Fig. 4.

Metformin inhibits PKR, reduces RAN proteins, and ameliorates disease in C9-BAC mice. (A) Protein blots of HEK293T cells transiently transfected with CAG, CCTG, CAGG, and G₄C₂ expansion constructs (n = 3 per group) treated with or without 5 mM metformin. (B) Protein blots of total PKR, p-PKR (T446), and p-PKR (T451) from HEK293T cells transfected with various repeat expansion constructs with or without metformin treatment. (C) Animal study design with metformin treatment for 2–5 (group A) or 6–10 mos (group B). (D) Quantification of GA aggregates by IHC. (E) Soluble GP levels measured by MSD. (F) Comparisons of C9-relevant DigiGait parameters in cohorts with or without metformin treatment at 3 mo of age. Gray boxes show parameters that significantly differ between C9 cohorts with and without metformin (Left) or metformin C9 and NT control groups (Right). (G) Example data of three DigiGait parameters. (H) Open-field analyses. (I) GFAP staining in metformin-treated vs. untreated C9-BAC animals. GFAP levels were separately normalized to NT levels for group A and group B. (J) ChAT-positive motor neurons in the lumbar spinal cord (L3–L6) in C9-BAC and NT mice with or without metformin treatment from 2 to 10 mo. Statistical analyses were performed using two-tailed t test (A, F, and I), one-way ANOVA with Tukey analyses for multiple comparisons (G), and one-way ANOVA with Holm–Sidak analyses for multiple comparisons (H and J) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All analyses were done in a blinded fashion.