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

Abstract

PurposeTo 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.MethodsHumanin 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.ResultsA 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.ConclusionsHumanin 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.

Humanin analogue, S14G-humanin, has neuroprotective effects against oxygen glucose deprivation/reoxygenation by reactivating Jak2/Stat3 signaling through the PI3K/AKT pathway

“…P revious repor ts have demonstrated that the Jak2/Stat3 signaling axis mediated neuroprotective functions of humanin (8,9). Using a Jak2/Stat3 inhibitor, the present study demonstrated the important role of the Jak2/Stat3 pathway in the effects of HNG against OGD/R injury.…”

“…Jak2, Janus kinase 2; Stat3, signal transducer and activator of transcription 3; OGD/R, oxygen glucose deprivation/reoxygenation; HNG, S14G-humanin; p, phosphorylated; Con, control group. Jak2/Stat3 signaling pathway underlies the multiple protective mechanisms of humanin (8,9). Thus, the present study detected the expression levels of Jak2 and p-Stat3 following treatment with HNG in OGD/R conditions.…”

“…Mitochondria thus become important targets for stroke therapy. It has been documented that humanin may alter mitochondrial oxidative phosphorylation and ATP production; however, the exact molecular mechanisms are not well understood (8). A study by Kumfu et al (35) reported that humanin may prevent brain mitochondrial dysfunction in a model of cardiac ischemia-reperfusion injury.…”

“…Previous results have revealed that humanin exerts various protective actions in multiple cell types and animal models. A study by Sreekumar et al (8) demonstrated that humanin functions to protect human retinal pigment epithelial cells from oxidative stress, senescence and mitochondrial dysfunction partly by Janus kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) signal activation and caspase-3 inhibition. Similarly, humanin rescues Stat3 from amyloid β (Aβ)-induced disruption and causes the downregulation of caspase-3, thereby exerting neuroprotective actions against Aβ toxicity in rats (9).…”

Mitochondrial Transcription Factor A's role in heart failure.

“…Humanin is a natural mitochondrial peptide that significantly increased mtDNA copy number, up-regulated TFAM, activated STAT3, and decreased caspase-3 activation and intrinsic apoptosis (Sreekumar et al 2016). …”