Figure 6 – Neuronal Effects of Ndufs4 Deficiency.

(A-C) VGlut2 specific knockout of Ndufs4 leads to shortened lifespan (A), progressive weight loss after approximately post-natal day 37, and (C) onset of neurologic symptoms by approximately P40 roughly equivalent to that observed in the whole-body Ndufs4(KO) mouse. (A) Log-rank test p=0.1. (B) No significant differences at any age. (C) Log-rank test ***p<0.0001. (D) Western blot analysis of whole-brain lysates from Ndufs4(KO) and control animals as a function of age. (E-N) Quantification of protein abundance. (E, G, I, K, M) Analysis of genotypes with all ages pooled, *p<0.05, **p<0.005 by pairwise t-test. (F, H, J, L, N) Scatter-plots of target abundance by genotype and age. GAD1 (Glutamate Decarboxylase 1); GAD2 (Glutamate Decarboxylase 2); MBP (myelin basic protein). (O-P) VGlut2-Cre promoter driven ZsGreen (Ai6 construct) expression in P60 control (O) and Ndufs4(KO) (whole-body knockout) (P) brains. Green = ZsGreen, blue = DAPI (DNA dye). Arrows indicate known lesion-associated areas of the cerebellum (right) and brainstem (left). An advanced lesion is visible in the brainstem region of the Ndusf4(KO) sample (P), as determined by the abnormal accumulation of nuclei (DAPI, blue) at this location. No overt changes to VGlut2-Cre expressing cell populations are present in the Ndufs4(KO) compared to control. Representative images, n>3 brains imaged per genotype. (Q-R) Higher magnification imaging of brainstem (Q-R) and cerebellum (S-T) in these control (Q, S) and Ndusf4(KO) (R, T) brains. In each set of panels, a-ZsGreen, b-DAPI, c-overlay.