Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28.

RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.

Rod Photoreceptors Avoid Saturation in Bright Light by the Movement of the G Protein Transducin.

Rod photoreceptors can be saturated by exposure to bright background light, so that no flash superimposed on the background can elicit a detectable response. This phenomenon, called increment saturation, was first demonstrated psychophysically by Aguilar and Stiles and has since been shown in many studies to occur in single rods. Recent experiments indicate, however, that rods may be able to avoid saturation under some conditions of illumination. We now show in ex vivo electroretinogram and single-cell recordings that in continuous and prolonged exposure even to very bright light, the rods of mice from both sexes recover as much as 15% of their dark current and that responses can persist for hours. In parallel to recovery of outer segment current is an ∼10-fold increase in the sensitivity of rod photoresponses. This recovery is decreased in transgenic mice with reduced light-dependent translocation of the G protein transducin. The reduction in outer-segment transducin together with a novel mechanism of visual-pigment regeneration within the rod itself enable rods to remain responsive over the whole of the physiological range of vision. In this way, rods are able to avoid an extended period of transduction channel closure, which is known to cause photoreceptor degeneration.SIGNIFICANCE STATEMENT Rods are initially saturated in bright light so that no flash superimposed on the background can elicit a detectable response. Frederiksen and colleagues show in whole retina and single-cell recordings that, if the background light is prolonged, rods slowly recover and can continue to produce significant responses over the entire physiological range of vision. Response recovery occurs by translocation of the G protein transducin from the rod outer to the inner segment, together with a novel mechanism of visual-pigment regeneration within the rod itself. Avoidance of saturation in bright light may be one of the principal mechanisms the retina uses to keep rod outer-segment channels from ever closing for too long a time, which is known to produce photoreceptor degeneration.

Non-photopic and photopic visual cycles differentially regulate immediate, early, and late phases of cone photoreceptor-mediated vision.

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters to 11-cis-retinol is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early, and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, while the emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaptation displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 min of light, early photopic vision was recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the nonphotopic or photopic visual cycles for mediating vision in bright light.

Light-Driven Regeneration of Cone Visual Pigments through a Mechanism Involving RGR Opsin in Müller Glial Cells.

While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Müller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Müller cell visual cycle and that regeneration of cone visual pigment can be driven by light.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

Peropsin modulates transit of vitamin A from retina to retinal pigment epithelium.

Peropsin is a non-visual opsin in both vertebrate and invertebrate species. In mammals, peropsin is present in the apical microvilli of retinal pigment epithelial (RPE) cells. These structures interdigitate with the outer segments of rod and cone photoreceptor cells. RPE cells play critical roles in the maintenance of photoreceptors, including the recycling of visual chromophore for the opsin visual pigments. Here, we sought to identify the function of peropsin in the mouse eye. To this end, we generated mice with a null mutation in the peropsin gene (Rrh). These mice exhibited normal retinal histology, normal morphology of outer segments and RPE cells, and no evidence of photoreceptor degeneration. Biochemically, Rrh-/- mice had ∼2-fold higher vitamin A (all-trans-retinol (all-trans-ROL)) in the neural retina following a photobleach and 5-fold lower retinyl esters in the RPE. This phenotype was similar to those reported in mice that lack interphotoreceptor retinoid-binding protein (IRBP) or cellular retinol-binding protein, suggesting that peropsin plays a role in the movement of all-trans-ROL from photoreceptors to the RPE. We compared the phenotypes in mice lacking both peropsin and IRBP with those of mice lacking peropsin or IRBP alone and found that the retinoid phenotype was similarly severe in each of these knock-out mice. We conclude that peropsin controls all-trans-ROL movement from the retina to the RPE or may regulate all-trans-ROL storage within the RPE. We propose that peropsin affects light-dependent regulation of all-trans-ROL uptake from photoreceptors into RPE cells through an as yet undefined mechanism.

Blue light regenerates functional visual pigments in mammals through a retinyl-phospholipid intermediate.

The light absorbing chromophore in opsin visual pigments is the protonated Schiff base of 11-cis-retinaldehyde (11cRAL). Absorption of a photon isomerizes 11cRAL to all-trans-retinaldehyde (atRAL), briefly activating the pigment before it dissociates. Light sensitivity is restored when apo-opsin combines with another 11cRAL to form a new visual pigment. Conversion of atRAL to 11cRAL is carried out by enzyme pathways in neighboring cells. Here we show that blue (450-nm) light converts atRAL specifically to 11cRAL through a retinyl-phospholipid intermediate in photoreceptor membranes. The quantum efficiency of this photoconversion is similar to rhodopsin. Photoreceptor membranes synthesize 11cRAL chromophore faster under blue light than in darkness. Live mice regenerate rhodopsin more rapidly in blue light. Finally, whole retinas and isolated cone cells show increased photosensitivity following exposure to blue light. These results indicate that light contributes to visual-pigment renewal in mammalian rods and cones through a non-enzymatic process involving retinyl-phospholipids.It is currently thought that visual pigments in vertebrate photoreceptors are regenerated exclusively through enzymatic cycles. Here the authors show that mammalian photoreceptors also regenerate opsin pigments in light through photoisomerization of N-ret-PE (N-retinylidene-phosphatidylethanolamine.

Diacylglycerol O-acyltransferase type-1 synthesizes retinyl esters in the retina and retinal pigment epithelium.

Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1(-/-) mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1(-/-) mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE's) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1(-/-) mice. Further, total retinyl-ester synthase activity was reduced in both dgat1(-/-) retina and RPE.

Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT).

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.

Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-α (TNF-α) in the interphotoreceptor matrix and retinas of Irbp(-/-) mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-α receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp(-/-) retina, and was colocalized with peanut agglutinin to the Irbp(-/-) cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp(-/-) retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp(-/-) retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp(-/-) retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp(-/-) mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp(-/-) mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

Autosomal recessive retinitis pigmentosa due to ABCA4 mutations: clinical, pathologic, and molecular characterization.

PURPOSE: Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous condition characterized by progressive loss of retinal photoreceptor cells. In order to gain new insights into the pathogenesis of ARRP, we evaluated the morphological, biochemical, and gene expression changes in eyes from a human donor with ARRP due to mutations in the ABCA4 gene. METHODS: Eyes were obtained postmortem from a donor with end-stage retinitis pigmentosa. The coding sequences of the RDS, RHO, and ABCA4 genes were screened for disease-causing mutations. Morphological changes in different regions of the retina were examined histologically, and levels of lipofuscin-associated bisretinoids were measured. Gene expression was examined in retinal/choroidal tissue using microarray analysis, and all parameters were compared to those in unaffected control donors. RESULTS: Genetic analysis of the donor's DNA identified two mutations in the ABCA4 gene, IVS14+1G > C and Phe1440del1 cT, each on a separate allele. Morphological evaluation revealed complete loss of the outer nuclear layer, remodeling of the inner retina, loss of retinal vasculature, and regional neovascularization. The retinal pigment epithelium and choriocapillaris exhibited regional preservation. Microarray analysis revealed loss of photoreceptor cell-associated transcripts, with preservation of multiple genes expressed specifically in inner retinal neurons. CONCLUSIONS: The persistence of transcripts expressed by inner retinal neurons suggests that despite significant plasticity that occurs during retinal degeneration, bipolar cells and ganglion cells remain at least partially differentiated. Findings from this study suggest that some forms of therapy currently under investigation may have benefit even in advanced retinal degeneration.

Receptor-mediated cellular uptake mechanism that couples to intracellular storage.

Cells are known to take up molecules through membrane transport mechanisms such as active transport, channels, and facilitated transport. We report here a new membrane transport mechanism that employs neither cellular energy like active transport nor a preexisting electrochemical gradient of the free substrate like channels or facilitated transport. Through this mechanism, cells take up vitamin A bound with high affinity to retinol binding protein (RBP) in the blood. This mechanism is mediated by the RBP receptor STRA6, which defines a new type of cell-surface receptor. STRA6 is essential for the proper functioning of multiple human organs, but the mechanisms that enable and control its cellular vitamin A uptake activity are unknown. We found that STRA6-mediated vitamin A uptake is tightly coupled to specific intracellular retinoid storage proteins, but no single intracellular protein is absolutely required for its transport activity. By developing sensitive real-time monitoring techniques, we found that STRA6 is not only a membrane receptor but also catalyzes vitamin A release from RBP. However, vitamin A released from RBP by STRA6 inhibits further vitamin A release by STRA6 unless specific intracellular retinoid storage proteins relieve this inhibition. This mechanism is responsible for its coupling to intracellular storage proteins. The coupling of uptake to storage provides high specificity in cellular uptake of vitamin A and prevents the excessive accumulation of free vitamin A. We have also identified a robust small-molecule-based technique to specifically stimulate cellular vitamin A uptake. This technique has implications in treating human diseases.

Complement system dysregulation and inflammation in the retinal pigment epithelium of a mouse model for Stargardt macular degeneration.

Accumulation of vitamin A-derived lipofuscin fluorophores in the retinal pigment epithelium (RPE) is a pathologic feature of recessive Stargardt macular dystrophy, a blinding disease caused by dysfunction or loss of the ABCA4 transporter in rods and cones. Age-related macular degeneration, a prevalent blinding disease of the elderly, is strongly associated with mutations in the genes for complement regulatory proteins (CRP), causing chronic inflammation of the RPE. Here we explore the possible relationship between lipofuscin accumulation and complement activation in vivo. Using the abca4(-/-) mouse model for recessive Stargardt, we investigated the role of lipofuscin fluorophores (A2E-lipofuscin) on oxidative stress and complement activation. We observed higher expression of oxidative-stress genes and elevated products of lipid peroxidation in eyes from abca4(-/-) versus wild-type mice. We also observed higher levels of complement-activation products in abca4(-/-) RPE cells. Unexpectedly, expression of multiple CRPs, which protect cells from attack by the complement system, were lower in abca4(-/-) versus wild-type RPE. To test whether acute exposure of healthy RPE cells to A2E-lipofuscin affects oxidative stress and expression of CRPs, we fed cultured fetal-derived human RPE cells with rod outer segments from wild-type or abca4(-/-) retinas. In contrast to RPE cells in abca4(-/-) mice, human RPE cells exposed to abca4(-/-) rod outer segments adaptively increased expression of both oxidative-stress and CRP genes. These results suggest that A2E accumulation causes oxidative stress, complement activation, and down-regulation of protective CRP in the Stargardt mouse model. Thus, Stargardt disease and age-related macular degeneration may both be caused by chronic inflammation of the RPE.

Rod outer segment retinol formation is independent of Abca4, arrestin, rhodopsin kinase, and rhodopsin palmitylation.

PURPOSE: The reactive aldehyde all-trans retinal is released in rod photoreceptor outer segments by photoactivated rhodopsin and is eliminated through reduction to all-trans retinol. This study was undertaken to determine whether all-trans retinol formation depends on Abca4, arrestin, rhodopsin kinase, and the palmitylation of rhodopsin, all of which are factors that affect the release and sequestration of all-trans retinal. METHODS: Experiments were performed in isolated retinas and single living rods derived from 129/sv wild-type mice and Abca4-, arrestin-, and rhodopsin kinase-deficient mice and in genetically modified mice containing unpalmitylated rhodopsin. Formation of all-trans retinol was measured by imaging its fluorescence and by HPLC of retina extracts. The release of all-trans retinal from photoactivated rhodopsin was measured in purified rod outer segment membranes according to the increase in tryptophan fluorescence. All experiments were performed at 37°C. RESULTS: The kinetics of all-trans retinol formation in the different types of genetically modified mice are in reasonable agreement with those in wild-type animals. The kinetics of all-trans retinol formation in 129/sv mice are similar to those in C57BL/6, although the latter are known to regenerate rhodopsin much more slowly. The release of all-trans retinal from rhodopsin in purified membranes is significantly faster than the formation of all-trans retinol in intact cells and is independent of the presence of the palmitate groups. CONCLUSIONS: The regeneration of rhodopsin and the recycling of its chromophore are not strongly coupled. Neither the activities of Abca4, rhodopsin kinase, and arrestin, nor the palmitylation of rhodopsin affects the formation of all-trans retinol.

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

Analysis of the retinoid isomerase activities in the retinal pigment epithelium and retina.

Light sensitivity in the vertebrate retina is mediated by the opsin visual pigments inside rod and cone photoreceptor cells. These pigments consist of a G protein-coupled receptor and the photo-sensitive ligand, 11-cis-retinaldehyde (11-cis-RAL). Absorption of a photon by an opsin pigment induces isomerization of the 11-cis-RAL chromophore to all-trans-retinaldehyde (all-trans-RAL), rendering the pigment insensitive to light. The bleached opsin regains light sensitivity by recombining with another 11-cis-RAL. The vertebrate eye contains a biochemical mechanism for regenerating 11-cis-RAL chromophore from all-trans-RAL, called the visual cycle. The visual cycle takes place within cells of the retinal pigment epithelium (RPE). A second visual cycle also appears to be present in Müller glial cells of the retina. A critical step in the regeneration of 11-cis-RAL chromophore is thermal re-isomerization to the 11-cis configuration of an all-trans-retinyl ester (all-trans-RE) or an all-trans-retinol (all-trans-ROL). In RPE cells, this step is carried out by an enzyme called Rpe65 isomerase. This chapter provides methods for assaying Rpe65 isomerase. Although Rpe65 utilizes an all-trans-RE such as all-trans-retinyl palmitate (all-trans-RP) as substrate, it can be assayed in RPE homogenates by providing all-trans-ROL substrate and allowing the endogenous lecithin:retinol acyl transferase (LRAT) to synthesize all-trans-REs using fatty acids from phosphatidylcholine in the membranes. Alternatively, all-trans-RP can be provided directly as substrate, although this requires the isomerase reaction to be carried out in the presence of detergent, since fatty-acyl esters of all-trans-ROL are insoluble. Methods are provided in this chapter for assaying Rpe65 in RPE homogenates with both all-trans-ROL and all-trans-RP substrates. A second visual cycle appears to be present in the retinas of cone-dominant species such as chicken. This retinal pathway may augment the RPE to provide 11-cis-RAL to cone photoreceptors under conditions of bright light where the rate of opsin photoisomerization is high. The isomerase in this pathway (isomerase-2) utilizes all-trans-ROL and palmitoyl coenzyme A (palm CoA) as substrates to synthesize 11-cis-retinyl palmitate (11-cis-RP). Isomerase-2 appears to be present in Müller cells but has not yet been identified. Methods are provided in this chapter for assaying isomerase-2 in chicken retina homogenates.

Flow of energy in the outer retina in darkness and in light.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Rpe65 isomerase associates with membranes through an electrostatic interaction with acidic phospholipid headgroups.

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

Predicting the pathogenicity of RPE65 mutations.

To assist in distinguishing disease-causing mutations from nonpathogenic polymorphisms, we developed an objective algorithm to calculate an "estimate of pathogenic probability" (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127%, and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available.