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

Abstract

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.

• Authors: Parameswaran G Sreekumar, Keijiro Ishikawa, Chris Spee, Hemal H Mehta, Junxiang Wan, Kelvin Yen, Pinchas Cohen, Ram Kannan, David R Hinton
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 57 Issue 3 Pg. 1238-53 (Mar 2016) ISSN: 1552-5783 [Electronic] United States
• PMID: 26990160 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Intracellular Signaling Peptides and Proteins
• humanin
• RNA

Topics

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