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Desiree Alexander, Anuradha Dhingra, William C Gordon, Bokkyoo Jun, Nicolas G Bazan, Kathleen Boesze-Battaglia, Alvina Bragin; Loss of MREG dependent LC3 associated phagocytosis (LAP) by the RPE leads to altered intracellular lipid processing.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6043.
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© ARVO (1962-2015); The Authors (2016-present)
RPE cells utilize a hybrid process that incorporates aspects of phagocytosis and autophagy to efficiently degrade ingested photoreceptor outer segments. Our previous studies have established that this LC3 dependent process is mediated by a cargo sorting protein, melanoregulin (MREG). The purpose of this study was to determine if defective phagosome maturation as observed in the absence of MREG and LC3B contributes to alterations in lipid deposition in the RPE.
In all studies we utilized RPE isolated from Mreg -/- , LC3B -/- and C57Bl6/J mice. Accumulation of cholesterol and cholesterol esters was assessed by filipin staining followed by multi-color confocal microscopy. Levels of cholesterol and 7-keto-cholesterol were determined using LC/MS and docosahexaenoic acid (DHA) levels by LC MS/MS. iPLA2-VIA activity was determined using a BODIPY® FL-based fluorometric assay.
Cholesterol accumulated in the Mreg -/- RPE as indicated by the prevalence of filipin positive structures. Biochemical analyses also showed elevated cholesterol levels in Mreg -/- RPE, however the most significant increases were in 7-ketocholesterol, in which case loss of MREG was associated with 5- fold increase in this immuno-modulatory lipid. Moreover, an increase in cholesterol accumulation (or filipin positive structures) was also seen in LC3B-/- RPE. OS lipids provide a massive daily supply of DHA esterified to phospholipids for cleavage by the iPLA2-VIA phospholipase A2 isoform. Defective phagosome maturation in Mreg -/- RPE also contributed to a decrease in free DHA, with a majority of the DHA found to be esterified to phospholipids. This decrease in phospholipid hydrolysis was not due to alterations in iPLA2-VIA activity but likely due to defective phagocyte processing.
Our results suggest that defective LC3 associated phagocytosis, specifically regulated by MREG, leads to accumulation of cholesterol over the long term. Furthermore, loss of MREG leads to a decrease in the intracellular DHA pool, likely compromising the generation of protective docosanoids.
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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