A Decline in Autophagic Efficiency Is Associated With AMD and Chronic Exposure to Oxidative Stress

Abstract

Purpose: : To determine if decreased autophagy plays a role in the pathogenesis of age-related macular degeneration and if this is a result of oxidative stress.

Methods: : The fractional volume of autophagic vacuoles was determined in electron micrographs from patients with AMD and age-matched controls. For in vitro studies cultured human RPE cells were transfected with an RFP-GFP-LC3 construct (a probe that can distinguish between autophagosomes and autolysosomes). After 24 hours cells were treated with sublethal 100, 200 or 400µM H₂O₂ for up to 7 days and toxic 1.5mM H₂O₂ for 3 hr in the presence or absence of a) 3-Methyl adenine (3MA) which inhibits autophagy and b) Rapamycin which promotes autophagy. Cells were examined by fluorescence microscopy and the number of autophagosomes determined. In a parallel experiment untransfected RPE cells were treated as above and the expression of the autophagy markers Atg7, LC3-I and LC3-II determined by Western blot and RT-PCR. Finally, the effect of regulation of autophagy on cell survival was determined using the MTT and crystal violet assays.

Results: : The volumes of autophagic vacuoles in the RPE of AMD donors was reduced by >25% compared to age-matched controls. Acute exposure of RPE cells to H₂O₂ at sub-lethal doses induced a greater than 3- and 2-fold increase in autophagosomes at 3 and 6 hr respectively compared to untreated controls and this could be inhibited by 3MA. This was associated with increased expression of both Atg7 and LC3 mRNA and protein and an increase in LC3-II:LC3-I ratio indicating efficient autophagic flux. By contrast, chronic exposure to oxidative stress for 7 days decreased autophagic efficiency and caused a reduction in Atg7 protein levels and in the LC3-II:LC3-I ratio. Stimulation of autophagy by Rapamycin enhanced autophagy and protected RPE cells against cell death caused by lethal concentrations of H₂O₂.

Conclusions: : Our findings support a role for autophagy in the pathogenesis of AMD and that this is, in part, regulated by the degree of oxidative stress. Acute oxidative stress promotes autophagy and the removal of damaged organelles while chronic oxidative stress reduces autophagic efficiency and thus leads to an accumulation of damaged intracellular organelles and cell death. These results suggest that targeting autophagy may offer an alternative therapeutic strategy for treatment of AMD.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • age-related macular degeneration