Figure 3: T61I point mutation preferentially increases accumulation of CHCHD2 and CHCHD10 in mitochondria in SN DA neurons.

(A) Representative fluorescence images of CHCHD2 (red), CHCHD10 (green), and merge in SN (top) and VTA (bottom) subregions in the midbrain of CHCHD2 T61I mice at 16 months. Punctae was semi-automatically annotated by Cellprofiler. CHCHD2 denoted in red and CHCHD10 in green. The region of cell body is indicated with dotted lines, based on TH signal. (B) Quantification of CHCHD2 and CHCHD10 intensity, area of CHCHD2 and D10 punctae, and area of co-localization between CHCHCHD2 and D10 punctae in SN (top panel) and VTA (bottom), normalized to the averaged cell area of each group. (C) Representative fluorescence images of mitochondria (PDH, red), CHCHD2 (green), and merge in SN (top) and VTA (bottom). PDH denoted in red, total CHCHD2 in green and CHCHD2 punctae in blue. The region of cell body is indicated with dotted lines, based on TH signal. (D) Quantification of cell area, mitochondrial content, CHCHD2 punctae area in the mitochondrial, and total CHCHD2 level in the mitochondria in SN (top panel) and VTA (bottom), normalized to averaged cell area of each group. In all fluorescence staining (A-D), n = 6–8 randomly selected TH-positive cells from 6 fields in each region in each mouse, 4 mice/genotype. Examiner was blinded to mouse genotype. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test, Scale bars indicate 1μm. (E) Impact of T61I mutation on mitochondrial morphology. DA neurons were labeled with primary TH antibody followed by secondary gold particle-conjugated antibody. N = 71 – 189 mitochondria/mouse, 3 mice/genotype. Data represent mean ± SEM. **P < 0.01 by t test with Welch’s correction. Images taken at 19,000X magnification, and scale bar indicates 1 μm.