Abstract
Purpose :
Galectin-3 (gal-3) is a member of the large family of secreted galectins with diverse tissue functions that often depend on specific galectin carbohydrate-binding activity. Adding gal-3 may alter apoptotic cell or debris phagocytosis in cell culture assays via the engulfment receptor MerTK. Diurnal retinal pigment epithelial (RPE) phagocytosis of distal photoreceptor outer segment tips (POS) is a MerTK-dependent process essential for photoreceptor function and survival in the mammalian retina that involves phosphatidylserine (PS) exposure by POS. Here, we explore gal-3-/- mice and primary wild-type (WT) and MerTK-/- RPE in culture to determine the contribution of gal-3 to outer segment renewal.
Methods :
To detect externalized PS, we used biosensor live imaging on freshly dissected retina. To localize gal-3, rhodopsin-positive phagosomes, and cell type marker proteins, we used immunofluorescence microscopy on fixed cells, retina tissue sections, and flatmounts. Protein levels were determined by immunoblotting. Retinal function was assessed by recording electroretinograms (ERGs). Protein binding and phagocytosis assays of purified POS were conducted serum-free in the presence of added recombinant proteins using unpassaged polarized primary mouse RPE in culture. Animal experiments used male and female mice to compare age- and strain-matched cohorts of at least 5 mice.
Results :
Gal-3 localizes to the neural retina and RPE in mice. Both WT and gal-3-/- retina exhibit the same increase in frequency of PS-exposing POS and decrease in length of PS-marked tips after light onset. Gal-3-/- RPE and WT RPE in vivo contain the same number of rhodopsin-positive phagosomes both 1.5 and 7 hours after light onset. Photoresponses and levels of opsins of WT and gal-3-/- retina are indistinguishable. Recombinant mouse gal-3 binds to the apical surface of primary mouse RPE in culture regardless of MerTK expression. Unlike addition of the MerTK ligand protein-S, addition of gal-3 does not promote engulfment of POS by primary mouse RPE in culture.
Conclusions :
Our results demonstrate that lack of gal-3 has no effect on diurnal photoreceptor outer segment renewal in mice. Extracellular gal-3 binds to the apical surface of the RPE independently of MerTK and does not promote MerTK-dependent phagocytosis of outer segment fragments by RPE cells in culture.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.