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The title of the chapter is "Melatonin as the Possible Link Between Age-Related Retinal Degeneration and the Disrupted Circadian Rhythm in Elderly" but degeneration was incorrectly published as regeneration. Now this has been corrected to degeneration.

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

Retinal degeneration (RD) affects millions of people and is a major cause of ocular impairment and blindness. With a wide range of mutations and conditions leading to degeneration, targeting downstream processes is necessary for developing effective treatments. Ceramide and sphingosine-1-phosphate, a pair of bioactive sphingolipids, are involved in apoptosis and its prevention, respectively. Apoptotic cell death is a potential driver of RD, and in order to understand the mechanism of degeneration and potential treatments, we studied rhodopsin mutant RD model, P23H-1 rats. Investigating this genetic model of human RD allows us to investigate the association of sphingolipid metabolites with the degeneration of the retina in P23H-1 rats and the effects of a specific modulator of sphingolipid metabolism, FTY720. We found that P23H-1 rat retinas had altered sphingolipid profiles that, when treated with FTY720, were rebalanced closer to normal levels. FTY720-treated rats also showed protection from RD compared with their vehicle-treated littermates. Based on these data, we conclude that sphingolipid dysregulation plays a secondary role in retinal cell death, which may be common to many forms of RDs, and that the U.S. Food and Drug Administration-approved drug FTY720 or related compounds that modulate sphingolipid metabolism could potentially delay the cell death.

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.

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

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

Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.

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.

The morphology of dendrites constrains and reflects the nature of synaptic inputs to neurons. The visual system has served as a useful model to show how visual function is determined by the arborization patterns of neuronal processes. In retina, light ON and light OFF responding ganglion cells selectively elaborate their dendritic arbors in distinct sublamina, where they receive, respectively, inputs from ON and OFF bipolar cells. During neonatal maturation, the bilaminarly distributed dendritic arbors of ON-OFF retinal ganglion cells (RGCs) are refined to more narrowly localized monolaminar structures characteristic of ON or OFF RGCs. Recently, brain-derived neurotrophic factor (BDNF) has been shown to regulate this laminar refinement, and to enhance the development of dendritic branches selectively of ON RGCs. Although other related neurotrophins are known to regulate neuronal process formation in the central nervous system, little is known about their action in maturing retina. Here, we report that overexpression of neurotrophin-3 (NT-3) in the eye accelerates RGC laminar refinement before eye opening. Furthermore, NT-3 overexpression increases dendritic branch number but reduces dendritic elongation preferentially in ON-OFF RGCs, a process that also occurs before eye opening. NT-3 overexpression does affect dendritic maturation in ON RGCs, but to a much less degree. Taken together, our results suggest that NT-3 and BDNF exhibit overlapping effects in laminar refinement but distinct RGC-cell-type specific effects in shaping dendritic arborization during postnatal development.

The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30-33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capillary profiles in the SP remained no different from controls in any of the lines, despite long-standing loss of photoreceptors; and (4) neovascularization of the RPE by retinal capillaries occurred with a latency of 60-180 days after the loss of photoreceptors, except in S334ter-7 rats, where neovascularization essentially did not occur. Analysis of RCS rats was carried out for comparison.

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.

Glutamatergic neurotransmission requires vesicular glutamate transporters (VGLUTs) to sequester glutamate into synaptic vesicles. Generally, VGLUT1 and VGLUT2 isoforms show complementary expression in the CNS and retina. However, little is known about whether isoform-specific expression serves distinct pathways and physiological functions. Here, by examining visual functions in VGLUT1-null mice, we demonstrate that visual signaling from photoreceptors to retinal output neurons requires VGLUT1. However, photoentrainment and pupillary light responses are preserved. We provide evidence that melanopsin-containing, intrinsically photosensitive retinal ganglion cells (RGCs), signaling via VGLUT2 pathways, support these non-image-forming functions. We conclude that VGLUT1 is essential for transmitting visual signals from photoreceptors to second- and third-order neurons, but VGLUT1 is not necessary for intrinsic visual functions. Furthermore, melanopsin and VGLUT2 expression in a subset of RGCs immediately after birth strongly supports the idea that intrinsic vision can function well before rod- and cone-mediated signaling has matured.

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD., Author Summary Human inherited photoreceptor degenerations (IPDs) are genetically heterogeneous and exhibit significant unexplained phenotypic variability. Mutation of hundreds of known genes can cause these disorders, but variability still occurs among individuals with mutations in the same gene. Possible explanations include environmental factors, mutation-specific effects, and sequence variants of modifier genes that influence the impact of disease-causing mutations. We have identified a modifier locus associated with profound suppression of photoreceptor degeneration in a mouse model of a human IPD. A gene similar to the IPD gene lies within the modifier locus, and the protein encoded by this putative modifier gene seems to substitute for the IPD protein. Our results link modest variation in the quantity of the modifier transcript, and a consequent change in the quantity of the protein, with variation in degeneration severity. Many common human genetic variants cause differences in the transcript levels of individual genes, but a significant fraction of such variants manifest only in subsets of tissues, and ocular tissues have not been surveyed. Our work suggests that variation in the transcript levels of modifier genes may contribute to variability in human IPDs, and that the retina should be assessed for genetic variants that affect gene expression.

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

MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.

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.

© 2015 The Association for Research in Vision and Ophthalmology, Inc. PURPOSE. Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes. METHODS. We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway. RESULTS. We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho-/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age. CONCLUSIONS. Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP’s inherent stability.

© 2014, Springer Science+Business Media New York. Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

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.

Rod photoreceptors are susceptible to light-induced cell death. Previous results have suggested that the neurotrophin receptor p75 in Müller cells controls photoreceptor cell death during light-exposure by suppressing trophic factor release; and consequently, if p75 is blocked or eliminated during light-exposure, apoptosis is delayed. We explored this question by examining photoreceptor cell survival in albino p75(-/-) mice as well as their heterozygous and homozygous littermates. Photoreceptor cell death was examined in semi-thin sections by counting the remaining rows of photoreceptors. No difference in the amount of cell death was found between p75(+/+) and p75(-/-) animals, whereas the single copy of p75 in the heterozygous p75(+/-) mice provided significant neuroprotection. Cell death in the wild-type animals may indeed be mediated by p75, whereas other known apoptosis pathways may be activated in the p75(-/-) mice. The pro-apoptotic activity of the p75 receptor may have been partially suppressed in the heterozygous p75(+/-) mice by the silencing effect of the Trk receptor. Thus, our results suggest that p75 signaling does not mediate the main apoptosis pathway activated during light-damage.

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.

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.

Chronic ER stress down-regulates XIAP by activating the PERK branch of the UPR. PERK attenuates Xiap translation via eIF2α phosphor­ylation. PERK promotes XIAP degradation via ATF4. CHOP induction and XIAP suppression act in parallel to sensitize cells to ER stress–induced apoptosis., Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.

Rhodopsin is a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and is essential for phototransduction and vision. Over 150 missense mutations in rhodops...

Chronic endoplasmic reticulum (ER) stress causes cell death and contributes to disease pathogenesis and progression. ER stress activates intracellular signaling pathways termed the Unfolded Protein Response (UPR). The mechanisms by which chronic ER stress and UPR promote cell death are poorly understood. The PERK arm of the UPR has been implicated in the induction of cell death through its upregulation of proapoptotic CHOP transcription factor. Chop-/- cells are only partially resistant to ER stress-induced apoptosis, and CHOP overexpression alone does not induce apoptosis. These findings strongly suggest that additional mechanisms are involved in regulating cell death downstream of PERK. Here, we find chronic ER stress dramatically suppresses XIAP protein levels. We find that PERK signaling is responsible for suppressing XIAP by eIF2a-dependent translational attenuation and ATF4-mediated proteasomal degradation. PERK’s down-regulation of XIAP occurs independently of CHOP. Loss of XIAP leads to increased ...

Focal mechanical injury to the retina substantially increases basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) mRNA expression, accompanied by a transient increase in FGFR-1 mRNA, and this response is thought to protect photoreceptors near the injury site from inherited and light-induced retinal degenerations. We have now examined retinal gene expression of the principal survival factors involved in the response to injury in normal rats as a function of postnatal age both in normal and injured retinas. Sprague–Dawley rats were injured in one eye by needle incision through the retina at postnatal day (P) 10, 22, 35, 60, 90, 120 and 180. The other eye was uninjured and served as the control. Retinas were taken 1 day post-injury. Northern blot analysis was performed to determine the mRNA expression of the following factors and receptors: bFGF and acidic fibroblast growth factors (aFGF) and FGF receptor-1 (FGFR-1); CNTF and CNTF receptor alpha (CNTFR-alpha); brain-derived neurotrophic factor (BDNF) and its receptor trk B; and insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR); glial fibrillary acidic protein (GFAP) and opsin. In the uninjured control eyes, mRNA expression of most of the factors increased with postnatal age, with little expression at P10 and maximal expression levels reached at P22 (opsin), P35 (aFGF), P60 (BDNF) or P90 (bFGF, FGFR-1, CNTF and GFAP). In contrast, IGF-1 mRNA rapidly decreased from a high level of expression at P10 to about 55% of that level by P22, reaching a stable 45–50% of the P10 level at P35 and thereafter. The response to injury of bFGF, FGFR-1, CNTF and GFAP mRNAs increased with postnatal age. Unexpectedly, only minimal increases in bFGF, FGFR-1, CNTF and GFAP over those seen in the control eyes were observed before P35. Thereafter, the increase of bFGF mRNA after injury reached a maximum of three-fold at P60, maintained this level to P120, and slightly decreased to 2.5-fold by P180. Expression of FGFR-1 mRNA showed a maximum increase of 2.6-fold at P90. Expression of CNTF and GFAP mRNAs followed a time course similar to that of bFGF. Mechanical injury did not alter the mRNA levels of aFGF, BDNF, IGF-I, and receptors, CNTFR-alpha, trk B and IGF-IR. These data show that the response to injury is minimal at early postnatal ages but increases with age and peaks at P60–90 for most potential survival factors.

We have recently noted that the inner nuclear layer (INL) and the inner plexiform layer (IPL) were significantly thinner in mice homozygous for the nervous defect (nr / nr) than in control (nr /+ or +/+) littermates. Here, we have carried out a series of anatomical studies to further understand these inner retinal changes. At postnatal day (P) 13, there was no difference in the inner retina between nervous and control mice, while a significant difference was observed at P30. Similar changes were not seen in other mouse models of photoreceptor degeneration. There was a significant reduction in the density of cells in the INL that were stained by antibodies against the inhibitory neurotransmitters GABA and glycine. These results indicate that the nervous defect causes a degeneration of one or more sub-types of amacrine cells, in addition to the loss of cerebellar Purkinje cells and retinal photoreceptors that is known to occur in these mutant animals. Finally, evidence is provided that photoreceptors die by an apoptotic pathway in nervous mice.

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.

The nervous (nr) mutation induces a progressive and severe degeneration of cerebellar Purkinje cells and retinal photoreceptors that is virtually complete within the first few months of life. Previous studies of the retina in nervous (nr/nr) mice have focused primarily on the structural abnormalities seen at the level of the photoreceptor cell bodies and outer segments. Here, we have carried out a series of functional studies of the visual pathway in nervous mice and have quantified the status of the inner retinal cell and plexiform layers. Affected animals were obtained by mating nr/+ heterozygotes and screening the offspring for the ataxia characteristic of nervous animals; phenotypically normal littermates (i.e. nr/+ or +/+) were used as controls. As described previously, there is a substantial loss of photoreceptors cells in the nervous retina and a marked shortening of the inner and outer segments. These changes are accompanied by a more modest decline in the thickness of the inner plexiform and inner nuclear layers. These anatomic abnormalities were accompanied by reproducible changes in visual function, as measured with the electroretinogram (ERG) and visual evoked potential (VEP). The dark-adapted ERGs of nervous and control mice had similar waveforms, although the nervous responses were substantially smaller in amplitude. The reductions in the amplitude of the ERG a-wave corresponded to the loss of photoreceptor cells and shortened outer segments seen histologically. Nevertheless, the kinetics of the leading edge of the a-wave did not differ between nervous and control mice, indicating that the rod outer segments of nervous mice continue to respond to light in a normal fashion. The amplitudes of cone ERGs were also reduced in nervous mice, although the extent of this reduction in any given animal was always less than that for rod-mediated ERG components. Overall, this result is consistent with cone involvement occurring only as a secondary effect of rod photoreceptor degeneration. The peak latencies of VEPs of nervous mice were slower than those of control littermates. These functional abnormalities correspond well to the structural changes induced by the nervous mutation, which does not appear to prevent visual signals from being transmitted centrally, beyond the limitations imposed by the degenerative process.

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.

Brain-derived neurotrophic factor (BDNF) acts through TrkB, a receptor with kinase activity, and mitigates light-induced apoptosis in adult mouse rod photoreceptors. To determine whether TrkB signaling is necessary for rod development and function, we examined the retinas of mice lacking all isoforms of the TrkB receptor. Rod migration and differentiation occur in the mutant retina, but proceed at slower rates than in wild-type mice. In postnatal day 16 (P16) mutants, rod outer segment dimensions and rhodopsin content are comparable with those of photoreceptors in P12 wild type (WT). Quantitative analyses of the photoreceptor component in the electroretinogram (ERG) indicate that the gain and kinetics of the rod phototransduction signal in dark-adapted P16 mutant and P12 WT retinas are similar. In contrast to P12 WT, however, the ERG in mutant mice entirely lacks a b-wave, indicating a failure of signal transmission in the retinal rod pathway. In the inner retina of mutant mice, although cells appear anatomically and immunohistochemically normal, they fail to respond to prolonged stroboscopic illumination with the normal expression of c-fos. Absence of the b-wave and failure of c-fos expression, in view of anatomically normal inner retinal cells, suggest that lack of TrkB signaling causes a defect in synaptic signaling between rods and inner retinal cells. Retinal pigment epithelial cells and cells in the inner retina, including Müller, amacrine, and retinal ganglion cells, express the TrkB receptor, but rod photoreceptors do not. Moreover, inner retinal cells respond to exogenous BDNF with c-fos expression and extracellular signal-regulated kinase phosphorylation. Thus, interactions of rods with TrkB-expressing cells must be required for normal rod development.

Interactions among growth factors are important in a variety of physiological and pathological processes. The regulation of IGF-I mRNA expression by bFGF was investigated in cultured rat Müller cells and the mechanism of regulation studied. Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured with Eagle MEM+10% FCS. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, carbonic anhydrase C, and glutamine synthetase. Cells of passage 1-4 were treated with bFGF, the PKC inhibitor H-7, calphostin C, the PKC activator PMA or the PKA inhibitor H-89, as well as the adenylate cyclase activator forskolin, or adenylate cyclase inhibitor SQ22536. IGF-I and bFGF expression levels were assessed by Northern blot analysis. The addition of bFGF to culture medium down-regulated IGF-I expression in a dose- and time-dependent manner. Decrease of IGF-I expression started at a bFGF concentration of 1 ng ml-1. IGF-I mRNA level declined to 44% of baseline level at 10 ng ml-1 of bFGF, and reached a trough of 40% at 50 ng ml-1. At 10 ng ml-1 of bFGF, down-regulation of IGF-I expression was observed as early as 4 hr (60%) after treatment, and reached a trough of 42% by 8 hr. The temporal and concentration dependence of IGF-I expression by addition of the PKC activator PMA, to culture medium was similar to that due to the addition of bFGF. The down-regulation of IGF-I expression by bFGF (10 ng ml-1) and PMA (0.1 microM) was blocked by the PKC inhibitors H-7 (30 microM) and calphostin C (1 microM). Forskolin (5 microM), an adenylate cyclase activator, had activator, had no effect on IGF-I expression. SQ22536 (100 microM), an adenylate cyclase inhibitor, and H-89, a PKA inhibitor, had no inhibitory effect on bFGF-induced down-regulation of IGF-I expression. These results indicate that bFGF down-regulates IGF-I expression in cultured rat M uller cells through PKC activation.

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.

© 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.