*4.1. Prevents Apoptosis*

Humanin binds to the pro-apoptotic protein BAX and prevents its translocation to the nucleus, thereby antagonizing the apoptotic activity of BAX and inhibiting the release of cytochrome c [36]. This finding reported by Guo et al. in the journal *Nature* was the first thorough study that delineated and reported the interactions of Humanin with the pro-apoptotic BAX protein. Since Humanin is encoded by the mitochondria, this report also suggested a possible retrograde signaling mechanism between the mitochondria and the nucleus that might be contributing to regulation of BAX-mediated apoptosis.

Humanin also binds and inactivates BAX-like proteins, such as Bid and BidEL [37]. Exogenously added HNG exerts substantive protective e ffects in transmitochondrial AMD RPE cells in vitro by (a) rescue of mitochondrial structure and function, (b) inhibiting the action of pro-apoptotic genes/proteins, and (c) intracellular and extracellular humanin receptor modulation (Figure 2) [38].

**Figure 2.** Effects of Humanin/Humanin analogs in RPE/AMD.

Humanin is cytoprotective against amyloid-β-mediated toxicity in neuronal cells, both in vitro and in vivo [17,18]; against cerebral ischemia and cardiac damage in mouse models [19,20]; and in numerous neurodegenerative disease models for Alzheimer's disease, Parkinson's disease, Huntington's disease, and Prion diseases [18,19,39,40]. Moreover, Hinton et al. in a comprehensive study published in the highly reputed *IOVS* eye journal demonstrated that Humanin rescues primary RPE cells from oxidative damage and subsequent death in vitro [41]. This showed that Humanin conferred RPE cell protection via two mechanisms: by enhancing mitochondrial biogenesis and function and by activation of STAT3. Further, Humanin mediated suppression of oxidative stress-induced cell senescence and maintained transepithelial resistance in human RPE monolayers. In summary, this study by Hinton et al. suggested the potential of Humanin as a therapeutic candidate for the treatment of geographic atrophy in dry AMD.

Each of the 24 amino acids in the Humanin peptide have a specific function. Serine at position 14 confers neuroprotection [22], but its substitution with glycine generates a variant called Humanin G/HNG that is 1000-fold more potent than its parent analog Humanin [16]. HNG is known to protect against cell death by preventing mitochondrial dysfunction [26].

Humanin potentially inhibits silver nanoparticles-induced cell death in human neuroblastoma cells by (a) protecting against redox imbalance and oxidative stress-induced DNA damage, (b) increasing mitochondrial membrane potential and enhancing the activity of mitochondrial succinate

dehydrogenase, and (c) deactivation of the ER stress pathways that were upregulated by the silver nanoparticles [42].
