Formation of lipofuscin-like autofluorescent granules in the retinal pigment epithelium requires lysosome dysfunction

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

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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.

Original languageEnglish
Article number39
JournalInvestigative ophthalmology & visual science
Volume62
Issue number9
DOIs
Publication statusPublished - Jul 2021

Keywords

  • Autofluorescent granules
  • Lipofuscin
  • Lysosome dysfunction
  • Photoreceptor outer segments
  • Retinal pigmented epithelium

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