We are studying RPE-specific mechanisms, at both the regulatory and functional levels, and have been studying the function and regulation of RPE65, the key retinol isomerase enzyme of the visual cycle. 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, over the past few reporting periods, focused on determining how RPE65 catalyzes all-trans to cis isomerization of retinol, supporting a retinyl cation-mediated mechanism. We are currently addressing a further aspect of the complex mechanism of RPE65, that of the O-alkyl bond cleavage that results in leaving of the fatty acid moiety. We hypothesize that this is the acquired primary activity of RPE65, with cis isomerization being a secondary but crucial ancillary activity. We are now focusing on i) defining the mechanism of O-alkyl cleavage and ii) 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. In this reporting period we also completed a project that identified the lipid analog triacsin C as an inhibitor of RPE65. Initial experiments showed that RPE65 activity was reduced by co-expression of ACSLs fatty acyl:CoA ligases (ACSLs) 1, 3 or 6, or SLC27A family fatty acyl-CoA synthase FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently competitively inhibited RPE65 isomerase activity in cellulo (IC50=500 nM). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 M atRE. In conclusion, as triacsin C lacks structural features comparable with retinoids it probably competes with binding of the acyl moiety of atRE. These results were submitted for publication in this reporting period. 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. 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 completed phenotypic analysis of the P25L hypomorphic knock-in of the mouse Rpe65 gene in this reporting period. This models the mild phenotype of a homozygous P25L LCA2 patient with well-preserved cone vision. 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. We found that under typical mouse husbandry and light conditions, the retinas of P25L KI/KI are normal out to 20 months, retinoid levels in the normally maintained KI/KI P25L are close to those seen in WT, and 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, unlike WT but similar to Rpe65 KO, P25L mice were completely protected against light-induced retinal damage, and chromophore levels after acute high light exposure recovered much more slowly than wildtype, suggesting that visual cycle chromophore turnover is much slower, despite its close to normal physiology under low light level regime. These results were published in this reporting period. We are currently studying D477G (a putative dominant-acting RPE65 mutation) knockin mice made by CRISPR/Cas9 technology. d) We continued a study on homeostatic metabolic responses of RPE to normal quotidian bisretinoid 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. Our cellular model is designed to study homeostatic mechanisms of ARPE-19 RPE cells in response to A2E accumulation at low micromolar amounts of A2E over several weeks and comparing 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 previously found that A2E-treated ARPE-19 cells showed an increase in a melanin-containing lysosome fraction upon challenge with ROS. Treatment with low doses of bafilomycin A (BfA) replicates A2E findings. In spite of lysosomal alkalinization, as previously seen, this putative homeostatic mechanism may protect cells from death. 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. Current plans are to identify changes in gene regulation associated with these changes.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Poliakov, Eugenia; Soucy, Joseph; Gentleman, Susan et al. (2017) Phylogenetic analysis of the metazoan carotenoid oxygenase superfamily: a new ancestral gene assemblage of BCO-like (BCOL) proteins. Sci Rep 7:13192
Redmond, T Michael (2017) RPE65 takes on another role in the vertebrate retina. Proc Natl Acad Sci U S A 114:10818-10820
Poliakov, Eugenia; Samuel, William; Duncan, Todd et al. (2017) Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets ?-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide ?4-desaturase 1. PLoS One 12:e0176487
Eroglu, Abdulkerim; Gentleman, Susan; Poliakov, Eugenia et al. (2016) Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism. J Biol Chem 291:4966-73
Kutty, R Krishnan; Samuel, William; Abay, Rebecca et al. (2015) Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells. Cytokine 74:335-8
Wright, Charles B; Redmond, T Michael; Nickerson, John M (2015) A History of the Classical Visual Cycle. Prog Mol Biol Transl Sci 134:433-48
Li, Yan; Yu, Shirley; Duncan, Todd et al. (2015) Mouse model of human RPE65 P25L hypomorph resembles wild type under normal light rearing but is fully resistant to acute light damage. Hum Mol Genet 24:4417-28
Poliakov, Eugenia; Strunnikova, Natalya V; Jiang, Jian-kang et al. (2014) Multiple A2E treatments lead to melanization of rod outer segment-challenged ARPE-19 cells. Mol Vis 20:285-300
Ma, Hongwei; Thapa, Arjun; Morris, Lynsie et al. (2014) Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration. Proc Natl Acad Sci U S A 111:3602-7
Wright, Charles B; Chrenek, Micah A; Foster, Stephanie L et al. (2013) Complementation test of Rpe65 knockout and tvrm148. Invest Ophthalmol Vis Sci 54:5111-22

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