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Jared Iacovelli, Glenn C. Rowe, Arogya Khadka, Daniel Diaz-Aguilar, Carrie Spencer, Zoltan Arany, Magali Saint-Geniez; PGC-1α Induces Human RPE Oxidative Metabolism and Antioxidant Capacity. Invest. Ophthalmol. Vis. Sci. 2016;57(3):1038-1051. doi: https://doi.org/10.1167/iovs.15-17758.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress and metabolic dysregulation of the RPE have been implicated in AMD; however, the molecular regulation of RPE metabolism remains unclear. The transcriptional coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a powerful mediator of mitochondrial function. This study examines the ability of PGC-1α to regulate RPE metabolic program and oxidative stress response.
Primary human fetal RPE (hfRPE) and ARPE-19 were matured in vitro using standard culture conditions. Mitochondrial mass of RPE was measured using MitoTracker staining and citrate synthase activity. Expression of PGC-1 isoforms, RPE-specific genes, oxidative metabolism proteins, and antioxidant enzymes was analyzed by quantitative PCR and Western blot. Mitochondrial respiration and fatty-acid oxidation were monitored using the Seahorse extracellular flux analyzer. Expression of PGC-1α was increased using adenoviral delivery. ARPE-19 were exposed to hydrogen peroxide to induce oxidative stress. Reactive oxygen species were measured by CM-H2DCFDA fluorescence. Cell death was analyzed by LDH release.
Maturation of ARPE-19 and hfRPE was associated with significant increase in mitochondrial mass, expression of oxidative phosphorylation (OXPHOS) genes, and PGC-1α gene expression. Overexpression of PGC-1α increased expression of OXPHOS and fatty-acid β-oxidation genes, ultimately leading to the potent induction of mitochondrial respiration and fatty-acid oxidation. PGC-1α gain of function also strongly induced numerous antioxidant genes and, importantly, protected RPE from oxidant-mediated cell death without altering RPE functions.
This study provides important insights into the metabolic changes associated with RPE functional maturation and identifies PGC-1α as a potent driver of RPE mitochondrial function and antioxidant capacity.
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