Abstract
A comprehensive review of humanin, the first mitochondria-derived peptide discovered, examining its cytoprotective mechanisms, neuroprotective effects against Alzheimer disease, cardiovascular and metabolic benefits, and therapeutic potential for age-related conditions.
Humanin is a 24-amino acid peptide encoded by the 16S ribosomal RNA region of mitochondrial DNA, making it the first identified member of a new class of bioactive molecules called mitochondrial-derived peptides. Its discovery in 2001 by Hashimoto and colleagues emerged from a functional screen of a cDNA library derived from the surviving brain tissue of a patient with familial Alzheimer's disease, where researchers sought to identify factors capable of protecting neuronal cells from amyloid precursor protein mutant-induced cell death. The identification of humanin represented a paradigm shift in our understanding of the mitochondrial genome, demonstrating that mitochondrial DNA encodes not only structural and enzymatic proteins for oxidative phosphorylation but also small bioactive signaling peptides with systemic effects.
The amino acid sequence of humanin is MAPRGFSCLLLLTSEIDLPVKRRA, utilizing the mitochondrial genetic code which differs from the standard nuclear code. Despite being encoded by mitochondrial DNA, humanin functions when translated cytoplasmically, and it is highly conserved across mammalian species, suggesting strong evolutionary pressure to maintain its biological activity. Circulating humanin levels decline progressively with age, and this decline correlates with increasing susceptibility to the very diseases that humanin protects against in experimental models, including Alzheimer's disease, cardiovascular disease, and metabolic dysfunction.
The mechanisms through which humanin exerts its protective effects are multiple and interconnected. One of the best characterized is activation of the STAT3 signaling pathway. Upon binding to its receptor complex, which consists of the trimeric assembly of CNTFR, WSX-1, and gp130, humanin triggers phosphorylation of STAT3, which then translocates to the nucleus to regulate gene expression programs that promote cell survival, reduce inflammation, and enhance mitochondrial function. In retinal pigment epithelial cells exposed to oxidative stress, STAT3 activation by humanin inhibits caspase-3 (the executioner enzyme of apoptosis), reduces intracellular reactive oxygen species, restores mitochondrial bioenergetics, increases mitochondrial DNA copy number and mitochondrial transcription factor A levels (indicating enhanced mitochondrial biogenesis), and suppresses senescence markers including beta-galactosidase, ApoJ, and p16INK4a.
Another critical mechanism involves humanin's interaction with IGFBP-3, or insulin-like growth factor binding protein 3. IGFBP-3 normally promotes apoptosis through IGF-independent mechanisms, and humanin directly binds to IGFBP-3 to neutralize its pro-apoptotic activity, thereby enhancing cell survival. Additionally, humanin reduces levels of BAX, the pro-apoptotic protein that forms pores in the outer mitochondrial membrane to trigger cytochrome c release and initiate the intrinsic apoptotic cascade. In models of myocardial infarction followed by reperfusion, humanin treatment significantly reduced BAX levels and diminished cardiomyocyte apoptosis.
The neuroprotective effects of humanin have been extensively studied, particularly in the context of Alzheimer's disease. The peptide protects neurons from amyloid-beta oligomer toxicity, the primary pathological mechanism in Alzheimer's, as well as from serum deprivation, stroke-induced ischemia, NMDA excitotoxicity, and other AD-related insults. In rodent models, humanin administration improves cognitive function and reduces amyloid burden. Research has also revealed that astrocytes are a significant source of humanin in the brain, and that humanin expression in the hippocampus is regulated by ovarian hormones. In ovariectomized rats, modeling the hormonal changes of menopause, hippocampal humanin expression drops significantly, colocalizing with astroglial markers. This finding provides a potential mechanistic link between menopause, declining estrogen levels, reduced brain humanin, and the increased susceptibility of post-menopausal women to Alzheimer's disease.
Cardiovascular protection by humanin extends beyond the acute anti-apoptotic effects in ischemia-reperfusion injury. In ApoE-deficient mice fed high-cholesterol diets, treatment with the humanin analog HNGF6A reduced atherosclerotic plaque size, preserved endothelial function, boosted endothelial nitric oxide synthase activity, and counteracted oxidative stress. Circulating humanin levels in humans inversely correlate with cardiovascular disease risk, suggesting that endogenous humanin may serve as a natural protective factor against vascular pathology.
The metabolic effects of humanin are equally significant. Intracerebroventricular administration enhances insulin sensitivity in rats by reducing hepatic glucose production and boosting peripheral glucose uptake. The HNGF6A analog normalizes glucose levels in Zucker diabetic fatty rats, a model of type 2 diabetes. In NOD mice, a model of type 1 diabetes, humanin improves glucose tolerance, suppresses pancreatic inflammation and lymphocyte infiltration, and protects beta cells from cytokine-induced destruction. In aging models, humanin treatment reduces midlife adiposity, increases lean mass, lowers visceral fat and markers of inflammation including IBA-1, IL-6, and IL-10, and modestly extends lifespan in transgenic C. elegans worms through the daf-16/FOXO pathway, which is the worm homolog of the insulin/IGF-1 signaling cascade.
The most potent humanin analog is HNG, also known as S14G-humanin, which carries a single amino acid substitution of serine for glycine at position 14. This modification increases biological potency approximately 1000-fold compared to native humanin, making it the preferred compound for research applications where lower dosing and higher efficacy are desired. Research dosages for humanin and its analogs range from 0.01 to 1 micromolar in cell culture systems, while animal studies have used varying doses of HNGF6A over periods of up to 16 weeks.
Humanin's clinical research status remains entirely preclinical. No human therapeutic trials have been completed, though endogenous humanin levels are being investigated as biomarkers for aging and disease. Elevated humanin levels have been documented in mitochondrial diseases such as MELAS and CPEO, where it may represent a compensatory protective response to mitochondrial dysfunction. The safety profile appears favorable in all published research, with no reported toxicity in animal studies at therapeutic doses, though the theoretical concern exists that sustained anti-apoptotic activity could promote survival of damaged or pre-malignant cells. Long-term human safety data is absent and will require formal clinical evaluation.
