GPR143 SIGNALING ALTERS INTRACELLULAR TRAFFICKING OF PHOTORECEPTOR OUTER SEGMENTS

Abstract

Photoreceptors undergo a daily renewal process by shedding the distal 10% of their outer segments (POS), which are phagocytosed and degraded by the retinal pigment epithelium (RPE). Inefficient digestion of POS could play a role in the pathogenesis of various retinopathies and be a sign of RPE aging and reduced function. L-DOPA, a drug that appears to delay onset of age-related macular degeneration (AMD), is the ligand of a receptor in the pigmentation pathway involved in vesicular trafficking, GPR143. Here we explore whether GPR143 signaling alters the rate of POS uptake and degradation by the RPE. We isolated POS from bovine retinas via differential ultracentrifugation and sucrose gradient sedimentation. For the uptake assay, POS were labeled with a green dye. Primary porcine RPE were challenged with POS in low tyrosine DMEM containing dialyzed FBS ± 1 μM L-DOPA for 2 hours. After the excess POS were removed, phase-contrast and fluorescent images were captured. For the degradation assay, POS were labelled with a pH sensitive dye that increases fluorescence intensity as pH decreases, such that the POS are only visible in the acidic lysosomal compartment. Primary porcine RPE were challenged with POS for 4 hours to allow endocytosis. We removed the excess POS, then added fresh low tyrosine DMEM with dialyzed FBS ± 1 μM L-DOPA. Phase-contrast and fluorescent images were captured every 8 hours for 28 hours. For both assays, the number and area of fluorescent POS were analyzed using ImageJ and statistical analysis was performed using Prism Graph. Our results indicate that L-DOPA had no effect on POS uptake. Our data also showed that once the POS were in the lysosomal compartment, L-DOPA had no effect on the rate of degradation past 12 hours. However, the reduced number and area of POS in the RPE after 12 hours suggest L-DOPA had a significant effect between hours 4 and 12, which most likely relates to more efficient endosomal trafficking to the lysosomal compartment. These findings may highlight a mechanism by which L-DOPA protects from AMD. This project is supported by the National Institutes of Health (NIH) under award number 5RO1 EY026544-05 (Brian S. McKay) and the Undergraduate Biology Research Program with funds from the Senior Vice Provost for Research.