Figure 5. Impaired cytochrome C oxidase increases glycolysis in astrocytes through a Warburg-like effect.

(A) Heat map of transcriptional differences in Surf1^+/+ versus Surf1^-/- brains. n=4. Significance denoted next to gene. (B) Western blots of phosphorylated PDHE1α at Ser293 and αTubulin from Surf1^+/+ and Surf1^-/- brains, 7 days post-injury. (C) Dot plot of single nuclei RNAseq of glycolytic and Warburg-related genes in neuronal cell subtypes from Surf1^+/+ and Surf1^-/- brains. (D) Dot plot of single nuclei RNAseq of glycolytic genes in astrocytes from Surf1^+/+ and Surf1^-/- brains. (E) Quantification of phosphorylated PDHE1α at Ser293 and αTubulin from Surf1^+/+ and Surf1^-/- brains after NaCl and dichloroacetate (DCA) treatment. (F) Quantification of cleaved caspase-3 immunostaining in NaCl and DCA treated Surf1^+/+ (n=3) and Surf1^-/- (n=4) brains 7 days post-injury. (G) Post-trauma retention of GFP fluorescence in dopaminergic neurons of C. elegans treated with pdp-1 RNAi and measured by large-particle flow cytometry. n=1758 worms (EV RNAi), n=2307 worms (pdp-1 RNAi) across two independent trials. (H) Representative micrographs of GFP dopaminergic neuronal morphology in injured C. elegans treated with pdp-1 RNAi. Scale bar=20 μm. Data are mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 5—figure supplement 1. Warburg effect in concussive brain injury.

(A) Relative ATP levels from Surf1^+/+ and Surf1^-/- brain single cell suspensions. n=5 Surf1^+/+, n=4 Surf1^-/-. (B to E) Metabolic cage analysis of Surf1^+/+ and Surf1^-/- mice subject to concussive injury show: (B) respiratory exchange rate, (C) food intake, (D) body temperature, and (E) body weight. n=3 Surf1^+/+ , n=3 Surf1^-/-. (F) Acidification of extracellular media measured from absorbance at 585nm from Surf1^+/+ and Surf1^-/- mouse embryonic fibroblasts, n=3 per group. (G) Relative transcript abundance of ldh-1 in C. elegans with cox-5b RNAi. n=3 per group. (H) Transcript abundance from RNAseq in reads per kilobase million. n=3 Surf1^+/+ , n=4 Surf1^-/-. (I) Relative pdp-1 transcript abundance by RNAseq in C. elegans subject to blunt force injury. n=3 per group. (J) Western blot of HIF1α from HEK293 cells treated with paraquat at varying concentrations. (K) Western blot of HIF1α from Surf1^+/+ and Surf1^-/- mouse embryonic fibroblasts treated with 0 and 0.625 mM paraquat. (L) Relative nhr-57 transcript abundance by RNAseq in C. elegans with cox-5b RNAi. n=3 per group. (M) UMAP clustering of snRNAseq from midbrain and striatal brain regions. (N) Gene enrichment in cell clusters defined in M. (O) Percent distribution of clustered cell types in Surf1^+/+ and Surf1^-/- mice. (P) Immunostaining of astrocytes (GFAP, green) and PDK2 (red) in the midbrain. Scale bar=5 μm. (Q) Immunostaining of astrocytes (GFAP, gray), PDK2 (red), and dopaminergic neurons (TH, green) in the midbrain. Scale bar=5 μm. (R) Western blot of phosphorylated PDHE1α at Ser293 and αTubulin from Surf1^+/+ and Surf1^-/- brains, 24 hr post NaCl or DCA treatment. Data are mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 5—figure supplement 2. Pyruvate dehydrogenase complex dynamics in blunt force trauma and concussive brain injury.

(A) Western blot of phosphorylated PDHE1α at Ser293 and αTubulin from Surf1^+/+ and Surf1^-/- brains 7 days post-injury. (B) Western blot of total baseline PDH and αTubulin from Surf1^+/+ and Surf1^-/- brains. (C) Western blot densitometry of PDH normalized to αTubulin from B. (D) Post-trauma retention of GFP fluorescence in dopaminergic neurons of PMD63 C. elegans treated with pdp-1 RNAi and measured by large-particle flow cytometry. n=5226 worms (EV RNAi), n=1703 worms (pdp-1 RNAi) across three independent trials. Data are mean ± SEM. ****p < 0.0001.