Fig. 8: Nato3 is essential for mitochondrial integrity in midbrain DA neurons.

a Mitochondria were visualized by anti-COX4 immunostaining (magenta) in TH⁺ neurons (green) of SN in 11- and 16- to 18-month-old control and Nato3 cKO mice. Scale bar, 10 µm. b Distribution of the circularity of COX4⁺ mitochondria particles in TH⁺ neurons from (a). Two-tailed Kolmogorov-Smirnov test, ****p < 0.0001. 700 COX4⁺ particles per group were analyzed from 3 brains per genotype. c Electron micrographs of mitochondria from TH-labeled DA neurons from the SN of 16- to 18-month-old control and Nato3 cKO mice. M, normal mitochondria. DM, degenerative mitochondria. n = 3 mice per group. Scale bar, 0.5 µm. d Classification criteria for mitochondria integrity (left) and mitochondria distribution in TH⁺ neurons in 6-month-old and 16- to18-month-old control and Nato3 cKO mice (right). 200 to 350 mitochondria from TH⁺ neurons in SN from 3 mice per group were analyzed. Scale bar, 0.25 µm. e Measurement of size (top) and roundness (bottom) of mitochondria. 200 to 350 mitochondria from SN TH⁺ neurons from 3 mice per group were analyzed. The horizontal line across the violin plot is the median. One-way ANOVA followed by a Tukey’s test for multiple group comparison, *p = 0.0280. ns, not significant. f Representative images of anti-SOD2 (magenta) immunostaining in ventral midbrain coronal sections of 11- and 16 to 18-month-old control and Nato3 cKO mice. mDA neurons were detected by anti-TH antibodies (green). Scale bar, 10 µm. g SOD2 fluorescence intensity in TH⁺ neurons from (f) were quantified. The horizontal line across the violin plot is the median. 350 (ctrl and cKO at 11 months) and 450 (ctrl and cKO at 16–18 months) TH⁺ neurons from 3 brains per group were analyzed. Two-tailed Mann–Whitney test, ****p < 0.0001. h, i RT-qPCR analysis of Sod2 (h) and neuroprotective transcription factors (i) mRNA relative levels in ventral midbrains of 16- to 18-month-old control and Nato3 cKO mice. n = 4 mice per genotype. Box boundaries are the 25th and 75th percentiles, the horizontal line across the box is the median, and the whiskers indicate the minimum and maximum values. Two-tailed Mann–Whitney test, *p < 0.0286. ns not significant.