title: PGC-1α Induces Human RPE Oxidative Metabolism and Antioxidant Capacity.
abstract: OBJECTIVE
  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.
  
  
  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-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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Jared
authors.initials: J
authors.lastname: Iacovelli
authors.affiliation: Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, Alabama, United States.
authors.forename: Glenn C
authors.initials: GC
authors.lastname: Rowe
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Arogya
authors.initials: A
authors.lastname: Khadka
authors.affiliation: Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Diaz-Aguilar
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States.
authors.forename: Carrie
authors.initials: C
authors.lastname: Spencer
authors.affiliation: Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Arany
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Magali
authors.initials: M
authors.lastname: Saint-Geniez
mesh_terms: D015153:Blotting, Western; D016923:Cell Death; D002460:Cell Line; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism; D010085:Oxidative Phosphorylation; D018384:Oxidative Stress / Q000502:physiology; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D006360:Heat-Shock Proteins; C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D012313:RNA