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Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction
Cristina Escrevente 1, Ana S Falcão 1, Michael J Hall 2, Mafalda Lopes-da-Silva 1, Pedro Antas 1, Miguel M Mesquita 1, Inês S Ferreira 1, M Helena Cardoso 1, Daniela Oliveira 1, Ana C Fradinho 1, Thomas Ciossek 3, Paul Nicklin 3, Clare E Futter 2, Sandra Tenreiro 1, Miguel C Seabra 1 2
Abstract:
Purpose: 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.
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.
PMID: 34313720
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