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Kathleen Boesze-Battaglia, Anuradha Dhingra, Desiree Alexander, Alvina Bragin, Daniel Lamond, Vanda Sofia Lopes, David Williams; Loss of ATG5 in the RPE slows phagosome degradation contributing to altered intracellular lipid processing.. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
RPE cells utilize a hybrid degradative pathway which utilizes components of both autophagy and phagocytosis called LC3 associated phagocytosis (LAP). In the absence of the autophagic protein, ATG5, LAP is inhibited. In these studies we sought to determine the role of ATG5-dependent LAP in the breakdown of ingested outer segments (OS) and to assess the consequences of a defect in this process.
Using a cre/lox recombination system we generated Atg5ΔRPE (BEST1-cre/+;Atg5flox/Atg5flox) and littermate controls of the following genotypes; BEST1-cre/+;Atg5+/+ and Atg5flox/Atg5flox. RPE-specific Cre expression was determined by immunofluorescence, using anti-CRE, with deletion of ATG5 confirmed by double labeling with anti-ATG5 in whole mounts and eyecups. ATG5 and LC3 levels in retinal and RPE lysates were compared by Western blot. The phagocytic profile of RPE cells was determined using ultrathin sections from retinas processed for conventional TEM from 3 month old Atg5ΔRPE, and control mice at -1h, 0h, 0.5h, 1h, and 3h relative to light onset. Accumulation of cholesterol and cholesterol esters was assessed by filipin staining followed by multi-color confocal microscopy.
In the Atg5ΔRPE mice, cre expression was consistently found in 85-90% of RPE cells, with only the RPE cells immunoreactive for CRE. Correspondingly, ATG5 was knocked down in approximately 90% of the RPE cells. As expected there was a concomitant decrease in LC3 lipidation. RPE isolated from Atg5ΔRPE mice 3 hours after light onset showed packets of whorled disk–like material; such structures were not seen in the littermate controls. The number of phagosomes in ultrathin sections of RPE in Atg5ΔRPE mice reached a sharp peak within an hour of lights on. Phagosome numbers rose similarly in the Atg5ΔRPE mutants but remained elevated for at least 3 hrs. Lastly, loss of ATG5 resulted in the accumulation of filipin positive structures in the RPE
In the absence of the autophagic protein, ATG5, degradation of phagosomes containing photoreceptor outer segment disk membranes is significantly inhibited, consistent with an essential role of the autophagic pathway in this critical non-autophagic event in the RPE. The resulting long-term pathology includes an abnormal accumulation of cholesterol.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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