Figure 5.

Humanin treatment exhibits enhanced mitochondrial biogenesis, restoring mitochondrial impairment in Parkinson's disease. (A) Real-time qPCR analysis showing expression of genes associated with mitochondrial biogenesis (NRF1, PGC-1α, and ESRRA), oxidative phosphorylation (SDHA, ATF5F1A, and ATF5F1D), fatty acid metabolism (ACC2 and ACLY), and TCA cycle (FASN and IDH3A) in SH-SY5Y cells after HN treatment (20 μM, 24 h). (B, C, D, E) Representative images (B) of stained mitochondria with MitoTracker Green in SH-SY5Y cells after HN treatment (20 μM, 24 h). The branch length (C), mitochondrial volume (D), and the number of branches (E) were calculated for analyzing the mitochondrial network. Scale bar, 10 μm. (F) Schematic diagram of experimental setup using ex vivo model of PD. The rat organotypic slices were pre-treated with HN (1 μM, 24 h) and then exposed with neurotoxin 6-OHDA (100 μM, 2 h). (G) Western blot analysis for proteins involved in mitochondrial biogenesis from ex vivo organotypic culture slices as indicated groups. (I) ATP determination based on luminescent signal proportion to the amount of ATP from ex vivo organotypic culture slices with humanin pre-treatment (24 h; 0.1 μM or 1 μM) and 6-OHDA exposure. (J, K) Dramatic loss of mitochondrial membrane potential stained with TMRM (100 nM) in SH-SY5Y cells following MPP⁺ exposure for indicated times. (L, M) Recovered mitochondrial membrane potential (L) and reduced mitochondrial superoxide production (M) by pre-treatment with HN in MPP⁺-challenged SH-SY5Y cells assessing with flow cytometry. The raw fluorescence for each probe was normalized against Hoechst 33342 (1 μg/ml) fluorescence to control for any variation in cell number. (N, O) Reversed mitochondrial membrane depolarization induced by FCCP (5 μM, 15 min) by HN pre-treatment (24 h; 20 μM) in SH-SY5Y cells. Data are expressed as means ± SEM. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). HN: humanin.