Metformin inhibits RAN translation through PKR pathway and mitigates disease in C9orf72 ALS/FTD mice

Abstract

Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and G₄C₂ expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in C9orf72 ALS/FTD human and mouse brains, and inhibiting PKR in C9orf72 BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for C9orf72 ALS/FTD and other expansion diseases.

Keywords: ALS/FTD; C9orf72; PKR; RAN translation; metformin.

Fig. 1.

RAN translation of CAG, CCTG, CAGG, G₄C₂ expansions is regulated by PKR. (A and B) In vitro PKR kinase assay showing levels of p-PKR in MEFs (A) and liver (B) from C9 BAC and NT mice. (C) Quantification of p-PKR immunofluorescence staining in CA hippocampal region elevated in a cohort of phenotypic C9-500 vs. NT mice. (D) IHC showing α-p-PKR staining in CA1 of hippocampus is elevated in C9 human autopsy brains. (E) Schematic diagrams showing PKR-WT, PKR-C-terminal, and PKR-K296R constructs. (F–I) Immunoblots of HEK293T lysates after transfection with CAG (F), CCTG (G), CAGG (H), and G₄C₂ (I) expansion constructs with or without constructs expressing PKR-WT, PKR-CT, or PKR-K296R. Protein blots were probed for tagged RAN proteins, p-PKR, p-eIF2α, and total PKR. PKR overexpression increases phospho-PKR (p-PKR) and phospho-eIF2α (p-eIF2α), whereas PKR-Cter has no effect and PKR-K296R decreases steady-state levels of p-PKR and p-eIF2α. RAN protein quantification is shown in the lower graphs. Statistical analyses were performed using the t test (B–D) or one-way ANOVA with Dunnett analyses for multiple comparisons (F–I); *P < 0.05, ***P < 0.001, ****P < 0.0001; n ≥ 3 per group. Bars show mean ± SEM.

Fig. 2.

RAN protein levels regulated by PKR/eIF2α pathways. (A–D) Protein blots comparing polyAla (A), polyLPAC (B), polyQAGR (C), and polyGP (D) RAN protein levels in PKR-KO compared to control HEK293T cells. (E) Protein blots showing that overexpression of TRBP reduces levels of RAN polyAla expressed from CAG and CUG expansion RNAs and RAN polyLPAC expressed from CCUG repeats. Additionally, TRBP reduces levels of p-eIF2α. (F) Protein blots of HEK293T WT and PKR-KO cells after cotransfection with plasmids expressing a CAG expansion and eIF2α-WT, eIF2α-S51D, or eIF2α-S51A. Protein blots were probed with α-HA to detect polyAla RAN protein levels, which are quantitated in Lower. (G) Protein blots after thapsigargin treatment of WT and PKR-KO cells transfected with CAG repeat constructs with polyAla quantification. (H) Protein blots of ISRIB treatment of HEK293T cells cotransfected with CAG repeat and PKR-WT constructs with polyAla quantification. Statistical analyses were performed using the two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001, n = 3 per group, Bars show mean ± SEM.

Fig. 3.

Inhibition of PKR decreases RAN proteins and improves behavior in C9-BAC mice. (A) Schematic diagrams of EGFP control and PKR-K296R AAV2/9 constructs used for ICV injections of C9-BAC and NT mice. (B) Representative IHC staining (brown color) of GA RAN protein aggregates in retrosplenial cortex from C9-EGFP and C9-PKR-K296R mice with quantification of GA RAN protein aggregates. Two-tailed t test; *P < 0.05, Bars show ± SEM. (C) MSD assay of brain lysates showing PKR-K296R–treated mice have lower levels of soluble GP RAN protein. Bars show ± SEM. (D) Open-field analyses of C9 mice treated with PKR-K296R or EGFP. (E and F) Comparisons of C9-relevant DigiGait parameters among AAV PKR-K296R and EGFP treatment cohorts at 3 mo of age. Gray boxes show parameters that significantly differ between PKR-K296R C9 and EGFP C9 cohorts (E) or PKR-K296R C9 and NT EGFP cohorts (F). (G and H) Example DigiGait data for Stance and Brake parameters. Statistical analyses were performed using two-tailed t tests (B–D), *P < 0.05, ****P < 0.0001 with corrections for multiple comparisons in G and H, *P < 0.025, **P < 0.005. Bars show ± SEM. All analyses were done in a blinded fashion.

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.

Fig. 5.

PKR activation model of RAN translation. Schematic diagram depicting chronic activation of PKR by repeat expansion RNAs increases RAN translation. Blocking the PKR pathway with PKR-K296R, metformin, or TRBP reduces RAN protein levels to a greater extent than blocking p-eIF2α with ISRIB. These results suggest PKR regulates RAN protein levels through both p-eIF2α–dependent and p-eIF2α–independent pathways. The accumulation of misfolded or aggregated RAN proteins may, in turn, activate the PERK pathway and exacerbate disease by leading to further eIF2α phosphorylation and a cycle of increased RAN protein production.