Type I PRMT Inhibition Protects Against C9ORF72 Arginine-Rich Dipeptide Repeat Toxicity

Abstract

Repeat expansion mutations in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG translation of this expansion produces dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here we demonstrated that small molecule inhibition of type I protein arginine methyltransferases (PRMT) protects against polyGR and polyPR toxicity. Furthermore, our findings suggest that asymmetric dimethylation of polyGR and polyPR by Type I PRMTs plays important roles in their cytotoxicity.

Keywords: ALS (Amyotrophic lateral sclerosis); C9ORF72 DPRs; FTD (Fronto-Temporal Dementia); arginine methylation; glycine-arginine; proline-arginine; protein arginine methyl transferase.

Figure 1

Type I PRMT inhibitors abrogate toxicity associated with GR₁₅ and PR₁₅ challenge in NSC-34 cells. (A) Relative signal of total ADMe in NSC-34 cells after having been incubated with Type I PRMT inhibitors for 24 hours. Quantification of the signal was done by ICW assay using an antibody against total ADMe and normalized using an antibody against total protein (MS023: df = 132, R² = 0.7441; MS049: df = 82, R² = 0.8218; EPZ020411: df = 83, R² = 0.7087; GSK3368715: df = 50, R² = 50). (B) Schematic of an ICW assay workflow and example visualization. We used a primary antibody against total ADMe, and a fluorescent green IRDye secondary antibody. A red, CellTag700 antibody was used to fluorescently label total protein. (C, D) Percent metabolic activity after challenging with 3 µM of GR₁₅ or PR₁₅ and dosing with a Type I PRMT inhibitor (GR₁₅ challenge: MS023: df = 67, R² = 0.8054; MS049: df = 10, R² = 0.9630; EPZ020411: df = 13, R² = 0.9237; GSK3368715: df = 19, R² = 0.8077; PR₁₅ challenge: MS023: df = 40, R² = 0.9034; MS049: df = 10, R² = 0.9571; EPZ020411: df = 13, R² = 0.9625; GSK3368715: df = 7, R² = 0.9330). (E, F) Percent LDH release after challenging with 3 µM of GR₁₅ or PR₁₅ and dosing with a Type I PRMT inhibitor (GR₁₅ challenge: MS023: df = 22, R² = 0.9056; MS049: df = 10, R² = 0.9414; EPZ020411: df = 13, R² = 0.8807; GSK3368715: df = 10, R² = 0.9313; PR₁₅ challenge: MS023: df = 22, R² = 0.9398; MS049: df = 10, R² = 0.9613; EPZ020411: df = 10, R² = 0.7078; GSK3368715: df = 10, R² = 0.9375). (G) Percent metabolic activity after challenging NSC-34 cells with 3 µM of GR₁₅ or PR₁₅ and dosing with GSK591 (two-way ANOVA with Dunnett’s multiple comparison; n = 6 untreated, n = 3 treated; P values > 0.1657, mean ± s.e.m). The 200 µM dose of GSK591 significantly decreased metabolic activity beyond the effect observed with 3 µM of GR₁₅ alone (two-way ANOVA with Sidak’s multiple comparison; n = 6 untreated, n = 3 treated; *P = 0.0261, mean ± s.e.m.) (H) Percent metabolic activity after challenging cells with 3 µM of GR₁₅ or PR₁₅ and dosing with MS094 (two-way ANOVA with Dunnett’s multiple comparison; NS P values >0.5496, mean ± s.e.m.). (I) Percent LDH release after challenging cells with 3 µM of GR₁₅ or PR₁₅ and dosing with MS094 (two-way ANOVA with Dunnett’s multiple comparison; NS P values >0.1650, mean ± s.e.m.). For (C, D, G, H), 100% activity represents untreated NSC-34 cells, and 0% activity represents metabolic activity after 3 µM of GR₁₅ or PR₁₅ challenge alone. For (C–F), full dose response plots can be found in Supplementary Figure 1 . For (A), (C–F), a full listing of n for each condition can be found in the Supplementary Statistics section of the Supplementary Material .

Figure 2

Asymmetrical arginine dimethylation of GR₁₅ prevents abrogation of toxicity by MS023. (A) Products of in-vitro methylation assay immunoblotted for asymmetrically dimethylated arginine 3 in Histone 4 and GR dipeptide (left) and total asymmetrical arginine dimethylation and GR dipeptide (right). Also shown is the peptide sequence of Histone 4 with the epitope of the H4R3me2a antibody highlighted. Both blots show ADMe of GR₁₅ and that it is increasingly dimethylated with increasing amounts of PRMT1. (B) Percent metabolic activity after challenging with ADMe-GR₁₅ compared to unmethylated GR₁₅ (****P < 0.0001, ***P = 0.0002, **P = 0.0013, *P = 0.0281) or ADMe-PR₁₅ compared to unmethylated PR₁₅ (^####P < 0.0001, ^##P = 0.004; two-way ANOVA with Sidak’s multiple comparison; n = 3 for each dose of DRP; mean ± s.e.m.). (C) Percent LDH release after challenging with ADMe-GR₁₅ compared to unmethylated GR₁₅ (**P = 0.0083, *P = 0.0117) or ADMe-PR₁₅ compared to PR₁₅ (^#P = 0.0239; two-way ANOVA with Sidak’s multiple comparison; n = 3 for each dose of DRP; NS P > 0.05 mean ± s.e.m.). (D) Percent metabolic activity after challenge with 3 µM of ADMe-GR₁₅ or unmethylated GR₁₅ and dosing with MS023, compared to activity after challenge of 3 µM of GR₁₅ alone (two-way ANOVA with Dunnett’s multiple comparison; n = 9 for each dosing group; NS P > 1.638, ****P < 0.0001, *P = 0.0411; mean ± s.e.m.). (E) Percent LDH release after challenge with 3 µM of ADMe-GR₁₅ or unmethylated GR₁₅ and dosing with MS023, compared to release after challenge of 3 µM of GR₁₅ alone (two-way ANOVA with Dunnett’s multiple comparison; n = 3 for each dosing group; NS, P > 0.0657, ****P < 0.0001, ***P = 0.0002; mean ± s.e.m.). (F) Percent metabolic activity after challenge with 3 µM of ADMe-PR₁₅ or unmethylated PR₁₅ and dosing with MS023, compared to activity after challenge of 3 µM of PR₁₅ alone (two-way ANOVA with Dunnett’s multiple comparison; n = 3 for each dosing group; NS, P > 0.1830, ****P < 0.0001, ***P = 0.0004, ^#P > 0.0242; mean ± s.e.m.). (G) Percent LDH release after challenge with 3 µM of ADMe-PR₁₅ or unmethylated PR₁₅ and dosing with MS023, compared to release after challenge of 3 µM of PR₁₅ alone (two-way ANOVA with Dunnett’s multiple comparison; n = 3 for each dosing group; NS, P > 0.0.2947, ****P < 0.0001, ***P = 0.0007; ^####P < 0.0001, ^###P = 0.0004, ^#P > 0.0273 mean ± s.e.m.). For (D, F), 100% activity represents untreated NSC-34 cells, and 0% activity represents metabolic activity after 3 µM of GR₁₅ or PR₁₅ challenge alone, respectively. For (F, G), *- PR₁₅ + MS023 compared to PR₁₅ alone. # - ADMe-PR₁₅ + MS023 compared to PR₁₅ alone.