The Role of Galectins in Photoreceptor Outer Segment Renewal
Diurnal outer segment renewal in the mammalian retina involves synchronized exposure at light onset of phosphatidylserine (PS) at the tips of photoreceptor outer segments, which serves as an “eat-me” signal stimulating the phagocytic machinery of the adjacent retinal pigment epithelium (RPE). Efficient clearance of shed, PS-marked photoreceptor outer segment fragments by RPE cells is essential for life-long function and viability of photoreceptors and hence for vision. Molecular signals that govern PS exposure on outer segments are poorly understood to date. Furthermore, extracellular molecular signals that serve as communicators between RPE cells and photoreceptors during phagocytosis have not been entirely discovered. In the wild-type mouse retina, the frequency of photoreceptors with PS-marked outer segment tips is high at light onset and shortly thereafter but low at all other times. In contrast, mice lacking the RPE phagocytic receptor integrin αvβ5 or its ligand MFG- E8 display no difference in the frequency of PS exposing outer segments between the morning and the afternoon. This suggests that RPE cells may influence this process through direct or indirect interaction with outer segments. Members of a family of β- galactoside binding proteins expressed by RPE cells called galectins can promote PS externalization by leukocytes in culture and can serve as phagocytic ligands for cells in culture. Therefore, I asked if two widely expressed galectins, gal-1 and gal-3, contribute to the regulation of PS exposure on spent outer segment tips and RPE phagocytosis in the mammalian retina. Immunofluorescence assays demonstrated that both gal-1 and gal-3 localize to the apical region of RPE cells in situ. Studies on knockout mice revealed that gal-1 -/- mice, but not gal-3 -/- mice, display fewer PS-exposing outer segments 1 hour after light onset compared to wild-type mice. Furthermore, comparison of phagosome content in wild-type vs gal-1 -/- RPE or gal-3 -/- RPE at different timepoints revealed a significant decrease in the number of phagosomes in gal-1 -/- RPE, but not gal-3 -/- RPE, at the peak of RPE phagocytosis. Incubation with recombinant galectins on freshly dissected wildtype retinas before staining with a fluorescent biosensor specific for PS showed that gal-1, but not gal-3, can induce PS exposure on wild-type outer segments ex vivo. Immunoblotting of wild-type mouse soluble interphotoreceptor space proteins revealed a significant increase in the level of gal-1 protein in the interphotoreceptor space at light onset compared to other times of day. Finally, specific gal-1 mutants were produced to test if carbohydrate binding or dimer formation by gal-1 are required for PS exposure on outer segments. Incubation with carbohydrate-binding deficient or monomeric gal-1 mutants revealed that neither mutant can induce PS exposure on outer segments. Taken together, these findings support a novel role for gal-1 in outer segment renewal as an extracellular molecular contributor to the synchronized increase in the vital process of outer segment PS exposure, and in turn, RPE phagocytosis.
Esposito, Nicholas John, "The Role of Galectins in Photoreceptor Outer Segment Renewal" (2019). ETD Collection for Fordham University. AAI13426898.