Studies of retinyl ester hydrolase in the visual cycle

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PROJECT SUMMARY/ABSTRACT Light-induced isomerization of 11-cis retinal, the chromophore of visual pigments, to all-trans retinal initiates vision. Efficient recycling of 11-cis retinal through the visual cycle is essential for the formation of visual pigments and maintaining normal vision. A key step in the visual cycle is the conversion of all-trans retinyl ester to 11-cis retinol, catalyzed by RPE65, the retinol isomerase. Documented studies have shown that retinoids are largely stored as all-trans retinyl ester, the substrate of the isomerase RPE65, in lipid droplets (retinosomes) in the RPE. However, RPE65 is located in the ER and not near the lipid droplets. It is unknown how hydrophobic all-trans retinyl ester, the substrate of RPE65, is transported from lipid droplets to the ER. This represents an important knowledge gap in the visual cycle. A retinyl ester hydrolase (REH) may be present in lipid droplets to mobilize all-trans retinyl ester to provide the substrate for the RPE65 isomerase in the ER. Patatine-like phospholipase domain containing protein 2 (PNPLA2) is known as an adipose triglyceride lipase with an REH activity in the liver. Its function in the RPE and its association with the visual cycle have not been investigated. Our preliminary studies identified PNPLA2 in the RPE and in lipid droplets. Importantly, PNPLA2-/- mice showed declined ERG responses and delayed regeneration of visual pigments. PNPLA2 KO also resulted in reduced 11-cis retinal generation and increased levels of all-trans retinyl ester in the RPE, suggesting a decreased isomerase activity. These findings suggest a potential role of PNPLA2 in the visual cycle. We hypothesize that PNPLA2 functions as an REH in the RPE and mobilizes all-trans retinyl ester from lipid droplets to provide sufficient substrate for the RPE65 isomerase in the ER to generate 11-cis retinol. In this project, we will define the role of PNPLA2 in maintaining the visual cycle and normal vision. We will use RPE-specific PNPLA2 conditional KO mice to study the impacts of PNPLA2 KO on retinal function, retinal degeneration, visual pigment formation and regeneration of 11-cis retinal. We also plan to determine if co-expression of PNPLA2 together with RPE65 in the RPE of RPE65-/- mice by gene delivery will enhance the effect of RPE65 on restoring visual function, visual pigment formation and 11-cis retinal regeneration, compared to RPE65 gene delivery alone. We will also elucidate the mechanism by which PNPLA2 promotes the RPE65 isomerase activity and accelerates 11-cis retinal regeneration in the visual cycle. We will investigate if knockout of PNPLA2 in RPE cells will decrease, while overexpression of PNPLA2 will promote, isomerase activity of RPE65 and reduce lipid droplets in RPE cells. We will also determine if a PNPLA2 activator will promote, while a PNPLA2 inhibitor will inhibit the isomerase activity in RPE cells. These studies have potential to identify a missing component in the visual cycle and address an important knowledge gap in vision. This project will also identify a new function of PNPLA2 in the RPE. PNPLA2 has potential to improve the RPE65 gene therapy for retinal dystrophies caused by RPE65 mutations.