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Falcao, A. S.

Publications and source records attributed to Falcao, A. S..

2 recordsLinked to original sources

Enhancing lysosome function via mTOR/TFEB activation reduces lipofuscin-like granules in early Age-related Macular Degeneration

Age-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be in the retinal pigment epithelium (RPE). RPE is responsible for the daily digestion of photoreceptor outer segments (POS), which imposes a heavy continuous burden on the lysosomal network. POS feeding assay in vitro suggested that the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo, derives from lysosomal dysfunction. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether induction of the mTOR/TFEB axis could improve digestion of POS and hence reduce AFG load. Treatment of POS-fed cells with rapamycin, an mTORC1 inhibitor after the appearance of AFG results in 30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the mTOR/TFEB axis in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.

molecular biology↗

FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT GRANULES IN THE RETINAL PIGMENT EPITHELIUM REQUIRES LYSOSOME DYSFUNCTION

PurposeWe aim to characterize the pathways required for autofluorescent granule (AFG) formation by retinal pigment epithelium (RPE) cells using cultured monolayers. MethodsWe 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. ResultsAFGs appear to derive from incompletely digested POS-containing phagosomes and are surrounded after 72h by a single membrane containing lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation but that impairment of lysosomal pH or catalytic activity, particularly Cathepsin D activity, enhances AF accumulation. ConclusionsWe conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.

cell biology↗