Characterization of the Effects of a Humanin Fragment Peptide (HNF₁₄) in Age-Related Macular Degeneration

Background: Age-related macular degeneration (AMD) is a leading cause of vision loss and is strongly associated with mitochondrial dysfunction in retinal pigment epithelial cells. Mitochondrial-derived peptides, including Humanin and its analogs, have demonstrated cytoprotective effects in AMD-related cellular models. However, the effects of shorter Humanin-derived fragments in disease-specific mitochondrial models remain incompletely characterized. Methods: Transmitochondrial retinal pigment epithelial cybrid cell lines containing mitochondria from AMD patients or age-matched normal donors were treated with HNF₁₄, a 14-amino acid Humanin fragment peptide. Cellular metabolic activity, cytotoxicity, oxidative stress, apoptotic signaling, inflammatory markers, angiogenic factor expression, and amyloid-β_1-42-induced apoptosis were evaluated using biochemical assays, protein analyses, and live-cell imaging approaches. Results: HNF₁₄ treatment was associated with improved metabolic activity and reduced cytotoxicity in AMD cybrids, with minimal effects in normal cybrids. HNF₁₄ significantly reduced intracellular and mitochondrial oxidative stress, suppressed apoptotic and inflammatory markers, and decreased VEGF-A protein expression in AMD cybrids. In addition, HNF₁₄ attenuated amyloid-β_1-42-induced apoptotic signaling in AMD cybrids. These effects were selective for cybrids containing AMD-derived mitochondria. Conclusions: This study demonstrates that HNF₁₄ mitigates mitochondrial and cellular stress responses in AMD transmitochondrial cybrid cells. The findings indicate that a short Humanin-derived fragment retains cytoprotective activity in a disease-specific mitochondrial context and support further investigation of mitochondrial-derived peptides as modulators of mitochondrial dysfunction relevant to AMD pathophysiology.