Fig. 1

a In the accelerating rotarod assay, rotation was accelerated from 5 to 40 rpm over the course of 5 min, and fall latency was recorded. The experiments were performed using mice from 10 to 120 weeks of age (n = 70). Accelerating rotarod tests were performed on a rotarod machine with automatic timers and falling sensors (MK-660D, Muromachi Kikai, Japan). Data are means ± SE (error bars). b Quantification of fall latency. Aged mice experiments were performed using mice 60, 80, and 120 weeks of age. c Kaplan–Meier analysis of survival of C57BL/6 mice (n = 20). d Body weight of mice 60, 80, and 120 weeks of age (60-week-old mice, n = 10; 80-week-old mice, n = 10; 120-week-old mice, n = 10). Data are presented as means ± SE (error bars); ** p < 0.01 (ANOVA, Tukey post hoc pairwise comparisons). N.S.; Mice 60, 80, and 120 weeks of age denote not significant. e Runway test of aged-mice. The runway test was performed using a narrow horizontally fixed beam. Aged-mice could hardly move on the beam, and their hindpaws frequently slipped. f The number of hindlimb slips was recorded for 60-, 80-, and 120-week-old mice crossing the 2 cm (left) and 3 cm (right) pole. Data are presented as means ± SE (60-week-old mice, n = 10; 80-week-old mice, n = 10; 120-week-old mice, n = 10); statistical significance was evaluated using ANOVA, Tukey post hoc pairwise comparisons. ** p < 0.01. g Histological analyses of SN in 70-week-old mice and 120-week-old mice. Paraffin sections were immunostained for TH. SNcl, lateral area of the substantia nigra pars compacta; SNcc, center area of the substantia nigra pars compacta; VTA, the ventral tegmental area. Scale bars, 20 μm. h For stereological quantification, three areas were selected. Every other 40-μm section of serial coronal brain slices for each genotype was stained for DAB. Quantification was performed with design-based stereology system (Stereo-Investigator version 2019, MBF Bioscience, Williston, VT, USA). Sampling parameters were set up according to the software guide to achieve the coefficient of error ranged between 0.06 and 0.09 using the Gundersen test. Data are means ± SE (70-week-old mice, n = 5; 120-week-old mice, n = 18); ** p < 0.01 (Student’s t-test). N.S.; Not significant. i HPLC analysis of dopamine (left), dihydroxyphenylacetic acid (DOPAC) (middle), and homovanillic acid (HVA) (right) levels in the dorsal striatum of 60-, 80-, and 120-week-old mice. Data are presented as means ± SE (60-week-old mice, n = 5; 80-week-old mice, n = 5; 120-week-old mice, n = 10); ** p < 0.01 (ANOVA, Tukey post hoc pairwise comparisons). N.S.; Not significant. j For conventional electron microscopy, mice were fixed by cardiac perfusion with 2.5% glutaraldehyde in 0.1 mol/L PB (pH 7.2). Brain slices were embedded in epoxy resin, and ultrathin sections (70 nm thickness) were prepared and imaged on an HT7700 electron microscope (Hitachi, Japan). Electron micrographs of dopaminergic neurons in the SN (a,b), cerebellum (c,d), cerebral cortex (e,f); 30- (n = 3) (left) and 120-week-old mice (n = 3) (right). Scale bars, 1 μm. k Quantitation of mitochondrial area (30-week-old mouse dopaminergic, cerebellar, and cerebral cortical cells, n = 20; each 120-week-old mouse dopaminergic, cerebellar, and cerebral cortical cells, n = 20). The mean mitochondrial area in dopaminergic neurons was smaller in 120-week-old mice (right) than in 30-week-old mice (left). Significance was evaluated using Student’s t-test. * p < 0.01. N.S.; Not significant