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Cristina Escrevente, Ana S. Falcão, Michael J. Hall, Mafalda Lopes-da-Silva, Pedro Antas, Miguel M. Mesquita, Inês S. Ferreira, M. Helena Cardoso, Daniela Oliveira, Ana C. Fradinho, Thomas Ciossek, Paul Nicklin, Clare E. Futter, Sandra Tenreiro, Miguel C. Seabra; Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction. Invest. Ophthalmol. Vis. Sci. 2021;62(9):39. doi: https://doi.org/10.1167/iovs.62.9.39.
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We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers.
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.
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.
We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.
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