Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction

Abstract

We aim to characterize the pathways required for autofluorescent granule (AFG) formation by retinal pigment epithelium (RPE) cells using cultured monolayers. 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. AFGs appear to derive from incompletely digested POS-containing phagosomes and are surrounded after 72h by a single membrane containing lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation but that impairment of lysosomal pH or catalytic activity, particularly Cathepsin D activity, enhances AF accumulation. We conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.