Pathomechanisms for aging of retinal pigment epithelium (RPE) and prophylactic therapy options in regard to AMD

An intact retinal pigment epithelium (RPE) represents an essential condition for the visual process. This post-mitotic RPE monolayer combines different functions such as degradation of photoreceptor outer segments, vitamin A cycle, support of retinal metabolism and maintenance of the outer blood-retina barrier. As a consequence of excessive metabolism, high oxygen levels, exposition to light of short wave length and ensuing radical formation, the RPE is highly dependent on protective systems. In spite of differentiated defence mechanisms, aging processes cause cumulative RPE damage, representing a major component of age-related macular degeneration (AMD), the leading cause of irreversible severe vision loss in people over 50 years old. A better understanding of the underlying pathophysiology will help to develop new prophylactic options which is becoming more and more important with increasing life expectancy.     

Successful RPE65 gene replacement and improved visual function in humans

Leber congenital amaurosis (LCA) is a group of severe autosomal recessive retinal dystrophies unified by the onset of blindness at birth and absence of ERG signals. Mutations in fourteen genes are currently associated with LCA, accounting for approximately 60% of patients. LCA genes encode retinal proteins that participate in a variety of significant retinal pathways ranging from photoreceptor development to replenishing vitamin A in the retinoid cycle. In some of the genetic subtypes of LCA (e.g. RPE65), viable photoreceptors and a relatively intact retina have been discovered. Consequently, successful photoreceptor and visual rescue have been achieved in mice and canine models of LCA. Two research groups, one in Philadelphia, USA (Maguire et al.) another in London, England (Bainbridge et al.) recently tested RPE65 replacement in two human trials with a total of six LCA patients. Remarkably, visual improvements were documented by ETDRS visual acuity measurements, pupillometry, nystagmus frequency, visual field measures, perimetry and an obstacle course. There were no local, or systemic side effects, nor improvements in ERG measures. These spectacular results will now stimulate further investigations of younger patients, higher dosages, and clinically or genetically related retinal disorders.     

RDH12 and RPE65, visual cycle genes causing leber congenital amaurosis, differ in disease expression

PURPOSE: 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.     

Klinische Manifestationen von Funktionsstörungen des retinalen Pigmentepithels

The retinal pigment epithelium (RPE) serves a variety of different functions and impairment of these functions can lead to a multitude of different diseases of the posterior segment of the eye. The RPE plays an important role as an ion and fluid pump for the reabsorption of subretinal fluid in retinal detachment. On the other hand, defects in this pump function and in the outer blood-retinal barrier formed by the RPE, lead to fluid retention in inflammatory diseases. Metaplasia of RPE cells to myofibroblasts can lead to proliferative vitreoretinopathy and tractive retinal detachment. Early age-related maculopathy is caused by disturbances of phagocytosis and metabolism of the RPE. Imbalance of the physiological equilibrium between vasoproliferative and vasoinhibitory factors secreted by the RPE is probably involved in the development of atrophic or neovascular forms of advanced age-related macular degeneration. Mutations in the different steps involved in regeneration of the visual pigment (visual cycle) may lead to retinal dystrophy. Finally, immunoregulatory properties of the RPE are responsible for the phenomenon of immunological privilege, which may facilitate clinical interventions such as gene therapy and RPE transplantation.     

Screening genes of the visual cycle RGR, RBP1 and RBP3 identifies rare sequence variations

The visual cycle is essential for vision and several genes encoding proteins of the cycle have been found mutated in various forms of inherited retinal dystrophy. We screened 3 genes of the visual cycle. RGR, encoding the retinal pigment epithelium (RPE) G protein-coupled receptor acting in vitro as a photoisomerase; RBP1, encoding the ubiquitous cellular retinol binding protein carrying intracellular all-trans retinoids; RBP3, encoding the interphotoreceptor retinoid binding protein, a retinal-specific protein which shuttles all-trans retinol from photoreceptors to RPE and 11-cis retinal from RPE to photoreceptors. We used denaturing high performance liquid chromatography (D-HPLC) and direct sequencing to screen 216 patients (134 with autosomal recessive or sporadic retinitis pigmentosa (RP) and 82 with other retinal dystrophies) for RBP1 and RBP3, and 331 patients for RGR (79 cases with autosomal dominant RP and 36 RP cases with undetermined inheritance were added to the 216 previous patients). Several variants were found in the 3 genes, including unique amino acid changes, but none of them showed evidence of pathogenicity. It is likely that mutations in RGR, RBP3, and possibly RBP1 occur rarely in inherited retinal dystrophies.     

Inherited multifocal RPE-diseases: mechanisms for local dysfunction in global retinoid cycle gene defects

Alterations of retinoid cycle genes are known to cause retinal diseases characterized by focal white dot fundus lesions. Fundus appearances reveal circumscribed RPE-changes, although generalized metabolic defects and global functional abnormalities are present. As a possible explanation, topographic inhomogeneities of the human photoreceptor mosaic and the role of a cone specific visual cycle will be discussed. Due to particular characteristics of photoreceptor subtypes as well as different pathways for photopigment regeneration the metabolic demand of individual RPE cells might differ. In "flecked retina diseases" heterogeneity of metabolic demand in individual RPE cells could therefore be responsible for their multifocal appearance.     

Leber congenital amaurosis: genes, proteins and disease mechanisms

Leber congenital amaurosis (LCA) is the most severe retinal dystrophy causing blindness or severe visual impairment before the age of 1 year. Linkage analysis, homozygosity mapping and candidate gene analysis facilitated the identification of 14 genes mutated in patients with LCA and juvenile retinal degeneration, which together explain approximately 70% of the cases. Several of these genes have also been implicated in other non-syndromic or syndromic retinal diseases, such as retinitis pigmentosa and Joubert syndrome, respectively. CEP290 (15%), GUCY2D (12%), and CRB1 (10%) are the most frequently mutated LCA genes; one intronic CEP290 mutation (p.Cys998X) is found in approximately 20% of all LCA patients from north-western Europe, although this frequency is lower in other populations. Despite the large degree of genetic and allelic heterogeneity, it is possible to identify the causative mutations in approximately 55% of LCA patients by employing a microarray-based, allele-specific primer extension analysis of all known DNA variants. The LCA genes encode proteins with a wide variety of retinal functions, such as photoreceptor morphogenesis (CRB1, CRX), phototransduction (AIPL1, GUCY2D), vitamin A cycling (LRAT, RDH12, RPE65), guanine synthesis (IMPDH1), and outer segment phagocytosis (MERTK). Recently, several defects were identified that are likely to affect intra-photoreceptor ciliary transport processes (CEP290, LCA5, RPGRIP1, TULP1). As the eye represents an accessible and immune-privileged organ, it appears to be uniquely suitable for human gene replacement therapy. Rodent (Crb1, Lrat, Mertk, Rpe65, Rpgrip1), avian (Gucy2D) and canine (Rpe65) models for LCA and profound visual impairment have been successfully corrected employing adeno-associated virus or lentivirus-based gene therapy. Moreover, phase 1 clinical trials have been carried out in humans with RPE65 deficiencies. Apart from ethical considerations inherently linked to treating children, major obstacles for the treatment of LCA could be the putative developmental deficiencies in the visual cortex in persons blind from birth (amblyopia), the absence of sufficient numbers of viable photoreceptor or RPE cells in LCA patients, and the unknown and possibly toxic effects of overexpression of transduced genes. Future LCA research will focus on the identification of the remaining causal genes, the elucidation of the molecular mechanisms of disease in the retina, and the development of gene therapy approaches for different genetic subtypes of LCA.     

Molecular genetics and emerging therapies for retinitis pigmentosa: Basic research and clinical perspectives

Retinitis Pigmentosa (RP) is a hereditary retinopathy that affects about 2.5 million people worldwide. It is characterized with progressive loss of rods and cones and causes severe visual dysfunction and eventual blindness in bilateral eyes. In addition to more than 3000 genetic mutations from about 70 genes, a wide genetic overlap with other types of retinal dystrophies has been reported with RP. This diversity of genetic pathophysiology makes treatment extremely challenging. Although therapeutic attempts have been made using various pharmacologic agents (neurotrophic factors, antioxidants, and anti-apoptotic agents), most are not targeted to the fundamental cause of RP, and their clinical efficacy has not been clearly proven. Current therapies for RP in ongoing or completed clinical trials include gene therapy, cell therapy, and retinal prostheses. Gene therapy, a strategy to correct the genetic defects using viral or non-viral vectors, has the potential to achieve definitive treatment by replacing or silencing a causative gene. Among many clinical trials of gene therapy for hereditary retinal diseases, a phase 3 clinical trial of voretigene neparvovec (AAV2-hRPE65v2, Luxturna) recently showed significant efficacy for RPE65-mediated inherited retinal dystrophy including Leber congenital amaurosis and RP. It is about to be approved as the first ocular gene therapy biologic product. Despite current limitations such as limited target genes and indicated patients, modest efficacy, and the invasive administration method, development in gene editing technology and novel gene delivery carriers make gene therapy a promising therapeutic modality for RP and other hereditary retinal dystrophies in the future.     

An overview of Leber congenital amaurosis: a model to understand human retinal development

Leber congenital amaurosis is a congenital retinal dystrophy described almost 150 years ago. Today, Leber congenital amaurosis is proving instrumental in our understanding of the molecular events that determine normal and aberrant retinal development. Six genes have been shown to be mutated in Leber congenital amaurosis, and they participate in a wide variety of retinal pathways: retinoid metabolism (RPE65), phototransduction (GUCY2D), photoreceptor outer segment development (CRX), disk morphogenesis (RPGRIP1), zonula adherens formation (CRB1), and cell-cycle progression (AIPL1). Longitudinal studies of visual performance show that most Leber congenital amaurosis patients remain stable, some deteriorate, and rare cases exhibit improvements. Histopathological analyses reveal that most cases have extensive degenerative retinal changes, some have an entirely normal retinal architecture, whereas others have primitive, poorly developed retinas. Animal models of Leber congenital amaurosis have greatly added to understanding the impact of the genetic defects on retinal cell death, and response to rescue. Gene therapy for RPE65 deficient dogs partially restored sight, and provides the first real hope of treatment for this devastating blinding condition.     

Screening genes of the visual cycle RGR,RBP1 and RBP3 identifies rare sequence variations

The visual cycle is essential for vision and several genes encoding proteins of the cycle have been found mutated in various forms of inherited retinal dystrophy. We screened 3 genes of the visual cycle. RGR, encoding the retinal pigment epithelium (RPE) G protein-coupled receptor acting in vitro as a photoisomerase; RBP1, encoding the ubiquitous cellular retinol binding protein carrying intracellular all-trans retinoids; RBP3, encoding the interphotoreceptor retinoid binding protein, a retinal-specific protein which shuttles all-trans retinol from photoreceptors to RPE and 11-cis retinal from RPE to photoreceptors. We used denaturing high performance liquid chromatography (D-HPLC) and direct sequencing to screen 216 patients (134 with autosomal recessive or sporadic retinitis pigmentosa (RP) and 82 with other retinal dystrophies) for RBP1 and RBP3, and 331 patients for RGR (79 cases with autosomal dominant RP and 36 RP cases with undetermined inheritance were added to the 216 previous patients). Several variants were found in the 3 genes, including unique amino acid changes, but none of them showed evidence of pathogenicity. It is likely that mutations in RGR, RBP3, and possibly RBP1 occur rarely in inherited retinal dystrophies.     

Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: a potential therapy for treatment of lipofuscin-based retinal diseases

PURPOSE: Excessive accumulation of lipofuscin is observed in numerous degenerative retinal diseases. A toxic vitamin A-based fluorophore (A2E) present within lipofuscin has been implicated in the death of RPE and photoreceptor cells. Here, we used an animal model that manifests accelerated lipofuscin accumulation (ABCA4-/- mutant) to evaluate the efficacy of a therapeutic approach based on reduction of serum retinol. METHODS: N-(4-hydroxyphenyl)retinamide (HPR) potently and reversibly reduces serum retinol. The interaction of HPR with retinol binding protein (RBP) and transthyretin was studied by spectrofluorometry and size-exclusion chromatography. To assess the effects of HPR on visual cycle retinoids and A2E biosynthesis, HPR was chronically administered to ABCA4-/- mice. Mice were evaluated using biochemical, electrophysiological, and morphologic techniques. RESULTS: Administration of HPR to ABCA4-/- mice caused immediate, dose-dependent reductions in serum retinol and RBP. Chronic administration produced commensurate reductions in visual cycle retinoids and arrested accumulation of A2E and lipofuscin autofluorescence in the RPE. Physiologically, HPR treatment caused modest delays in dark adaptation. Chromophore regeneration kinetics, light sensitivity of photoreceptors, and phototransduction processes were normal. Histologic examinations showed no alteration of retinal cytostructure or morphology. CONCLUSIONS: These findings demonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approach with potential to halt the accumulation of lipofuscin fluorophores in the eye.     

Uptake, processing and release of retinoids by cultured human retinal pigment epithelium

Upon absorption of a photon, the 11-cis retinaldehyde chromophore of rhodopsin is isomerized and reduced to all-trans retinol (vitamin A) in the photoreceptor outer segments, whereupon it leaves the photoreceptors, and moves to the retinal pigment epithelium (RPE). To clarify the function of the RPE in the regeneration of 11-cis retinaldehyde, we delivered all-trans retinol to monolayer cultures of human RPE. During delivery the retinol was associated with its putative natural carrier, interphotoreceptor retinoid binding protein (IRBP). IRBP has been proposed as a carrier protein involved in the exchange of retinoids between the photoreceptors and the retinal pigment epithelium. The retinoid composition of RPE cells and culture medium was analyzed by HPLC following several incubation periods. The RPE monolayer was found to process all-trans retinol into two distinct end-products: all-trans retinyl palmitate, which remained within the RPE monolayer: and 11-cis retinaldehyde which was released into the culture medium. These results demonstrate retinoid isomerase, retinol oxidoreductase and retinyl ester synthetase activity in human RPE cells cultured under the appropriate conditions. They show that IRBP can serve as a carrier of retinol through an aqueous medium to the RPE, and they illustrate that the visual cycle can be studied in vitro.     

Age-related alterations in vitamin A metabolism in the rat retina

Vitamin A plays a central role in visual transduction and in maintaining the structural integrity of the retina. It is possible that age-related alterations in vitamin A metabolism in the eye could contribute to the impairment of visual function that occurs during senescence. Therefore, investigations were conducted to determine whether the metabolism of this vitamin in the rat retina was altered during aging. Pigmented rats aged 12-, 22-, and 32 months were dark-adapted, and one eye from each animal was enucleated under dim red light. The neural retinas were separated from the retinal pigment epithelium (RPE)-choroid-scleral complexes, and the amounts and forms of vitamin A in both tissues were determined. The animals were then fully light-adapted, and the same measurements were performed on the tissues from the remaining eye of each rat. A number of age-related alterations in the vitamin A composition and content of the retina and RPE were observed. The most pronounced of these changes were significant increases in the ratios of retinyl palmitate to retinyl stearate with advancing age in both the neural retina and RPE. The total vitamin A ester contents of the RPEs increased during senescence in the dark-adapted state, but not in the light-adapted state. Retinyl ester levels in the neural retinas, on the other hand, did not differ significantly between 12- and 32-month-old animals in either the light-adapted or dark-adapted states. The amounts of all-trans retinol in the neural retinas decreased during aging, mainly in the dark-adapted state, whereas aging had no influence on RPE all-trans retinol content. The age-related alterations in metabolism of vitamin A that these observations reflect may be related to certain changes in visual function that occur during senescence.     

Development and cellular functions of the iris pigment epithelium

A number of studies have shown that transplantation of retinal pigment epithelial (RPE) cells to the subretinal space offers a promising treatment modality for retinal degenerative diseases. However, it is necessary to transplant autologous cells to avoid rejection; unfortunately, obtaining autologous RPE cells necessitates such traumatic surgical intervention as to make this approach irrelevant. It has been hypothesized that iris pigment epithelial (IPE) cells may be a possible substitute for RPE cells for transplantation into the subretinal space. The iris pigment epithelium, which has the same embryonic origin as retinal pigment epithelium, has not received much attention from visual scientists. Even though it forms a highly specialized tissue, it is not clear whether the iris pigment epithelium contributes critical functions to the health of the visual system. In vivo the IPE does not appear to have any of the functions characteristic of RPE; however, in vitro cultured IPE cells do acquire functions, such as specific phagocytosis of rod outer segments, that are characteristic of RPE cells, and have been shown to have the potential to carry out many functions characteristic of RPE cells, e.g., retinol metabolism. This review outlines the development and cellular functions of the IPE with special emphasis on the modulation of those functions that can allow the IPE cells to be transplanted to the subretinal space where they appear to acquire differentiated properties of retinal pigment epithelium (RPE).     

The bisretinoids of retinal pigment epithelium

The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.     

Reduced phagosomal content of the retinal pigment epithelium in response to retinoid deprivation.

Previous investigations have shown that lipofuscin accumulation in the retinal pigment epithelium (RPE) is reduced greatly as a consequence of vitamin A deprivation. The mechanism by which vitamin A regulates RPE lipofuscin deposition remains to be determined. It is possible that retinoids are direct precursors of this substance. Alternatively, vitamin A deficiency may reduce the uptake and processing of other potential precursors. In retinas lacking photoreceptor cells, RPE lipofuscin accumulation is decreased substantially. This finding suggested that components of phagocytosed photoreceptor outer segments may be precursors for RPE lipofuscin. The effect of vitamin A deprivation on RPE lipofuscin content therefore could be the result of reduced outer segment phagocytosis by the RPE of vitamin A-deprived animals. To evaluate this possibility, experiments were conducted to determine whether vitamin A deprivation altered the phagosomal content of the RPE. Rats were fed diets containing or lacking retinoid precursors of 11-cis retinal. Retinoic acid was included in the diets of the vitamin A-deprived animals. After both 10 and 26 weeks, the RPE phagosomal contents were determined in animals from each dietary group. Photoreceptor cell densities also were measured in these rats. At both time points, the RPE phagosomal content was lower significantly in the retinoid-deprived animals than in those fed a vitamin A precursor of the visual pigment chromophore. This reduction was not the result of photoreceptor cell death; the density of these cells was not affected significantly by dietary vitamin A. Thus, it appears that retinoid deprivation reduces the rate of photoreceptor outer segment turnover and, consequently, outer segment phagocytosis by the RPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Rpe65 knockout on accumulation of lipofuscin fluorophores in the retinal pigment epithelium.

PURPOSE: In all mammalian species examined to date the retinal pigment epithelium (RPE) has been found to accumulate autofluorescent lysosomal storage bodies (lipofuscin) during senescence. Substantial evidence indicates that retinoids in the RPE-retina complex play a major role in RPE lipofuscin formation. Indeed, at least one RPE lipofuscin fluorophore is derived in part from vitamin A aldehyde. However, the precise mechanisms by which retinoids modulate RPE lipofuscin accumulation have not been elucidated. In mice without a functional Rpe65 gene, isomerization of all-trans- to 11-cis-retinol is blocked. Experiments were performed to determine whether this impairment of retinoid metabolism alters RPE lipofuscin accumulation. METHODS: RPE lipofuscin fluorophore content was compared in 12- to 13-month-old Rpe65(+/+), Rpe65(+/-), and Rpe65(-/-) mice. Lipofuscin fluorophore content was determined using quantitative fluorometric measurements. RPE lipofuscin content was also estimated with quantitative ultrastructural techniques. RESULTS: In the Rpe65(-/-) mice, RPE lipofuscin fluorophore accumulation was almost abolished. In addition, a significantly reduced accumulation of lipofuscin fluorophores was also observed in the Rpe65(+/-) animals. The inability of the RPE of)Rpe65(-/-) mice to supply 11-cis-retinal from the RPE to the retinal photoreceptors was accompanied by a massive accumulation of lipid droplets in the RPE that appeared to contain substantial amounts of retinoids. CONCLUSIONS: These findings indicate that formation of RPE lipofuscin fluorophores is almost completely dependent on a normal visual cycle. The absence of retinal (both all-trans and 11-cis) in Rpe65 knockout mice drastically reduced formation of lipofuscin fluorophores in these animals. Even an excessive accumulation of retinyl fatty acid esters in the RPE of Rpe65 knockout mice did not contribute to lipofuscin accumulation.     

The retinal pigmentation pathway in human albinism: Not so black and white

Albinism is a pigment disorder affecting eye, skin and/or hair. Patients usually have decreased melanin in affected tissues and suffer from severe visual abnormalities, including foveal hypoplasia and chiasmal misrouting. Combining our data with those of the literature, we propose a single functional genetic retinal signalling pathway that includes all 22 currently known human albinism disease genes. We hypothesise that defects affecting the genesis or function of different intra-cellular organelles, including melanosomes, cause syndromic forms of albinism (Hermansky-Pudlak (HPS) and Chediak-Higashi syndrome (CHS)). We put forward that specific melanosome impairments cause different forms of oculocutaneous albinism (OCA1-8). Further, we incorporate GPR143 that has been implicated in ocular albinism (OA1), characterised by a phenotype limited to the eye. Finally, we include the SLC38A8-associated disorder FHONDA that causes an even more restricted “albinism-related” ocular phenotype with foveal hypoplasia and chiasmal misrouting but without pigmentation defects. We propose the following retinal pigmentation pathway, with increasingly specific genetic and cellular defects causing an increasingly specific ocular phenotype: (HPS1-11/CHS: syndromic forms of albinism)-(OCA1-8: OCA)-(GPR143: OA1)-(SLC38A8: FHONDA). Beyond disease genes involvement, we also evaluate a range of (candidate) regulatory and signalling mechanisms affecting the activity of the pathway in retinal development, retinal pigmentation and albinism. We further suggest that the proposed pigmentation pathway is also involved in other retinal disorders, such as age-related macular degeneration. The hypotheses put forward in this report provide a framework for further systematic studies in albinism and melanin pigmentation disorders.     

RPE65 is essential for the function of cone photoreceptors in NRL-deficient mice

PURPOSE: 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.