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Miguel Seabra, Cristina Escrevente, Ana Sofia Falcão, Michael J Hall, Mafalda Lopes-da-Silva, Pedro Antas, Miguel Mesquita, Inês Ferreira, Helena Cardoso, Ana Fradinho, Clare E Futter, Sandra Tenreiro; FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT GRANULES IN THE RETINAL PIGMENT EPITHELIUM REQUIRES LYSOSOME DYSFUNCTION. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3278.
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© ARVO (1962-2015); The Authors (2016-present)
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 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.
We conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.
This is a 2021 ARVO Annual Meeting abstract.
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