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Inherited retinal diseases encompass a large group of clinically and genetically heterogeneous diseases estimated to affect two million people worldwide. Among these people, approximately 80,000 are or will become blind in their first decades of life due to mutations in both alleles of the Crumbs homologue-1 (CRB1) gene. Microglia are the resident immune surveyor cells in the retina, and their roles have been heavily studied in several retinal diseases, including retinitis pigmentosa (RP), age-related macular degeneration, and diabetic retinopathy. However, very little is known about the role of microglia in CRB1-associated retinopathies. Thus, we here summarize the main findings described in the literature concerning inflammation and the role of microglia in CRB1-patients and CRB1-rodent models.

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to

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

Retinal degenerations, including age-related macular degeneration and the retinitis pigmentosa family of diseases, are among the leading causes of legal blindness in the United States. We previously found that Stanniocalcin-1 (STC-1) reduced photoreceptor loss in the S334ter-3 and Royal College of Surgeons rat models of retinal degeneration. The results were attributed in part to a reduction in oxidative stress. Herein, we tested the hypothesis that long-term delivery of STC-1 would provide therapeutic rescue in more chronic models of retinal degeneration. To achieve sustained delivery, we produced an adeno-associated virus (AAV) construct to express STC-1 (AAV-STC-1) under the control of a retinal ganglion cell targeting promoter human synapsin 1 (hSYN1). AAV-STC-1 was injected intravitreally into the P23H-1 and S334ter-4 rhodopsin transgenic rats at postnatal day 10. Tissues were collected at postnatal day 120 for confirmation of STC-1 overexpression and histologic and molecular analysis. Electroretinography (ERG) was performed in a cohort of animals at that time. Overexpression of STC-1 resulted in a significant preservation of photoreceptors as assessed by outer nuclear thickness in the P23H-1 (P&nbsp

The P23H mutation within the rhodopsin gene ( RHO ) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2α and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2α, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

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

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

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

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

Past studies have shown that acute administration of ciliary neurotrophic factor (CNTF) can prolong the survival of retinal photoreceptor cells that have undergone phototoxic injury or that express gene mutations. Adenovirus-vectored CNTF has also been effective but for all of these treatments, the effect has been transient. On the other hand, adeno-associated virus-vectored minigenes offer considerable promise for long-term survival. The authors sought to provide long-term, CNTF-based protection of mouse photoreceptors expressing a dominant-negative point mutation in the rds gene by using recombinant adeno-associated virus (rAAV) to deliver minigenes that code for a secreted form of CNTF.Secreted CNTF, under control of a cytomegalovirus (CMV) or chick beta actin (CBA) promoter provided long-term, panretinal rescue of photoreceptors following single injections of rAAV vectors into the subretinal compartment. Rescue was much less effective and less reproducible when the vectors were placed in the vitreous compartment. However, there were unexpected side effects that appeared to be dose-related. One side effect was a change in rod photoreceptor nucleus phenotype, featuring an increase in euchromatin and an increase in nuclear size following subretinal injections but not intravitreal injections. These nuclear changes were panretinal when the putatively stronger CBA promoter was used but not panretinal when the CMV promoter was used. In the latter case, the nuclear changes were much more pronounced at the site of injection. Thus, chronic hyperstimulation of retinal cells with CNTF may up-regulate gene expression in photoreceptors. Based on current knowledge of retinal cell targets for CNTF, this effect may be indirect and may not represent direct stimulation of photoreceptors by CNTF.A second side effect was a paradoxical decrease in scotopic a- and b-wave amplitudes and a decrease in photopic b-wave amplitudes in the injected, rescued retina when compared to its contralateral, uninjected counterpart, in spite of the fact that these retinas had more photoreceptors than their untreated mates. The basis for these decreased ERG amplitudes may be related to changes in gene expression. The mechanisms for these side effects and proper doses of CNTF administration should be determined before human clinical trials are considered for the amelioration of inherited retinal degenerations with CNTF.

© Springer Science+Business Media, LLC 2014. Rhodopsin mutations cause many types of heritable retinitis pigmentosa (RP). Biochemical and in vitro studies have demonstrated that many RPlinked mutant rhodopsins produce misfolded rhodopsin proteins, which are prone to aggregation and retention within the endoplasmic reticulum, where they cause endoplasmic reticulum stress and activate the Unfolded Protein Response signaling pathways. Many vertebrate models of retinal degeneration have been created through expression of RP-linked rhodopsins in photoreceptors including, but not limited to, VPP/GHL mice, P23H Rhodopsin frogs, P23H rhodopsin rats, S334ter rhodopsin rats, C185R rhodopsin mice, T17M rhodopsin mice, and P23H rhodopsin mice. These models have provided many opportunities to test therapeutic strategies to prevent retinal degeneration and also enabled in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death. Here, we examine and compare the contribution of endoplasmic reticulum stress to retinal degeneration in several vertebrate models of RP generated through expression of mutant rhodopsins.

The design, delivery and preclinical testing of ribozymes specific for opsin mutations are discussed with particular focus on their use in the treatment of dominantly inherited retinal disease. There are several preliminary experimental stages that must be completed in order to maximize the chances for a therapeutic result. The first consideration of critical importance for therapy is the way the therapeutic gene is introduced into the retina and how its expression is controlled. The properties of the adeno-associated virus (AAV) as a viral vector are surveyed with emphasis on those properties best suited for ribozyme-based therapies in the retina. The second key consideration is the design and initial in vitro screening of ribozymes, since we have seen that the behavior of appropriately designed RNA enzymes in cell-free systems is a reasonable predictor of their in vivo activity in the animal. Finally, recent experimental results employing AAV-vectored ribozyme genes are summarized to provide a tangible example of how ribozyme gene therapy for an autosomal dominant retinitis pigmentosa (ADRP)-like disease in a rodent model can be approached. Here, we emphasize the need to analyze effects of the therapy at three levels, biochemical, morphological and electrophysiological, in order to gain a full appreciation of any retinal rescue and to understand the mechanism whereby it was achieved.

Vertebrate photoreceptor cells are the basic sensory apparatus of the retina, capable of converting the energy of absorbed photons into neuronal signals. The proximal portions of mammalian photoreceptor outer segments are synthesized daily by cell bodies, and outer segment tips are shed with a circadian rhythm, resulting in a complete turnover of outer segments about every 9 days. The shed outer segments are phagocytosed by adjacent retinal pigment epithelial (RPE) cells, and metabolites are recycled to photoreceptors. The Royal College of Surgeons (RCS) rat is a widely studied, classic model of recessively inherited retinal degeneration in which the RPE fails to phagocytose shed outer segments, and photoreceptor cells subsequently die. We have used a positional cloning approach to study the rdy (retinal dystrophy) locus of the RCS rat. Within a 0.3 cM genetic inclusion interval, we have discovered a small deletion of RCS DNA that disrupts the gene encoding the receptor tyrosine kinase Mertk. The deletion includes the splice acceptor site upstream of the second coding exon of Mertk and results in a shortened transcript that lacks this exon. The aberrant transcript joins the first and third coding exons, leading to a frameshift and a translation termination signal 20 codons after the AUG. The concordance of these and other data indicate that Mertk is probably the gene for rdy. Our results provide genetic evidence for an essential role of a receptor tyrosine kinase in a specialized form of phagocytosis and suggest a molecular model for ingestion of outer segments by RPE cells.

In the dystrophic Royal College of Surgeons (RCS) rat retina, there is a progressive loss of photoreceptors. As a result, the retinal circulation becomes apposed to the retinal pigment epithelium (RPE) and neovascular formations develop. RPE and inner nuclear layer cells migrate along these vessels towards the retinal ganglion cell (RGC) layer. The retinal layers gradually become disrupted, and some of the RGC axon bundles involute into the retina. These bundles are always associated with blood vessels, and there is evidence of axon damage where they juxtapose. In wholemount preparations of dystrophic retinae (≥6 months of age), abrupt changes are observed in the trajectory of RGC axon bundles, where they are crossed by circumferential vessels. Degenerative profiles can be seen at these locations. Visualisation of RGCs with Fluoro-gold shows wedge-shaped sectors in the dystrophic retina devoid of labelling, initially in the ventral retina but later spreading dorsally. It is hypothesised that the vessels supplying the neovascular formations contract and pull surface vessels into the retina, thus displacing any axon bundles that lie beneath them into the inner plexiform layer. The contractility may be an intrinsic property of the vessels or it may be conferred by the cells migrating along them. Axonal transport becomes blocked at the points of tension, thereby causing retrograde degeneration of the parent RGCs. Because RGC loss is also a feature of human retinitis pigmentosa, the RCS rat may provide a model to test interventions devised to prevent such loss following photoreceptor degeneration. This model also may be useful for testing methods designed to control blood vessel and matrix formation. J. Comp. Neurol. 392:58–77, 1998. © 1998 Wiley-Liss, Inc.

Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD).

Small interfering RNA (siRNA) is a promising tool for the treatment of dominant diseases. Autosomal dominant eye diseases like retinitis pigmentosa are a leading cause of blindness. Mutations in rds/peripherin lead to the degeneration of photoreceptors and are associated with several autosomal retinal diseases. Our goal is to develop a gene therapy for rds mutations. We describe an siRNA-based mutation-independent approach, targeting rds in which levels of endogenous mutant and wild-type mRNA were reduced, and an siRNA-resistant version of rds gene was supplied simultaneously. siRNAs and resistant rds were delivered to the photoreceptors by recombinant adeno-associated virus (rAAV) vector through subretinal injections. The retinal phenotype was examined, both structurally and functionally at different time points after rAAV delivery. We demonstrate suppression of rds transcript by up to 50% with concomitant expression of replacement transcript in the retina of mice in vivo. These results validate the concept of suppression of rds and replacement strategies of gene therapy with rAAV vectors containing siRNA.

The mechanism of autosomal dominant retinitis pigmentosa (ADRP) caused by the P23H mutation in rhodopsin is tightly associated with misfolded rhodopsin (RHO) which causes endoplasmic reticulum overload (ER stress), activates the unfolded protein response (UPR) and triggers apoptosis. In efforts to create a therapy for ADRP caused by the P23H mutation, we have explored different approaches leading to survival of photoreceptor (PR) cells. The direct approach involves the modulation of the level of wild-type RHO, while the indirect approach involves reprogramming the UPR and increasing the expression of heat shock proteins (HSPs). Taking the direct approach, we found that over-expression of wild-type RHO rescues scotopic ERG responses partially. However, greater therapeutic effects were obtained by manipulation of the UPR in P23H RHO rat PRs treated with the endoplasmic reticulum protein BiP/Grp78. In vitro study revealed that the pro-survival effect of Bip gene was not associated with its function as a molecular chaperone, but rather with its regulation of the UPR. Another indirect approach was the over-expression of the Hsf-1 gene, a transcriptional regulator of the heat shock response. AAV-delivery of Hsf-1 resulted in an increase of scotopic ERG amplitudes by over 35%. Taken together these data suggest viable therapeutic treatments for ADRP.

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

Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death by activating the Unfolded Protein Response, a set of conserved intracellular signaling pathways that detect protein misfolding within the endoplasmic reticulum and control protective and proapoptotic signal transduction pathways. Here, we review the misfolded proteins associated with select types of retinitis pigmentosa, Stargadt-like macular degeneration, and Doyne Honeycomb Retinal Dystrophy and discuss the role that endoplasmic reticulum stress and UPR signaling play in their pathogenesis. Last, we review new therapies for these diseases based on preventing protein misfolding in the retina.

Preface.- About The Editors.- Travel Awardees.- Part I Keynote Lectures.- Curing Blindness With Stem Cells: Hope, Reality, And Challenges.- Retinal Degenerations: Planning For The Future.- Part Ii Neuroprotection.- Neurotrophins Induce Neuroprotective Signaling In The Retinal Pigment Epithelial Cell By Activating The Synthesis Of The Anti-Inflammatory And Anti-Apoptotic Neuroprotectin D1.- On The Role Of Cntf As A Potential Therapy For Retinal Degeneration.- Nanoceria Particles Prevent Roi-Induced Blindness.- An In-Vivo Assay To Identify Compounds Protective Against Light Induced Apoptosis.- Role Of Bcl-Xl In Photoreceptor Survival.- The Hypoxic Transcriptome Of The Retina.- Part Iii Gene Therapy And Neuroprotection.- Lentiviral Gene Transfer-Mediated Cone Vision Restoration In Rpe65 Knockout Mice.- In Vitro Analysis Of Ribozyme-Mediated Knockdown Of An Adrp Associated Rhodopsin Mutation.- Gene Therapy For Mouse Models Of Adrp.- Development Of Viral Vectors With Optimal Transgene Expression For Ocular Gene Therapies.- Adeno-Associated Viral Vectors And The Retina.- Genetic Supplementation Of Rds Alleviates A Loss-Of-Function Phenotype In C214s Model Of Retinitis Pigmentosa.- Morphological Aspects Related To Long-Term Functional Improvement Of The Retina In The 4 Years Following Raav-Mediated Gene Transfer In The Rpe65 Null Mutation Dog.- Virus-Mediated Gene Delivery To Neuronal Progenitors.- Part Iv Animal Models Of Retinal Degeneration.- Loss Of Visual And Retinal Function In Light-Stressed Mice.- Erg Responses And Microarray Analysis Of Gene Expression In A Multifactorial Murine Model Of Age-Related Retinal Degeneration.- Oxygen Supply And Retinal Function: Insights From A Transgenic Animal Model.- Characterization Of Gene Expression Profiles Of Normal Canine Retina And Brain Using A Retinal Cdna Microarray.- Toward A Higher Fidelity Model Of Amd.- The Potential Of Ambient Light Restriction To Restore Function To The Dengerating P23h-3 Rat Retina.- Part V Molecular Genetics And Candidate Genes.- Mutations In Known Genes Account For 58% Of Autosomal Dominant Retinitis Pigmentosa (Adrp).- Genetics Of Age-Related Macular Degeneration.- Retinal Phenotype Of An X-Linked Pseudo-Usher Syndrome In Association With The G173r Mutation In The Rpgr Gene.- Mutation In The Pyk2-Binding Domain Of Pitpnm3 Causes Autosomal Dominant Cone Dystrophy (Cord5) In Two Swedish Families.- Identification And Characterization Of Genes Expressed In Cone Photoreceptors.- Clinical And Genetic Characterization Of A Chinese Family With Csnb1.- 10q26 Is Associated With Increased Risk Of Age-Related Macular Degeneration In The Utah Population.- Part Vi Diagnostic, Clinical, Cytopathological And Physiologic Aspects Of Retinal Degeneration.- Carboxyethylpyrrole Adducts, Age-Related Macular Degeneration And Neovascularization.- A Possible Impaired Signaling Mechanism In Human Retinal Pigment Epithelial Cells From Patients With Macular Degeneration.- Expression and Cell Compartmentalization of Efemp1, A Protein Associated with Malattia Leventinese.- Role of Elovl4 in Fatty Acid Metabolism.- Organization and Molecular Interactions of Retinoschisin in Photoreceptors.- Part VII Basic Science Underlying Retinal Degeneration.- Proteomics Profiling of the Cone Photoreceptor Cell Line, 661W.- G -Secretase Regulates Vegfr-1 Signalling in Vascular Endothelium and RPE.- Analysis of the Rate of Disk Membrane Digestion by Cultured RPE Cells.- Functional Expression of Cone Cyclic Nucleotide-Gated Channel in Cone Photoreceptor-Derived 661W Cells.- Phosphorylation of Caveolin-1 in Bovine Rod Outer Segments in Vitro by an Edogenous Tyrosine Kinase.- Regulation of Neurotrophin Expression and Activity in the Retina.- Involvement of Guanylate Cyclases in Transport of Photoreceptor Peripheral Membrane Proteins.- Rod Progenitor Cells in the Mature Zebrafish Retina.- Avss5 Integrin Receptors at the Apical Surface of The RPE: One Receptor, Two Functions.- Implantation of Mouse Eyes

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

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

Transgenic animals, in which the transgene has been engineered to mimic mutations known to cause photoreceptor degenerations in humans, provide powerful models of the diseases known as retinitis pigmentosa. Here we describe the time course of photoreceptor death in rats in which degeneration is caused by a rhodopsin transgene containing the P23H or S334ter mutations.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy ( rdy ) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

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

The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported.An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively.Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months.This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

Retinal degenerations, including retinitis pigmentosa and age-related macular degeneration (AMD), affect the sight of nearly 10 million people in the United States, and treatment options for those affected are limited to nonexistent. The understanding of retinal degenerations has been greatly enhanced through the use of environmental exposures that result in damage to photoreceptors or the Bruch membrane. Environmentally induced retinal degenerations, such as exposure to intense constant light or laser photocoagulation, in normal rodents1 have provided useful models for the study of retinal degeneration and choroidal neovascularization. Studies of light exposure in rodents with mutations that cause photoreceptor degeneration have provided further insight into the complex relationship between light exposure and retinal degeneration (reviewed by Wenzel et al2 and Paskowitz et al3).

The effects of eye pigmentation and light deprivation on the rate of photoreceptor degeneration have been explored in rodents with inherited photoreceptor degenerations. In pink-eyed RCS rats with inherited retinal dystrophy, the rate of degeneration is slowed when the animals are dark-reared. Fully pigmented RCS rats reared in cyclic light show the same degree of slowing as seen in dark-reared, pink-eyed RCS rats. Dark-rearing in fully pigmented rats fails to result in any further slowing of the degeneration. In albino nervous mutant mice with a slow form of photoreceptor degeneration, darkrearing fails to slow the rate of degeneration. The findings are compared with those of similar experiments in mice with the retinal degeneration mutation and human retinitis pigmentosa patients. In all forms of inherited retinal degeneration tested thus far, light deprivation fails to retard the disease, at least if the eyes are pigmented and in some cases even if they are not.

Physiological consequences of early stages of photoreceptor degeneration were examined in heterozygous P23H rhodopsin transgenic (Tg) and in aging normal Sprague–Dawley rats. Rod photoreceptor and rod bipolar (RB) cell function were estimated with maximum value and sensitivity parameters of P3 and P2 components of the electroretinogram. In both Tg and aging normal rats, the age-related rate of decline of P3 amplitude was steeper than that of the P2 amplitude. Tg rats showed greater than normal sensitivity of the rods. A new model of distal RB pathway connectivity suggested photoreceptor loss could not be the sole cause of physiological abnormalities; there was an additional increase of post-receptoral sensitivity. We propose that changes at rod-RB synapses compensate for the partial loss of rod photoreceptors in senescence and in early stages of retinal degeneration. © 2001 Elsevier Science Ltd. All rights reserved.

Chimeric rats were produced by the aggregation of embryos of the pinkeyed, retinal dystrophic RCS strain with those of pigmented, normal rats. In the mosaic eyes, patches of neural retina with abnormal and degenerated photoreceptors were present only opposite patches of nonpigmented, mutant pigment epithelium. This indicates that the retinal dystrophy gene acts in the pigment epithelial cell rather than in the photoreceptor cell.

HUMAN retinitis pigmentosa represents a class of diseases, the principal characteristic of which is the slow and progressive degeneration of photoreceptor cells. In several human neurological syndromes, photoreceptor degeneration accompanies cerebellar ataxia, deafness, mental retardation or other abnormalities. Some examples of such syndromes are BattenSpielmeyer–Vogt disease, Refsum's disease, Laurence–Moon Biedl syndrome, Bassen–Kornzweig disease, Usher's syndrome and several of the mucopolysaccharidoses1–4. As far as we know laboratory animals with neurological syndromes that include photoreceptor degeneration have not been available.