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Aparna Lakkaraju, Kimberly A Toops, Li Xuan Tan; Live imaging reveals multiple regulators of canonical autophagy in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4564.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy is an evolutionarily conserved mechanism for clearing damaged proteins and organelles. Fusion of autophagosomes with lysosomes is essential for completion of autophagy. Decreased autophagic efficiency in post-mitotic retinal pigment epithelium (RPE) could contribute to age-related macular degeneration (AMD). Here, we first investigated autophagosome biogenesis, trafficking and autophagic flux in polarized monolayers of primary adult RPE after inhibition of mammalian target of rapamycin (mTOR); next we examined how outer segment (OS) phagocytosis and visual cycle metabolites regulate autophagy in the RPE.
Live imaging of autophagosome biogenesis and trafficking was performed by high-speed spinning disk confocal microscopy of primary porcine RPE expressing GFP-LC3. We measured endogenous LC3 and WIPI2 puncta after immunostaining to quantify autophagosome biogenesis. Tandem fluorescent LC3 (tf-LC3) was used to follow autophagic flux in real time. Lysosome-autophagic clearance after OS phagocytosis or mTOR inhibition was monitored by immunoblotting for lipidated LC3, opsin and p62. Cells exposed to the bisretinoid A2E, a component of RPE lipofuscin, were used to study the influence of visual cycle metabolites on canonical autophagy.
Analysis of live imaging and immunofluorescence data showed that in healthy RPE, mTOR inhibition and OS phagocytosis induce autophagosome biogenesis. Autophagosomes undergo rapid, bidirectional, tubulovesicular traffic with long-range displacement. Cells with A2E have fewer autophagosomes with shorter displacement and constrained motility. Consequently, tfLC3 flux, a measure of autophagosome-lysosome fusion, is decreased in the presence of A2E. We showed previously that A2E causes RPE cholesterol storage. Treatment with an LXR agonist removes excess cholesterol and corrects autophagy defects in RPE with A2E.
Our data show that in the RPE, canonical autophagy is regulated by mTOR inhibition and OS phagocytosis. We identify two additional modulators of autophagy: bisretinoid metabolites of the visual cycle like A2E and membrane cholesterol, which interfere with this critical housekeeping function at multiple steps, including autophagosome biogenesis, trafficking and autophagic flux. As with neurodegenerative diseases, impaired autophagy and decreased cellular clearance can compromise RPE health and promote retinal disease.
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