The Mitochondrial-Derived Peptide Humanin Protects RPE Cells From Oxidative Stress, Senescence, and Mitochondrial Dysfunction

Invest Ophthalmol Vis Sci. 2016 Mar;57(3):1238-53. doi: 10.1167/iovs.15-17053.


Purpose: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Cells, Cultured
  • Cellular Senescence / genetics*
  • Gene Expression Regulation, Developmental*
  • Humans
  • In Situ Nick-End Labeling
  • Intracellular Signaling Peptides and Proteins / biosynthesis
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Microscopy, Confocal
  • Microscopy, Electron, Transmission
  • Mitochondria / metabolism*
  • Mitochondria / ultrastructure
  • Oxidative Stress / genetics*
  • Phosphorylation
  • RNA / genetics*
  • Retinal Pigment Epithelium / metabolism*
  • Retinal Pigment Epithelium / ultrastructure


  • Intracellular Signaling Peptides and Proteins
  • humanin
  • RNA