(A and B) Dose-dependent impact of the CSF1R inhibitor pexidartinib (A) and rapamycin (ABI-009 formulation) (B) on the fraction of Iba1⁺ leukocytes (likely microglia given cell origins, see Methods) in mixed primary brain cultures. Error bars show ± SEM. Dashed lines, 95% confidence interval for inhibitor vs. response (3 parameters) least squares fit. n = 3 replicates/concentration for rapamycin, 6/condition for pexidartinib (data representative of 3 independent experiments). (C) Representative images of mixed primary brain cultures from A and B stained with an anti-Iba1 antibody (red) and DAPI (blue, nuclei). Scale bar in rapamycin image is representative for all. (D) Representative pictures of control and Ndufs4(KO) animals fed control diet or ~300 mg/kg/d pexidartinib chow. (E and F) Brainstem (E) and cerebellar peduncle (F) lesion size (area of lesion in central slice in serial sectioning, see Methods) in control-treated (Untreated) and 300 mg/kg/d pexidartinib (Pex300) treated control (Ctl) and Ndufs4(KO) (KO) animals. Quantification and representative images provided for each region. Representative images are only provided for Ndufs4(KO) animals as control mice do not develop lesions. See Figure 3 for quantification of Iba1⁺ leukocytes and GFAP⁺ astrocytes in control and Ndufs4(KO) mice. Anti-Iba1 antibody staining in red, DAPI (nuclei) in grayscale (see Methods). Lesion areas indicated by dashed white lines. Ages are noted in representative images (P78, etc.). ****P < 0.0001 by unpaired, unequal variances (Welch’s) t test. n = 3 animals per condition. (G–I) Onset of clasping (G), ataxia (H), and circling (I) in Ndufs4(KO) mice fed control diet (black lines, n as in Figure 1; see Methods) or administered pexidartinib at 100 mg/kg/d (dotted red lines, n = 9, 9, 9), 200 mg/kg/d (dashed red lines, n = 11, 11, 11), or 300 mg/kg/d (solid red lines, n = 9, 9, 9). ***P < 0.0005, and ****P < 0.0001 by log-rank test vs. untreated Ndufs4(KO) animals (Bonferroni significance threshold = P < 0.0167). (J) Performance of control- and pexidartinib-treated animals on a rotarod assay. **P < 0.005, and ****P < 0.0001 by unpaired, unequal variances (Welch’s) t test (BCST = *P < 0.0167 for both comparisons between pexidartinib-treated and control untreated mice and for comparisons to baseline within same treatment). Replicates (ns) are shown by vertically oriented numbers within/above bars. For rotarod, where animals do not survive to P80, t test undefined but interpreted as highly significant (see Methods). (K) Representative traces of respiratory (breathing) activity in P70 (±2 days) control and Ndufs4(KO) mice control treated or fed 300 mg/kg/d pexidartinib chow. (L) Multivariable plotting of respiratory amplitude irregularity, frequency, and frequency irregularity in P70 (±2 days) control and Ndufs4(KO) mice fed control chow or administered 300 mg/kg/d pexidartinib. (M) Frequency of breathing (representative data in K). (N and O) Single-variable analysis of data in L. (M–O) Data points represent individual animals, *P < 0.05, **P < 0.005, and ****P < 0.0005 by 2-way ANOVA with Tukey’s multiple-testing correction–adjusted P values for pairwise comparisons. Each comparison is pairwise. (P and Q) Respiratory responses to increased environmental CO₂. Pairwise data shown for responses in individual mice. **P < 0.005 by Wilcoxon matched pairs signed-rank test. For all panels, data represent mean, error bars ± SEM, unless otherwise stated. (M–Q) n = 9 for control animal data sets, 10 for Ndufs4(KO) data sets.