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P. Thampi, H. Vittal Rao, S. Jarrett, J. Cai, C. Romano, M. E. Boulton; The 5HT1A Agonist 8OH DPAT Decreases Oxidative Stress-Induced Mitochondrial Damage and Reduces Accumulation of Lipofuscin Granules in ARPE19 Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):461.
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To investigate whether the 5HT1A agonist, 8OH DPAT, decreases oxidative stress-induced mitochondrial damage and reduces lipofuscin accumulation in cultured human retinal pigment epithelial (RPE) cells.
ARPE19 cells in basal media were 1) maintained for 4 weeks to generate autophagy derived-lipofuscin, 2) fed photoreceptor outer segments (POS) or peroxidized POS (oxPOS) every two days for 14 days to induce phagocytosis-derived lipofuscin, 3) fed mature lipofuscin granules and 4) treated with oxidative stressors 200µM H2O2 (1 hr) or 40µg/ml 7-ketocholesterol (7-kCh) (8 hr). In all conditions, 8OH DPAT was replenished every two days while controls received vehicle only. Lipofuscin accumulation and superoxide (O2.) anion generation were quantified by FACS and fluorescence microscopy, lipofuscin phototoxicity was assessed by the MTT assay, REDOX activity was measured by ELISA and mitochondrial DNA damage assessed by long chain QPCR.
8OH DPAT induced a dose-dependent decrease in both autophagy- and POS-derived lipofuscin granules in RPE cells. Maximal inhibition (>50%) of lipofuscin formation was observed at 10 µM 8OH DPAT and this was used as the concentration of choice for all subsequent experiments. 8OH DPAT decreased autophagy-derived lipofuscin by 30% at 3 weeks and 67% at 4 weeks. 8OH DPAT induced a 28% and 20% decrease at 14 days in phagocytosis-derived lipofuscin accumulation in POS and oxPOS fed cells respectively compared to untreated controls fed POS or oxPOS. Similar changes were observed by fluorescence microscopy. However, 8OH DPAT failed to reduce the existing mature lipofuscin granules. In cells fed oxPOS for 14 days phototoxicity was decreased by 56% in 8OH DPAT treated cells compared to untreated controls. H2O2 and 7-kCh induced a 102% and 37% increase in O2. generation respectively and this increase was blocked by 67% and 50% respectively following treatment with 8OH DPAT. In addition, 8OH DPAT protected cells from loss of REDOX potential caused by exposure to H2O2 and 7-kCh. H2O2-induced mitochondrial DNA damaged was significantly reduced by >70% in the presence of 8OH DPAT.
5HT1A agonists have the capacity to reduce the generation of both autophagy- and phagocytosis-derived lipofuscin in RPE cells. These agonists can also protected against oxidative damage to mitochondria thus reducing the autophagic load. 5HT1a agonists may prove useful in the treatment of AMD.
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