Purpose: Extracellular accumulation of all-trans-retinaldehyde (atRAL), a highly reactive visual cycle intermediate, is toxic to cells of the outer retina and contributes to retinal and macular degenerations. However, the contribution of atRAL to retinal capillary function has not been studied. We hypothesized that atRAL released from the outer retina can contribute to retinal vascular permeability. We, therefore, tested the contribution of atRAL to retinal ischemia-reperfusion (IR)-induced vascular permeability.

Methods: IR was induced in mice by transient increase in intraocular pressure followed by natural reperfusion. The visual cycle was ablated in the Lrat-/- mice, reduced by dark adaptation or the use of the RPE65 inhibitor and atRAL scavenger emixustat. Accumulation of FITC-BSA was used to assess vascular permeability and DNA fragmentation quantified cell death after IR. Primary bovine retinal endothelial cell (BREC) culture was used to measure the direct effects of atRAL on endothelial permeability and cell death.

Results: Inhibition of the visual cycle by Lrat-/-, dark adaptation, or with emixustat, all reduced approximately half of IR induced vascular permeability at 48 hours. An increase in BREC permeability with atRAL coincided with lactate dehydrogenase (LDH) release, a measure of cell death. Both permeability and toxicity were blocked by emixustat.

Conclusions: Outer retinal pathology may contribute to vascular permeability by release of atRAL, which can act directly on vascular endothelial cells to alter barrier properties and induce cell death. These studies may have implications for a variety of blinding eye diseases that include outer retinal damage and retinal vascular permeability.

BACKGROUND

The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.

RESULTS

The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency.

CONCLUSIONS

The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal maculopathies.

In retinal degenerative disorders, when neural retinal cells are damaged, cell transplantation is one of the most promising therapeutic approaches. Optogenetic technology plays an essential role in the neural differentiation of stem cells via membrane depolarization. This study explored the efficacy of blue light stimulation in neuroretinal differentiation of Opto-mGluR6-engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs were selected for optogenetic study due to their capability to differentiate into retinal-specific neurons. BMSCs were isolated and phenotypically characterized by the expression of mesenchymal stem cell-specific markers, CD44 (99%) and CD105 (98.8%). mRPE culture identity was confirmed by expression of RPE-specific marker, RPE65, and epithelial cell marker, ZO-1. mRPE cells and BMSCs were transduced with AAV-MCS-IRES-EGFP-Opto-mGluR6 viral vector and stimulated for 5 days with blue light (470 nm). RNA and protein expression of Opto-mGluR6 were verified. Optogenetic stimulation-induced elevated intracellular Ca²⁺ levels in mRPE- and BMS-treated cells. Significant increase in cell growth rate and G1/S phase transition were detected in mRPE- and BMSCs-treated cultures. Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rxrg, Nr2f2, Ascl1, Hes5, and Sox8 were overexpressed in treated BMSCs and Barlh2, Rorb, and Sox8 were overexpressed in treated mRPE cells. Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal-specific neuron markers, in mRPE and BMS cell cultures were demonstrated. Differentiation of ganglion, amacrine, photoreceptor cells, and bipolar and Muller precursors were determined in BMSCs-treated culture and were compared with mRPE. mRPE cells represented more abundant terminal Muller glial differentiation compared with BMSCs. Our results also demonstrated that optical stimulation increased the intracellular Ca²⁺ level and proliferation and differentiation of Opto-mGluR6-engineered BMSCs. It seems that optogenetic stimulation of mRPE- and BMSCs-engineered cells would be a potential therapeutic approach for retinal degenerative disorders.

Keywords: mesenchymal stem cells; optogenetic stimulation; retinal degenerative disorders; retinal differentiation; retinal pigment epithelial cells.

Retinal dystrophies (RD) are a rare genetic disorder with high genetic heterogeneity. This study aimed at identifying disease-causing variants in fifteen consanguineous Tunisian families. Full ophthalmic examination was performed. Index patients were subjected to IROme analysis or whole exome sequencing followed by homozygosity mapping. All detected variations were confirmed by direct Sanger sequencing. Mutation analysis in our patients revealed two compound heterozygous mutations p.(R91W);(V172D) in RPE65, and five novel homozygous mutations: p.R765C in CNGB1, p.H337R in PDE6B, splice site variant c.1129-2A > G and c.678_681delGAAG in FAM161A and c.1133 + 3_1133 + 6delAAGT in CERKL. The latter mutation impacts pre-mRNA splicing of CERKL. The other changes detected were six previously reported mutations in CNGB3 (p.R203*), ABCA4 (p.W782*), NR2E3 (p.R311Q), RPE65 (p.H182Y), PROM1 (c.1354dupT) and EYS (c.5928-2A > G). Segregation analysis in each family showed that all affected individuals were homozygotes and unaffected individuals were either heterozygote carriers or homozygous wild type allele. These results confirm the involvement of a large number of genes in RD in the Tunisian population.

OBJECTIVE

Spatially resolved functional assessment of rods and cones under photopic and scotopic conditions is desirable to evaluate the treatment outcome of gene therapeutic applications in inherited retinal disorders, such as early- onset severe retinal dystrophy (EOSRD) or achromatopsia.

METHODS

A sample of 3 healthy subjects, 6 patients with RPE65 deficiency (aged 11-45 years), and 3 patients with cone dysfunction disorders underwent spectral sensitivity testing (SST) under conditions of dark and light adaptation using a Humphrey Field Analyzer modified perimeter.

RESULTS

SST in healthy subjects revealed sensitivity curves corresponding well with the CIE (International Commission on Illumination) standard fundamentals. Absence of cone function was observed in patients with cone dysfunction disorders. In patients with RPE65 mutations, SST under conditions of both dark and light adaptation revealed similar curves at typical cone sensitivities. S cone-related thresholds were diminished in young patients (11-14 years) and absent in adults (19 years and over).

CONCLUSIONS

In the present study, residual vision was cone mediated both under photopic and scotopic conditions in young patients with EOSRD associated with RPE65 mutations, but S cone function was severely reduced early on. In rod monochromats, vision was rod mediated both under conditions of dark and light adaptation. These observations are important for ongoing and future clinical trials employing gene therapeutic strategies in both rod-cone dystrophies and achromatopsia.

Inherited retinal diseases (IRDs) are a group of progressive disorders that lead to severe visual impairment or even complete blindness. IRDs display a vast heterogeneity, clinically as well as genetically, with over 250 genes identified in which mutations can cause one or more clinical subtypes of IRD. Long considered incurable diseases, intense research over the last two decades, combined with major technological advancements, have enabled the development of the first therapeutic approaches for these diseases. The approval of LuxturnaTM (voretigene neparvovec), a gene augmentation therapy vector for RPE65-associated IRD, by the US Food and Drug Administration and the European Medicines Agency, is considered a true milestone in the field, and has led to the development of similar, or different therapeutic strategies for many other subtypes of IRD. Despite these major achievements, there are still many aspects that can-and need to-be improved, including more insights into the relationship between genetic variation and cellular dysfunction, optimization of the vectors and sequences used, improving delivery methods, as well as understanding and modulating the (local) immune response. In addition, the extreme rarity of some genetic subtypes of IRDs poses an enormous challenge on the development of novel therapies, in terms of e.g., costs and regulatory affairs.[...].

Visual pigment consists of opsin covalently linked to the vitamin A-derived chromophore, 11-cis-retinaldehyde. Photon absorption causes the chromophore to isomerize from the 11-cis- to all-trans-retinal configuration. Continued light sensitivity necessitates the regeneration of 11-cis-retinal via a series of enzyme-catalyzed steps within the visual cycle. During this process, vitamin A aldehyde is shepherded within photoreceptors and retinal pigment epithelial cells to facilitate retinoid trafficking, to prevent nonspecific reactivity, and to conserve the 11-cis configuration. Here we show that redundancy in this system is provided by a protonated Schiff base adduct of retinaldehyde and taurine (A1-taurine, A1T) that forms reversibly by nonenzymatic reaction. A1T was present as 9-cis, 11-cis, 13-cis, and all-trans isomers, and the total levels were higher in neural retina than in retinal pigment epithelium (RPE). A1T was also more abundant under conditions in which 11-cis-retinaldehyde was higher; this included black versus albino mice, dark-adapted versus light-adapted mice, and mice carrying the Rpe65-Leu450 versus Rpe65-450Met variant. Taurine levels paralleled these differences in A1T. Moreover, A1T was substantially reduced in mice deficient in the Rpe65 isomerase and in mice deficient in cellular retinaldehyde-binding protein; in these models the production of 11-cis-retinal is compromised. A1T is an amphiphilic small molecule that may represent a mechanism for escorting retinaldehyde. The transient Schiff base conjugate that the primary amine of taurine forms with retinaldehyde would readily hydrolyze to release the retinoid and thus may embody a pool of 11-cis-retinal that can be marshalled in photoreceptor cells.

Keywords: photoreceptor cells; retinal pigment epithelium; taurine; visual cycle; vitamin A.

Purpose: Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE).

Methods: ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After seven days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin's effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content.

Results: Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported.

Conclusions: This is the first study to demonstrate that supplementing the culture media with sericin promotes viability of iPSC-RPE and ARPE-19. Sericin's viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Keywords: ARPE-19; Sericin; induced pluripotent stem-cell derived RPE; maturation; retinal pigment epithelium; viability.

Purpose: Pentosan polysulfate sodium (PPS; Elmiron) is a FDA-approved heparanase inhibitor for the treatment of bladder pain and interstitial cystitis. The chronic use of PPS has been associated with a novel pigmentary maculopathy, associated with discrete vitelliform deposits that exhibit hyperfluorescence, macular hyper-pigmentary spots, and foci of nodular RPE enlargement. Therefore, this study aimed to investigate the retinal morphology of heparanase knockout mice. Material and methods: The retinal morphology of heparanase knock-out and age-matched control wild type mice of 3-, 9- and 15-weeks old was characterized by means of histological evaluation. Immuno-histological stains for RPE65, F4/80 and Ki67 were performed for investigating the RPE, inflammatory and proliferating cells, respectively. Results: Histological analysis showed no changes in age-matched wild-type controls, whereas the eyes of heparanase null mice were characterized by alterations in RPE and neural retina, as manifest by RPE folds and choroidal thickening, detached RPE cells, thickening of the photoreceptor layer and retinal disorganization. The presence of discrete hyperfluorescent foci, however, was absent. The prevalence of the RPE/choroidal changes or protrusions seemed to progress over time and were correlated with more RPE65 signal rather than influx of F4/80- or Ki67-positive cells. These results indicate that the subretinal alterations were mostly RPE driven, without influx of inflammatory or proliferating cells. Conclusions: Our results indicate that heparanase deficiency in the mice leads to RPE folds, choroidal thickening, and retinal disorganization. The presence of discrete hyperfluorescent foci, a key characteristic of the human disease, was not observed. However, it can be concluded that some of the observations in mice are similar to those seen after chronic use of PPS in humans. These findings indicate that the toxicity observed in the presence of heparanase inhibitors is target-related and will preclude the clinical use of heparanase inhibition as a therapeutic intervention.

Keywords: Heparanase; retina; retinal pigment epithelium [RPE].

Anti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells.

Senescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept.

Clinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells.

Clinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Over the past two decades we have developed techniques and models to investigate the ways in which known molecular defects affect visual performance. Because molecular defects in retinal signalling invariably alter the speed of visual processing, our strategy has been to measure the resulting changes in flicker sensitivity. Flicker measurements provide not only straightforward clinical assessments of visual performance but also reveal fundamental details about the functioning of both abnormal and normal visual systems. Here, we bring together our past measurements of patients with pathogenic variants in the GNAT2, RGS9, GUCA1A, RPE65, OPA1, KCNV2 and NR2E3 genes and analyse the results using a standard model of visual processing. The model treats flicker sensitivity as the result of the actions of a sequence of simple processing steps, one or more of which is altered by the genetic defect. Our analyses show that most defects slow down the visual response directly, but some speed it up. Crucially, however, other steps in the response sequence can make compensatory adjustments to offset the abnormality. For example, if the abnormal step slows down the visual response, another step is likely to speed up or attenuate the response to rebalance system performance. Such compensatory adjustments are probably made by steps in the sequence that usually adapt to changing light levels. Our techniques and modelling also allow us to tease apart stationary and progressive effects, and the localised molecular losses help us to unravel and characterise individual steps in the normal and abnormal processing sequences.

Keywords: Clinical vision; Flicker sensitivity; GNAT2; GUCA1A; Gene defects; KCNV2; Light adaptation; Linear systems; Molecular loss; NR2E3; OPA1; RGS9; RPE65; Temporal processing; Visual psychophysics.

Purpose: To report surgical observations formulated during the first 120 cases of subretinal gene therapy in patients with inherited retinal degenerations (IRDs).

Methods: A two-surgeon team compiled surgical observations and formulated surgical pearls based on the consecutive cases of subretinal viral vector injection in patients enrolled in clinical trials focusing on choroideremia, achromatopsia, and RPGR-associated retinitis pigmentosa, as well as patients with RPE65-associated Leber congenital amaurosis receiving FDA-approved voretigene neparvovec-rzyl therapy.

Results: 120 subretinal surgeries were performed by a two-surgeon team. Key anatomical features pertinent to surgical management were noted and are described in this manuscript. Surgical decision making for successful subretinal administration of viral vectors and management of potential surgical challenges were formulated.

Conclusion: Lessons learned during subretinal gene therapy cases may be helpful to other surgeons entering clinical trials or performing post-approval gene therapy administration. Surgical pearls outlined in this manuscript may also be helpful for other targeted subretinal therapies such as cellular transplantation or retinal prosthesis implantation.

OBJECTIVE

To develop a transgene expression system in retinal pigment epithelial cells with the aim of enhancing the transcriptional activity of a weak RPE-specific/preferential promoter.

METHODS

The transgene expression system was established by introducing a chimeric transcriptional activator (GAL4-VP16) and its DNA binding sequence and using truncated human and mouse RPE65 promoters in combination with a luciferase reporter gene. Two groups of expression plasmids were constructed for transfection. The group for co-transfection contained two DNA constructs where the reporter and GAL4-VP16 were separately expressed in pLuc and pGV series. The other group, pLuc-GV series, was prepared as single DNA constructs expressing both the reporter and GAL4-VP16. The transcriptional activities of the DNA constructs were assayed by transfection of human RPE cells (RPE51 and D407) and other cell lines (HEK293, COS-1, Hela, HepG2, and F2000).

RESULTS

We found that the transcriptional activity of the human RPE65 promoter was dramatically enhanced 10-13 fold in RPE cells co-transfected with DNA constructs phR65luc and phR65GV when compared to the human RPE65 promoter alone. A comparatively lower, 4-5 fold, increase was observed following transfection with the single DNA construct phR65luc-GV. In RPE cells, when the transcriptional responses to GAL4-VP16 expression were compared between the RPE65 promoter of phR65luc and the minimal promoter of pLuc, the increase in transcriptional activity was about 10 fold higher in phR65luc constructs. Low or non-significant enhancement of promoter activity was observed with these constructs following transfection of the non-RPE cell lines.

CONCLUSIONS

Our results indicate that the current transgene expression system dramatically amplifies transcriptional activity of weak and cell-specific/preferential promoters (e.g., the hRPE65 promoter) whilst retaining relative cell specificity.

RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65's membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.

Leber congenital amaurosis is a retinal dystrophy with several forms of presentation due to its genetic variability. Case of a female girl followed up from 4 to 11 years old is presented, with positive clinical data of nyctalopia, myopia and choroid ocular fundus. Electroretinogram was not measurable in all phases but diagnostic was confirmed by RPE65 mutation genetic study. RPE65 Leber congenital amaurosis is particularly important as it has been researched for a gene therapy treatment with good functional outcomes up to now, awaiting to offer hope and a better quality of life to people with this disease.

The retinal pigment epithelium-specific 65 kDa (RPE65) isomerase plays a pivotal role in photoreceptor survival and function. RPE65-catalyzed synthesis of 11-cis-retinol from all-trans-retinyl esters in the visual cycle is negatively regulated, through a heretofore unknown mechanism, by the fatty acid transport protein FATP4, mutations in which are associated with ichthyosis prematurity syndrome (IPS). Here, we analyzed the interaction between deletion mutants of FATP4 and RPE65 as well as the impacts of IPS-associated FATP4 mutations on RPE65 expression, 11-cis-retinol synthesis, and all-trans-retinyl ester synthesis. Our results suggest that the interaction between FATP4 and RPE65 contributes to inhibition of RPE65 function and that IPS-associated nonsense and missense mutations in FATP4 have different effects on the visual cycle.

The ability of iron to transfer electrons enables the contribution of this metal to a variety of cellular activities even as the redox properties of iron are also responsible for the generation of hydroxyl radicals (^•OH), the most destructive of the reactive oxygen species. We previously showed that iron can promote the oxidation of bisretinoid by generating highly reactive hydroxyl radical (^•OH). Now we report that preservation of iron regulation in the retina is not sufficient to prevent iron-induced bisretinoid oxidative degradation when blood iron levels are elevated in liver-specific hepcidin knockout mice. We obtained evidence for the perpetuation of Fenton reactions in the presence of the bisretinoid A2E and visible light. On the other hand, iron chelation by deferiprone was not associated with changes in postbleaching recovery of 11-cis-retinal or dark-adapted ERG b-wave amplitudes indicating that the activity of Rpe65, a rate-determining visual cycle protein that carries an iron-binding domain, is not affected. Notably, iron levels were elevated in the neural retina and retinal pigment epithelial (RPE) cells of Abca4^-/- mice. Consistent with higher iron content, ferritin-L immunostaining was elevated in RPE of a patient diagnosed with ABCA4-associated disease and in RPE and photoreceptor cells of Abca4^-/- mice. In neural retina of the mutant mice, reduced Tfrc mRNA was also an indicator of retinal iron overload. Thus iron chelation may defend retina when bisretinoid toxicity is implicated in disease processes.

Keywords: Fenton reaction; bisretinoid; iron; light; lipofuscin; retina; retinal pigment epithelium.

Voretigene neparvovec (VN) is the first gene therapy in ophthalmology for patients with RPE65-mediated hereditary retinal dystrophy. It has recently obtained European market approval, which is subject to strict regulatory and organizational conditions for its use. Here, we analyze the main studies supporting the authorization of this new therapy and describe the necessary steps to take at a hospital level for optimal administration to patients following current regulations.

Keywords: Adeno-associated viral vectors; Amaurose congénitale de Leber; Dystrophie rétinienne héréditaire; Gene therapy; Gène RPE65; Hereditary retinal dystrophy; Leber's congenital amaurosis; RPE65 gene; Retinitis pigmentosa; Rétinite pigmentaire; Thérapie génique; Vecteur Adénoviral; Voretigene neparvovec.

OBJECTIVE

To investigate whether previously reported changes in protein expression of middle and long (M/L) and short (S) wavelength cone opsin pigments in cultured retina are correlated with changes in their gene expression. Additionally, to elucidate the importance of a functional retinal pigment epithelium for the development of photoreceptor outer segments.

METHODS

Neonatal rat retinas were maintained in culture for 11 days and either fixed in 4% paraformaldehyde for immunohistochemistry or prepared for RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR. S-cone and M/L-cone photoreceptors as well as rod photoreceptors were immunohistochemically identified using specific antibodies. Peanut agglutinin (PNA)-lectin histochemistry was used to identify interphotoreceptor matrix associated with cone photoreceptors. Immunolabeling for ED-1 and RPE65 was performed in combination with PNA-lectin staining to examine interactions between photoreceptor cells and the retinal pigment epithelium. Relative estimates of mRNA expression levels for M/L-opsin, S-opsin, recoverin, and rhodopsin in normal and cultured retina were determined by using quantitative RT-PCR.

RESULTS

Strong immunolabeling for recoverin and rhodopsin accumulated in outer segments as well as photoreceptor somata in vitro. Cultured and normal retinas showed similar relative expression levels of recoverin and rhodopsin mRNA. In cultured rat retina, the density of S-cones was high and M/L-cones could not be immunohistochemically detected. However, M/L-cone photoreceptor mRNA was detectable, but at a fourfold lower level in cultured than in vivo retinas. The S-cone photoreceptor mRNA level was almost twofold lower than in vivo. Retinal pigment epithelium cells in cultured specimens showed no RPE65 immunolabeling, but expressed immunolabeling for ED-1 indicating phagocytic activity of these cells in vitro.

CONCLUSIONS

We assume that the high density of S-cones and virtually no M/L-cones seen in in vitro retinas might represent an immature stage with numerous S-cones and suppressed transdifferentiation into M/L-cone phenotype. A non-functional relationship between photoreceptor cells and a dysfunctional retinal pigment epithelium may have severe consequences for the development of outer segments.

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.

Leber congenital amaurosis (LCA) is an early onset retinal dystrophy that causes severe visual impairment. With the development of molecular genetics and the therapeutic gene replacement technology, the adeno-associated viral (AAV) vector-mediated gene therapy for LCA achieved encouraging progress in the past decade. The success of the Phase I clinical trials of human RPE65 gene therapy for LCA II patients makes it a pioneer in the field of retinal gene therapy and brings light to the cure of other hereditary retinopathy. This article briefly reviews the recent developments in the preclinical animal experiments and Phase I clinical trials for LCA.

<strong class="sub-title"> Purpose: </strong> Intrinsically photosensitive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and can signal light continuously for many hours. Melanopsin is excited when its chromophore <i>11-cis-</i>retinal absorbs a photon and becomes <i>all-trans-</i>retinal, which must be reisomerized to <i>11-cis-</i>retinal to regenerate photoexcitable melanopsin. Due to the great distance separating ipRGCs from the retinal pigment epithelium (RPE) whose retinoid cycle produces <i>11-cis-</i>retinal, ipRGCs had been assumed to regenerate all melanopsin molecules autonomously. Surprisingly, we previously found that pharmacologically inhibiting the retinoid cycle rendered melanopsin-based responses to prolonged illumination less sustained, suggesting that the RPE may supply retinoids to help ipRGCs regenerate melanopsin during extended photostimulation. However, the specificity of those drugs is unclear. Here, we reexamined the role of the retinoid cycle, and tested whether the RPE-to-ipRGC transport of retinoids utilizes cellular retinaldehyde-binding protein (CRALBP), present throughout the RPE and Müller glia.

Methods: To measure melanopsin-mediated photoresponses in isolation, all animals were 8- to 12-month-old rod/cone-degenerate mice. We genetically knocked out RPE-specific 65 kDa protein (RPE65), a critical enzyme in the retinoid cycle. We also knocked out the CRALBP gene rlbp1 mainly in Foxg1-expressing Müller cells. We obtained multielectrode-array recordings from ipRGCs in a novel RPE-attached mouse retina preparation, and imaged pupillary light reflexes in vivo.

Results: Melanopsin-based ipRGC responses to prolonged light became less tonic in both knockout lines, and pupillary light reflexes were also less sustained in RPE65-knockout than control mice.

<strong class="sub-title"> Conclusions: </strong> These results confirm that ipRGCs rely partly on the retinoid cycle to continuously regenerate melanopsin during prolonged photostimulation, and suggest that CRALBP in Müller glia likely transports <i>11-cis-</i>retinal from the RPE to ipRGCs - this is the first proposed functional role for CRALBP in the inner retina.

Keywords: Müller glia; Retinal pigment epithelium; ipRGC; melanopsin; pupillary light reflex.