Abstract
Purpose :
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 may lead to buildup of lipids and proteins within the RPE and play a role in retinopathies such as age-related macular degeneration (AMD). L-DOPA, a drug that appears to delay onset of AMD, is the ligand of a receptor in the pigmentation pathway involved in vesicular trafficking, GPR143. Here we test whether GPR143 signaling alters the rate of POS degradation by the RPE.
Methods :
We isolated POS from bovine retinas via differential ultracentrifugation and sucrose gradient sedimentation. 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 was challenged with POS for 4 hours to allow endocytosis with and without L-DOPA. We removed the excess POS, then added fresh DMEM with dialyzed FBS ± 1μM L-DOPA. Phase-contrast and fluorescent images were captured every 8 hours for 28 hours. 6 experiments were conducted with multiple replicates in each. The number and area of fluorescent POS were analyzed using ImageJ and statistical analysis was performed using Prism Graph.
Results :
4 hours after POS introduction, the number of fluorescent particles and the area they occupied were similar regardless of L-DOPA treatment. At 12 hours, RPE treated with L-DOPA had significantly fewer fluorescent particles (79.1 ± 4.36% of control, p<0.001, n=58) that occupied a significantly smaller area (79.5 ± 5.03% of control, p<0.001, n=58). This trend continued at 20 hours, where RPE treated with L-DOPA had significantly fewer fluorescent particles (78.6 ± 5.09% of control, p<0.001, n=58) that occupied a significantly smaller area (77.7 ± 5.20% of control, p<0.001, n=58) than the control.
Conclusions :
Our results indicate that L-DOPA had no effect on POS phagocytosis measured at 4 hours. 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 abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.