The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels, and we have been studying the function and regulation of RPE65, the key retinol isomerase enzyme of the visual cycle, a gene whose expression is restricted to the RPE, and mutations in which cause severe blindness in humans, known as Leber Congenital Amaurosis 2 (LCA2). Disruption of the RPE-based vitamin A visual cycle blocking regeneration of visual pigment chromophore is the common phenotype shared by humans with RPE65 gene defects (LCA2) and the Rpe65 knockout mouse (overaccumulation of all-trans-retinyl esters and total absence of 11-cis retinal, resulting in extreme insensitivity to light). We have established a catalytic role for RPE65 in the synthesis of 11-cis retinol, identifying it as the long-sought visual cycle isomerohydrolase. Current work is focused on establishing the molecular mechanism of RPE65 catalysis, as well as its regulation and activity in the context of retinal development and in disease. We are also studying the effects of bisretinoid byproducts of the visual cycle (e.g., A2E) on RPE lysosomal metabolism. In the past year we have made the following progress: a) We have published several papers over the past few reporting periods focused on determining how RPE65 catalyzes all-trans to cis isomerization of retinol. Our experiments support a retinyl cation-mediated mechanism causing a general polyene bond delocalization rather than a nucleophilic substitution mechanism targeting only the C11-C12 double bond. Our analysis specifically favors a radical cation intermediate rather than a carbocation intermediate as the former alternative allows for the early loss of bond order crucial for docking of cis retinyl esters. We are currently addressing another aspect of the complex mechanism of RPE65, that of the O-alkyl bond cleavage that results in leaving of the fatty acid moiety. It is our hypothesis that this is the acquired primary enzymatic activity of RPE65, with cis isomerization being a secondary but crucial function. This O-alkyl bond cleavage is simplistically, but erroneously, characterized as a hydrolase reaction. We are now focusing on a) defining the mechanism of O-alkyl cleavage and b) determining the fate of the palmitate product. In this reporting period we have tested possible mechanisms for cleavage and fate of the palmitate product to answer these questions. b) We continued a project to study to establish (or disprove, as the case may be) palmitoylation of RPE65 cysteine(s), a controversial aspect of RPE65 biochemistry. Different groups have used mass spectrometry (MS) to definitively establish that RPE65 is palmitoylated, or that it is not. Clearly, only one of these alternatives is true. We are using bioorthogonal methods to determine if RPE65 is acylated by metabolic labeling in vitro, as well as in physiologically relevant cell culture models. Existence of labeled cysteine(s) will be established by labeling of protein and secondarily by MS of RPE65 peptides. Two separate approaches are being used to validate the presence or absence of a palmitoyl group. Progress in the establishment of palmitoylation as a valid process has been made and current work is focused on dissecting its functional relevance. c) We are revisiting our project on the origins of the vertebrate visual cycle. We have already disproved the notion that primitive chordates, such as tunicates and cephalochordates, anticipated a true vertebrate visual cycle and concluded that the crucial transition from the typical carotenoid double bond cleavage functionality (BCMO) to the isomerase functionality (RPE65), coupled with the origin of LRAT, occurred subsequent to divergence of the more primitive chordates (tunicates, etc.) in the last common ancestor of the jawless and jawed vertebrates. The new work in this area will address how a retinoid isomerase evolved from the carotenoid double bond cleavage functionality by assessing newly acquired sequences, including from hagfish, a species that is either a basal jawless vertebrate or a secondarily degenerated jawless vertebrate. d) We continued phenotypic analysis of hypomorphic knock-in mice in the mouse Rpe65 gene in this reporting period. The P25L knockin mouse models the mild phenotype of a homozygous P25L LCA2 patient with well-preserved cone vision. Milder human RPE65 missense mutations have better preserved cone function. Preserving cone function is a key concern in managing RPE65 retinal dystrophy, and an important objective of RPE65 gene therapy. The P25L line (Ki/Ki) had RPE65 mRNA levels identical to wildtype (WT) but its RPE65 protein levels are significantly lower. The retinas of P25L Ki/Ki are normal at 2 months and 8 months compared to WT. Retinoid levels in the normally maintained Ki/Ki are close to those seen in wildtype mice. We found that under typical mouse husbandry and light conditions, P25L Ki/Ki electrophysiological rod and cone function are close to WT, but with slower recovery kinetics. Importantly, there was no evidence of cone opsin mislocalization in P25L retina at 7 months suggestive of extended cone viability, unlike in the Rpe65 KO where this occurs by 1 month. However, compared to WT, P25L mice were protected against severe light damage, and retinoid levels after acute high light exposure are altered compared to wildtype, suggesting that visual cycle chromophore turnover is negatively affected by the P25L mutation, despite its close to normal physiology under low light level regime. These results are being prepared for publication. e) We continued a study on homeostatic responses of RPE to lipofuscin accumulation. Daily phagocytosis of outer segments (OS) leads to the accumulation in the RPE of autofluorescent lipofuscin, consisting of lipids and bisretinoids, such as A2E and its oxidation products, visual cycle byproducts implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in RPE during normal aging. We developed a cell model to study homeostatic mechanisms of RPE cells in response to A2E accumulation. To distinguish between pathologic and normal responses of RPE we treated ARPE-19 cells with low micromolar amounts of A2E over several weeks and compared lysosomal function and pH, degree of phagocytosis and melanization of treated to untreated differentiated ARPE-19 cells in response to a challenge of purified rod OS. We found that differentiated post-confluent ARPE-19 cells uptake, accumulate and partially degrade A2E under dim light conditions. The A2E uptake in lysosomes leads to an increase in lysosomal pH. Upon challenge with ROS, A2E-treated ARPE-19 cells showed an increase in a lysosome fraction containing melanin and are not impaired in OS phagocytosis. In spite of lysosomal alkalinization, as previously seen, this putative homeostatic mechanism may protect cells from death. A paper describing these results was published during the reporting period. We have also detected melanin in lysosomal fractions of BfA1-treated cells. In collaboration with Prof. Ulrich Schraermeyer (Univ. of Tuebingen, Germany), we analyzed melanized A2E-treated and BfA-treated ARPE19 cells by electron microscopy to identify the subcellular location of the melanin to corroborate the biochemical findings.
Showing the most recent 10 out of 26 publications