFD. To facilitate in vivo investigation of mutant TIMP3, the authors generated a knock-in mouse carrying a disease-related Ser156Cys mutation in the orthologous murine Timp3 gene.. Site-directed mutagenesis and homologous recombination in embryonic stem (ES) cells was used to generate mutant ES cells carrying the Timp3(S156C) allele. Chimeric animals were obtained, of which two displayed germline transmission of the mutated allele. Molecular genetic, biochemical, electron microscopic, and electrodiagnostic techniques were used for characterization.. At 8 months of age, knock-in mice showed abnormalities in the inner aspect of Bruch's membrane and in the organization of the adjacent basal microvilli of the retinal pigment epithelium (RPE). Changes resembling those in the mutant animals were also present to some extent in normal littermates, but only at an advanced age of 30 months. Long-term electrodiagnostic recordings indicated normal retinal function throughout life. The biochemical characteristics of the mutant protein appear similar in humans and knock-in mice, suggesting common molecular pathways in the two species. The localization of the mutant protein in the eye is normal, although there is evidence of increased Timp3 levels in Bruch's membrane of mutant animals.. The knock-in mice display early features of age-related changes in Bruch's membrane and the RPE that may represent the primary clinical manifestations of SFD. In addition, our immunolabeling studies and biochemical data support a model proposing that site-specific excess rather than absence or deficiency of functional Timp3 may be the primary consequence of the known Timp3 mutations.

Topics: Animals; Blotting, Northern; Blotting, Western; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Fundus Oculi; Genetic Vectors; Macular Degeneration; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutagenesis, Site-Directed; Ophthalmoscopy; Radiation Injuries, Experimental; Retina; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Tissue Inhibitor of Metalloproteinase-3

Treatment of rats with monocrotaline (MCT) leads to pulmonary hypertension, right ventricular (RV) hypertrophy, and finally to RV heart failure. This is associated with characteristic changes in right ventricular beta-adrenoceptors (beta-AR), neuronal noradrenaline transporter (NAT) density and activity (uptake1), and G protein-coupled receptor kinase (GRK) activity. This study aimed to find out factors that determine beta-AR, uptake1, and GRK changes. Thus, 6-week-old rats were treated with 50 mg/kg MCT subcutaneous or 0.9% saline. Within 13 to 19 days after MCT application (group A), RV weight (222+/-6 versus 147+/-5 mg) and RV/left ventricular (LV) weight ratio (0.42+/-0.01 versus 0.29+/-0.01) were significantly increased, whereas plasma noradrenaline, RV beta-AR density, RV NAT density and activity, and RV GRK activity were not significantly altered. Twenty-one to twenty-eight days after MCT (group B), however, not only RV weight (316+/-4 versus 148+/-2 mg) and RV/LV weight ratio (0.61+/-0.01 versus 0.3+/-0.01) were markedly increased but also plasma noradrenaline (645+/-63 versus 278+/-18 pg/mL); now, RV beta-AR density (13.4+/-1.3 versus 26.5+/-1.1 fmol/mg protein), RV NAT density (50.9+/-11.3 versus 79.6+/-2.9 fmol/mg protein), and RV NAT activity (65.4+/-7.4 versus 111.8+/-15.9 pmol [3H]-NA/mg tissue slices/15 min) were significantly decreased and RV-membrane GRK activity (100+/-15 versus 67+/-6 [32P]-rhodopsin in cpm) significantly increased. LV parameters of MCT-treated rats were only marginally different from control LV. We conclude that in MCT-treated rats ventricular hypertrophy per se is not sufficient to cause characteristic alterations in the myocardial beta-AR system often seen in heart failure; only if ventricular hypertrophy is associated with neurohumoral activation beta-ARs are downregulated and GRK activity is increased.

Topics: Animals; Binding, Competitive; Cell Membrane; Disease Models, Animal; Eye Proteins; Fluoxetine; G-Protein-Coupled Receptor Kinase 1; Heart Failure; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Male; Monocrotaline; Myocardium; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Organ Size; Protein Kinases; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Receptors, Neurotransmitter; Rhodopsin; Symporters

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

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

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

A dominant form of human congenital nightblindness is caused by a gly90-->asp (G90D) mutation in rhodopsin. G90D has been shown to activate the phototransduction cascade in the absence of light in vitro. Such constitutive activity of G90D rhodopsin in vivo would desensitize rod photoreceptors and lead to nightblindness. In contrast, other rhodopsin mutations typically give rise to nightblindness by causing rod cell death. Thus, the proposed desensitization without rod degeneration would be a novel mechanism for this disorder. To explore this possibility, we induced mice to express G90D opsin in their rods and then examined rod function and morphology, after first crossing the transgenic animals with rhodopsin knock-out mice to obtain appropriate levels of opsin expression. The G90D mouse opsin bound the chromophore and formed a bleachable visual pigment with lambda(max) of 492 nm that supported rod photoresponses. (G+/-, R+/-) retinas, heterozygous for both G90D and wild-type (WT) rhodopsin, possessed normal numbers of photoreceptors and had a normal rhodopsin complement but exhibited considerable loss of rod sensitivity as measured electroretinographically. The rod photoresponses were desensitized, and the response time to peak was faster than in (R+/-) animals. An equivalent desensitization resulted by exposing WT retinas to a background light producing 82 photoisomerizations rod(-1) sec(-1), suggesting that G90D rods in darkness act as if they are partially "light-adapted." Adding a second G90D allele gave (G+/+, R+/-) animals that exhibited a further increase of equivalent background light level but had no rod cell loss by 24 weeks of age. (G+/+, R-/-) retinas that express only the mutant rhodopsin develop normal rod outer segments and show minimal rod cell loss even at 1 year of age. We conclude that G90D is constitutively active in mouse rods in vivo but that it does not cause significant rod degeneration. Instead, G90D desensitizes rods by a process equivalent to light adaptation.

Topics: Adaptation, Ocular; Alleles; Amino Acid Substitution; Animals; Cell Count; Disease Models, Animal; Dose-Response Relationship, Radiation; Electroretinography; Genes, Dominant; Genotype; Heterozygote; Homozygote; Humans; Immunohistochemistry; Light; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Night Blindness; Retina; Retinal Rod Photoreceptor Cells; Rhodopsin

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

Physiological consequences of early stages of photoreceptor degeneration were examined in heterozygous P23H rhodopsin transgenic (Tg) and in aging normal Sprague-Dawley rats. Rod photoreceptor and rod bipolar (RB) cell function were estimated with maximum value and sensitivity parameters of P3 and P2 components of the electroretinogram. In both Tg and aging normal rats, the age-related rate of decline of P3 amplitude was steeper than that of the P2 amplitude. Tg rats showed greater than normal sensitivity of the rods. A new model of distal RB pathway connectivity suggested photoreceptor loss could not be the sole cause of physiological abnormalities; there was an additional increase of post-receptoral sensitivity. We propose that changes at rod-RB synapses compensate for the partial loss of rod photoreceptors in senescence and in early stages of retinal degeneration.

Topics: Aging; Animals; Animals, Genetically Modified; Confidence Intervals; Disease Models, Animal; Electroretinography; Linear Models; Rats; Rats, Sprague-Dawley; Regression Analysis; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Visual Pathways

To determine the extent to which rhodopsin mis-sorting and constitutive activation of the phototransduction cascade contribute to retinal degeneration in a transgenic rat model of retinitis pigmentosa.. Retinas from transgenic rats expressing truncated rhodopsin (Ser334ter) were examined by light and electron microscopic immunocytochemistry at several time points. Retinal degeneration in transgenic rats raised in darkness was evaluated by quantification of outer nuclear layer thickness and by electroretinography.. Mutant rhodopsin was found at inappropriately high levels in the plasma membrane and cytoplasm of Ser334ter rat photoreceptors. When the cell death rate was high this mis-sorting was severe, but mis-sorting attenuated greatly at later stages of degeneration, as the cell death rate decreased. The distributions of two other outer segment proteins (the cGMP-gated channel and peripherin) were examined and found to be sorted normally within the photoreceptors of these rats. Raising Ser334ter transgenic rats in darkness resulted in minimal rescue from retinal degeneration.. Because dark rearing Ser334ter rats results in little rescue, it is concluded that constitutive activation of the phototransduction cascade does not contribute significantly to photoreceptor cell death in this rat model. The nature of the rhodopsin sorting defect and the correlation between the severity of mis-sorting and rate of cell death indicate that truncated rhodopsin may cause apoptosis by interfering with normal cellular machinery in the post-Golgi transport pathway or in the plasma membrane.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Biological Transport; Blotting, Western; Disease Models, Animal; Electroretinography; Fluorescent Antibody Technique, Indirect; Point Mutation; Rats; Rats, Sprague-Dawley; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Topics: Animals; Cell Line; Cricetinae; Disease Models, Animal; Genetic Therapy; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Inbred CBA; Mice, Transgenic; Nerve Tissue Proteins; Peripherins; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

A combined total of approximately 100 mutations have been encountered within the rhodopsin gene in retinitis pigmentosa (RP) and congenital night blindness. Mice carrying a targeted disruption of the rhodopsin gene phenotypically mimic RP, losing their photoreceptors over a period of 3 months and having no recordable rod electroretinogram. These animals will serve as a model for both recessive and dominant disease (in the latter case, the presence of normal and mutant human rod opsin transgenes on the murine Rho(-/-)background). Precise knowledge of apoptotic photoreceptor cell death, together with factors which may influence apoptosis will be required for optimum utility of Rho(-/-)mice as a model for therapeutic genetic intervention. A peak phase of apoptosis of the photoreceptors of Rho(-/-)mice was shown to occur at 24 days post-birth. The extent of apoptosis appeared to be similar, irrespective of whether or not the rod opsin knockout was present on a c-fos(+/+)or c-fos(-/-)genetic background, the latter known to favor survival of photoreceptors following exposure of mouse retinas to excessive light. These data clearly support the existence in animals of distinct apoptotic pathways in light-induced, as opposed to mutation-induced apoptosis, and together with similar observations recently reported in studies of the naturally occurring rd mouse, may assist in focusing future research on precisely defining the distinct molecular pathways giving rise to such dichotomy.

Topics: Animals; Apoptosis; Disease Models, Animal; Genes, fos; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Knockout; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP), a type of retinal degeneration involving first rod and then slow cone photoreceptor degeneration, can be caused by any of a number of mutations in different genes. In the cases of mutations affecting rod-specific genes such as rhodopsin, it is unclear how the mutations may cause degeneration of cones. We have used the porcine retina, which is rod-dominated and has an abundance of cones, to study the mutation-induced changes in both rod and cone photoreceptors. Like patients with the same mutation, rhodopsin P347L transgenic swine manifest rod-cone degeneration. In addition, the rod bipolar cells fail to form synaptic connections with rods; instead, they form ectopic synapses with cones. The mechanisms that prevent the formation of the rod-rod bipolar cell synaptic connection are not known. We used specific antibodies and immunocytochemistry to show that the synaptic protein, PSD-95, is present in both normal and transgenic porcine retinas. During neonatal development, however, PSD-95 is lost from rod terminals in the transgenic swine. This loss is virtually complete (90%) by postnatal day 5, at a time when greater than 80% of rod cell bodies still remain. Furthermore, the remaining rods retain their outer segments and their gross morphology appears relatively normal. In contrast, PSD-95 expression continues in cone terminals, even in 10-month-old transgenic swine, where the rods have all disappeared and the cones show signs of severe degeneration. These results suggest that loss of PSD-95 may not be a general consequence of the deteriorating cell. Rather, the very early and selective loss of PSD-95 from the rod terminals may be causally related to the absence of rod-rod bipolar cell synapses in the rhodopsin P347L transgenic retina.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Antibodies; Disease Models, Animal; Nerve Tissue Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Synapses

To further characterize the retinas of Pro3471Leu rhodopsin transgenic pigs, a model for human retinitis pigmentosa.. Retinas from normal and transgenic pigs, newborn to 20 months old, were processed for light and electron microscopic immunocytochemical examination.. At birth, rod numbers were normal in the transgenic retinas, but their outer segments were short and disorganized and their inner segments contained stacks of rhodopsin-positive membranes. The newborn rod synapses lacked synaptic vesicles and ribbons and had numerous rhodopsin-positive, filopodia-like processes that extended past the cone synapses into the outer plexiform layer. Rod cell death was apparent by 2 weeks and was pronounced in the mid periphery and central regions by 6 weeks. Far peripheral rods were initially better preserved, but by 9 months virtually all rods had degenerated. Cones degenerated more slowly than rods, but by 4 weeks the cone synapses were shrunken and some mid peripheral cones had lost their immunoreactivity for phosphodiesterase-gamma, arrestin, and recoverin. From 9 months to 20 months, the cone outer segments shortened progressively, and more cones lost immunoreactivity for these proteins.. The rhodopsin transgenic pig retina shares many cytologic features with human retinas with retinitis pigmentosa and provides an opportunity to examine the earliest stages in photoreceptor degeneration, about which little is known in humans. The finding of abnormal rhodopsin localization in newborn rods is consistent with misrouting of mutant rhodopsin as an early process leading to rod cell death. Novel changes in the photoreceptor synapses may correlate with early electrophysiological abnormalities in these retinas.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Cell Count; Cell Death; Disease Models, Animal; Eye Proteins; Fluorescent Antibody Technique, Indirect; Neuroglia; Photoreceptor Cells; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Swine

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Topics: Animals; Animals, Genetically Modified; Dependovirus; Disease Models, Animal; Genes, Dominant; Genetic Therapy; Histidine; Photoreceptor Cells; Point Mutation; Proline; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Catalytic; Rod Opsins

Basic fibroblast growth factor (FGF2) is constitutively expressed in the retina and its expression is increased by a number of insults, but its role in the retina is still uncertain. This study was designed to test the hypothesis that altered expression of FGF2 in the retina affects the development of retinal neovascularization. Mice with targeted disruption of the Fgf2 gene had no detectable expression of FGF2 in the retina by Western blot, but retinal vessels were not different in appearance or total area from wild-type mice. When FGF2-deficient mice were compared with wild-type mice in a murine model of oxygen-induced ischemic retinopathy, they developed the same amount of retinal neovascularization. Transgenic mice with a rhodopsin promoter/Fgf2 gene fusion expressed high levels of FGF2 in retinal photoreceptors but developed no retinal neovascularization or other abnormalities of retinal vessels; in the ischemic retinopathy model, they showed no significant difference in the amount of retinal neovascularization compared with wild-type mice. These data indicate that FGF2 expression is not necessary nor sufficient for the development of retinal neovascularization. This suggests that agents that specifically antagonize FGF2 are not likely to be useful adjuncts in the treatment of retinal neovascularization and therapies designed to increase FGF2 expression are not likely to be complicated by retinal neovascularization.

Topics: Animals; Disease Models, Animal; DNA Primers; Fibroblast Growth Factor 2; Humans; Immunoenzyme Techniques; Infant, Newborn; Mice; Mice, Knockout; Mice, Transgenic; Polymerase Chain Reaction; Recombinant Fusion Proteins; Retina; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Rhodopsin

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11-cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Disease Models, Animal; Genes, Dominant; Mice; Mice, Transgenic; Photochemistry; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Spectrophotometry; Vision, Ocular

Several mutations causing both photoreceptor degeneration and malfunction have been identified in humans and animals. Although intraocular injection of trophic factors has been shown to reduce photoreceptor death in a few conditions of rapid photoreceptor loss, it is unclear whether long-term beneficial changes in functional properties of affected photoreceptors can be obtained by treatment with these factors. The rds/rds mouse is a spontaneous mutant bearing a null mutation in the rds/peripherin gene, which is linked to many forms of dominant retinal degenerations in humans. Here, we report that intraocular adenovirus-mediated gene transfer of ciliary neurotrophic factor (CNTF) in this mutant reduces photoreceptor loss, causes a significant increase in the length of photoreceptor segments, and results in a redistribution and an increase in the retinal content of the photopigment rhodopsin. These effects are accompanied by a significant increase in the amplitude of the a- and b-waves of the scotopic electroretinogram. These results suggest that continuous administration of CNTF could potentially be useful for the treatment of some forms of retinal degeneration.

Topics: Adenoviridae; Animals; Apoptosis; Ciliary Neurotrophic Factor; Disease Models, Animal; Electroretinography; Gene Transfer Techniques; Homozygote; Lac Operon; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Nerve Growth Factors; Nerve Tissue Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Disease Models, Animal; Genetic Engineering; Humans; Mutation; Phenotype; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rhodopsin; Rodentia; Swine

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

To evaluate the consequences of the expression of a mutant mouse opsin gene on rod- and cone-mediated function. Experimental conditions were chosen to provide a basis of comparison to the results reported for patients with autosomal dominant retinitis pigmentosa (ADRP) in whom the proline at position 23 has been replaced by a histidine (P23H).. The mutated gene product resulted in three substitutions in the rhodopsin molecule: P23H, glycine for valine at position 20 (V20G), and leucine for proline at position 27 (P27L). Mice positive for the transgene were differentiated from normal littermates by the polymerase chain reaction. Electroretinograms (ERGs) were obtained from anesthetized mice between 1 and 9 months of age. After photically bleaching approximately 18% of the available rhodopsin, the time course of rod dark adaptation was examined by monitoring rod ERG amplitude recovery. Rhodopsin densitometry was used to determine the relative amounts of rhodopsin in the retinae of normal and transgenic mice.. ERGs obtained from transgenic mice showed a significant reduction in rod-mediated response amplitude at 1 month of age and a relatively slow progressive decrease thereafter. Cone-mediated ERGs, on the other hand, were nearly normal in amplitude for approximately the first 5 months after birth, but at later ages response amplitudes also underwent a progressive decline. In the normal retina, rod ERG amplitudes returned to prebleach levels within 30 minutes, whereas in transgenic mice response amplitudes did not recover within a 2-hour test period. The age-related decline in rod-mediated electroretinal potentials seen in transgenic mice was paralleled by a concomitant fall in rhodopsin density, and the sensitivity losses obtained electroretinographically could be accounted for solely on the basis of reduced quantal absorption.. The pattern of functional changes seen in the transgenic mice are in good agreement with those reported in patients with ADRP with the P23H mutation in the rhodopsin gene. Particularly noteworthy is the fact that the changes in rhodopsin density and visual sensitivity are associated with a progressive shortening of the rod outer segments; the histologic changes induced by the disease process in patients with ADRP have yet to be determined.

Topics: Animals; Dark Adaptation; Densitometry; Disease Models, Animal; Electroretinography; Gene Expression; Mice; Mice, Mutant Strains; Mice, Transgenic; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

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

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

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

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.

Topics: Animals; Codon; Disease Models, Animal; Electroretinography; Fundus Oculi; Humans; Mice; Mice, Transgenic; Microscopy, Electron; Mutation; Ophthalmoscopy; Photoreceptor Cells; Proline; Restriction Mapping; Retina; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

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

Rhodopsin, a membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) in Lewis rats. Synthetic peptides derived from rhodopsin sequences that cover hydrophilic, exposed regions of the protein were tested for their capacity of eliciting in vitro T cell proliferation and their ability for inducing EAU in Lewis rats. Rats were injected with rhodopsin's peptides mixed in complete Freund's adjuvant containing M. tuberculosis H37Ra (5 mg/ml) three days after pretreatment with cyclophosphamide (20 mg/kg). ELISA results indicate that all peptides induce antibody responses; however antibody titers differ among sera tested. Immunization with four peptides--the amino-terminus (2-32), loop I-II (61-75), loop V-VI (230-251), and the carboxyl-terminus (324-348 and 331-342) induced both antibody and T cell responses. In all cases, the proliferative responses of cells derived from peptide-injected rats were stronger against the immunizing peptide than against native protein. Three distinct uveitogenic epitopes were identified on rhodopsin's cytoplasmic surface--within the rhodopsin carboxyl-terminus (324-348), loop I-II (61-75), and loop V-VI (230-250). Histopathologically, at the immunized doses, total destruction of the photoreceptor cell layer was observed as compared to the control group. Loop V-VI caused severe inflammation of the retina while the other pathogenic peptides produced less severe destruction with few inflammatory cells present. Our study indicates that the major immunodominant T cell epitope (331-342) is also involved in EAU induction but is not the primary uveitogenic site.

Topics: Amino Acid Sequence; Animals; Autoimmune Diseases; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Immunodominant Epitopes; Injections; Lymphocyte Activation; Molecular Sequence Data; Peptides; Rats; Rats, Inbred Lew; Rhodopsin; T-Lymphocytes; Uveitis

Since lipid peroxidation induced by oxygen free radicals is believed to play an important role in retinal photic injury and high doses of certain steroids have been demonstrated to inhibit lipid peroxidation, we evaluated the effect of high-dose methylprednisolone sodium succinate on retinal injury inflicted by green light (490 to 580 nm, 160 to 180 foot-candles [1721.6 to 1936.8 lux]) for 24 hours in 36 rats. Animals received either intraperitoneal injections of the drug (80 mg/kg twice daily) or saline as a control, twice daily for 2 days, commencing with light exposure. The retinal photic injury was assessed 6 hours, 6 days, and 14 days after light exposure by light and electron microscopy, morphometric study of outer nuclear layer thickness and count of subretinal macrophages, and spectrophotometric measurement of rhodopsin. A beneficial effect of high-dose methylprednisolone was observed in retinal photic injury.

Topics: Animals; Cell Count; Disease Models, Animal; Injections, Intraperitoneal; Light; Macrophages; Male; Methylprednisolone; Radiation Injuries, Experimental; Rats; Rats, Inbred Lew; Retina; 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

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

Ocular malformations and visual deficits are pathognomic of fetal alcohol syndrome (FAS). However, there are no reports on retinal visual function. To determine if prenatal (human second-trimester equivalent) or prenatal plus 10 days of postnatal (human third-trimester equivalent) ethanol exposure produced scotopic and/or photopic deficits in adult hooded rats, electroretinography (ERG) was used to examine rod and cone, increment threshold, dark adaptation, and paired-flash amplitude recovery functions. The rhodopsin content per eye also was determined. Five main results were found. First, voltage-log intensity and latency-log intensity functions, generated from single-flash ERGs in fully dark-adapted rats, showed increases in absolute threshold and latency and decreases in response amplitude. Second, cone ERGs had latency increases. Third, there were decreases in the scotopic and photopic critical flicker-fusion frequencies, increment threshold functions, and absolute and relative refractory periods. Fourth, rod sensitivity, range, and rate of dark adaptation were decreased. Fifth, rhodopsin content per eye was decreased. These data showed that prenatal ethanol exposure produces long-term deficits in retinal sensitivity, amplitude, light and dark adaptation, temporal processing, and excitability. Larger deficits occurred in the scotopic than photopic system and were produced with an additional 10 days of postnatal ethanol exposure. Alterations in photoreceptors and other cells of the distal retina probably contributed to these deficits. The relevance and applicability of these data to FAS and subclinical alcohol embryopathy have yet to be demonstrated; however, they suggest that similar retinal alterations may occur in human FAS.

Topics: Animals; Dark Adaptation; Disease Models, Animal; Electroretinography; Ethanol; Female; Fetus; Flicker Fusion; Photoreceptor Cells; Pregnancy; Rats; Retina; 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

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: 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

24 different antigenic preparations from bovine or guinea pig retina and 3 from bovine uvea were tested for their ability to induce uveo-retinitis in guinea pigs. Each animal received one injection into the hind foot pads of 0.1 ml og the tissue preparation mixed with an equal volume of complete Freund's adjuvant. The intensity of the disease was assessed by clinical and histological criteria. Homogenates and extracts from whole guinea pig retina are more active than the same preparations from bovine retina. Autologous retinal extract is slightly more active than homologous in low doses. In bovine retina, the autoantigen(s) is localized in the photoreceptor structures and the pigment epithelium. Bovine uveal preparations seem to be inactive when the epithelium has been removed. Purified outer segments are very active, as well as soluble extracts of outer segments. Highly purified bovine rhodopsin has no immunopathogenic activity. A soluble autoantigen (autoantigen S) has been isolated by preparative isoelectrofocusing from retinas of several species. Autoantigen S from guinea pig induces the disease in guinea pigs at a dose of a few micrograms.

Topics: Animals; Antigens; Autoantigens; Autoimmune Diseases; Cattle; Disease Models, Animal; Eye Proteins; Freund's Adjuvant; Guinea Pigs; Isoelectric Focusing; Phosphoric Diester Hydrolases; Photoreceptor Cells; Pigment Epithelium of Eye; Retina; Retinitis; Rhodopsin; Solubility; Species Specificity; Uvea; Uveitis

Topics: Animals; Darkness; Disease Models, Animal; Fatty Acids; Haplorhini; Hyperbaric Oxygenation; Iodoacetates; Light; Macaca mulatta; Phospholipids; Photoreceptor Cells; Rabbits; Rats; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Temperature

Topics: Animals; Autoimmune Diseases; Blindness; Disease Models, Animal; Haplorhini; Macaca mulatta; Retinal Degeneration; Retinal Pigments; Rhodopsin