FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT GRANULES IN THE RETINAL PIGMENT EPITHELIUM REQUIRES LYSOSOME DYSFUNCTION

Abstract

Purpose : We aim to characterize the pathways required for autofluorescent granule (AFG) formation by retinal pigment epithelium (RPE) cells using cultured monolayers.

Methods : We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24h the cells started accumulating AFGs similar to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking Cathepsin D by gene editing.

Results : AFGs appear to derive from incompletely digested POS-containing phagosomes and after 72h are surrounded by a single membrane containing lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AF granule formation but that impairment of lysosomal pH or catalytic activity, particularly Cathepsin D activity, enhances AF intensity.

Conclusions : We conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.

This is a 2021 ARVO Annual Meeting abstract.