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Parameswaran G Sreekumar, Keijiro Ishikawa, Christine Spee, Hemal Mehta, Kelvin Yen, Pinchas Cohen, Ram Kannan, David R Hinton; Humanin protects RPE cells from oxidative stress induced cell death. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3445.
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© ARVO (1962-2015); The Authors (2016-present)
Humanin (HN) is a 24-amino acid peptide encoded from the mitochondrial chromosome. Recent studies have reported a neuroprotective role for HN in vitro and in various animal models. Yet, the role of HN in age-related macular degeneration is hitherto unknown. Hence, in this study, we investigated the protective role of HN in human fetal RPE cells (hfRPE) and evaluated the participation of mitochondrial respiration in this process.
To study the protective effect of HN, primary hfRPE cells were co-treated with varying doses of HN (0.5-10ug/ml) and 150 µM of tert-Butyl hydroperoxide (tBH) for 24 h in serum-free medium. HN localization in RPE cells was assessed by confocal microscopy. Mitochondrial respiration was measured in RPE cells grown on 96-well plates using XF96 analyzer (Seahorse Bioscience Inc, MA). RPE cell death and caspase-3 activation induced by tBH were studied by TUNEL staining and immunoblot analysis.
Our studies showed a prominent expression of HN in the cytoplasm and nucleus of hfRPE cells. In the cytoplasm, HN co-localized with mitochondria. A similar pattern of expression was found in human polarized RPE monolayers. Mitochondrial respiration was significantly decreased with oxidative stress (p<0.05 vs control) while HN co-treatment upregulated mitochondrial respiration significantly (p<0.01 vs tBH treated cells). HN protected RPE cells from oxidative stress-induced cell death and prevented caspase-3 activation.
HN protects RPE cells from oxidant injury by attenuating cell death and restoring mitochondrial function as evidenced by increased mitochondrial respiration. These data suggest a potential role for humanin therapy in the prevention and treatment of age-related macular degeneration.
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