An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11-cis-retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an all-trans to 11-cis-retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11-cis-retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all-trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11-cis-retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11-cis-retinal for cone vision.

The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C(11) of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to approximately 75% of wild type, W331F to approximately 50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-pi carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C(15) oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11- and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal.

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.

OBJECTIVE

To describe the clinical phenotypes associated with various genotypes known to cause Leber congenital amaurosis (LCA).

METHODS

One hundred ten LCA patients were screened for various probable disease-causing gene sequence variations. Those patients with a probable disease-causing sequence variation in one of six genotypes were recalled for a follow-up examination. Evaluations included assessment of visual acuity, slit-lamp biomicroscopy, and dilated fundus examination. When possible, Goldmann perimetry was also performed.

RESULTS

Of the 37 LCA patients with suspected disease-causing sequence variations, 7 had an AIPL1 variation, 8, a CRB1 variation, 2, a CRX variation, 4, a GUCY2D variation, 11, an RPE65 variation, and 5, an RPGRIP1 variation. Across the 6 genotypes, we observed a wide range of visual acuities from 20/40 to no light perception. The widest range of vision was noted for patients with a CRB1 or RPE65 variation. Younger patients with an AIPL1 or RPGRIP1 variation were found to have severely reduced vision. Drusenlike deposits were more selectively observed in patients with mutations in the AIPL1, CRB1, RPE65, and RPGRIP1 genes, whereas focal regions of peripheral chorioretinal atrophy were observed only in patients with AIPL1 or RPE65 variations. Neurologic, intellectual, or psychomotor developmental delay was noted in 8.1% of our cohort.

CONCLUSIONS

There was considerable overlap of phenotypic expression in six genetic subtypes in our LCA cohort. However, phenotypic trends were noted in our patients' visual acuities and posterior segment findings within genotypes. These findings have practical value for genetic screening strategies for LCA patients based upon phenotype as well as for counseling patients on their visual prognosis.

In animals, successful production of the visual chromophore (11-cis-retinal or derivatives thereof such as 11-cis-3-hydroxy-retinal) is essential for photoreceptor cell function and survival. These carotenoid-derived compounds must combine with a protein moiety (the opsin) to establish functional visual pigments. Evidence from cell culture systems has implicated that the retinal pigment epithelium protein of 65 kDa (RPE65) is the long-sought all-trans to 11-cis retinoid isomerase. RPE65 is structurally related to nonheme iron oxygenases that catalyze the conversion of carotenoids into retinoids. In vertebrate genomes, two carotenoid oxygenases and RPE65 are encoded, whereas in insect genomes only a single representative of this protein family, named NinaB (denoting neither inactivation nor afterpotential mutant B), is encoded. We here cloned and functionally characterized the ninaB gene from the great wax moth Galleria mellonella. We show that the recombinant purified enzyme combines isomerase and oxygenase (isomerooxygenase) activity in a single polypeptide. From kinetics and isomeric composition of cleavage products of asymmetrical carotenoid substrates, we propose a model for the spatial arrangement between substrate and enzyme. In Drosophila, we show that carotenoid-isomerooxygenase activity of NinaB is more generally found in insects, and we provide physiological evidence that carotenoids such as 11-cis-retinal can promote visual pigment biogenesis in the dark. Our study demonstrates that trans/cis isomerase activity can be intrinsic to this class of proteins and establishes these enzymes as key components for both invertebrate and vertebrate vision.

OBJECTIVE

Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells.

METHODS

Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins.

RESULTS

One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A.

CONCLUSIONS

Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.

OBJECTIVE

Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. The functional consequences of the novel P25L RPE65 mutation was correlated with its early-childhood phenotype and compared with other pathogenic missense mutations.

METHODS

In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and two-color threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids.

RESULTS

A very mild phenotype was detected in a now 7-year-old boy homozygous for the P25L mutation in RPE65. Although abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5 years and 20/30 at age 7 years. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated responses in scotopic conditions, and light-adapted cone responses were approximately 1.5 log units below normal. High-resolution spectral domain OCT revealed morphologic changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wild-type RPE65-transfected cells, whereas RPE65/L22P-transfected cells had 13.5%.

CONCLUSIONS

The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to <10% of wild-type RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. Functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy.

OBJECTIVE

Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD) are genetically heterogeneous, with 11 genes currently implicated. The LCA chip may be used to interrogate many variants in one hybridization reaction. The purpose of this study was to assess the utility of this technology.

METHODS

One hundred fifty-three patients with LCA and EOSRD were screened using an array (Asper Ophthalmics, Tartu, Estonia) containing 344 published disease-causing variants and polymorphisms in eight genes: AIPL1, GUCY2D, CRB1, CRX, RPGRIP1, RPE65, MERTK, and LRAT. One hundred thirty-six probands underwent bidirectional sequencing of the full coding region of the RPE65 gene. The same technique was also used to confirm CRB1 and AIPL1 mutations initially identified with the Apex chip (Asper Ophthalmics). Single nucleotide polymorphism (SNP) analysis within control populations was performed for two variants, P701S and W21R, on the chip for GUCY2D.

RESULTS

Of the possible 109,392 interrogations, 3,346 (3.06%) failed on one strand whereas 259 (0.47%) failed on both. The chip reported mutations in 68 (44%) patients; 26 patients had two alleles identified (17%). Direct sequencing of RPE65 showed no discrepancies, whereas sequencing of AIPL1 and CRB1 revealed seven samples called erroneously. The SNP analysis of both GUCY2D variants revealed equal prevalence in the EOSRD panel and the normal population. Subsequent reanalysis, after excluding these polymorphisms, revealed one (18.3%) or two (11.7%) mutations identified in 46 patients. When evaluated by diagnosis, 46% of patients with LCA had one or two mutations identified, compared with 24% of patients with EOSRD.

CONCLUSIONS

This approach is a rapid and reasonably low-cost technique for identifying both previously identified mutations and common polymorphisms. The addition of further genes and mutations to the chip will improve its utility, though it is advised that all results be checked by direct sequencing.

OBJECTIVE

The purpose of this study was to perform a comprehensive survey of all known Leber congenital amaurosis (LCA) genes and loci in a collection of 37 consanguineous LCA families from Saudi Arabia.

METHODS

Direct PCR and sequencing were used to screen 13 known LCA genes (GUCY2D, CRX, RPE65, TULP1, AIPL1, CRB1, RPGRIP1, LRAT, RDH12, IMPDH1, CEP290, RD3, LCA5). In addition, families without mutations identified were further screened with STR markers around these 13 known LCA genes and two loci.

RESULTS

Disease-causing mutations were identified in nine of the 37 families: five in TULP1, two in CRB1, one in RPE65, and one in GUCY2D. Mutations in known genes only accounted for 24% of the Saudi families--much less than what has been observed in the European population (65%). Phenotype-genotype analysis was carried out to investigate the LCA disease penetrance for all families whose mutations identified. All identified mutations were found to segregate perfectly with the disease phenotype. On the other hand, severity of the disease varies for different patients carrying the same mutation and even within the same family. Furthermore, based on homozygosity mapping with both STR and SNP markers, one family is likely to map to the LCA3 locus.

CONCLUSIONS

These results underscore the importance of studying LCA disease families from different ethnic backgrounds to identify additional novel LCA disease genes. Furthermore, perfect segregation between mutation and disease indicates that LCA is fully penetrant. However, phenotypic variations among patients carrying the same mutation suggest that at least some of the variations in the clinical phenotype is due to modification from the genetic background, environment, or other factors.

OBJECTIVE

To examine the long-term effects of acute photooxidative stress in the retina, retinal pigment epithelium (RPE), and choroid.

METHODS

Albino rats injected with either the protective antioxidant phenyl-N-tert-butylnitrone (PBN) or saline 30 minutes before exposure to 5 klx white fluorescent light for 6 hours were kept for up to 3 months in 5 lux cyclic light. Electroretinograms were recorded, and the outer nuclear layer (ONL) and the choroidal thickness and area were measured after hematoxylin-eosin (H&E) staining. The expression of rod, cone, and RPE cell markers was detected by Western blotting, and apoptosis was analyzed by TUNEL staining. Oxidative stress was analyzed by immunohistochemistry against 4-hydroxynonenal (4-HNE)-modified proteins. Retinal and choroidal ultrastructures were observed by transmission electron microscopy (TEM). Choroidal circulation was analyzed by in vivo staining of the choroidal layer by trypan blue.

RESULTS

In the saline-injected animals, TUNEL- and 4-HNE-labeling in the ONL, RPE, and choroid were higher 24 hours and 7 days after light exposure, and ERG amplitude, ONL and choroidal thickness and area, and rhodopsin and RPE65 expression were lower 7 or more days after light exposure than in phenyl-N-tert-butylnitrone (PBN)-injected animals. In the saline-injected animals, the expression of mid-wavelength opsin and the presence of cone cells in the ONL and the choroidal circulation were preserved for 7 days after light exposure but started to decrease by 1 month and continued to decrease for 3 months after light exposure. An increase in TUNEL-positive cells was observed in the ONL at the inferior peripheral retina, just behind the iris, by 3 months after light exposure. Delayed loss of cone cells, remaining rod cells, and choroidal circulation were counteracted by PBN treatment.

CONCLUSIONS

Although cone cells are resistant to cell damage induced by acute photooxidative stress, progressive loss of cone cells continued for up to 3 months after light exposure. Impaired choroidal circulation is likely to be involved in the mechanism of delayed photoreceptor cell death after light exposure. Preserving choroidal circulation may provide a novel target for preserving the cone and the remaining rod cells in patients with retinal degeneration such as retinitis pigmentosa.

The history of the North African Jewish community is ancient and complicated with a number of immigration waves and persecutions dramatically affecting its population size. A decade-long process in Israel of clinical-molecular screening of North African Jews with incurable autosomal recessive blindness led to the identification of a homozygous splicing mutation (c.95-2A>> T; IVS2-2A>> T) in RPE65, the gene encoding the isomerase that catalyzes a key step in the retinoid-visual cycle, in patients from 10 unrelated families. A total of 33 patients (four now deceased) had the severe childhood blindness known as Leber congenital amaurosis (LCA), making it the most common cause of retinal degeneration in this population. Haplotype analysis in seven of the patients revealed a shared homozygous region, indicating a population-specific founder mutation. The age of the RPE65 founder mutation was estimated to have emerged 100-230 (mean, 153) generations ago, suggesting it originated before the establishment of the Jewish community in North Africa. Individuals with this RPE65 mutation were characterized with retinal studies to determine if they were candidates for gene replacement, the recent and only therapy to date for this otherwise incurable blindness. The step from molecular anthropological studies to application of genetic medicine was then taken, and a representative of this patient subgroup was treated with subretinal rAAV2-RPE65 gene therapy. An increase in vision was present in the treated area as early as 15 days after the intervention. This process of genetically analyzing affected isolated populations as a screen for gene-based therapy suggests a new paradigm for disease diagnosis and treatment.

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.

OBJECTIVE

The final step in the retinoid visual cycle is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs) that oxidize 11-cis-retinol (11cROL) to 11-cis-retinaldehyde (11cRAL). Genetic studies in mice indicate that the full repertoire of 11-cis-RDH enzymes remains to be identified. This study was conducted to characterize the 11-cis-RDH activity of RDH10 in vitro and specifically to determine whether RDH10 can functionally and physically interact with visual cycle proteins.

METHODS

Human RDH10 was expressed in COS1 cells to measure its 11-cis-RDH activity in the presence or absence of purified recombinant cellular retinaldehyde-binding protein (CRALBP). The RPE visual cycle was reconstituted in HEK-293A cells by co-expressing RDH10, CRALBP, RPE-specific 65-kDa protein (RPE65) and lecithin retinol acyltransferase (LRAT). The cells were subsequently treated with all-trans-retinol (atROL), and retinoid profiles were quantified by HPLC. Immunocytochemical and co-immunoprecipitation analyses were performed to determine whether RDH10 physically interacts with other visual cycle proteins.

RESULTS

RDH10 oxidized 11cROL to generate 11cRAL in vitro in the presence of CRALBP. RDH10 can use both NAD(+) and NADP(+) as cofactors for 11-cis-RDH activity, although NAD(+) cofactor confers more robust activity. In a cell culture model co-expressing RDH10 with RPE65, LRAT and CRALBP, the visual chromophore 11cRAL was generated from atROL. Immunohistochemistry showed that RDH10 co-localizes with RPE65 and CRALBP in vivo in primary bovine RPE cells. Immunoprecipitation analysis demonstrated that RDH10 physically interacts with CRALBP and RPE65.

CONCLUSIONS

RDH10 may function in the RPE retinoid visual cycle as an 11-cis-RDH, and thereby partially compensate for the loss of RDH5 function in patients with fundus albipunctatus.

OBJECTIVE

Cultured retinal pigment epithelium (RPE) may become a therapeutic option for transplantation in retinal disease. However maintaining a native RPE phenotype in vitro has proven challenging. The human RPE cell-line ARPE-19 is used widely as an alternative to primary RPE. It is grown in DMEM/F12 medium as standard, but its phenotype is dependent on culture conditions, and many differentiation markers are usually absent. The purpose of this study was to examine how this sensitive phenotype of ARPE-19 can be modulated by growth media with or without the metabolite pyruvate to elucidate better RPE growth conditions.

METHODS

ARPE-19 cells at passages p22 to p28 were cultured on filters for up to 3 months in DMEM/F12 or DMEM media with or without pyruvate and 1% fetal calf serum. Assessment of differentiation was performed using pigmentation, immunocytochemistry, protein/mRNA expression, transepithelial resistance, VEGF secretion, and ultrastructure.

RESULTS

Pyruvate, in combination with DMEM, induced dark pigmentation and promoted differentiation markers such as CRALBP and MerTK. Importantly, RPE65 protein was detected by Western blotting and was enhanced by pyruvate, high glucose, and DMEM. ARPE-19 cells maintained in this medium could also phagocytose human photoreceptor outer segments (POS). VEGF secretion was greater in DMEM cultures and was affected by glucose but not by pyruvate. Pigmentation never occurred in DMEM/F12.

CONCLUSIONS

This study demonstrated important differentiation markers, including pigmentation and Western blots of RPE65 protein, and showed human POS phagocytosis in ARPE-19 cultures using a simple differentiation protocol. The results favor the use of high-glucose DMEM with pyruvate for future RPE differentiation studies.

OBJECTIVE

To identify possible mutations in known candidate genes in patients with autosomal recessive (ar) and simplex retinitis pigmentosa (RP), by using an established strategy of flexible, multiplexed, microsatellite-based homozygosity mapping.

METHODS

A total of 78 microsatellite markers corresponding to 16 genes known to be responsible for arRP were selected and used in 18 multiplex amplifications, followed by genotyping. Twelve consanguineous probands and 47 nonconsanguineous probands (59 patients with arRP or simplex RP) agreed to the screening.

RESULTS

Of the 59 probands examined, 24 had a mean of 1.4 genes showing homozygosity for all markers within the corresponding gene region. Subsequent direct sequencing revealed three homozygous mutations. Two of them were novel mutations in the genes TULP1 (c.1145T->>C, F382S) and CNGB1 (c.3444 + 1G->>A). The other was a mutation in RPE65 (c.1543C->>T, R515W), which is known to cause Leber's congenital amaurosis. The clinical features of each patient, together with the cosegregation analysis, strongly support the pathogenicity of these mutations.

CONCLUSIONS

This systematic approach facilitated the identification of genes that cause arRP, and the results provide a widened spectrum of the mutation severity associated with a broader range of phenotypic manifestations of arRP.

RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark- and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12(-/-) animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.

Mice lacking the visual cycle enzymes RPE65 or lecithin-retinol acyl transferase (Lrat) have pupillary light responses (PLR) that are less sensitive than those of mice with outer retinal degeneration (rd/rd or rdta). Inner retinal photoresponses are mediated by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs), suggesting that the melanopsin-dependent photocycle utilizes RPE65 and Lrat. To test this hypothesis, we generated rpe65(-/-); rdta and lrat(-/-); rd/rd mutant mice. Unexpectedly, both rpe65(-/-); rdta and lrat(-/-); rd/rd mice demonstrate paradoxically increased PLR photosensitivity compared with mice mutant in visual cycle enzymes alone. Acute pharmacologic inhibition of the visual cycle of melanopsin-deficient mice with all-trans-retinylamine results in a near-total loss of PLR sensitivity, whereas treatment of rd/rd mice has no effect, demonstrating that the inner retina does not require the visual cycle. Treatment of rpe65(-/-); rdta with 9-cis-retinal partially restores PLR sensitivity. Photic sensitivity in P8 rpe65(-/-) and lrat(-/-) ipRGCs is intact as measured by ex vivo multielectrode array recording. These results demonstrate that the melanopsin-dependent ipRGC photocycle is independent of the visual retinoid cycle.

Cones function in constant light and are responsible for mediating daytime human vision. Like rods, cones use the photosensitive molecule 11-cis-retinal to detect light, and in constant illumination, a continuous supply of 11-cis-retinal is needed. A retina visual cycle is thought to provide a privileged supply of 11-cis-retinal to cones by using 11-cis-retinol generated in Müller cells. In the cycle, 11-cis-retinol is transported from Müller cells to cone inner segments, where it is oxidized to 11-cis-retinal. This oxidation step is only performed in cones, thus rendering the cycle cone-specific. Interphotoreceptor retinoid-binding protein (IRBP) is a retinoid-binding protein in the subretinal space that binds 11-cis-retinol endogenously. Cones in Irbp(-/-) mice are retinoid-deficient under photopic conditions, and it is possible that 11-cis-retinol supplies are disrupted in the absence of IRBP. We tested the hypothesis that IRBP facilitates the delivery of 11-cis-retinol to cones by preserving the isomeric state of 11-cis-retinol in light. With electrophysiology, we show that the cone-like photoreceptors of Nrl(-/-) mice use the cone visual cycle similarly to wild-type cones. Then, using oxidation assays in isolated Nrl(-/-)Rpe65(-/-) retinas, we show that IRBP delivers 11-cis-retinol for oxidation in cones and improves the efficiency of the oxidation reaction. Finally, we show that IRBP protects the isomeric state of 11-cis-retinol in the presence of light. Together, these findings suggest that IRBP plays an important role in the delivery of 11-cis-retinol to cones and can facilitate cone function in the presence of light.

OBJECTIVE

RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE.

METHODS

Human RPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.

RESULTS

Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed by Western blot analysis and by partial peptide sequencing. rHRPE65 from the nonmembrane fraction has a mass of 64,867 +/- 80 which is close to the calculated molecular weight from the amino acid sequence including the His-tag (64,663), whereas the membrane-associated rHRPE65 has a molecular mass of 65,380 +/- 150, which is significantly higher than that of the non-membrane-associated form and the calculated molecular weight, suggesting posttranslational modifications. Similarly, native RPE65 was detected in the cytosolic and microsomal fractions of the bovine RPE, with an average level of 3.8 +/- 1.3 and 7.2 +/- 0.4 microg RPE65 per eye, respectively. The cytosolic form had a molecular mass of 61,161 +/- 60, which is close to the calculated value (60,944), whereas that of the microsomal form was 61,961 +/- 170.

CONCLUSIONS

RPE65 is expressed in two forms, one of which is membrane associated and contains significant posttranslational modifications, similar to the native membrane-associated form.

Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat(-/-) and Rpe65(-/-) mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat(-/-) and Rpe65(-/-) mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases.

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.

Retinitis pigmentosa (RP) is a heterogeneous group of retinal dystrophies characterized by photoreceptor cell degeneration. RP causes night blindness, a gradual loss of peripheral visual fields, and eventual loss of central vision. Advances in molecular genetics have provided new insights into the genes responsible and the pathogenic mechanisms of RP. The genetics of RP is complex, and the disease can be inherited in autosomal dominant, recessive, X-linked, or digenic modes. Twenty-six causative genes have been identified or cloned for RP, and an additional fourteen genes have been mapped, but not yet identified. Eight autosomal dominant forms are due to mutations in RHO on chromosome 3q21-24, RDS on 6p21.1-cen, RP1 on 8p11-21, RGR on 10q23, ROM1 on 11q13, NRL on 14q11.1-11.2, CRX on 19q13.3, and PRKCG on 19q13.4. Autosomal recessive genes include RPE65 on chromosome 1p31, ABCA4 on 1p21-13, CRB1 on 1q31-32.1, USH2A on 1q41, MERTK on 2q14.1, SAG on 2q37.1, RHO on 3q21-24, PDE6B on 4p16.3, CNGA1 on 4p14-q13, PDE6A on 5q31.2-34, TULP1 on 6p21.3, RGR on 10q, NR2E3 on 15q23, and RLBP1 on 15q26. For X-linked RP, two genes, RP2 and RP3 (RPGR), have been cloned. Moreover, heterozygous mutations in ROM1 on 11q13, in combination with heterozygous mutations in RDS on 6p21.1-cen, cause digenic RP (the two-locus mechanism). These exciting molecular discoveries have defined the genetic pathways underlying the pathogenesis of retinitis pigmentosa, and have raised the hope of genetic testing for RP and the development of new avenues for therapy.

OBJECTIVE

Mutations in either retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) lead to Leber congenital amaurosis (LCA). By using the Lrat(-/-) mouse model, previous studies have shown that the rapid cone degeneration in LCA was caused by endoplasmic reticulum (ER) stress induced by S-opsin aggregation. The purpose of this study is to examine the efficacy of an ER chemical chaperone, tauroursodeoxycholic acid (TUDCA), in preserving cones in the Lrat(-/-) model.

METHODS

Lrat(-/-) mice were systemically administered with TUDCA and vehicle (0.15 M NaHCO(3)) every 3 days from P9 to P28. Cone cell survival was determined by counting cone cells on flat-mounted retinas. The expression and subcellular localization of cone-specific proteins were analyzed by western blotting and immunohistochemistry, respectively.

RESULTS

TUDCA treatment reduced ER stress and apoptosis in Lrat(-/-) retina. It significantly slowed down cone degeneration in Lrat(-/-) mice, resulting in a ∼3-fold increase in cone density in the ventral and central retina as compared with the vehicle-treated mice at P28. Furthermore, TUDCA promoted the degradation of cone membrane-associated proteins by enhancing the ER-associated protein degradation pathway.

CONCLUSIONS

Systemic injection of TUDCA is effective in reducing ER stress, preventing apoptosis, and preserving cones in Lrat(-/-) mice. TUDCA has the potential to lead to the development of a new class of therapeutic drugs for treating LCA.