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 RPE cells using cultured monolayers. METHODS.We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable 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 seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS.We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

Deacidification of endolysosomes by neuronal aging drives synapse loss

“…TEM was essentially performed as previously 107,108 . Glass coverslips containing cell monolayers were fixed in 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer for 30 min at room temperature.…”

“…TEM was essentially performed as previously. 107,108 Glass coverslips containing cell monolayers were fixed in 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer for 30 min at room temperature. Specimens were post-fixed for 1 h on ice using 1% osmium tetroxide and 1.5% potassium ferrocyanide in distilled water, and then incubated with 1% tannic acid in distilled water for 20 min at room temperature.…”