Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3.

Purpose: Galectin-3 (gal-3) is a soluble glycoprotein that has been associated with diverse forms of phagocytosis, including some mediated by the engulfment receptor MerTK. Retinal pigment epithelium (RPE) in vivo uses MerTK (or the related Tyro3) for phagocytosis of shed outer segment fragments during diurnal outer segment renewal. Here, we test if gal-3 plays a role in outer segment renewal in mice and if exogenous gal-3 can promote MerTK-dependent engulfment of isolated outer segment fragments by primary RPE cells in culture. Methods: We explored age- and strain-matched wild-type (wt), lgals3-/- and mertk-/- mice. Immunofluorescence and immunoblotting characterized gal-3 and RPE/retina protein expression, respectively. Outer segment renewal was investigated by live imaging of phosphatidylserine (PS) exposure on photoreceptor outer segment distal tips and by microscopy of rhodopsin-labeled RPE phagosomes in tissue sections. Retinal function was assessed by recording electroretinograms (ERGs). Phagocytosis assays feeding purified outer segment fragments (POS) were conducted with added recombinant proteins testing unpassaged primary mouse RPE. Results: Gal-3 localizes to neural retina and RPE in wt mice. The lgals3-/- photoreceptor outer segments display normal diurnal PS exposure at distal tips. The number of rhodopsin-positive phagosomes in wt and lgals3-/- RPE does not differ at peak or trough of diurnal phagocytosis activity. lgals3-/- mice show light responses like wt, and their eyes contain wt levels of retinal and RPE proteins. Unlike purified protein S, recombinant gal-3 fails to promote POS engulfment by mouse primary RPE in culture. Conclusions: Gal-3 has no essential role in MerTK-dependent outer segment renewal in mice.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.