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Prajitha Thampi, Haripriya Vittal Rao, Sayak K. Mitter, Jun Cai, Carl Romano, Michael E. Boulton; The 5HT1a Receptor Agonist 8OH DPAT-Induced Sustained Protection of RPE Cells From Lipofuscin Accumulation by Increasing Cellular Antioxidants and Decreasing Autophagy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4783.
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We have previously shown that the 5HT1A receptor agonist 8OH DPAT reduces accumulation of lipofuscin granules in ARPE19 cells. The aim of this study was to determine whether 5HT1A receptor agonist-induced prevention of lipofuscin accumulation in the RPE is due to increased cellular antioxidants and/or decreased autophagy.
Confluent ARPE 19 cells in basal media (Ham’s F-10 + 2% FCS) with or without 8OH DPAT (0.1, 1 and 5 µM; replenished every 2 days) were 1) maintained for 4 weeks to generate autophagy induced lipofuscin accumulation, 2) fed isolated photoreceptor outer segments (POS) every two days for 14 days to induce phagocytosis-induced lipofuscin accumulation, 3) treated with rotenone (1 or 10 uM) to induce mitochondrial damage and 4) treated with oxidative stressors 200 µM H2O2 (1 hr) or 40µg/ml 7ketocholesterol (7KC) (8 hr). 8OH DPAT specificity was concerned using the 5HT1A receptor antagonist S(-)-UH-301. Lipofuscin accumulation and superoxide (SO) anion generation was quantified by flow cytometry and fluorescence microscopy, Superoxide dismutase (SOD) and the autophagy proteins Beclin1, LC3, Atg5, Atg7 and Atg9, were confirmed by qRT PCR and Western blot. Autophagosomes were determined by fluorescence microscopy.
5HT1A receptor inhibition blocked the protective effects of 8OH DPAT. The ability of 8OH DPAT to reduce accumulation of autophagy- and phagocytosis-derived lipofuscin was sustained by at least 50% of control for up to 28 days following cessation of 8OH DPAT treatment (p<0.005). Interestingly, 8OH DPAT was able to reduce pre-generated lipofuscin derived from ingestion of POS but not autophagy. Rotenone induced mitochondrial damage, elevated ROS generation and increased autophagy flux that resulted in significant lipofuscin accumulation. These detrimental effects were significantly reduced by 8OH DPAT treatment (p<0.005). 8OH DPAT alone was able to reduce oxidative damage in RPE cultures by upregulation of SOD2 and glutathione with levels of antioxidant enzymes eventually returning to baseline once oxidative damage had been resolved.
5HT1A agonists have a sustained effect on lipofuscin accumulation in the RPE and this is a combination of enhanced endogenous antioxidant protection and reduced autophagy. 5HT1A agonists may prove a useful clinical adjunct in the treatment of oxidative stress and lipofuscin accumulation associated with AMD.
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