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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

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.

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 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.

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.

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.

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.

We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene ( gfp ) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to −385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10–20% of the total retinal area after a single 2-μl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp -containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) play an essential role in both normal and pathological extracellular matrix degradation, and a TIMP has been associated with at least one type of retinal degeneration. We have studied expression of MMP-2 and TIMP-1 by zymography, immunocytochemistry, and immunoblotting in the retinal pigment epithelium (RPE) from normal, aged and diseased retinas. MMPs and TIMPs were found in the rat RPE, interphotoreceptor matrix (IPM), and in media conditioned by human and rat RPE in culture. In other polarized cells. MMPs and TIMP-2 are secreted vectorially towards the basal lamina. In the RPE, however, MMP-2 and TIMP-1 were secreted preferentially from the apical surface, the surface bordering the IPM. These findings provide new evidence that MMPs and TIMPs could play a role in the turnover of IPM components. Cell homogenates and conditioned media from RPE isolated from mutant Royal College of Surgeons (RCS) rats with inherited retinal dystrophy had similar amounts of MMP-2 and TIMP-1 as those from congenic control rats. The secretion of MMP-2 and TIMP-1 from RPE cell cultures isolated from young and aged human donors varied widely. However, with increasing cell passage number, secretion of MMPs and TIMPs from human RPE increased dramatically. Also, growing human RPE on bovine corneal endothelial cell-generated extracellular matrix instead of plastic reduced the secretion of both MMPs and TIMPs. These data suggest that the integrity of Bruch's membrane may serve to regulate RPE functions in MMP and TIMP secretion and that extracellular matrices contain signals that regulate MMP and TIMP synthesis and/or secretion by the RPE.

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.

Aims: Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI). Methods: ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting of human RPE cell extracts. Lipid transport assays were performed using radiolabelled photoreceptor outer segments (POS). ABCA1 and SR-BI expression was examined in normal mouse eyes by immunofluorescence staining. BrMs of ABCA1 and SR-BI heterozygous mice were examined microscopically. Results: Human RPE cells expressed ABCA1 mRNA and protein. The ABCA1 and SR-BI inhibitor glyburide (also known as glibenclamide) abolished basal transport of POS-derived lipids in RPE cells in the presence of high-density lipoprotein. Mouse retina and RPE expressed ABCA1 and SR-BI. SR-BI was highly expressed in RPE. BrMs were significantly thickened in SR-BI heterozygous mice, but not in ABCA1 heterozygous mice. Conclusion: RPE cells express ABCA1 and SR-BI. This implies a significant role for SR-BI and ABCA1 in lipid transport and RCT in the retina and RPE.

The normal function of vertebrate photoreceptor cells depends on multiple interactions and transfer of substances between the photoreceptors and the retinal pigment epithelium (RPE), but the mechanisms of these interactions are poorly understood. Many are thought to be mediated by the interphotoreceptor matrix (IPM), a complex extracellular matrix that surrounds the photoreceptors and lies between them and the RPE. Histochemical, immunocytochemical, and lectin probes for several IPM constituents revealed that components of the IPM in the rat undergo a major shift in distribution or molecular conformation after the transition between light and dark. In the light, various IPM constituents concentrated in bands at the apical and basal regions of the outer segment zone; in the dark, they distributed much more uniformly throughout the zone. The change in IPM distribution was triggered by the light-dark transition; it was not a circadian event, and it was not driven by a systemic factor. The light-evoked change in IPM distribution may facilitate the transfer of substances between the photoreceptors and the RPE.

The retinas of both normal and Royal College of Surgeons (RCS) rats with inherited retinal dystrophy have been examined using lectin histochemistry to determine the developmental and degenerative changes of the glycoconjugates in the interphotoreceptor matrix (IPM) between postnatal day (P) 10 and P25, when the adult lectin binding patterns are seen in normal rats. Wheat germ agglutinin (WGA; recognizing sialic acid and/or N-acetyl-D-glucosamine) bound to the apical surface of the retinal pigment epithelium (RPE) sparsely at P10 and prominently at P12 in both strains. In both strains at P14, WGA also stained the basal outer segment zone at the inner segment-outer segment junction. Between P14 and P16 in both strains, there was a dramatic increase in the binding of the interstitial region, the space alongside the outer segments and between the apical and basal outer segment zones. The binding pattern of WGA in normal rats remained basically unchanged from P16 to P25, although the intensity of binding was increased somewhat. Ricinus communis agglutinin-1 (RCA-1; specific for galactosyl residues) bound to the outer segment zone prominently and diffusely with increasing intensity with age at P10, P12 and P14 in both strains. At P16 and older, the intense binding of the interstitial zone was dramatically reduced and the RCA-1 bound primarily to the inner and outer segment junctional region, with weak binding to the apical surface of the RPE in both strains. At P25, the binding of the inner and outer segment junctional region was even more restricted, limited to punctate sites in this zone in normal rats and almost missing in RCS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo. The AAV experiments made use of a "stabilized double-stranded" version of these vectors with rapid onset of gene expression. In the rat retinal pigment epithelium, shRNAs delivered by either vector reduced bFGF immunoreactivity to undetectable levels in transduced cells, whereas a nonfunctional control construct incorporating a two-base pair mutation had no measurable effect on bFGF expression. Silencing commenced within a few days after injection of virus and remained stable throughout the period of observation, as long as 60 days. Viral delivery of RNAi constructs offers a powerful and versatile approach for both gene therapy and the analysis of fundamental questions in retinal biology.

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

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.

We characterized the survival-factor response of the normal mouse retina to mechanical injury by examining the expression of mRNAs for basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), and their receptors, FGF receptor-1 (FGFR-1) and CNTF receptor alpha (CNTFR-alpha). The retina was injured by making an incision through the choroid and retinal pigment epithelium that penetrated the subretinal space of each eye of an adult BALB/c mouse. Retinas were taken 6 hr, 12 hr, 1, 2, 4, 7, 10 and 16 days post-injury. Control animals were without injury. Northern blot analysis was performed to determine bFGF, CNTF and their receptor mRNA levels. A significant increase in bFGF and CNTF mRNAs was observed after injury, along with an increase in glial fibrillary acidic protein (GFAP) expression. More than 2-fold of upregulation of bFGF mRNA was seen as early as 6 hr after injury. This increase reached a maximum of more than 5-fold at day 2 post-injury and then declined slowly, and was still about 2.5-fold of the control level by day 16. Expression of CNTF showed a small increase of about 1.6-fold at 6 hr after injury. The upregulation reached a peak level of about 2.7-fold at day 4 after injury, then declined to control level by day 16. There was only a very small increase in FGFR-1 at 6, 12 and 24 hr after injury, and no significant increases in FGFR-1 at time points longer than 1 day post-injury. Expression of GFAP followed a time course similar to that of bFGF. We conclude that mechanical injury induces bFGF, CNTF, and GFAP expression in the mouse retina with time courses similar to the upregulation of these molecules in rat retina. Compared to the upregulation in rat retina, however, the injury-induced upregulation of bFGF and GFAP is much less in the mouse retina. In addition, there was only a very small induction of FGFR-1 expression in the mouse retina. These findings may explain, at least in part, the lack of injury-induced photoreceptor protection in the mouse retina.

Our objective was to determine quantitatively whether herpes simplex virus infects preferentially the apical or basolateral surfaces of two well-differentiated cell types, human retinal pigment epithelial cell and Madin-Darby canine kidney epithelial cells. Secondarily, we sought to localise the mannose 6-phosphate/insulin-like growth factor II receptor, a putative receptor for herpes simplex virus, in the membrane domains of the retinal pigment epithelial cells. Although it has been suggested that receptors utilized by the herpesviruses are heterogeneously distributed on epithelial cells, no quantitative evidence of preferential polarized uptake of wild-type herpes simplex virus into an epithelial cell has yet appeared. Moreover, no evidence has appeared of the distribution of mannose-6-phosphate/insulin-like growth factor II receptor in human retinal pigment epithelial cells. We hypothesized that the preferred pole of uptake and infection by HSV would correlate with the distribution of the receptor. Understanding the preferred site of entry in these cells may shed light on the mechanism of pathological infection and spread of this and related viruses, such as cytomegalovirus, in acute retinal necrosis and herpetic encephalitis. The efficiency of viral infection was assayed two ways. First, using permeable filters on which the monolayer of polarized epithelial cells was grown, we compared the number of foci of infected cells that resulted from an apical infection with that resulting from application of virus to the underside of the filter from which the virus could reach the basolateral surface of the cells. Second, we compared the number of infected cell foci that resulted from an apical infection to the number formed following infection at both the apical and basolateral surfaces of the cells. Both surfaces were exposed to virus following disruption of the tight junctions between cells with a Ca2+ chelator. After the efficiency of infection was normalized for relative surface areas, we found that both cell types were equally infectable with the F strain of the virus. However, there was a difference in the degree of polarized uptake of virus by the two cell types. Virus infected the basolateral surface of the retinal cells only about 6.5 times as effectively as it infected the apical surface of those cells, whereas virus infected the basolateral surface of the kidney epithelial cells about 435 times as effectively as it infected the apical surface of the same cells. These data suggest that herpes simplex virus can efficiently enter either the apical or basolateral surface of retinal pigment epithelial cells, unlike its more polarized preference for the basolateral surface of the kidney epithelial cell type. The mannose 6-phosphate/insulin-like growth factor II receptor was present in human retinal pigment epithelial cells, as determined by Western blotting. Surface biotinylation experiments revealed the presence of the receptor in both the apical and basolateral membranes of the retinal epithelial cells. Our evidence is consistent with the hypothesis that the virus may utilize the mannose 6-phosphate/insulin-like growth factor II receptor to facilitate entry.

The development of the retinal pigment epithelium (RPE) was studied in rhesus monkey (Macaca mulatta) fetuses, neonates, and juveniles exposed to a pulse of (3H-TdR) between embryonic day (E) 25 and postnatal day (P) 204 and examined at short and long intervals after the injection of the isotope. The RPE develops from the outer layer of the optic cup which by E45 consists of a multistratified epithelium. The outer layer appears immature near the retina's edge and gradually becomes monostratified and more mature centrally. Even at this early stage, all cells contain pigmented melanosomes, although peripherally the pigment is limited to the apical portion of the cells. Examination of autoradiograms from animals allowed to survive for several postnatal months shows that monkey RPE cell genesis begins just after E27, increasing to a peak frequency of 0. 38 cells/mm at E43. Between E30 and E85 the density of radiolabelled cells varies within a restricted range of from 0.2 to 0.4 cells/mm (mean = 0.25 ± 0.09). From the density of radiolabelled cells, and data on the overall density of RPE cells in the juvenile retina, we determined the labelling index. During the first half of gestation, between 0. 38% and 0. 99% (mean = 0. 65 ± 0. 22) of RPE cells are generated during the short interval of isotope availability after pulse injection. Approximately 5% of RPE cells were generated by E33, and 50% by E71. After E85, RPE cytogenesis begins gradually to decrease, and 95% of the cells have been generated by the time of birth. Continued, very low density (0. 01 cells/mm) cytogenesis in the RPE is seen at P17, and persists at least until seven months postnatally. RPE cell genesis begin near the fovea, and proceeds towards the periphery. Cell division largely ceases in both foveal and perifoveal regions by E56, at which time labelled cells first begin to appear peripheral to the equator. Besides the timing differences, RPE genesis in the central retina differs from that in the peripheral retina in that it proceeds at a higher rate, and lasts for a shorter time period. A prolonged postnatal period of low density RPE cell genesis persists in both central and peripheral retina. Comparison of the pattern of expansion of the area containing radiolabelled cells in the RPE and neuroretina demonstrates a remarkable spatial and temporal correspondence. Close analysis suggests that at any point on the retina, the last cells are generated in the neuroretina slightly before the last cells in the RPE. © 1995 Wiley-Liss, Inc.

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.

Nervous mutant mice (nr/nr) show a rapid loss of most of cerebellar Purkinje cells between the ages of 3 and 7 weeks, as well as a progressive photoreceptor cell degeneration that occurs most rapidly between postnatal days (P) 13 and 19, but with a much slower attrition during the subsequent months. We have carried out an electron microscopic analysis of nr/nr and littermate control mice at representative ages to characterize the subcellular cytopathological changes in this form of retinal degeneration, to gain insight into photoreceptor cell degeneration mechanisms by comparing these changes to those in other rodent forms of retinal degeneration, and to compare the photoreceptor changes with those of cerebellar Purkinje cells. Ultrastructural observations were limited to rod photoreceptors, since the number of cones was limited in our micrographs. The retinas of nr/nr mutant mice can be distinguished from those of normal littermates as early as postnatal day (P) 6. At this time, some of the mitochondria in rod inner segments are larger and more rounded than normal. This represents the earliest cytopathological change thus far observed in this mutant. As early as P9 and thereafter, the volume and integrity of rod outer segment membranes are reduced from normal. In the inner segments of some rod photoreceptor cells, there is a reduction in the volume or number of polyribosomes as early as P11, a reduction in rough endoplasmic reticulum as early as P13, and reduced incidence and less organized Golgi membranes as early as P16. Qualitative evaluation and quantitative stereological analysis show that the enlarged mitochondria in rod inner segments never become normal in shape or size. No changes are seen in the inner retinal layers at any age. Despite similarities with inherited retinal dystrophy in the Royal College of Surgeons rat, as noted in the original description of retinal degeneration in nr/nr mice, ultrastructural features clearly distinguish these mutants. Moreover, nr/nr mice can be distinguished from all other murine forms of retinal degeneration by electron microscopy.

Nervous is an autosomal recessive mutation in mice (gene symbol, nr) that produces a progressive cerebellar and retinal degeneration. We have examined various cytopathological features of the photoreceptor degeneration by light microscopy. An increase in the number of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) is first seen at postnatal day (P) 11. Between P13 and P19 there is a rapid loss of photoreceptors, with the ONL about 60% the thickness of littermate controls at P19. Between P19 and 2.5 months of age, photoreceptor cell loss is minimal, and there is a relatively slow loss of these cells between 3 and 7.5 months of age. At 7.5 months, the ONL consists of single row of nuclei, most of which are lost over the ensuing months, although a few photoreceptor nuclei persist at 17 months of age and older. Both rods and cones are lost at comparable rates for the first 2 months of life, but rods are somewhat preferentially lost at later ages. A very slight central-to-peripheral gradient of photoreceptor degeneration exists in the nr/nr retina, but no superior-inferior hemispheric differences are evident. The rate, spatiotemporal gradient, and hemispheric similarity in photoreceptor degeneration are the same in albino nr/nr mice reared either in cyclic light or in the dark, and in pigmented nr/nr mice. Autoradiographic analysis of rod outer segment renewal shows that outer segment membranes are synthesized in nervous homozygotes. Rhythmic outer segment disc shedding and phagocytosis by the retinal pigment epithelium occur at approximately normal rates in nr/nr mice. Histochemical and immunocytochemical study of the interphotoreceptor matrix (IPM) reveals the exclusion of stainable IPM from the outer segment zone by lamellar whorls of outer segment membrane, accumulation of stainable IPM in the basal region of the outer segment zone, and the absence of an intense band of stainable IPM at the apical surface of the retinal pigment epithelium. These changes in the IPM are similar to those seen in the Royal College of Surgeons rat. However, comparison of cytopathological changes in these two mutants reveal that the IPM defect probably is not the primary cause of photoreceptor cell death in nr/nr mice, and that similar phenotypic appearance does not necessarily signify similar pathological processes.

Photoreceptor cells in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy can be rescued by the transplantation of normal, wild-type retinal pigment epithelial (RPE) cells, if done before photoreceptor cell death. In the present study, we have examined several metabolic features of rescued photoreceptors and transplanted RPE cells at 2.3–3.3 months after transplantation. Rescued photoreceptors with a structurally normal RPE interface showed a rod outer segment renewal rate similar to that of normal control rats of 2.2 μm day−1, as measured in autoradiograms. Rod outer segment disc shedding had values idistinguishable from those in normal controls, as measured by the number of phagosomes in the transplanted RPE cells both during the burst of disc shedding soon after the onset of light in the morning and during the middle of the light cycle when disc shedding is low. The interphotoreceptor matrix, which is synthesized by both photoreceptors and the RPE, was distributed normally in the regions where normal-appearing photoreceptors were present underlying normal, transplanted RPE cells. Thus, the rescued photoreceptors show normal metabolic rates and normal interactions with the RPE in each of the parameters examined. These findings, combined with the previous demonstration of opsin and Na+,K+-ATPase expression by the rescued photoreceptors, support our interpretation that the surviving, normal-appearing photoreceptors may function normally. Moreover, transplanation of normal RPE cells reversed pathological changes in the photoreceptors that had already occurred by the time of transplantation. Thus, it appears that if photoreceptors are capable of being rescued from cell death, at least some of their metabolic processes can be restored to normal levels by RPE cell transplantation.

Experimental mouse chimeras and Cattanach's translocation mice, both with alternating pigmented and non-pigmented cells of the retinal pigment epithelium (RPE), have been exposed to various periods of constant fluorescent light. Photoreceptor degeneration was always more severe in the central retina than in the peripheral retina, and it was independent of the pigmentation phenotype of the immediately overlying RPE. The findings suggest that although RPE melanosomes lower total retinal irradiance by absorption of light, they do not provide direct protection from light damage for the immediately underlying photoreceptor cells by a biochemical mechanism as previously suggested.

The interphotoreceptor matrix (IPM) is a mixture of proteoglycans and glycoproteins through which metabolites must pass in transit between the retinal pigment epithelium (RPE) and the photoreceptor cells. In order to localize various species of chondroitin sulfates in the IPM of the normal and dystrophic rat retina, we have used monoclonal antibodies directed against 6-sulfated chondroitin sulfate (6S), 4-sulfated chondroitin sulfate (4S) and unsulfated chondroitin (0S). Immunofluorescence and immuno-peroxidase methods were carried out on frozen and wax-embedded sections of rat eyes. In the normal rat retina, strong labeling with the 6S antibody was observed in the basal IS/OS zone and, to a lesser extent, in the photoreceptor interstices extending to the apical RPE surface. In the RCS retina, the IPM in the basal IS/OS zone and much of the outer segment debris zone were labeled intensely with 6S antibody, but no strong labeling was present at the apical RPE surface. The labeling pattern with 0S antibody was similar to that of the 6S antibody for both normal and RCS retinas. We failed to detect any 4S antibody labeling of the IPM in both the normal and RCS retinas using these methods. These results indicate that in the rat retina, the IPM contains 6-sulfated chondroitin sulfate and unsulfated chondroitin proteoglycans that are most concentrated in the basal IS/OS zone and, to a lesser degree, between the photoreceptor outer segments. Furthermore, it appears that the rat IPM contains little or no 4-sulfated chondroitin sulfate or dermatan sulfate proteoglycan.

We have assessed the effects of 15 pigmentation mutations on the development of retinal ganglion cell projections in mice in two ways: (1) by analyzing the pattern of innervation of the ipsilateral lateral geniculate nucleus as mapped in autoradiograms of brains of animals killed 12 days after intravitreal injection of 3H-proline into one eye and (2) by determining the ratio of axonally transported radioactive protein in the contralateral and ipsilateral optic tracts after similar intravitreal injections. Analysis of the ratio of transported protein in the two optic tracts provides a new and useful assay of the degree of decussation in experimental animals. The effects of the mutations on eye pigmentation, whole eye melanin content and relative tyrosinase activity also were examined. The degree of ipsilateral innervation generally correlates with the degree of pigmentation of the retinal pigment epithelium and with tyrosinase activity. However, discrepancies have been found in ch and ce mutants. In these animals the pigment epithelium is well pigmented, and the area of ipsilateral innervation in the lateral geniculate nucleus is extensive, despite a high ratio of label in contralateral to ipsilateral optic tracts and low tyrosinase activity. Furthermore, mice heterozygous for the c2J allele have pigmentation and optic projections that are normal even though tyrosinase is reduced to 40% of normal. The few anomalous results suggest that alternative or additional factors may control optic axon projections.

Photoreceptor destruction is known to occur as a result of exposure for various lengths of time to low (less than about 1000 lux) (1, 2), moderate (about 1000–3000 lux) (3, 4) and higher (5, 6) levels of visible light, as well as by the radiant energy from lasers of different wavelengths (7, 8). In the case of high levels of visible light (6) and laser-induced damage to the retina, the mechanism appears to be the absorption of energy by melanosomes in the pigment epithelium and, indeed, the extent of laser-induced retinal destruction is directly related to the degree of retinal pigmentation (7, 8).