Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD

Abstract

GGGGCC (G₄C₂) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, we identified a number of genetic modifiers of poly(GR) toxicity. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G₄C₂ repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.

Keywords: ALS/FTD; C9ORF72; DNA damage; Drosophila; iPSC.

Fig. 1.

Identification of genetic suppressors of poly(GR) toxicity in Drosophila nonneuronal cells. (A) Representative images of adult fly wings of UAS-(GR)₈₀/+ and Vg-Gal4/+; UAS-(GR)₈₀/+ flies. (B) Schematic of primary and secondary genetic modifier screens. Df, deficiency lines. (C) Summary table of newly identified genes that suppress poly(GR) toxicity in Drosophila. (D) Quantification of the effects of some suppressor genes on poly(GR)-induced loss of nonneuronal cells. ***P < 0.001 by χ² analysis. Flies (80–130) were examined for each genotype.

Fig. 2.

Elevated Ku80 expression contributes to poly(GR) toxicity in Drosophila neuronal cells. (A) Representative images of Drosophila eye with various degrees of poly(GR) toxicity, as revealed by dissecting microscope (Top row) and scanning electron microscope (Bottom row). (B) Partial loss of Ku80 activity suppresses poly(GR)-induced retinal degeneration. ***P < 0.001 by χ² analysis. (C) ELISA analysis of poly(GR) protein levels in the eyes of 1-wk-old flies. Values are mean ± SEM of three independent experiments, analyzed by one-way ANOVA and Tukey’s test. (D) Ku80 mRNA level is elevated by poly(GR) expression. Values are mean ± SD from three independent experiments, analyzed by Student’s t test.

Fig. 3.

Ku80 levels are greatly elevated in C9ORF72 iPSC-derived motor neurons. (A) Schematic of the motor neuron differentiation. (B) Ku80 mRNA levels in control and C9ORF72 motor neurons. The average of Ku80 mRNA levels in all control neuron cultures was defined as 1. Values for each iPSC line are mean ± SEM from two independent differentiations. (C–F) Western blot analysis and quantification of Ku80 and Ku70 protein levels in 2-wk-old (C and D) and 3-mo-old (E and F) motor neurons. The average of Ku80 protein levels in all control neuron cultures was defined as 1. Values for each column are mean ± SEM from three independent differentiations. Two-tailed Student’s t test was used in B, D, and F to compare three control subjects and four C9ORF72 patients. (G) Increased level of Ku80 in C9ORF72 iPSC-derived ChAT-positive motor neurons. (Scale bar, 20 µm.) (H and I) Ku80 immunostaining (H) and quantification (40 cells per line) (I) in control and C9ORF72 astrocytes positive for glial fibrillary acidic protein (GFAP). Values are mean ± SEM analyzed by two-tailed Student’s t test. (Scale bar, 10 µm.)

Fig. 4.

Activation of the DNA damage pathway in C9ORF72 iPSC-derived neurons. (A) Western blot analysis of DNA-PKcs and pATM in control and C9ORF72 iPSC-derived motor neuron cultures. (B) DNA-PKcs protein levels from two independent differentiations and pATM protein levels from three independent differentiations. Values for each iPSC line are mean ± SEM. Two-tailed Student’s t test was used to compare three control subjects and four C9ORF72 patients. (C) Schematic representation of the CRISPR-Cas9 strategy to delete the expanded G₄C₂ repeats in C9ORF72 iPSC lines. (D) Nuclear RNA foci are present in 26L6 and 27L11, but not in 26Z90 and 27M91 iPSCs. (Scale bar, 5 μm.) (E) Western blot analysis of Ku80, pATM, and p-P53 in iPSC-derived motor neuron cultures.

Fig. 5.

Activation of the proapoptotic pathway in C9ORF72 iPSC-derived motor neurons. (A) Western blot analysis of apoptotic markers PUMA, BAX, and cleaved caspase 3 in iPSC-derived motor neuron cultures. (B–D) Levels of PUMA (B), Bax (C), and cleaved caspase-3 (D) protein in iPSC-derived motor neuron cultures from three independent differentiations. Values for each iPSC line are mean ± SEM. Two-tailed Student’s t test was used to compare three control subjects and four C9ORF72 patients. (E and F) Immunostaining analysis of increased pATM (E) and cleaved caspase 3 (F) levels in C9ORF72 iPSC-derived ChAT-positive motor neurons. (Scale bar, 20 μm.)

Fig. 6.

Genetic inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD. (A) Suppression of poly(GR) toxicity in the eye by partial loss of function of Tefu, the Drosophila homolog of ATM. ***P < 0.001 by χ² analysis. Flies (80–130) were analyzed for each genotype. (B) ELISA analysis of poly(GR) protein level. Values are mean ± SEM of three independent experiments analyzed by one-way ANOVA and Tukey’s test. (C) Partial loss of p53 activity rescues poly(GR) toxicity in the fly eye. ***P < 0.001 by χ² analysis. Flies (80–130) were analyzed for each genotype. (D) Schematic representation of CRISPR-Cas9 deletion of one copy of Ku80. (E) PCR analysis of Ku80 deletion. (F–H) Western blot analysis of the relative levels of several proteins in motor neurons derived from the parental C9ORF72 iPSC line 26L6 and its isogenic daughter lines 26L6-34E and 26L6-64A. (I) Quantification of Western blot analysis. Values are mean ± SEM of two independent differentiation experiments. *P < 0.05, **P < 0.01 by one-way ANOVA and Tukey’s test.