Figure 5.

AIF deficiency stimulates Akt and mTOR activity. (A–B) Immunoblot analyses were performed on (A) muscle and (B) cerebellar tissues from 3 to 6 month-old animals. Densitometry is relative to wt littermates and reported as mean ± SEM. Antibodies against total and phosphorylated forms of Akt, 40-kDa proline-rich Akt substrate (PRAS40), p70 S6 kinase (P70-S6K), ribosomal Protein S6 (RPS6) were used (n = 4–9 per genotype). GAPDH and actin were used as loading control (***p < 0.001, **p < 0.01, *p < 0.05). Color code is: black = wt (for KI); red = KI; dark grey = wt (for Hq); green = Hq. (C) Immunoblot analyses on muscle and cerebellar tissue from Hq mice and littermate controls following 2 weeks of rapamycin or vehicle treatment. Compared to vehicle-treated mice, animals of the rapamycin-group showed reduced mTOR activity, as revealed by decreased pRPS6. (D) Grip strength test was performed in wt and Hq mice injected with vehicle or rapamycin. Compared to mock treatment, rapamycin improved muscular performance in Hq animals (n = 9–10 per group). (E) RT-PCR analysis of Mthfd2 expression in wt and Hq mice treated with rapamycin. Compared to vehicle-treated animals, rapamycin-injected mice displayed a significant decrease in Mthfd2 mRNA expression within the quadriceps muscle and cerebellum (n = 5–6 per group). (F) Schematic comparison of molecular and phenotypic defects in AIF deficient animals. Compared to Harlequin mutant mice, Aifm1 (R200 del) knockin animals have a more homogeneous phenotype, with common pathological traits with AIFM1-linked human patients, such as COX defects associated with early-onset myopathy. In Aifm1 (R200 del) knockin animals, molecular defects include CI and CIV impairment, altered carbon metabolism and aberrant Akt/mTOR activity.