Age-dependent decline in SN and VTA dopaminergic neurons following K320E-Twinkle expression. (A) Schematic of the generation of K320E-Twinkle^DaN mice. (B) Schematic showing the location of midbrain sections harbouring dopaminergic neurons in sagittal (left) and coronal (right) plane (images modified from Paxinos and Franklin³²). (C) Representative images of simultaneous green fluorescent protein (GFP) and tyrosine hydroxylase (TH) immunostaining in the midbrain of 5-month-old control and homozygous K320E-Twinkle^DaN mice. Scale bar = 500 μm. (D) Quantification of TH-positive cells expressing GFP and GFP-positive cells expressing TH (brain sections from three distinct levels relative to bregma from three animals per group). (E) TH immunohistochemistry of coronal midbrain sections of 20-month-old control (grey), K320E/Wt^DaN (light blue) and K320E/K320E^DaN mice (dark blue). Scale bar = 500 µm. (F) Stereological quantification of midbrain dopaminergic neurons and (G) relative number of remaining neurons in the substantia nigra (SN, green) and ventral tegmental area (VTA, magenta; 5 months, 3–5 mice; 10 months, 4–5 mice; 20 months, 4–5 mice). Significant differences to controls are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences between hetero- and homozygous K320-Twinkle^DaN mice are indicated by hash marks: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001. P-values were calculated using one-way (F) or two-way ANOVA (D and G) with Bonferroni’s correction for multiple comparison. Data are presented as mean ± standard error of the mean (SEM).