title: The Mitochondrial-Derived Peptide Humanin Protects RPE Cells From Oxidative Stress, Senescence, and Mitochondrial Dysfunction.
abstract: OBJECTIVE
  To investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress-induced cell death, mitochondrial bioenergetics, and senescence.
  
  
  METHODS
  Humanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5-10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress-induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment.
  
  
  RESULTS
  A prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress-induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death.
  
  
  CONCLUSIONS
  Humanin protected RPE cells against oxidative stress-induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.
authors.affiliation: Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States.
authors.forename: Parameswaran G
authors.initials: PG
authors.lastname: Sreekumar
authors.affiliation: Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States.
authors.forename: Keijiro
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Department of Ophthalmology, University of Southern California, Los Angeles, California, United States.
authors.forename: Chris
authors.initials: C
authors.lastname: Spee
authors.affiliation: USC Leonard Davis School of Gerontology, University of Southern California, Los Angeles, California, United States.
authors.forename: Hemal H
authors.initials: HH
authors.lastname: Mehta
authors.affiliation: USC Leonard Davis School of Gerontology, University of Southern California, Los Angeles, California, United States.
authors.forename: Junxiang
authors.initials: J
authors.lastname: Wan
authors.affiliation: USC Leonard Davis School of Gerontology, University of Southern California, Los Angeles, California, United States.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Yen
authors.affiliation: USC Leonard Davis School of Gerontology, University of Southern California, Los Angeles, California, United States.
authors.forename: Pinchas
authors.initials: P
authors.lastname: Cohen
authors.affiliation: Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States.
authors.forename: Ram
authors.initials: R
authors.lastname: Kannan
authors.affiliation: Department of Ophthalmology, University of Southern California, Los Angeles, California, United States 4Department of Pathology, University of Southern California, Los Angeles, California, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
mesh_terms: D017209:Apoptosis; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D018384:Oxidative Stress / Q000235:genetics*; D010766:Phosphorylation; D012313:RNA / Q000235:genetics*; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C427211:humanin; D012313:RNA