Involvement of LC3-dependent phagocytosis and MREG in the degradation of disk membranes by the RPE

Abstract

Purpose: Several studies suggest a biphasic degradation of ingested photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE). In 1993, Bosch,et.al. (J. Histochem Cytochem 41:253), proposed that two lysosomal pools are required for complete digestion of ingested POS. Earlier, Remé, et al., identified an autophagic pool of degradative vesicles that followed a diurnal pattern (IOVS 1977,9:807). Our own studies of RPE phagocytosis have identified a novel cargo-sorting protein melanoregulin (MREG) that is required for digestion of POSs. Here we explore the hypothesis that MREG-dependent processing of POS phagosomes links aspects of both the autophagic and phagocytic processes in a pathway known as LC3-dependent phagocytosis (LAP).

Methods: The diurnal pattern of degradative processes as reflected in lysosomal and proteolytic enzymes was compared in Mreg+/+ and Mreg-/- mouse RPE at mRNA( qPCR) and protein (western blot) levels. MREG and LC3 localization in RPE cells was followed using Immuno-EM and confocal microscopy in vivo, and in vtiro respectively. To determine if MREG-mediated degradation affects lipid digestion, cholesterol accumulation was followed using filipin staining and LC-MS. In addition, docosahexaenoic acid levels (DHA) were determined by LC-MS-MS

Results: In RPE cells isolated from Mreg+/+ mice, Cathepsin-S and Cathepsin-D were upregulated 2h after light onset, while autophagy associated proteins, Atg5/Atg12 and LC3II were upregulated 4-6h after lights on. This biphasic expression pattern was lost in RPE from Mreg-/- mice, with an excessive accumulation of LC3II measured during the 12h period after light onset. The excessive LC3II accumulation temporally mirrored the accumulation of POS described previously. MREG was found to co-localize with LC3 in vivo and in ARPE 19 cells with both proteins associated with POS positive vesicles as detected by Immuno-EM and confocal microscopy. Loss of MREG led to cholesterol accumulation seen as increased filipin binding and confirmed as enhanced cholesterol by LC-MS-MS. Lastly, more DHA was found esterified to phospholipids in Mreg-/- retinas than in Mreg+/+ retinas.

Conclusions: POS degradation is a complex process likely requiring convergence of the phagocytic and autophagic pathways in a process that involves MREG. Loss of MREG-dependent POS degradation leads to the accumulation of toxic lipids in the RPE.

Keywords: 701 retinal pigment epithelium • 645 phagocytosis and killing • 583 lipids