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Nady Golestaneh, Yi Chu, Yang-Yu Xiao, Gianna Storelu, Alexander Constantine Theos; Reduced autophagy dynamics and flux, dilated LAMP-1 vesicles and dysfunctional mitochondria in RPE of AMD donor eyes.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):812.
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Purpose: Age-related macular degeneration (AMD) is a major cause of blindness. Drusen formation beneath the RPE is a hallmark of AMD. However, the mechanisms inducing lipid accumulation and drusen formation remain elusive. We hypothesize that dysfunctional autophagy causes lipid accumulation and subsequently induces mitochondrial dysfunction and disintegration resulting in drusen formation and RPE degeneration in AMD.
Methods: RPE from AMD and normal donor eyes (n=5) were isolated, purified with MACS, and cultured on transwells to generate RPE monolayers. Electron microscopy (EM) was performed. Cell viability and reactive oxygen species (ROS) were measured under oxidative stress conditions. Mitochondrial activity was analyzed by measuring ATP production in the presence and absence of hexokinase inhibitor to measure ATP produced by mitochondria or glycolysis respectively. Autophagy dynamics was analyzed using LC3-II/LC3-I ratios, and flux determined using p62 antibody in RPE pre-treated with IGF-1 and starved in HBSS in the presence or absence of IGF-1, and in the presence of lysosomal protease inhibitors. Anti-LAMP-1 staining was performed for cytological analysis of autophagolysosomes.
Results: EM analysis revealed distinct disease phenotypes including autophagosome accumulation, disintegrated mitochondria, lipid and glycogen accumulation in AMD compared to normal RPE. Functional studies revealed increased susceptibility to oxidative stress and higher levels of ROS production in AMD compared to normal RPE. ATP produced by mitochondria was significantly lower in AMD RPE. Whereas, glycolytic ATP production was significantly higher in AMD compared to normal RPE, suggesting that in AMD RPE, ATP is produced primarily through glycolysis. Autophagy analysis revealed that normal RPE rapidly induced autophagy after starvation in the absence of IGF-1, whereas AMD RPE failed to increase the ratio of LC3-II/LC3-I, under the same conditions. Autophagic flux was reduced in AMD RPE as shown by their inability to reduce p62 levels under starvation. LAMP-1-positive organelles were swollen in AMD RPE, as compared with normal cells.
Conclusions: Our data are consistent with our hypothesis that dysfunctional autophagy contributes to AMD-like phenotypes in RPE of AMD donor eyes, and propose autophagy as a contributor factor in AMD pathophysiology.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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