Moderately Reduced ATP Levels Promote Oxidative Stress and Debilitate Autophagic and Phagocytic Capacities in Human RPE Cells

PURPOSE. Aging of the RPE is associated with a decrease of intracellular ATP levels and increased oxidative stress. We investigated the effects of moderate energy deficit on intracellular glutathione levels, oxidative damage of cellular proteins and DNA, and autophagy rates using an RPE cell culture model. Additionally, phagocytosis of photoreceptor outer segments was assayed as an example of an ATP-dependent normal function of the RPE. METHODS. ATP synthesis of primary human RPE cells was moderately inhibited by atractyloside. Oxidative stress was induced by tert-butyl hydroperoxide (tBH). ATP, reduced glutathione (rG), malondialdehyde (MDA) adduct formation and 8-hydroxydeoxyguanosine (8OHdG) levels were measured. Autophagy and phagocytosis of photoreceptor outer segments were assayed by radiometric methods. RESULTS. Atractyloside-treatment reduced cellular ATP levels by 30%, mimicking the energy status of aged RPE. tBH decreased rG in RPE cells with lowered ATP levels whereas cells with normal ATP content were not affected. tBH-induced oxidative stress resulted in substantial accumulation of MDA protein adducts in cells with lowered ATP while cells with regular ATP levels were only modestly affected. tBH induced more oxidative DNA damage (8OHdG formation) in cells with lowered ATP levels than in cells with regular ATP. In atractyloside-treated cells, autophagy rates decreased 3-fold as compared with controls. Phagocytic capacity for uptake and degradation of photoreceptor segments was reduced in RPE with low ATP. CONCLUSIONS. Moderately decreased ATP levels such as seen in aged individuals might contribute to the vulnerability of RPE to oxidative stress damage and to dysfunction. (Invest Ophthalmol Vis Sci. 2012;53:5354-5361) DOI: 10.1167/iovs.12-9845