Publicação

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

Detalhes bibliográficos
Resumo:	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.
Autores principais:	Escrevente, Cristina
Outros Autores:	Falcão, Ana S.; Hall, Michael J.; Lopes-Da-Silva, Mafalda; Antas, Pedro; Mesquita, Miguel M.; Ferreira, Inês S.; Helena Cardoso, M.; Oliveira, Daniela; Fradinho, Ana C.; Ciossek, Thomas; Nicklin, Paul; Futter, Clare E.; Tenreiro, Sandra; Seabra, Miguel C.; C Seabra, Miguel
Assunto:	Autofluorescent granules Lipofuscin Lysosome dysfunction Photoreceptor outer segments Retinal pigmented epithelium Ophthalmology Sensory Systems Cellular and Molecular Neuroscience
Ano:	2021
País:	Portugal
Tipo de documento:	artigo
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade Nova de Lisboa
Idioma:	inglês
Origem:	Repositório Institucional da UNL

Descrição
Resumo:	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.