Purpose: 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 1a (PGC-1a) is a powerful mediator of mitochondrial function. This study examines the ability of PGC-1a to regulate RPE metabolic program and oxidative stress response.

Methods: 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-1a 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.

Results: Maturation of ARPE-19 and hfRPE was associated with significant increase in mitochondrial mass, expression of oxidative phosphorylation (OXPHOS) genes, and PGC-1a gene expression. Overexpression of PGC-1a increased expression of OXPHOS and fatty-acid ß-oxidation genes, ultimately leading to the potent induction of mitochondrial respiration and fatty-acid oxidation. PGC-1a gain of function also strongly induced numerous antioxidant genes and, importantly, protected RPE from oxidant-mediated cell death without altering RPE functions.

Conclusions: This study provides important insights into the metabolic changes associated with RPE functional maturation and identifies PGC-1a as a potent driver of RPE mitochondrial function and antioxidant capacity.

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