Poly(GR) in C9ORF72-Related ALS/FTD Compromises Mitochondrial Function and Increases Oxidative Stress and DNA Damage in iPSC-Derived Motor Neurons

Abstract

GGGGCC repeat expansions in C9ORF72 are the most common genetic cause of both ALS and FTD. To uncover underlying pathogenic mechanisms, we found that DNA damage was greater, in an age-dependent manner, in motor neurons differentiated from iPSCs of multiple C9ORF72 patients than control neurons. Ectopic expression of the dipeptide repeat (DPR) protein (GR)₈₀ in iPSC-derived control neurons increased DNA damage, suggesting poly(GR) contributes to DNA damage in aged C9ORF72 neurons. Oxidative stress was also increased in C9ORF72 neurons in an age-dependent manner. Pharmacological or genetic reduction of oxidative stress partially rescued DNA damage in C9ORF72 neurons and control neurons expressing (GR)₈₀ or (GR)₈₀-induced cellular toxicity in flies. Moreover, interactome analysis revealed that (GR)₈₀ preferentially bound to mitochondrial ribosomal proteins and caused mitochondrial dysfunction. Thus, poly(GR) in C9ORF72 neurons compromises mitochondrial function and causes DNA damage in part by increasing oxidative stress, revealing another pathogenic mechanism in C9ORF72-related ALS and FTD.

Keywords: ALS; C9ORF72; DNA damage; DPR; FTD; RAN translation; iPSC; mitochondria; oxidative stress; repeats.

Figure 1. Characterization of Motor Neuron Cultures Differentiated from Control and C9ORF72 iPSC Lines

(A) Schematic of the motor neuron differentiation protocol. (B) Representative images of TUJ1⁺ neurons from one control and one C9ORF72 iPSC line. Scale bar: 50 μm. (C) Average percentage of TUJ1⁺ neurons in cultures of three control and three C9ORF72 patients. No statistically significant difference was found. (D) Immunostaining of TUJ1⁺ and ChAT⁺ motor neurons in control and C9ORF72 neuron cultures. Scale bar: 50 μm. (E) Average percentage of ChAT⁺ motor neurons among all neurons of three control and three C9ORF72 patients. (F) RNA foci are present in iPSC-derived ChAT⁺ motor neurons of three C9ORF72 patients but not control subjects. Representative images of neurons from one control (35L11) and one patient (16L15) are shown. DAPI: blue; ChAT: green; RNA foci: red. Scale bar: 20 μm. (G) Percentage of motor neurons with foci. Data are from two independent cultures. (H) Average number of foci per ChAT⁺ motor neuron. Data are from two independent cultures. All the values in panels C, E, G and H are mean ± S.E.M. (I) Poly(GP) is present in 2-week-old C9ORF72 but not control iPSC-derived motor neuron cultures. (J) Dot blot analysis of Poly(GR) in 2-month-old high-yield motor neuron cultures. See also Figure S1 and Table S1.

Figure 2. iPSC-Derived C9ORF72 Motor Neurons Show Increased DNA Damage

(A) γH2AX expression is increased in 4-month-old C9ORF72 motor neuron cultures. (B) Immunostaining for γH2AX in 4-month-old ChAT⁺ C9ORF72 motor neurons. Scale bar: 20 μm. (C) Total p53 expression is elevated in 4-month-old C9ORF72 motor neuron cultures. (D) P53 expression is increased in 4-month-old iPSC-derived ChAT⁺ C9ORF72 motor neurons. Scale bar: 20 μm. (E) DNA strand breaks are more abundant in 4-month-old C9ORF72 motor neuron cultures than in control motor neurons, as measured by comet assay. Representative images are shown. (F) Relative tail length from 50 4-month-old cells of each subject. (G) Percentage of DNA in the tail in 50 cells per subject from 4-month-old motor neuron cultures. (H) Age-dependent increase in DNA damage in C9ORF72 motor neuron cultures. Values in panels F–H are mean ± S.E.M. *: p < 0.05; **: p < 0.01; ***: p < 0.0001 by one-way ANOVA and Tukey’s multiple-comparison test. (I) Increased RNA damage in 3-month-old C9ORF72 motor neurons as shown by anti-8-OHG antibody staining. Scale bar: 10 μm. See also Figure S2.

Figure 3. (GR)₈₀ Expression Induces DNA Damage in Control iPSC-Derived Human Motor Neurons

(A,B) Subcellular localization of HA-tagged (GA)₈₀ (A) and Flag-tagged (GR)₈₀ (B) in control iPSC-derived motor neurons. DAPI: blue; ChAT: red; Flag: green. Scale bar: 20 μm. (C) (GA)₈₀ expression has little effect on the level of p-p53 in control iPSC-derived human motor neurons. (D) The level of p-p53 is increased in (GR)₈₀-transduced ChAT⁺ human motor neurons. (E) (GA)₈₀ expression does not affect γH2AX level. (F) (GR)₈₀ expression increases the level of γH2AX in control iPSC-derived motor neurons. Scale bar in C–F: 20 μm. (G) (GR)₈₀ but not (GA)₈₀ expression increases DNA fragmentation in control motor neurons as measured by the comet assay. Scale bar: 20 μm. (H, I) Tail length (H) and percentage of DNA in the tail (I) are increased in (GR)₈₀- but not (GA)₈₀-expressing human motor neurons. 50 cells from each condition were analyzed. Values are mean ± S.E.M. ***: p < 0.0001, by one-way ANOVA and Tukey’s multiple-comparison test. See also Figure S3.

Figure 4. Increased ROS Contributes to the DNA Damage Phenotype in 4-Month-Old iPSC-Derived C9ORF72 Motor Neuron Cultures

(A–C) Mitochondrial ROS levels in 2-week-old (A), 2-month-old (B) and 4-month-old (C) iPSC-derived high-yield motor neuron cultures of three controls (red) and three C9ORF72 patients (blue). **: p < 0.01 by Student’s t test. (D) (GR)₈₀ expression (blue) increases mitochondrial ROS in 2-week-old iPSC-derived control motor neuron cultures. **: p < 0.01 by Student’s t test. (E) (GR)₈₀-induced increase in mitochondrial ROS is partially rescued by Trolox treatment. *: p < 0.05; **: p < 0.01 by one-way ANOVA and Tukey’s multiple-comparison test. (F) Trolox partially rescues the DNA damage phenotype caused by (GR)₈₀ in 2-week-old control iPSC-derived motor neuron cultures. *: p < 0.05; **: p < 0.01 by one-way ANOVA and Tukey’s multiple-comparison test. (G) DNA damage was partially reduced when Trolox was added to 3-month old C9ORF72 motor neuron cultures for additional 3 weeks. **: p < 0.01; ***: p < 0.001 by one-way ANOVA and Tukey’s multiple-comparison test. (H) Ectopic expression of both hSOD1 and catalase suppresses (GR)₈₀ toxicity in the fly wing. *: p < 0.05; **: p < 0.01; ***: p < 10⁻¹² by chi-square test for categorical data. (I) The interactome of top 100 proteins that bind to (GR)₈₀ after RNase A treatment. The protein names are visible after zoom-in. (J) The majority of top 100 (GR)₈₀-interacting proteins are ribosomal proteins. (K) Mitochondrial membrane potential is increased in 4-month-old C9ORF72 motor neurons. Values are mean ± S.E.M. ***: p < 0.001. See also Figure S4 and Table S2.