C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

Abstract

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question, we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats, but not stop codon-interrupted "RNA-only" repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.

Fig. 1. Generation and characterisation of expanded pure and RNA-only GGGGCC repeats.

(A) Agarose gel showing pure GGGGCC repeats and stop codon-interrupted RNA-only (RO) repeats. (B) Circular dichroism (CD) spectra of both 24 pure and 24 RO repeats showed characteristic RNA G-quadruplex structure with minima and maxima at 237 and 262 nm respectively (23). (C) Confocal microscope images of nuclei (blue) in RNA FISH-labelled SH-SY5Y cells showed that 103 pure and 107 RO repeats both produced nuclear RNA foci (red). Scale bar represents 5 µm. (D) Quantification of the number of SH-SY5Y cells containing RNA foci after transfection with pure and RO repeats of different lengths, and empty vector (E). No difference was observed between equivalent length pure and RNA-only repeats (36 vs. 36 RO, 103 vs. 107 RO, 1 way ANOVA with Bonferroni test (selected pairs), n > 3, error bars represent SEM).

Fig. 2. Pure GGGGCC repeats caused toxicity via DPR proteins.

(A) Dot blot showing that 36 and 103 pure repeats generated poly-(GR) proteins while 3 pure repeats, and 36, 108 and ~288 RNA-only (RO) repeats did not. Genotypes were: w; UAS-3/hsGal4, w; UAS-36/hsGal4, w; UAS-103/ hsGal4, w; UAS-36 RO/hsGal4, w; UAS-108 RO/ hsGal4, w; UAS-288 RO/hsGal4. (B) Stereomicroscopy images of representative Drosophila eyes expressing pure or RO repeats using the GMR-GAL4 driver. 36 pure repeats were mildly toxic. 103 pure repeats showed more overt toxicity. 3 repeats and 36 and 108 RO repeats had no effect. Genotypes were: w; GMR-Gal4/+, w; GMR-Gal4/UAS-3, w; GMR-Gal4/UAS-36, w; GMR-Gal4/UAS-103, w; GMR-Gal4/UAS-36RO, w; GMR-Gal4/UAS-108RO, w; GMR-Gal4/UAS-288RO. Scale bar represents 200 µm. (C) Quantification of egg-to-adult viability showed that 36 and 103 pure repeats were lethal at higher temperatures, whereas RO repeats had no effect (Kruskal Wallis test with Dunn's multiple comparison (selected pairs), ***p<0.001, **p<0.01, *p<0.05, error bars represent SEM). Genotypes were as in (B). (D) Survival of female flies expressing repeats in adult neurons using the elav-GeneSwitch (elavGS) driver. 36 and 103 pure repeats substantially decreased survival, while 36, 108 and 288 RO repeats had no effect (p<0.0001, log-rank test). Genotypes were: w; UAS-3/+; elavGS/+, w; UAS-36/+; elavGS/+, w; UAS-103/+; elavGS/+, w; UAS-36 RO/+; elavGS/+, w; UAS-108 RO/+; elavGS/+, w; UAS-288 RO/+; elavGS/+. (E) Flies expressing 36 and 103 pure repeats survived longer in the presence of cycloheximide than in its absence (p<0.001, log-rank test). Genotypes were: w; UAS-36/+; elavGS/+, w; UAS-103/+; elavGS/+.

Fig. 3. DPR toxicity was caused by poly-GR and poly-PR proteins.

“Protein-only” constructs for individual DPR proteins were expressed in the Drosophila eye (A-C), and the adult nervous system (D). (A) (GR)₃₆ and (PR)₃₆ caused eye degeneration, while (GA)₃₆ and (PA)₃₆ had no effect. Genotypes were: w; UAS-PA36/GMR-Gal4, w; UAS-GA36/GMR-Gal4, w; UAS-GR36/GMR-Gal4, w; UAS-PR36/GMR-Gal4. Scale bar represents 200 µm. (B) (GR)₁₀₀ and (PR)₁₀₀ caused extensive eye degeneration, while (GA)₁₀₀ and (PA)₁₀₀ had no effect. Genotypes were: w; UAS-PA100/GMR-Gal4, w; UAS-GA100/GMR-Gal4, w; UAS-GR100/GMR-Gal4, w; UAS-PR100/GMR-Gal4. (C) Quantification of egg-to-adult viability showed (GR)₁₀₀ and (PR)₁₀₀ caused a substantial reduction in survival, whereas (GA)₁₀₀ and (PA)₁₀₀ had no effect (Kruskal Wallis test with Dunn's multiple comparison, selected pairs, ***p<0.001, **p<0.01, error bars represent SEM). Genotypes were as in (A) and (B). (D) Expression of (GR)₁₀₀ and (PR)₁₀₀ in adult neurons using the elav-GeneSwitch (elavGS) driver caused a substantial decrease in viability (p<0.001, log-rank test); (GA)₁₀₀ caused a late-onset decrease in survival, and (PA)₁₀₀ or elavGS driver alone had no effect. Genotypes were: w; elavGS/+, w; UAS-PA100/+; elavGS/+, w; UAS-GA100/+; elavGS/+, w; UAS-GR100/+; elavGS/+, w; UAS-PR100/+; elavGS/+.