Regeneration of visual pigments is essential for sustained visual function. Although the requirement for non-photochemical regeneration of the visual chromophore, 11-cis-retinal, was recognized early on, it was only recently that the trans to cis retinoid isomerase activity required for this process was assigned to a specific protein, a microsomal membrane enzyme called RPE65. In this review, we outline progress that has been made in the functional characterization of RPE65. We then discuss general concepts related to protein-membrane interactions and the mechanism of the retinoid isomerization reaction and describe some of the important biochemical and structural features of RPE65 with respect to its membrane-binding and enzymatic properties.

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. We used Lrat(-)(/-), a murine model for LCA, to investigate the mechanism of rapid cone degeneration. Although both M and S cone opsins mistrafficked as reported previously, mislocalized M-opsin was degraded whereas mislocalized S-opsin accumulated in Lrat(-)(/-) cones before the onset of massive ventral/central cone degeneration. As the ventral and central retina express higher levels of S-opsin than the dorsal retina in mice, our results may explain why ventral and central cones degenerate more rapidly than dorsal cones in Rpe65(-)(/-) and Lrat(-)(/-) LCA models. In addition, human blue opsin and mouse S-opsin, but not mouse M-opsin or human red/green opsins, aggregated to form cytoplasmic inclusions in transfected cells, which may explain why blue cone function is lost earlier than red/green-cone function in patients with LCA. The aggregation of short-wavelength opsins likely caused rapid cone degenerations through an endoplasmic reticulum stress pathway, as demonstrated in both the Lrat(-)(/-) retina and transfected cells. Replacing rhodopsin with S-opsin in Lrat(-)(/-) rods resulted in mislocalization and aggregation of S-opsin in the inner segment and the synaptic region of rods, ER stress, and dramatically accelerated rod degeneration. Our results demonstrate that cone opsins play a major role in determining the degeneration rate of photoreceptors in LCA.

OBJECTIVE

To identify mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa.

METHODS

Mutation analysis was carried out in a group of 35 unrelated patients with juvenile autosomal recessive retinitis pigmentosa (ARRP), Leber's congenital amaurosis (LCA), or juvenile isolated retinitis pigmentosa (IRP), by denaturing high performance liquid chromatography followed by direct sequencing.

RESULTS

All three groups of patients showed typical combinations of eye signs associated with retinitis pigmentosa: pale optic discs, narrow arterioles, pigmentary changes, and nystagmus. Mutations were found in 34% of

METHODS

in CRB1 (11%), GUCY2D (11%), RPE65 (6%), and RPGRIP1 (6%). Nine mutations are reported, including a new combination of two mutations in CRB1, and new mutations in GUCY2D and RPGRIP1. The new GUCY2D mutation (c.3283delC, p.Pro1069ArgfsX37) is the first pathological sequence change reported in the intracellular C-terminal domain of GUCY2D, and did not lead to the commonly associated LCA, but to a juvenile retinitis pigmentosa phenotype. The polymorphic nature of three previously described (pathological) sequence changes in AIPL1, CRB1, and RPGRIP1 was established. Seven new polymorphic changes, useful for further association studies, were found.

CONCLUSIONS

New and previously described sequence changes were detected in retinitis pigmentosa in CRB1, GUCY2D, and RPGRIP1; and in LCA patients in CRB1, GUCY2D, and RPE65. These data, combined with previous reports, suggest that LCA and juvenile ARRP are closely related and belong to a continuous spectrum of juvenile retinitis pigmentosa.

Uniparental disomy (UPD) is a rare condition in which a diploid offspring carries a chromosomal pair from a single parent. We now report the first two cases of UPD resulting in retinal degeneration. We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient. In both families, the gene defect was present in the patient's heterozygous father but not in the patient's mother. Analysis of haplotypes in each nuclear kindred, by use of DNA polymorphisms distributed along both chromosomal arms, indicated the absence of the maternal allele for all informative markers tested on chromosome 1 in the first patient and on chromosome 2 in the second patient. Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles. Our findings provide evidence for the first time, in the case of chromosome 2, and confirm previous observations, in the case of chromosome 1, that there are no paternally imprinted genes on chromosomes 1 and 2 that have a major effect on phenotype.

RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.

Proliferation and epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy. This study aims at clarifying the role of growth factors, such as epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and transforming growth factor-β1 (TGF-β1), in controlling how RPE proliferates while undergoing EMT. When contact inhibition of post-confluent ARPE-19 cells was disrupted by EGTA, an increase of BrdU labeling was noted only in the presence of EGF and/or FGF-2, and was accompanied by EMT as evidenced by the loss of a normal RPE phenotype (altered cytolocalization of RPE65, N-cadherin, ZO-1, and Na,K-ATPase) and the gain of a mesenchymal phenotype (increased expression of vimentin, S100A4, and α-smooth muscle actin). EMT with proliferation by EGTA+EGF+FGF-2 was accompanied by activation of canonical Wnt signaling (judged by the TCF/LEF promoter activity, increased nuclear levels of and interaction between β-catenin and LEF1 proteins, and the replication by overexpression of β-catenin), abolished by concomitant addition of XAV939, a Wnt inhibitor, but not associated with suppression of Hippo signaling (negative expression of nuclear TAZ or YAP and cytoplasmic p-TAZ or p-YAP). The causative role of Wnt signaling on EMT with proliferation was confirmed by overexpression of stable S33Y β-catenin with EGTA treatment. In addition, contact inhibition disrupted by EGTA in the presence of TGF-β1 also led to EMT, but suppressed proliferation and Wnt signaling. The Wnt signaling triggered by EGF+FGF-2 was sufficient and synergized with TGF-β1 in activating the Smad/ZEB1/2 signaling responsible for EMT. These findings establish a framework for further dissecting how RPE might partake in a number of proliferative vitreoretinopathies characterized by EMT.

OBJECTIVE

RPE65 has been shown to be essential for the production of 11-cis retinal by the retinal pigment epithelium. Mutations in RPE65 are known to be associated with severe forms of early-onset retinal dystrophy. This project was designed to determine the amount of regenerable opsin in Rpe65-/- mice during development and aging, and to examine the function of this rhodopsin by electroretinography (ERG).

METHODS

Young and aged Rpe65-/- and wild-type (WT) mice were dark adapted. Endogenous rhodopsin and regenerable opsin were measured using absorption-difference spectrophotometry. Photoreceptor function was assessed with scotopic single-flash ERGs and photoreceptors were counted in histologic sections. Opsin's primary structure was analyzed by mass-spectrometric mapping.

RESULTS

Unlike WT mice, amounts of regenerable opsin in Rpe65-/- mice decreased significantly with age, which correlated with a decrease in the number of photoreceptors and a decline in ERG amplitudes. Opsin structure, however, did not change. No endogenous levels of rhodopsin were measurable in the Rpe65-/- mice (detection limit: 0.225 pmol). 11-cis Retinal injections resulted in the regeneration of similar amounts of rhodopsin and improved rod function in a comparable way, irrespective of age.

CONCLUSIONS

In the aged Rpe65-/- mouse, opsin levels decrease because of the loss of photoreceptors. The remaining opsin is structurally intact, and the components of the phototransduction cascade and the retinal circuitry remain functional, despite the absence of normal photoreceptor activity.

Several groups have reported the results of clinical trials of gene augmentation therapy for Leber congenital amaurosis (LCA) because of mutations in the RPE65 gene. These studies have used subretinal injection of adeno-associated virus (AAV) vectors to deliver the human RPE65 cDNA to the retinal pigment epithelial (RPE) cells of the treated eyes. In all of the studies reported to date, this approach has been shown to be both safe and effective. The successful clinical trials of gene augmentation therapy for retinal degeneration caused by mutations in the RPE65 gene sets the stage for broad application of gene therapy to treat retinal degenerative disorders.

OBJECTIVE

Phototransduction in cones is initiated by the bleaching of their visual pigment, which comprises a protein component-cone opsin-and a vitamin A derivative-11-cis retinal. Little is known about the source of 11-cis retinal for cones. In the current study, neural retina leucine zipper-deficient (Nrl(-/-)) and rod opsin (Rho(-/-))-deficient mice were used, two mouse models that have been described as having a "cone-only" retina, to analyze the retinoid metabolism of cones. In addition, these mice were bred to retinal pigment epithelial protein 65 (Rpe65(-/-))-deficient mice to study the role of RPE65.

METHODS

Mice were analyzed using morphology, Western blot analysis, immunohistochemistry, electroretinography (ERG), and retinoid profiling by HPLC.

RESULTS

In comparison to wild-type mice, the retina of Nrl(-/-) mice contained elevated levels of RPE65 and cellular retinaldehyde-binding protein (CRALBP), suggesting a particular role of these two proteins for the retinoid metabolism of cones. In Nrl(-/-) mice, different retinoid species were present in proportions similar to wild type. Ablation of RPE65 in Nrl(-/-) and Rho(-/-) mice led to the absence of 11-cis retinal, but increased the total retinoid content, with retinyl esters representing the most abundant retinoid species. In the absence of RPE65, retinal sensitivity in Nrl(-/-) mice dropped by a factor of a thousand.

CONCLUSIONS

The data show that RPE65, previously shown to be essential for rod function, is also indispensable for the production of 11-cis retinal for cones and thus for cone function.

Leber's congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65(-/-)) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4(+) T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials.

OBJECTIVE

The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians.

METHODS

The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections.

RESULTS

RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested.

CONCLUSIONS

RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.

OBJECTIVE

The induced pluripotent stem (iPS) cell is expected to be a powerful tool for research and development in regenerative medicine. Previously, the authors reported that human iPS cells differentiated into retinal cells, including photoreceptors and retinal pigment epithelial cells. In this study, they produced iPS cell lines from monkeys to investigate their ability to differentiate into retinal cells.

METHODS

To generate iPS cells, the fibroblasts derived from cynomolgus monkey abdominal skin were infected with retroviruses carrying Oct3/4, Sox2, Klf4, and c-Myc genes and then were cultured on STO feeder cells. Next, the established iPS cells were cultured with the conditioned medium of PA6 cells to induce RPE cells. The properties of the differentiated RPE cells were analyzed.

RESULTS

Approximately 1 month after viral infection, some epithelial-like colonies appeared among the fibroblasts. These colonies were morphologically similar to the cynomolgus embryonic stem (ES) cell and expressed ES cell-specific markers. By producing teratomas in SCID mice, these cells were confirmed to have the ability to differentiate into three germ layers. In addition, the RPE cells induced from the monkey iPS cells had characteristic polygonal shapes and pigments. These cells expressed RPE cell-specific markers such as RPE65, CRALBP, Bestrophin 1, and MERTK and exhibited phagocytotic function in vitro.

CONCLUSIONS

The RPE cells derived from monkey skin with iPS cell technology can be used for autologous or allogeneic transplantation to test the possibility of immune rejection and to evaluate their function in vivo with the same techniques that will be used in clinical trials.

BACKGROUND

To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice.

METHODS

Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation.

RESULTS

RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue.

CONCLUSIONS

ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

Rpe65(-/-) mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65(-/-) mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65(-/-) mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65(-/-) mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.

Adeno-associated viral gene therapy has shown promise for the treatment of inherited and acquired retinal disorders. In most applications, regulation of expression is a critical concern for both safety and efficacy. The purpose of our study was to evaluate the ability of the tetracycline-regulatable system to establish long-term transgene regulation in the retina of nonhuman primates. Three rAAV vectors expressing the tetracycline-dependent transactivator (rtTA) under the control of either the ubiquitous CAG promoter or the specific RPE65 promoter (AAV2/5.CAG.TetOn.epo, AAV2/4.CAG.TetOn.epo, and AAV2/4.RPE65.TetOn.epo) were generated and administered subretinally to seven macaques. We demonstrated that repeated inductions of transgene expression in the nonhuman primate retina can be achieved using a Tet-inducible system via rAAV vector administration over a long period (2.5 years). Maximum erythropoietin (EPO) secretion in the anterior chamber depends upon the rAAV serotype and the nature of the promoter driving rtTA expression. We observed that the EPO isoforms produced in the retina differ from one another based on the transduced cell type of origin within the retina and also differ from both the physiological EPO isoforms and the isoforms produced by AAV-transduced skeletal muscle.

Defects in the RPE65 gene, which is selectively expressed in the retinal pigment epithelium (RPE), result in blindness and gradual photoreceptor cell degeneration. Experiments were conducted to assess the efficacy of gene replacement therapy in restoring retinal function in RPE65-/- dogs. Long-term effects of RPE65 gene therapy were assessed using visual behavioral testing and electroretinographic (ERG) recordings at 10-12 weeks and 6-9 months after surgery in five affected dogs. Subretinal injections of similar dosages of two constructs were performed in affected dogs at the ages of 4-30 months: rAAV.RPE65 into one eye and, in four of five dogs, rAAV.GFP contralaterally. Before surgery all RPE65-/- dogs were behaviorally blind with either no or very low-amplitude ERG responses to light stimuli. Marked improvements in visual behavior and ERG responses were observed as early as 4 weeks after surgery in affected animals. Except for light-adapted 50 Hz ERG flicker responses, all ERG parameters tested increased significantly in the eyes treated with the rAAV.RPE65 construct at the early follow-up. Gradual progressive improvements in ERG responses were observed in the RPE65-treated eyes over time. An unexpected finding was that on long-term follow-up, marked improvement of photopic ERG responses were also observed in the contralateral control eye in both young and older dogs. These results are promising for future clinical trials of human patients with retinal degenerative diseases, such as Leber congenital amaurosis, that result from RPE65 gene defects.

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

OBJECTIVE

To investigate genetic determinants of macular pigment optical density in women from the Carotenoids in Age-Related Eye Disease Study (CAREDS), an ancillary study of the Women's Health Initiative Observational Study.

METHODS

1585 of 2005 CAREDS participants had macular pigment optical density (MPOD) measured noninvasively using customized heterochromatic flicker photometry and blood samples genotyped for 440 single nucleotide polymorphisms (SNPs) in 26 candidate genes related to absorption, transport, binding, and cleavage of carotenoids directly, or via lipid transport. SNPs were individually tested for associations with MPOD using least-squares linear regression.

RESULTS

Twenty-one SNPs from 11 genes were associated with MPOD (P ≤ 0.05) after adjusting for dietary intake of lutein and zeaxanthin. This includes variants in or near genes related to zeaxanthin binding in the macula (GSTP1), carotenoid cleavage (BCMO1), cholesterol transport or uptake (SCARB1, ABCA1, ABCG5, and LIPC), long-chain omega-3 fatty acid status (ELOVL2, FADS1, and FADS2), and various maculopathies (ALDH3A2 and RPE65). The strongest association was for rs11645428 near BCMO1 (βA = 0.029, P = 2.2 × 10(-4)). Conditional modeling within genes and further adjustment for other predictors of MPOD, including waist circumference, diabetes, and dietary intake of fiber, resulted in 13 SNPs from 10 genes maintaining independent association with MPOD. Variation in these single gene polymorphisms accounted for 5% of the variability in MPOD (P = 3.5 × 10(-11)).

CONCLUSIONS

Our results support that MPOD is a multi-factorial phenotype associated with variation in genes related to carotenoid transport, uptake, and metabolism, independent of known dietary and health influences on MPOD.

OBJECTIVE

To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.

METHODS

Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67.

RESULTS

In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells).

CONCLUSIONS

Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.

OBJECTIVE

RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued.

METHODS

Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin-specific antibodies.

RESULTS

Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin-positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin-positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths.

CONCLUSIONS

The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials.

OBJECTIVE

Human blindness caused by mutation of visual cycle genes has been discussed as potentially treatable by retinoid replacement either through gene transfer or pharmacological bypass. Mutations in the RDH12 gene disrupt the visual cycle in vitro, but little is known of the in vivo effects of mutant RDH12, other than the association with severe early-onset autosomal recessive retinal disease. The relationship of retinal organization and visual function in patients with RDH12 mutations was determined and comparisons made with the disease from mutations in another visual cycle gene, RPE65.

METHODS

Young patients with RDH12 mutations were studied with optical coherence tomography (OCT) and colocalized measures of vision with dark-adapted absolute thresholds. Results were compared to those in patients with RPE65 mutations.

RESULTS

Retinal architecture of patients with RDH12 mutations was appreciably distorted, precluding identification of the normal laminae. Some RDH12-mutant retinas were remarkably thick and others were thin, but all had the same dysplastic pattern. A comparison with the structural and functional consequences in patients with mutations in RPE65 indicated that the pathogenesis of retinal degeneration in RDH12 mutations was distinctly different.

CONCLUSIONS

The results demand critical consideration of the human disease mechanism and the therapeutic approach in patients with mutations in the putative visual cycle gene RDH12.

The need for mouse models, with their well-developed genetics and similarity to human physiology and anatomy, is clear and their central role in furthering our understanding of human disease is readily apparent in the literature. Mice carrying mutations that alter developmental pathways or cellular function provide model systems for analyzing defects in comparable human disorders and for testing therapeutic strategies. Mutant mice also provide reproducible, experimental systems for elucidating pathways of normal development and function. Two programs, the Eye Mutant Resource and the Translational Vision Research Models, focused on providing such models to the vision research community are described herein. Over 100 mutant lines from the Eye Mutant Resource and 60 mutant lines from the Translational Vision Research Models have been developed. The ocular diseases of the mutant lines include a wide range of phenotypes, including cataracts, retinal dysplasia and degeneration, and abnormal blood vessel formation. The mutations in disease genes have been mapped and in some cases identified by direct sequencing. Here, we report 3 novel alleles of Crx(tvrm65), Rp1(tvrm64), and Rpe65(tvrm148) as successful examples of the TVRM program, that closely resemble previously reported knockout models.

OBJECTIVE

Leber Congenital Amaurosis (LCA) is one of the most severe inherited retinal dystrophies with the earliest age of onset. This study was a mutational analysis of eight genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, RPGRIP1, MERTK, and LRAT) in 299 unrelated Spanish families, containing 42 patients with initial diagnosis of LCA: 107 with early-onset autosomal recessive retinitis pigmentosa (ARRP; onset <10 years of age) and 150 with non-early-onset ARRP (onset, >10 years of age).

METHODS

Samples were studied by using a genotyping microarray (Asper Biotech, Ltd., Tartu, Estonia) followed by a family study in cases with potential digenism/triallelism.

RESULTS

The frequencies of alleles carrying disease-causing mutations found in the authors'cohort using the chip were 23.8% (20/84) for LCA with 13 families carrying mutations, 6.1% (13/214) for early-onset ARRP with 12 families carrying mutations, and 4.3% (13/300) for non-early-onset ARRP with 12 families carrying mutations. CRB1 was the most frequently found mutated gene in affected Spanish families. Five families with anticipated digenism or triallelism were further studied in depth. Digenism could be discarded in all these cases; however, triallelism could not be ruled out.

CONCLUSIONS

CRB1 is the main gene responsible for LCA in the Spanish population. Sequence changes p.Asp1114Gly (RPGRIP1), p.Pro701Ser (GUCY2D), and p.Tyr134Phe (AIPL1) were found at similar frequencies in patients and control subjects. The authors therefore suggest that these changes be considered as polymorphism or modifier alleles, rather than as disease-causing mutations. The LCA microarray is a quick and reasonably low-cost first step in the molecular diagnosis of LCA. The diagnosis should be completed by conventional laboratory analysis as a second step. This stepwise proceeding permits detection of novel disease-causing mutations and identification of cases involving potential digenism/triallelism. Previous accurate ophthalmic diagnosis was found to be indispensable.

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.