Humanin treatment protects neurons and ameliorates behavioral defects in PD. (A, B, C, D) Murine primary dopaminergic neurons were pre-treated with DMSO or HN (1 μM) for 12 h and then challenged with MPP+ (100 μM) for 24 h. (A) Photomicrographs show a protective effect of humanin. Scale bar, 10 μm. (B) Viability of primary dopaminergic neurons using MTT assay. Neurite length (C) and neurite number (D) were decreased after MPP+ exposure (#P < 0.05; ##P < 0.01; control versus MPP+ group). These damages were recovered by pre-treatment with HN peptide. (E) Schematic diagram of experimental schedule following intranasal injection of HN (0.1 mg/kg, 5 mg/kg) in MPTP-challenged mice at indicated time points. (F) Behavioral evaluation with pole test. (G) Representative images of tyrosine hydroxylase (TH) staining from substantia nigra of indicated groups. Scale bar, 100 μm. (H) Stereotaxic assessment of TH-positive neurons. (I) Western blot images of TH proteins from the striatum brain lysates of the indicated groups. (J) Three independent Western blots quantified by densitometry using Image Lab Software. Data are expressed as means ± SEM. (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). HN: humanin.