11-cis-retinal and Retinal-Diseases

Visual perception by photoreceptors relies on the interaction of incident photons from light with a derivative of vitamin A that is covalently linked to an opsin molecule located in a special subcellular structure, the photoreceptor outer segment. The photochemical reaction produced by the photon is optimal when the opsin molecule, a seven-transmembrane protein, is embedded in a lipid bilayer of optimal fluidity. This is achieved in vertebrate photoreceptors by a high proportion of lipids made with polyunsaturated fatty acids, which have the detrimental property of being oxidized and damaged by light. Photoreceptors cannot divide, but regenerate their outer segments. This is an enormous energetic challenge that explains why photoreceptors metabolize glucose through aerobic glycolysis, as cancer cells do. Uptaken glucose produces metabolites to renew that outer segment as well as reducing power through the pentose phosphate pathway to protect photoreceptors against oxidative damage.

Topics: Animals; Fatty Acids; Glucose; Humans; Oxidation-Reduction; Oxidative Stress; Pentose Phosphate Pathway; Photoreceptor Cells; Retina; Retinal Diseases; Rhodopsin; Signal Transduction

Topics: Animals; Humans; Optogenetics; Retina; Retinal Diseases; Rhodopsin

Topics: Acyltransferases; Animals; cis-trans-Isomerases; Coenzyme A-Transferases; Humans; Mevalonic Acid; Photoreceptor Cells; Retinal Diseases; Retinaldehyde; Retinoids; Retinol-Binding Proteins; Rhodopsin

Rhodopsin is the light receptor in rod photoreceptor cells of the retina that initiates scotopic vision. In the dark, rhodopsin is bound to the chromophore 11-cis retinal, which locks the receptor in an inactive state. The maintenance of an inactive rhodopsin in the dark is critical for rod photoreceptor cells to remain highly sensitive. Perturbations by mutation or the absence of 11-cis retinal can cause rhodopsin to become constitutively active, which leads to the desensitization of photoreceptor cells and, in some instances, retinal degeneration. Constitutive activity can arise in rhodopsin by various mechanisms and can cause a variety of inherited retinal diseases including Leber congenital amaurosis, congenital night blindness, and retinitis pigmentosa. In this review, the molecular and structural properties of different constitutively active forms of rhodopsin are overviewed, and the possibility that constitutive activity can arise from different active-state conformations is discussed.

Topics: Animals; Humans; Mutation; Phenotype; Retinal Diseases; Rhodopsin

A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.

Topics: Acyltransferases; Alcohol Oxidoreductases; Animals; Carrier Proteins; cis-trans-Isomerases; Eye Proteins; Humans; Mice; Phospholipids; Photoreceptor Cells, Vertebrate; Rats; Retina; Retinal Diseases; Retinaldehyde; Retinoids; Rhodopsin; Vision, Ocular

Topics: Dark Adaptation; G-Protein-Coupled Receptor Kinase 1; G-Protein-Coupled Receptor Kinases; Humans; Retinal Cone Photoreceptor Cells; Retinal Diseases; Retinal Rod Photoreceptor Cells; Rhodopsin

Membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC), a key enzyme for the recovery of photoreceptors to the dark state, has a topology identical to and cytoplasmic domains homologous to those of peptide-regulated GCs. However, under the prevailing concept, its activation mechanism is significantly different from those of peptide-regulated GCs: GC-activating proteins (GCAPs) function as the sole activator of ROS-GC in a Ca(2+)-sensitive manner, and neither reception of an outside signal by the extracellular domain (ECD) nor ATP binding to the kinase homology domain (KHD) is required for its activation. We have recently shown that ATP pre-binding to the KHD in ROS-GC drastically enhances its GCAP-stimulated activity, and that rhodopsin illumination, as the outside signal, is required for the ATP pre-binding. These results indicate that illuminated rhodopsin is involved in ROS-GC activation in two ways: to initiate ATP binding to ROS-GC for preparation of its activation and to reduce [Ca(2+)] through activation of cGMP phosphodiesterase. These two signal pathways are activated in a parallel and proportional manner and finally converge for strong activation of ROS-GC by Ca(2+)-free GCAPs. These results also suggest that the ECD receives the signal for ATP binding from illuminated rhodopsin. The ECD is projected into the intradiscal space, i.e., an intradiscal domain(s) of rhodopsin is also involved in the signal transfer. Many retinal disease-linked mutations are found in these intradiscal domains; however, their consequences are often unclear. This model will also provide novel insights into causal relationship between these mutations and certain retinal diseases.

Topics: Adenosine Triphosphate; Animals; Guanylate Cyclase; Guanylate Cyclase-Activating Proteins; Humans; Retinal Diseases; Rhodopsin; Rod Cell Outer Segment; Signal Transduction

Following exposure of our eye to very intense illumination, we experience a greatly elevated visual threshold, that takes tens of minutes to return completely to normal. The slowness of this phenomenon of "dark adaptation" has been studied for many decades, yet is still not fully understood. Here we review the biochemical and physical processes involved in eliminating the products of light absorption from the photoreceptor outer segment, in recycling the released retinoid to its original isomeric form as 11-cis retinal, and in regenerating the visual pigment rhodopsin. Then we analyse the time-course of three aspects of human dark adaptation: the recovery of psychophysical threshold, the recovery of rod photoreceptor circulating current, and the regeneration of rhodopsin. We begin with normal human subjects, and then analyse the recovery in several retinal disorders, including Oguchi disease, vitamin A deficiency, fundus albipunctatus, Bothnia dystrophy and Stargardt disease. We review a large body of evidence showing that the time-course of human dark adaptation and pigment regeneration is determined by the local concentration of 11-cis retinal, and that after a large bleach the recovery is limited by the rate at which 11-cis retinal is delivered to opsin in the bleached rod outer segments. We present a mathematical model that successfully describes a wide range of results in human and other mammals. The theoretical analysis provides a simple means of estimating the relative concentration of free 11-cis retinal in the retina/RPE, in disorders exhibiting slowed dark adaptation, from analysis of psychophysical measurements of threshold recovery or from analysis of pigment regeneration kinetics.

Topics: Animals; Dark Adaptation; Humans; Retinal Diseases; Retinoids; Rhodopsin; Rod Cell Outer Segment; Vision, Ocular

In the first part of the paper, the results of the investigation of the rhodopsin arrestin interaction are presented. The results were mainly obtained with the technique of the selective labelling of the rhodopsin and arrestin SH-groups and the rhodopsin limited proteolysis. These results are discussed in the frame of the latest data on the three-dimensional structure of arrestin. In the second part of the paper, results of the antigenic properties of arrestin (S-antigen) and its role in the pathogenesis of the retina diseases are summarized. The data on the role of the autoimmune processes in the pathogenesis of diabetic retinopathy are presented. We have also described the results of the use of the elaborated technique of the immune diagnostics in the prognosis of the diabetic retinopathy and retinopathy of the premature babies.

Topics: Animals; Arrestin; Autoimmune Diseases; Eye Diseases; Humans; Infant, Newborn; Infant, Premature; Protein Binding; Retinal Diseases; Rhodopsin; Vision, Ocular

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

Rhodopsin is the membrane receptor responsible for photoreception in the vertebrate retina. Its characteristic seven-transmembrane helical structural motif is today widely recognised as a paradigm in signal transduction. Rhodopsin and the phototransduction system are frequently used as structural and mechanistic models for the G-protein coupled receptor superfamily. Recent advances in the activation mechanism (as derived from the structural available data) and the implications for normal and pathological - in retinal disorders - visual function will be reviewed.

Topics: Amino Acid Sequence; Animals; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Night Blindness; Protein Structure, Secondary; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.

Topics: Amino Acid Sequence; Animals; Cattle; Cytoplasm; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Humans; Models, Chemical; Models, Molecular; Molecular Sequence Data; Photons; Protein Conformation; Receptors, Cell Surface; Retinal Diseases; Rhodopsin; Signal Transduction

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.

Topics: Animals; Group II Chaperonins; Humans; Models, Molecular; Molecular Chaperones; Photoreceptor Cells; Protein Conformation; Protein Folding; Retina; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin

The present models of phototransduction for vertebrates and invertebrates have been reviewed and the relative literature updated. The emerging picture for vertebrate phototransduction is a result of a better knowledge of its general outlines, although some important details such as the role of calcium ions are still lacking. The molecular events involved in the rising phase of the electrical response have basically been understood, whilst those involved in response inactivation and recovery remain to be elucidated. In an overall strategy, the phototransduction in invertebrates shares a great deal of similarity with that in vertebrates but differs in the underlying molecular events. However, a complete picture of phototransduction in invertebrate photoreceptors has not yet emerged. The available data on the structure of the visual pigment rhodopsin reveal further details on the present model of the retinal-binding pocket of the protein and consequently of the "red shift" of the absorbance of retinal. The problem of the energy supplied during photoreception, in particular, the availability of ATP in the rod outer segment and the presence in the disk membranes of a Ca-ATPase are discussed. Finally, recent progress in understanding the molecular mechanisms of inherited retinal diseases and relative gene identification are summarized.

Topics: Animals; Humans; Photoreceptor Cells, Invertebrate; Photoreceptor Cells, Vertebrate; Retinal Diseases; Rhodopsin; Vision, Ocular

Recent studies on rhodopsin structure and function are reviewed and the properties of vertebrate as well as invertebrate rhodopsin described. Open issues such as the 'red shift' of the absorbance spectra are emphasized in the light of the present model of the retinal-binding pocket. The processes that restore the rhodopsin content in photoreceptors are also presented with a comparison between vertebrate and invertebrate visual systems. The central role of rhodopsin in the phototransduction cascade becomes evident by examining the main reports on light-activated conformational changes of rhodopsin and its interaction with transducin. Shut-off mechanisms are considered by reporting the studies on the sites of rhodopsin phosphorylation and arrestin binding. Furthermore, recent findings on the energetics of phototransduction point out that the ATP needed for photoreception in vertebrates is synthesized in the outer segments where phototransduction events take place.

Topics: Amino Acid Sequence; Animals; Energy Transfer; Humans; Invertebrates; Molecular Sequence Data; Protein Structure, Secondary; Retinal Diseases; Rhodopsin; Vertebrates; Vision, Ocular

Topics: Adult; Aged; Apoptosis; Chromosome Mapping; Environment; Female; Genes, Dominant; Humans; Male; Middle Aged; Phenotype; Retinal Diseases; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

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

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

Topics: 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

To describe the clinical, perimetric, and electroretinographic (ERG) results of 4 patients with cone dysfunction following irofulven treatment including the histopathologic and immunocytochemical features of one patient's retinas.. Observational case series.. The patients were examined clinically, including perimetric and ERG evaluations. Eyes from patient 1 and healthy postmortem eyes were processed for histopathologic and immunocytochemistry studies with antibodies specific for cones, rods, and reactive Müller cells.. Clinical signs and symptoms, perimetry, ERG, retinal histopathologic and immunocytochemistry study results.. All 4 patients had ERG changes consistent with abnormal cone responses and relatively normal rod responses. Compared with control eyes, the retina of patient 1 had approximately half the normal numbers of macular cones and fewer peripheral cones. The number of rods were normal but all rod and cone outer segments were shortened.. High-dose irofulven treatment causes cone-specific damage with relative sparing of rods.

Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Glial Fibrillary Acidic Protein; Humans; Middle Aged; Neoplasms; Retinal Cone Photoreceptor Cells; Retinal Diseases; Rhodopsin; Sesquiterpenes; Visual Field Tests; Visual Fields

Ionising radiation interacts with lenses and retinae differently. In human lenses, posterior subcapsular cataracts are the predominant observation, whereas retinae of adults are comparably resistant to even relatively high doses. In this study, we demonstrate the effects of 2 Gy of low linear energy transfer ionising radiation on eyes of B6C3F1 mice aged postnatal day 2. Optical coherence tomography and Scheimpflug imaging were utilised for the first time to monitor murine lenses and retinae in vivo. The visual acuity of the mice was determined and histological analysis was conducted. Our results demonstrated that visual acuity was reduced by as much as 50 % approximately 9 months after irradiation in irradiated mice. Vision impairment was caused by retinal atrophy and inner cortical cataracts. These results help to further our understanding of the risk of ionising radiation for human foeti (∼ 8 mo), which follow the same eye development stages as neonatal mice.

Topics: Animals; Animals, Newborn; Calbindin 2; Cataract; Glial Fibrillary Acidic Protein; Immunohistochemistry; Lens, Crystalline; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Protein Kinase C-alpha; Radiation Dosage; Radiation Injuries, Experimental; Radiation, Ionizing; Retina; Retinal Diseases; Rhodopsin; Tomography, Optical Coherence; Vision Disorders; Visual Acuity

The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.

Topics: Animals; Cattle; Light; Models, Molecular; Mutation; Protein Conformation; Protein Folding; Retinal Diseases; Rhodopsin

Topics: DNA; Female; Humans; Middle Aged; Mutation; Retina; Retinal Diseases; Retinal Pigment Epithelium; 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

Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next-generation sequencing-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r

Topics: Gene Expression Regulation; Gene Library; Genetic Predisposition to Disease; Genetic Variation; Genomics; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Models, Biological; Retinal Diseases; Rhodopsin; Sequence Analysis, DNA

In retinal disease, despite the loss of light sensitivity as photoreceptors die, many retinal interneurons survive in a physiologically and metabolically functional state for long periods. This provides an opportunity for treatment by genetically adding a light sensitive function to these cells. Optogenetic therapies are in development, but, to date, they have suffered from low light sensitivity and narrow dynamic response range of microbial opsins. Expression of light-sensitive G protein coupled receptors (GPCRs), such as vertebrate rhodopsin , can increase sensitivity by signal amplification , as shown by several groups. Here, we describe the methods to (1) express light gated GPCRs in retinal neurons, (2) record light responses in retinal explants in vitro, (3) record cortical light responses in vivo, and (4) test visually guided behavior in treated mice.

Topics: Animals; Behavior, Animal; Genetic Therapy; Light; Mice; Mice, Inbred C57BL; Neurons; Optogenetics; Retina; Retinal Diseases; Rhodopsin

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

Delivery of antibodies to monitor key biomarkers of retinopathy in vivo represents a significant challenge because living cells do not take up immunoglobulins to cellular antigens. We met this challenge by developing novel contrast agents for retinopathy, which we used with magnetic resonance imaging (MRI). Biotinylated rabbit polyclonal to chick IgY (rIgPxcIgY) and phosphorylthioate-modified oligoDNA (sODN) with random sequence (bio-sODN-Ran) were conjugated with NeutrAvidin-activated superparamagnetic iron oxide nanoparticles (SPION). The resulting Ran-SPION-rIgPxcIgY carries chick polyclonal to microtubule-associated protein 2 (MAP2) as Ran-SPION-rIgP/cIgY-MAP2, or to rhodopsin (Rho) as anti-Rho-SPION-Ran. We examined the uptake of Ran-SPION-rIgP/cIgY-MAP2 or SPION-rIgP/cIgY-MAP2 in normal C57black6 mice (n = 3 each, 40 μg/kg, i.c.v.); we found retention of Ran-SPION-rIgP/cIgY-MAP2 using molecular contrast-enhanced MRI in vivo and validated neuronal uptake using Cy5-goat IgPxcIgY ex vivo. Applying this novel method to monitor retinopathy in a bilateral carotid artery occlusion-induced ocular ischemia, we observed pericytes (at d 2, using Gd-nestin, by eyedrop solution), significant photoreceptor degeneration (at d 20, using anti-Rho-SPION-Ran, eyedrops, P = 0.03, Student's t test), and gliosis in Müller cells (at 6 mo, using SPION-glial fibrillary acidic protein administered by intraperitoneal injection) in surviving mice (n ≥ 5). Molecular contrast-enhanced MRI results were confirmed by optical and electron microscopy. We conclude that chimera and molecular contrast-enhanced MRI provide sufficient sensitivity for monitoring retinopathy and for theranostic applications.

Topics: Animals; Brain Ischemia; Carotid Arteries; Contrast Media; Eye Injuries; Ischemia; Male; Mice; Mice, Inbred C57BL; Neurons; Retinal Diseases; Rhodopsin

The perception of light begins when photons reach retinal tissue located at the back of the eye and photoisomerize the visual chromophore 11-cis-retinal to all-trans-retinal within photoreceptor cells. Isomerization of 11-cis-retinal activates the protein rhodopsin located in photoreceptor outer segments, thereby inducing a phototransduction cascade leading to visual perception. To maintain vision, 11-cis-retinal is regenerated in the retinal pigmented epithelium (RPE) via the visual cycle and delivered back to the photoreceptor cells where it may again bind to rhodopsin. Distinct pathological mechanisms have been observed to contribute to inherited retinal degenerative diseases including severe delay in 11-cis-retinal regeneration and delayed clearance of all-trans-retinal, which leads to the accumulation of harmful retinoid by-products. In the last decade, our group has conducted several proof-of-concept (POC) studies with retinoid derivatives aimed at developing treatments for retinal degenerative diseases caused by an impaired visual cycle. Here, we will introduce experimental procedures, which have been developed for POC studies involving retinoid biology.

Topics: Animals; Diterpenes; Light Signal Transduction; Retina; Retinal Diseases; Retinal Pigment Epithelium; Rhodopsin; Vision, Ocular; Vitamin A

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration--defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.

Topics: Adenosine Triphosphate; Animals; Citrate (si)-Synthase; Drosophila; Drosophila Proteins; Electroretinography; Endocytosis; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Photoreceptor Cells, Invertebrate; Pyruvate Dehydrogenase Complex; Reactive Oxygen Species; Retinal Diseases; Rhodopsin; RNA-Binding Proteins; Vision, Ocular

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from light damage. There is potential in developing these compounds as preventative therapeutics for the treatment of human retinal degenerations in which the accumulation of lipofuscin may be pathogenic.

Topics: Animals; Cattle; cis-trans-Isomerases; Cyclic N-Oxides; Female; Injections, Intraperitoneal; Light; Male; Mice; Papio anubis; Rats; Retinal Diseases; Retinal Pigment Epithelium; Rhodopsin

Recent progress in molecular analysis has revealed the possible involvement of multiple inflammatory signaling pathways in pathogenesis of retinal degeneration. However, how aberrant signaling pathways cause tissue damage and dysfunction is still being elucidated. Here, we focus on 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), originally recognized as a key regulator of energy homeostasis. AMPK is also modulated in response to inflammatory signals, although its functions in inflamed tissue are obscure. We investigated the role of activated AMPK in the retinal neural damage and visual function impairment caused by inflammation. For this purpose, we used a mouse model of lipopolysaccharide-induced inflammation in the retina, and examined the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). During inflammation, activated AMPK in the neural retina was decreased, but AICAR treatment prevented this change. Moreover, the electroretinogram (ERG) a-wave response, representing photoreceptor function, showed visual dysfunction in this model that was prevented by AICAR. Consistently, the model showed shortened photoreceptor outer segments (OSs) with reduced levels of rhodopsin, a visual pigment concentrated in the OSs, in a post-transcriptional manner, and these effects were also prevented by AICAR. In parallel, the level of activated NF-κB increased in the retina during inflammation, and this increase was suppressed by AICAR. Treatment with an NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) preserved the rhodopsin level during inflammation, suppressing NF-κB. These findings indicated that AMPK activation by AICAR and subsequent NF-κB inhibition had a protective effect on visual function, and that AMPK activation played a neuroprotective role during retinal inflammation.

Topics: Adenylate Kinase; Aminoimidazole Carboxamide; Animals; Electroretinography; Inflammation; Male; Mice; NF-kappa B; Photoreceptor Cells; Retinal Diseases; Rhodopsin; Ribonucleotides; Signal Transduction

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2(-/-) mice carrying homozygous Pde6b(rd1) alleles or the Tg(RHO P347S) transgene. In the Edn2(-/-) background, PR survival increased 110% in Pde6b(rd1/rd1) mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b(rd1/rd1) ; Edn2(-/-) mice. This finding, together with systemic abnormalities in Edn2(-/-) mice, suggested that the increased survival of mutant PRs in the Edn2(-/-) background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b(rd1/rd1) ; Edn2(-/-) PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b(rd1/rd1) retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

Topics: Animals; Cell Hypoxia; Cell Survival; Cyclic Nucleotide Phosphodiesterases, Type 6; Endothelin-2; Fibroblast Growth Factor 2; Humans; Mice; Mice, Transgenic; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Diseases; Rhodopsin; RNA, Messenger; Signal Transduction; Up-Regulation

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

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

Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy. Here we report the crystal structures of both wild-type human CRALBP and of its mutant R234W as binary complexes complemented with the endogenous ligand 11-cis-retinal, at 3.0 and 1.7 A resolution, respectively. Our structural model of wild-type CRALBP locates R234 to a positively charged cleft at a distance of 15 A from the hydrophobic core sequestering 11-cis-retinal. The R234W structural model reveals burial of W234 and loss of dianion-binding interactions within the cleft with physiological implications for membrane docking. The burial of W234 is accompanied by a cascade of side-chain flips that effect the intrusion of the side-chain of I238 into the ligand-binding cavity. As consequence of the intrusion, R234W displays 5-fold increased resistance to light-induced photoisomerization relative to wild-type CRALBP, indicating tighter binding to 11-cis-retinal. Overall, our results reveal an unanticipated domino-like structural transition causing Bothnia-type retinal dystrophy by the impaired release of 11-cis-retinal from R234W.

Topics: Amino Acid Substitution; Binding Sites; Carrier Proteins; Crystallography, X-Ray; Humans; Isomerism; Ligands; Light; Mutant Proteins; Mutation, Missense; Protein Structure, Secondary; Retinal Diseases; Retinitis Pigmentosa; Retinoids; Time Factors

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

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

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

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches.. To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, GRM6, NYX, PDE6B, and SAG and spotted on the chip.. Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations.. This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects.

Topics: Adolescent; Calcium Channels, L-Type; Calcium-Binding Proteins; Child; Cyclic Nucleotide Phosphodiesterases, Type 6; DNA Mutational Analysis; Eye Proteins; Female; Gene Expression Profiling; Genotype; Heterotrimeric GTP-Binding Proteins; Humans; Male; Mutation; Night Blindness; Oligonucleotide Array Sequence Analysis; Pedigree; Polymerase Chain Reaction; Proteoglycans; Receptors, Metabotropic Glutamate; Retinal Diseases; Rhodopsin; Transducin

Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations.. Metabolic labeling with 33P-orthophosphate was used to study phosphorylation of NRL in transfected COS-1 cells. NRL or NRL mutants were expressed as glutathione S-transferase (GST)-fusion proteins and used as substrate to screen various kinases by in vitro phosphorylation assays. CV-1 cells were co-transfected with rhodopsin promoter-reporter construct and expression plasmids, with or without specific mitogen-activated protein kinase (MAPK) inhibitors, to examine their effect on NRL-mediated transactivation. Expression of activated MAPKs in postnatal mice retina was determined by immunoblot analysis.. Metabolic labeling of NRL produces multiple phosphorylated protein bands in transfected COS-1 cells. Fewer but more intense radiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutants compared to wild-type NRL. We show that MAPK2 and p38 induce specific phosphorylation of NRL, but this pattern is altered in NRL mutants. Immunoblot analysis of extracts from developing mouse retina reveals enhanced expression of activated MAPK2 at postnatal day 0-3, concordant with the reported phosphorylation pattern of NRL in vivo. Inhibition of MAPK signaling pathways decreases NRL and CRX-mediated synergistic activation of rhodopsin promoter in transfected CV-1 cells.. Our results suggest that multiple MAPKs can phosphorylate NRL and this phosphorylation pattern is altered by disease-associated NRL mutations. As inhibition of MAPK signaling pathways decreases NRL-mediated transactivation of rhodopsin promoter, we propose that phosphorylation changes associated with NRL mutations perturb gene expression in rods, leading to photoreceptor degeneration in retinopathies.

Topics: Animals; Animals, Newborn; Chlorocebus aethiops; COS Cells; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Leucine Zippers; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Promoter Regions, Genetic; Retina; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Transcriptional Activation; Transfection

Retinol dehydrogenase 12 (RDH12) is an NADP(+)-dependent oxidoreductase that in vitro catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol or the oxidation of retinol to retinaldehyde depending on substrate and cofactor availability. Recent studies have linked the mutations in RDH12 to severe early-onset autosomal recessive retinal dystrophy. The biochemical basis of photoreceptor cell death caused by mutations in RDH12 is not clear because the physiological role of RDH12 is not yet fully understood. Here we demonstrate that, although bi-directional in vitro, in living cells, RDH12 acts exclusively as a retinaldehyde reductase, shifting the retinoid homeostasis toward the increased levels of retinol and decreased levels of bioactive retinoic acid. The retinaldehyde reductase activity of RDH12 protects the cells from retinaldehyde-induced cell death, especially at high retinaldehyde concentrations, and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. Disease-associated mutants of RDH12, T49M and I51N, exhibit significant residual activity in vitro, but are unable to control retinoic acid levels in the cells because of their dramatically reduced affinity for NADPH and much lower protein expression levels. These results suggest that RDH12 acts as a regulator of retinoic acid biosynthesis and protects photoreceptors against overproduction of retinoic acid from all-trans-retinaldehyde, which diffuses into the inner segments of photoreceptors from illuminated rhodopsin. These results provide a novel insight into the mechanism of retinal degeneration associated with mutations in RDH12 and are consistent with the observation that RDH12-null mice are highly susceptible to light-induced retinal apoptosis in cone and rod photoreceptors.

Topics: Alcohol Oxidoreductases; Amino Acid Substitution; Animals; Apoptosis; Gene Expression Regulation, Enzymologic; Genetic Diseases, Inborn; Homeostasis; Humans; Light; Macaca mulatta; Mice; Mice, Mutant Strains; Mutation, Missense; NADP; Oxidation-Reduction; Photoreceptor Cells; Retinal Diseases; Retinaldehyde; Rhodopsin; Tretinoin

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

To investigate retinal involvement in chronic Chagas' disease, we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency, under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors, all related to the same subfamily of G-protein-coupled receptors.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adult; Amino Acid Sequence; Animals; Antibodies, Protozoan; Autoimmune Diseases; Cattle; Chagas Disease; Cross Reactions; Electroretinography; Female; Fluorescein Angiography; Humans; Immunoglobulin G; Male; Middle Aged; Molecular Mimicry; Molecular Sequence Data; Reaction Time; Receptors, Adrenergic, beta-1; Retinal Diseases; Retinal Rod Photoreceptor Cells; Rhodopsin; Rod Cell Outer Segment; Signal Transduction; Trypanosoma cruzi

Metastatic melanoma still has a very poor prognosis since it withstands conventional therapies like surgery or chemotherapy. A paraneoplastic autoimmune manifestation of this disease is melanoma-associated retinopathy (MAR). MAR has been associated with prolonged survival and may be an early marker of tumor progression. By screening a retina and a melanoma cDNA phage library by SEREX using sera of patients suffering from melanoma and, in some cases, clinical symptoms of MAR, we identified 20 new antigens (HD-MM-28-47), of which 14 clones had high homology to well-known genes. Six of these genes had previously been associated with retina: rhodopsin, visual arrestin, MEK1, SRPX, BBS1 and galectin-3. Individual clones were recognized by up to 43% of patients' sera, while sera of healthy volunteers were negative except in 2 cases. The expression profile of the antigens identified on the basis of homologous EST database entries in healthy tissues was ubiquitous to differential. Using RT-PCR, we found frequent expression of preselected antigens in melanoma cell lines. For rhodopsin, this could be quantified by quantitative PCR. Retinal proteins were recognized by serum antibodies of melanoma patients but not healthy controls. The role of these antigens in MAR awaits further investigation. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.)

Topics: Antigens, Neoplasm; Blotting, Northern; Cell Line, Tumor; DNA, Complementary; DNA, Neoplasm; Gene Library; Humans; Melanoma; Paraneoplastic Syndromes; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Neoplasm; Serologic Tests; Skin Neoplasms

The purpose of this study was to investigate the differentiation of neural progenitor cells (NPCs) following retinal transplantation in N-methyl-D-aspartate (NMDA)-treated eyes. NMDA was injected into the vitreous cavity of adult rat eyes. NPCs were prepared from telencephalic neuroepithelium of enhanced green fluorescence protein (EGFP) transgenic mice on embryonic day 14.5. A cell suspension was injected into the vitreous cavity in experimental eyes. Immunohistochemistry was conducted at 1, 2 or 4 weeks after transplantation of NPCs in an effort to determine the survival and differentiation of transplanted NPCs. Similar experiments were conducted using glycoprotein (gp)130-null (-/-) mice. Examination of retinal sections revealed that transplanted NPCs could survive for at least 4 weeks in NMDA-treated retinas. Immunohistochemical studies for specific cell-type markers revealed that, among the transplanted NPCs at 2 weeks after transplantation, the mean percentage (+/-standard deviation) of glial fibrillary acidic protein (GFAP)-positive (glial) cells was 63.5 +/- 7.4%, demonstrating the differentiation of transplanted NPCs with a preference for the glial lineage. Furthermore, the mean percentage of betaIII-tubulin-positive (mature neuronal) cells was 18.8 +/- 4.5%. Following transplantation of NPCs isolated from gp130-/- mice into NMDA-treated retinas, the mean percentage of GFAP-positive cells (17.6 +/- 7.0%), was significantly lower than that in NPCs isolated from wild-type mice (59.1 +/- 6.0%, P = 0.04, Mann-Whitney U test). Preferential differentiation of NPCs into the glial lineage is induced through gp130 signaling in NMDA-treated eyes.

Topics: Animals; Cell Count; Cell Differentiation; Cells, Cultured; Ciliary Neurotrophic Factor; Cytokine Receptor gp130; Embryo, Mammalian; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; Green Fluorescent Proteins; Immunohistochemistry; Intermediate Filament Proteins; Male; Mice; Mice, Transgenic; Microtubule-Associated Proteins; N-Methylaspartate; Nerve Tissue Proteins; Nestin; Neuroglia; Neurons; Rats; Rats, Sprague-Dawley; Retinal Diseases; Rhodopsin; Signal Transduction; Statistics, Nonparametric; Stem Cell Transplantation; Stem Cells; Time Factors; Tubulin

A novel paired-like homeobox gene, designated as Qrx, was identified by a yeast one-hybrid screen using the bovine Rhodopsin promoter Ret-1 DNA regulatory element as bait. Qrx is preferentially expressed in both the outer and inner nuclear layers of the retina. Its homeodomain is nearly identical to that of Rx/Rax, a transcription factor that is essential for eye development, but it shares only limited homology elsewhere. Although Qrx and Rx/Rax show similar DNA binding properties in vitro, the two proteins demonstrate distinct target selectivity and functional behavior in promoter activity assays. QRX synergistically increases the transactivating function of the photoreceptor transcription factors Crx and NRL and it physically interacts with CRX. Qrx is present in the bovine and human genomes, but appears to be absent from the mouse genome. Nonetheless, a 5.8 kb upstream region of human QRX is capable of directing expression in presumptive photoreceptor precursor cells in transgenic mice. These results indicate that Qrx may be involved in modulating photoreceptor gene expression. In addition, the finding of rare heterozygous QRX sequence changes in three individuals with retinal degeneration raises the possibility that QRX may be involved in disease pathogenesis.

Topics: Amino Acid Sequence; Animals; Basic-Leucine Zipper Transcription Factors; Cattle; Chromosome Mapping; Chromosomes, Human, Pair 9; Coatomer Protein; DNA Footprinting; DNA-Binding Proteins; Eye Proteins; Gene Expression Regulation; Genes, Homeobox; Homeodomain Proteins; Humans; Molecular Sequence Data; Point Mutation; Promoter Regions, Genetic; Protein Binding; Response Elements; Retinal Diseases; Rhodopsin; Sequence Alignment; Trans-Activators; Transcription Factors

Mutations in rhodopsin, the visual pigment found in rod cells, account for a large fraction of genetic changes underlying the human retinal diseases, Retinitis Pigmentosa (RP). The availability of rhodopsin sequences from a large number of vertebrates has allowed us to investigate factors important in the development of RP by contrasting interspecific differences (long-term evolutionary patterns) with RP disease mutation data. We find that disease mutations in rhodopsin are overabundant in highly conserved sites and that amino acid positions with any potential of variability among vertebrates are likely to harbour disease mutations less frequently. At any amino acid position in rhodopsin, the set of disease-associated amino acids does not show any commonality with the set of amino acids present among species. The disease mutations are biochemically four times more radical than the interspecific (neutral) variation. This pattern is also observed when disease mutations are categorized based on clinical classifications that reflect biochemical, physiological and psychophysical traits such as protein folding, cone electroretinogram (ERG) amplitude, pattern of visual field loss, and equivalent field diameter. We also found that for artificial mutations (those not observed in nature interspecifically), there was a positive relationship between the biochemical distance and the magnitude of blue shift in the absorption spectrum maximum. We introduce the concept of the expected chemical severity based on the normal human codon at a position. Results reveal that the analysis of disease mutations in the context of the original codon is very important for the practical application of evolutionary principles when comparing original and disease amino acid mutations. We conclude that the analysis of rhodopsin data clearly demonstrates the usefulness of molecular evolutionary analyses for understanding patterns of clinical as well as artificial mutations and underscores the biomedical insights that can be gained by using simple measures of biochemical difference in the context of evolutionary divergence.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Codon; Conserved Sequence; Evolution, Molecular; Gene Frequency; Genetic Variation; Genotype; Humans; Molecular Sequence Data; Mutation; Phenotype; Phylogeny; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Species Specificity

Psychophysical and electroretinographic (ERG) studies indicate that patients with Stargardt disease exhibit abnormally slow rod dark adaptation after illumination that bleaches a substantial fraction of rhodopsin. However, relatively little information is available concerning rod recovery in this disease after weaker adapting (i.e., conditioning) light. With the use of a paired-flash ERG method, properties of the derived rod response to a low-bleach (<1%) but rod-saturating conditioning flash were investigated in seven normal subjects and in five Stargardt patients with identified sequence variations in the ABCA4 gene.. In the first of two experiments, the interval between a fixed conditioning flash (67 or 670 scotopic cd s m(-2)) and a bright probe flash of fixed strength was varied to determine the falling-phase kinetics of the derived rod response to the conditioning flash. In the second, the instantaneous amplitude-intensity function for the rod response at an intermediate stage of recovery from the conditioning flash was determined by presenting a test flash of various strengths at a fixed time after the conditioning flash, and a probe flash at 200 ms after the test flash.. The maximum peak amplitude of the dark-adapted, rod-mediated a-wave determined in Stargardt patients (211 +/- 87 microV) was on average lower than that determined in normal subjects (325 +/- 91 microV; P = 0.06). The derived rod response to the 670 scotopic cd s m(-2) conditioning flash determined in normal subjects and Stargardt patients exhibited a biphasic recovery, and the kinetics of the early stage of this recovery were similar in the two subject groups. For both normal subjects and patients, normalized amplitude-intensity functions describing the dark-adapted derived rod response exhibited half-saturation at approximately 1.5 log scotopic troland second. In both groups, the normalized amplitude-intensity function determined at approximately 2 seconds after the 67 scotopic cd s m(-2) conditioning flash and at approximately 9 seconds after the 670 scotopic cd s m(-2) conditioning flash exhibited an average desensitization (i.e., an increase of test flash strength at half-saturation) of approximately 0.5 to 0.6 log unit relative to that determined under dark-adapted conditions.. The results indicate that, despite a reduction in the average dark-adapted maximum a-wave amplitude in the Stargardt/ABCA4 patients, the early-stage recovery kinetics of the derived rod response to a low-bleaching conditioning flash as well as the lingering rod desensitization produced by such a flash are similar to those determined in normal subjects.

Topics: Adult; ATP-Binding Cassette Transporters; Dark Adaptation; Electroretinography; Female; Humans; Male; Middle Aged; Mutation; Photic Stimulation; Retinal Diseases; Retinal Rod Photoreceptor Cells; Rhodopsin; Vitamin A

Topics: Humans; Retinal Diseases; Retinoids; Rhodopsin; Vision, Ocular

The natural ligand of the retinal photoreceptor rhodopsin, 11-cis-retinal, is isomerized to its all-trans configuration as a consequence of light absorption in the first step of the visual phototransduction process. Here we show, by means of difference spectroscopy and high-performance liquid chromatography analysis, that thermal denaturation of rhodopsin induces the same type of isomerization. This effect is likely due to thermally induced conformational rearrangements of amino acid residues in the retinal-binding pocket--possibly implying helical movements--and highlights the tight coupling between 11-cis-retinal and opsin. This effect could have implications in the instability and functional changes seen for certain mutations in rhodopsin associated with retinal disease, and in the stability of the different conformers induced by mutations in other G protein-coupled receptors.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Hot Temperature; Night Blindness; Protein Denaturation; Receptors, G-Protein-Coupled; Retinal Diseases; Retinaldehyde; Rhodopsin; Spectrum Analysis; Stereoisomerism

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.

Topics: Amacrine Cells; Animals; Astrocytes; Calbindins; Cell Differentiation; Cell Movement; Glial Fibrillary Acidic Protein; Graft Survival; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Immunohistochemistry; Leukocyte Common Antigens; Male; Neurons; Rats; Retina; Retinal Diseases; Retinal Rod Photoreceptor Cells; Rhodopsin; S100 Calcium Binding Protein G; Vimentin

To identify and characterize the lineage potential of rat neural retina progenitor cells (NRPCs) in vitro and engrafted into rats with retinal degeneration, NRPCs were isolated from neural retinas of embryonic day 17 Long Evans rats and cultured in serum-free or serum-containing media with fibroblast growth factor 2 and neurotrophin 3. After expansion, cellular differentiation was initiated by the withdrawal of these growth factors. Despite forming primary neurospheres, NRPCs cultured in serum-free medium survived poorly after passage. In contrast, NRPCs cultured in serum-containing medium could be expanded for up to 12 passages and differentiated into glial fibrillary acidic protein-positive glial cells and retina-specific neurons expressing rhodopsin, S-antigen, calbindin, recoverin, and calretinin. For in vivo analysis, passage 1 (P1) undifferentiated NRPCs were labeled with bromodeoxyuridine (BrdU), implanted into the subretinal space of Royal College of Surgeons (RCS) rats, and analyzed immunohistochemically 4 weeks postgrafting. The grafted NRPCs showed extensive glial differentiation, irrespective of their topographic localization. A few BrdU-labeled grafted NRPCs expressed protein kinase C, a marker for bipolar and amacrine interneuron-specific differentiation. Other retina-specific or oligodendrocytic differentiation was not detected in the grafted cells. Although NRPCs are capable of self-renewal and multilineage differentiation in vitro, they developed mostly into glial cells following engraftment into the adult retina. These data suggest that the adult retina retains epigenetic signals that are either restrictive for neuronal differentiation or instructive for glial differentiation. Induction of lineage-specific cell differentiation of engrafted NRPCs to facilitate retinal repair will likely require initiation of specific differentiation in vitro prior to grafting and/or modification of the host environment concomitantly with NRPC grafting.

Topics: Animals; Arrestin; Blood Proteins; Brain Tissue Transplantation; Calcium-Binding Proteins; Cell Differentiation; Cell Lineage; Cells, Cultured; Culture Media, Serum-Free; Female; Fetus; Glial Fibrillary Acidic Protein; Graft Survival; Neuroglia; Neurons; Pregnancy; Rats; Rats, Long-Evans; Retina; Retinal Diseases; Rhodopsin; Spheroids, Cellular; Stem Cell Transplantation; Stem Cells

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects retinal neurons from ischemia-reperfusion injury. Rats were injected intraperitoneally with either vehicle or alpha-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram was measured prior to ischemia and 5 days after reperfusion. Rats were killed 5 or 8 days after reperfusion and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia-reperfusion caused a significant reduction of the a- and b-wave amplitudes of the electroretinogram, a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease of retinal ganglion cell-specific mRNAs and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by alpha-lipoic acid. Moreover, in mixed rat retinal cultures, alpha-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that alpha-lipoic acid provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia-reperfusion injuries. alpha-Lipoic acid also displayed negligible affinity for voltage-dependent sodium and calcium channels.

Topics: Anesthetics, Local; Animals; Antioxidants; Binding, Competitive; Brain-Derived Neurotrophic Factor; Calcium; Calcium Channel Blockers; Cells, Cultured; Ciliary Neurotrophic Factor; Diltiazem; Dizocilpine Maleate; DNA Primers; Dose-Response Relationship, Drug; Drug Interactions; Electroretinography; Fibroblast Growth Factors; Glial Fibrillary Acidic Protein; Glyceraldehyde-3-Phosphate Dehydrogenases; N-Methylaspartate; Nifedipine; Rats; Reperfusion Injury; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Sodium; Tetrodotoxin; Thioctic Acid; Thy-1 Antigens; Veratridine

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

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

It was recently found that recoverin acts as an autoantigen recognized by sera of patients with cancer-associated retinopathy (CAR), and that CAR-like retinal dysfunction is produced by intravitreous administration of anti-recoverin antibody in Lewis rat eyes. To examine the pathologic molecular mechanism of CAR, and to elucidate an effective therapy for CAR, the function and morphology of CAR were compared with those of phototoxic retinal damage, another form of photoreceptor dysfunction, and the effect of nilvadipine, a Ca(2+) antagonist, on the retinal degenerations was studied, using these models.. Under different illumination conditions and/or medication with nilvadipine, the functional and morphologic properties of the retinas were evaluated after intravitreous injection of anti-recoverin antibody into Lewis rat eyes (six rats, 12 eyes in each experimental condition), using electroretinogram (ERG), rhodopsin phosphorylation, and light microscopy.. Anti-recoverin antibody administered into the vitreous of Lewis rat eyes induced a significant decrease and increase of ERG responses and rhodopsin phosphorylation levels, respectively, under cyclic or continuous light. Similar changes were observed in eyes of rats bred under continuous illumination that did not receive anti-recoverin antibodies. However, anti-recoverin antibody-induced retinal dysfunctions were not observed in rat eyes under dark conditions. Administration of nilvadipine, a Ca(2+) antagonist, to the anti-recoverin antibody-treated rats and rats with phototoxic retinal dysfunction caused significant improvement of the deterioration of ERG and normalization of rhodopsin phosphorylation.. The present data indicate that anti-recoverin antibody-induced retinal dysfunction was functionally similar to phototoxic retinal dysfunction and was markedly suppressed under dark conditions or by systemic administration of a Ca(2+) antagonist.

Topics: Animals; Antibodies; Antigens, Neoplasm; Calcium Channel Blockers; Calcium-Binding Proteins; Dark Adaptation; Electroretinography; Eye Proteins; Hippocalcin; Injections; Injections, Intraperitoneal; Light; Lipoproteins; Nerve Tissue Proteins; Nifedipine; Paraneoplastic Syndromes; Phosphorylation; Photoreceptor Cells, Vertebrate; Rats; Rats, Inbred BN; Rats, Inbred Lew; Recoverin; Retinal Diseases; Rhodopsin; Vitreous Body

Topics: Electroretinography; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells, Vertebrate; Retinal Diseases; Rhodopsin; Signal Transduction

Several ophthalmic conditions manifest a flecked retina. Developing an understanding of their clinical presentations will enable the practitioner to most appropriately manage these conditions.. A 27-year-old Middle Eastern woman manifested flecked retinas and nyctalopia. She had been given a diagnosis of retinitis punctata albescens, an inherited, progressive, night blindness; however, the medical history and clinical findings were not consistent with this disorder. Rather, they were consistent with fundus albipunctatus, an autosomal recessive, stationary, night blindness. The clinical presentation of fundus albipunctatus is characterized by discrete, white dots at the level of the retinal pigment epithelium and stable night blindness. A prolonged time for dark adaptation is required to produce normal amplitude electroretinograms in fundus albipunctatus as the result of a delay in the regeneration of rhodopsin. An electroretinogram administered after a prolonged dark adaptation time confirmed the diagnosis of stationary night blindness.. In order to ensure an accurate diagnosis for fundus albipunctatus, it is important to be aware of the clinical characteristics and appropriate electroretinogram protocol for this disorder.

Topics: Adult; Dark Adaptation; Diagnosis, Differential; Electroretinography; Eye Diseases, Hereditary; Female; Fundus Oculi; Humans; Night Blindness; Retinal Diseases; Retinal Rod Photoreceptor Cells; Rhodopsin; Syndrome

The histologic manifestations of rhodopsin-mediated versus short-wavelength classes of retinal phototoxicity were compared after spectral exposures of the albino rat retina. Animals were exposed to wave-bands of light centered at the peak of rhodopsin absorbance (green, 500 nm) or in the ultraviolet A (UVA; 360 nm). Intensity-damage curves generated for each wave-band indicated that UVA light was 50-80 times more effective than green light at causing photoreceptor cell losses. Examination of early ultrastructural changes in rod inner segments, outer segments, and retinal pigment epithelium revealed a remarkable degree of similarity between UVA and green light-induced damage. Furthermore, the two classes of damage were indistinguishable in terms of post-exposure recovery from threshold damage and regional distribution of photoreceptor cell loss along the vertical meridian. The finding of essentially identical histologic manifestations for the two classes of damage raises the possibility that they share a common biochemical etiology or pathway of cell destruction.

Topics: Animals; Differential Threshold; Dose-Response Relationship, Radiation; Female; Light; Photoreceptor Cells; Pigment Epithelium of Eye; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retinal Diseases; Rhodopsin; Ultraviolet Rays

There is considerable variety among the clinical features of autosomal dominant retinitis pigmentosa (ADRP). This is probably at least in part due to genetic heterogeneity. Recently, various mutations of the rhodopsin gene have been detected in some ADRP families. We report on six patients from two families with ADRP who were investigated by means of psychophysical and electrophysiological methods. All displayed the same rhodopsin gene mutation at codon 347, which exchanges the amino acid proline for leucine (pro-347-leu). The patients had early-onset night blindness and impaired side vision as of the end of their second life decade. They produced monophasic dark-adaptation curves, showing a lack of rod function and elevated cone thresholds. Dark-adapted two-color threshold perimetry using 500- and 650-nm stimuli revealed a diffuse loss of rod function and centrally preserved cone function. The electroretinogram was nonrecordable at the age of about 30 years. A certain variability of visual function loss was noted among patients in the overall severe course of the disease, but the clinical findings of this genotype corresponded to type 1 ADRP of Massof and Finkelstein in all cases.

Topics: Adult; Aged; Dark Adaptation; DNA Mutational Analysis; Electroretinography; Female; Fundus Oculi; Humans; Leucine; Male; Middle Aged; Mutation; Pedigree; Photoreceptor Cells; Proline; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Visual Fields

The protective effect of dimethylthiourea (DMTU) against retinal light damage was determined in albino rats reared in darkness or in weak cyclic light. Rats maintained under these conditions were treated with DMTU at different concentrations and dosing schedules and then exposed for various times to intense visible light, either intermittently (1 hr light and 2 hr dark) or continuously. The extent of retinal light damage was determined 2 weeks after light exposure by comparing rhodopsin levels in experimental rats with those in unexposed control animals. To determine the effect of DMTU on rod outer segment (ROS) membrane fatty acids, ROS were isolated immediately after intermittent light exposure, and fatty acid compositions were measured. The time course for DMTU uptake and its distribution in serum, retina, and the retinal pigment epithelium (RPE)/choroid complex was determined in other rats not exposed to intense light. After intraperitoneal injection of the drug (500 mg/kg body weight), DMTU appeared rapidly in the serum, retina, and the RPE and choroid. In the ocular tissues, it was distributed 70-80% in the retina and 20-30% in the RPE and choroid. This antioxidant appears to have a long half-life because it was present in these same tissues 72 hr after a second intraperitoneal injection. For rats reared in the weak cyclic light environment, DMTU (two injections) provided complete protection against rhodopsin loss after intense light exposures of up to 16 hr. Only 15% rhodopsin loss was found in cyclic-light DMTU-treated rats after 24 hr of intermittent or continuous light. For rats reared in darkness, DMTU treatment resulted in a rhodopsin loss of less than 20% after 8-16 hr of continuous light and approximately 40% after similar exposure to intermittent light. Irrespective of the type of light exposure, rhodopsin loss in the dark-reared DMTU-treated rats was nearly identical to that found in uninjected cyclic light-reared animals. In rats from both light-rearing environments, DMTU treatment prevented the light-induced loss of docosahexaenoic acid from ROS membranes. As measured by rhodopsin levels 2 weeks later, DMTU was most effective when given as two doses administered 24 hr before and just before intense light exposure. As a single dose given during continuous light exposure, DMTU protected cyclic light-reared rats for at least 4 hr after the start of exposure but was ineffective in dark-reared animals if injected 1 hr after the start of light

Topics: Animals; Choroid; Circadian Rhythm; Dark Adaptation; Fatty Acids; Light; Male; Pigment Epithelium of Eye; Rats; Rats, Inbred Strains; Retina; Retinal Diseases; Rhodopsin; Rod Cell Outer Segment; Thiourea

Retinal light damage in dark-reared rats supplemented with ascorbic acid and exposed to multiple doses of intermittent light was studied and compared with damage in unsupplemented dark-reared and cyclic-light-reared rats. The extent of photoreceptor cell loss from intense light exposure was determined by whole-eye rhodopsin levels and retinal DNA measurements two weeks after light treatment. Two weeks after 3 or 8 hr of intermittent light, ascorbate-supplemented animals had rhodopsin and retinal DNA levels that were two to three times higher than in unsupplemented dark-reared rats. In both types of rats rhodopsin levels were influenced by the number of light doses, the duration of light exposure, and to a lesser extent, by the length of the dark period between exposures. Rhodopsin levels in the dark-reared ascorbate-supplemented rats were significantly higher than in unsupplemented dark-reared rats, and were similar to the levels in unsupplemented cyclic-light-reared animals. Ascorbate treatment had no effect on the rate of rhodopsin bleaching. However, regeneration was greater in supplemented rats after multiple 1-hr light exposures. Intermittent light also resulted in lower ascorbate levels in the retinas of supplemented and unsupplemented rats, with dramatic losses from the retinal pigment epithelium (RPE)-choroid in both types of animals. We conclude that ascorbic acid protects the eye by reducing the irreversible Type I form of light damage in dark-reared rats. Ascorbate appears to shift light damage to the Type II form typical of cyclic-light-reared animals.

Topics: Animals; Ascorbic Acid; Darkness; DNA; Dose-Response Relationship, Radiation; Eye; Light; Male; Periodicity; Photoreceptor Cells; Radiation-Protective Agents; Rats; Rats, Inbred Strains; Regeneration; Retina; Retinal Diseases; Rhodopsin

A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.

Topics: Animals; Antibody Specificity; Autoantibodies; Autoimmune Diseases; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Immunoblotting; Photoreceptor Cells; Rabbits; Retinal Diseases; Retinal Pigments; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Sodium Dodecyl Sulfate

A healthy, 14-year-old girl presented with nyctalopia, good vision, and multiple, irregular, yellowish lesions of the fundus. Dark adaptometry showed prolonged cone and rod branches, elevated thresholds, and the cone-rod transition occurring after 50 minutes in darkness. Her scotopic electroretinogram (ERG) b-wave attained normal amplitudes after 45 minutes of dark adaptation. The half-time for regeneration of rhodopsin after an extensive bleach was 16 minutes, four times longer than normal, and the maximum density difference measured by fundus reflectometry was at the lower limit of the normal range. Although photopigment kinetics were significantly faster than observed in other reported cases of fundus albipunctatus, it appears likely that there is a wide spectrum of functional and funduscopic abnormalities in this disorder. However, fundus appearance, adaptometric findings, and rhodopsin determinations serve to distinguish fundus albipunctatus from other flecked retina diseases.

Topics: Adolescent; Dark Adaptation; Electroretinography; Female; Fluorescein Angiography; Fundus Oculi; Humans; Night Blindness; Retinal Diseases; Rhodopsin

Patients with acute and chronic central serous retinopathy (CSR) were studied by psychophysical and photochemical means to establish the extent of visual depression and to investigate the basis of rod dysfunction in this disorder. In acute disease with serous detachment of the retina, the loss of sensitivity attains 3 log units and parallels the height of retinal elevation as does its recovery with resolution of the episode. Immediately after resolution, there is a residual 0.5 log unit threshold elevation. In chronic disease, marked loss of function exists over areas of abnormal retinal pigment epithelium in the absence of clinically detectable serous detachment. Although rhodopsin levels are low in both acute and chronic CSR, this relative lack of visual pigment does not totally account for the functional deficits in either situation.

Topics: Fluorescein Angiography; Humans; Photoreceptor Cells; Retina; Retinal Detachment; Retinal Diseases; Retinitis; Rhodopsin; Visual Perception

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.

Topics: Aging; Animals; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Fluorescent Antibody Technique; Immune Sera; Molecular Weight; Rats; Rats, Mutant Strains; Retina; Retinal Diseases; Retinol-Binding Proteins; Rhodopsin

The presence and localization of autoantibodies was determined in strain 13 guinea pigs with experimental retinal autoimmunity (ERA) induced by immunization with rhodopsin and rod outer segments (ROS). Sera were obtained from rhodopsin-immunized and from ROS-immunized guinea pigs before, during, and after onset of clinical uveitis. Autoantibodies were detected by indirect immunofluorescent staining of autogenic retinas as well as normal guinea pig retinas. Sera from animals with clinical disease showed specific labeling of the photoreceptor cell layer of the retina. The rhodopsin autoantibody showed a more defined specificity than the ROS autoantibody staining, only the retinal photoreceptors and retinal pigment epithelium. Specific fluorescence was localized only in the retina, and not in any other ocular or nonocular tissues. Neither the rhodopsin nor the ROS antibodies stained the uvea. Sera from animals taken before the onset of clinical disease did not demonstrate the presence of retinal-binding autoantibodies. These findings suggest that photoreceptor-binding autoantibodies appear in the sera of animals immunized with rhodopsin and with ROS, but only in animals with clinical disease. However, these antibodies probably are not the primary cause of pathology, since previous passive transfer experiments (data not shown here) could not be achieved with anti-ROS or with anti-rhodopsin antibodies. These autoantibodies could occur secondarily as a response to the bovine antigens which cross-reacted with the autologous guinea pig antigens. Subsequently these antibodies could be of primary importance in further tissue alteration and destruction.

Topics: Animals; Autoantibodies; Autoimmune Diseases; Fluorescent Antibody Technique; Guinea Pigs; Photoreceptor Cells; Retinal Diseases; Rhodopsin

Strain 13 guinea pigs injected with either homologous or bovine rhodopsin, the visual pigment of the retinal outer segments, in complete Freund's adjuvant (CFA) developed experimental retinal autoimmunity (ERA). Initial clinical signs of disease were seen within 21 days after immunization. Pathologic examination of the eyes revealed the presence of inflammatory cells in the choroid and the destruction of the retinal rod outer segments. The unique feature of this disease is that despite the destruction of the inner and outer segments of the retina, at no time is there a substantial inflammatory cell infiltrate. Even as late as 45 days after immunization, when destruction of the retinal ganglion cell layer was noted, no inflammatory cells were detected in the retina. These findings suggest that the retinal inner and outer segments are the target of the autoimmune reaction subsequent to sensitization with purified rhodopsin-CFA.

Topics: Animals; Antigens; Autoimmune Diseases; Cattle; Dose-Response Relationship, Immunologic; Female; Freund's Adjuvant; Guinea Pigs; Herpes Simplex; Male; Retina; Retinal Diseases; Retinal Pigments; Rhodopsin

Night blindness is a frequent concomitant of retinal disorders, many of which are of genetic origin. Through the use of quantitative noninvasive test procedures it has been possible to study patients with these hereditary conditions and to show that the visual abnormalities often result from defects in the functional properties of the rod photoreceptors. More important, the uniqueness of the functional disturbance in the various types of night-blinding disorders suggests that each involves a specific aspect of the rod's internal machinery, i.e., the molecular processes devoted to transduction, intercellular communication, and the renewal of cellular components. Knowledge gained from the study of these clinical entities and from the investigation of experimental animals regarding the cellular events involved in these vital processes have enabled us to formulate tentative hypothesis as to the molecular bases of the hereditary defects.

Topics: Genetic Diseases, Inborn; Horseradish Peroxidase; Humans; Neuromuscular Junction; Neurotransmitter Agents; Night Blindness; Photoreceptor Cells; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Synapses; Synaptic Transmission

The levels of rhodopsin and opsin were investigated in relation to the maintenance of retinal structure in retinas of vitamin A--deficient rats in low levels of cyclic illumination (1.5 to 2 foot-candles). Rhodopsin levels decreased in the deficient retinas to approximately 20% of control at 9 weeks, and this level was retained through 39 weeks on the deficient diet. Opsin levels decreased at a slower rate but reached about 20% of control levels at 32 weeks. Despite the decrease in rhodopsin levels, obvious deterioration of disc structure was not observed until 16 weeks of deficiency, when opsin levels had already decreased to 60% to 70% of control. The structural disruption of photoreceptor outer segments was localized initially in discs of the distal third. Rod cell degeneration preceded cone cell degeneration in vitamin A--deficient retinas. Most of the rods and cones persisted in the posterior retina at 23 weeks on the deficient diet; however, by 40 weeks, only 11% of the rod nuclei remained. In contrast, about 63% of the cone nuclei were present at 40 weeks of deficiency. The photoreceptor cells were affected by the deficiency to a greater extent in the inferior hemisphere than in the superior hemisphere of the eye.

Topics: Animals; Female; Male; Photoreceptor Cells; Pregnancy; Rats; Retina; Retinal Diseases; Retinal Pigments; Rhodopsin; Vitamin A Deficiency

Systemic immunization with purified homologous rhodopsin from retinal outer segments induced blindness in primates (Macaca mulatta). Inflammation and characteristic retinal changes were the earliest clinical signs of the disease. Perivasculitis, subretinal exudations and bullous detachments of the retina were progressive and unrelenting pathological processes leading to rapid and irreversible visual deterioration. Electroretinographic responses (ERG) at this stage of the disorder became abolished. Antibodies and delayed hypersensitivity to rhodopsin were demonstrated only in the experimental diseased animals. Homologous visual purple appears to be organ and immunopathologically specific. Histological confirmation of these findings showed a pathological spectrum of destructive alterations confirmed specifically to the outer segments of the entire retina. The pathologic reaction was supported by a distinct and pronounced granulomatous inflammatory response.

Topics: Animals; Blindness; Electroretinography; Female; Haplorhini; Immunization; Macaca mulatta; Male; Pigment Epithelium of Eye; Retinal Diseases; Retinal Pigments; Rhodopsin; Uveitis

Retinal dystrophies, known in man, dog, mouse, and rat, involve progressive loss of photoreceptor cells with onset during or soon after the developmental period. Functional (electroretinogram), chemical (rhodopsin analyses) and morphological (light and electron microscopy) data obtained in the rat indicated two main processes: (a) overproduction of rhodopsin and an associated abnormal lamellar tissue component, (b) progressive loss of photoreceptor cells. The first abnormality recognized was the appearance of swirling sheets or bundles of extracellular lamellae between normally developing retinal rods and pigment epithelium; membrane thickness and spacing resembled that in normal outer segments. Rhodopsin content reached twice normal values, was present in both rods and extracellular lamellae, and was qualitatively normal, judged by absorption maximum and products of bleaching. Photoreceptors attained virtually adult form and ERG function. Then rod inner segments and nuclei began degenerating; the ERG lost sensitivity and showed selective depression of the a-wave at high luminances. Outer segments and lamellae gradually degenerated and rhodopsin content decreased. No phagocytosis was seen, though pigment cells partially dedifferentiated and many migrated through the outer segment-debris zone toward the retina. Eventually photoreceptor cells and the b-wave of the ERG entirely disappeared. Rats kept in darkness retained electrical activity, rhodopsin content, rod structure, and extracellular lamellae longer than litter mates in light.

Topics: Animals; Disease; Dogs; Electrons; Electroretinography; Humans; Light; Male; Mice; Microscopy; Microscopy, Electron; Photoreceptor Cells; Rats; Retina; Retinal Diseases; Retinal Dystrophies; Rhodopsin; Vision, Ocular

Topics: Light; Pharmaceutical Solutions; Photobiology; Retinal Diseases; Rhodopsin; Solutions