Biological impact of HNF_14, a Humanin fragment peptide, in ARPE-19 cells

Abstract

Purpose : AMD (Age-related Macular Degeneration) research on Humanin, a cytoprotective mitochondrial-derived peptide, has primarily focused on the intact Humanin peptide, but no study has investigated the effects of any Humanin fragment peptide in AMD. We tested the hypothesis that in ARPE-19 cell lines, treatment with HNF₁₄, a 1653.7 Da Humanin fragment peptide containing 14 amino acids i.e., “LLLTSEIDLPVKRR” (N-terminus→C-terminus), will improve RPE cell health and will prevent RPE cell loss.

Methods : The HNF₁₄ peptide was custom synthesized (Anaspec Inc.) with ≥96.0% purity (by HPLC) and its effects were examined on ARPE-19 cells. Untreated ARPE-19 cells served as naïve controls and Humanin G (HNG)-treated ARPE-19 cells as positive controls. HNF₁₄-treated and control ARPE-19 cells were analyzed to measure cell viability, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) activity; cell proliferation and Caspase-3/7-mediated apoptosis were measured using NucLight Red and Caspase-3/7 reagents, respectively (IncuCyte live-cell imaging).

Results : In ARPE-19 cells, we found that compared to their untreated counterparts, treatment with HNF₁₄ caused: a 27.7% (P=0.01) increase in cellular metabolic activity that reflects cell viability (MTT assay); a 18.6% (P=0.0001) decrease in ROS (H2DCFDA); 17% (P=0.028) reduction in activity of LDH that is released from damaged cells and is directly proportional to the number of lysed cells and cytotoxicity; 11% (P=0.02) higher cell proliferation and 36.5% (P=0.03) reduced Caspase-3/7-mediated apoptosis (IncuCyte). However, we found no statistically significant difference in cellular metabolic activity, ROS, LDH, cell proliferation, and apoptosis between HNF₁₄-treated versus HNG-treated ARPE-19 cells. Moreover, treatment of ARPE-19 cells with varying concentrations of HNF₁₄ resulted in similar cytoprotective effects.

Conclusions : In conclusion, HNF₁₄ is a biologically active peptide that has significant cytoprotective effects on ARPE-19 cells and its effects are equivalent to that of the full-length 24-aa HNG peptide. We successfully demonstrated HNF₁₄’s cytoprotective ability using ARPE-19 cells, and therefore this study will lay the groundwork for our future HNF₁₄ studies using AMD transmitochondrial RPE cybrid cell lines, which could lead to the development of mitochondria-targeting personalized treatment for AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.