C9orf72 expansion disrupts ATM-mediated chromosomal break repair

Abstract

Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA-RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.

Figure 1. Expression of C9orf72 expansions leads to R-loop-driven DSBs and cellular toxicity.

(a) MRC5 cells mock transfected or transfected with 10 or 102 RREs. FISH-IF was performed using a G4C2 fluorescent probe ‘RNA’ and S9.6 antibodies ‘R-Loops’. Cells were treated with RNase H1 ‘+RNASE H’. Left, Representative images shown, scale bar 5 μm. Right, The average (± SEM) number of nuclear S9.6 foci per cell was quantified from 3 cell culture replicates, 50 cells each. Significance assessed using a one-way ANOVA. (b) MRC5 cells mock transfected or transfected with 34 or 69 poly-GA DPRs. Cells examined by immunocytochemistry using anti-V5 ‘DPRs’ and S9.6 antibodies ‘R-Loops’. Left, Representative images are shown, scale bar 5μm. Right, S9.6 foci was quantified, presented, and analysed as described for (a). (c,d) Rat cortical neurons transduced with AAV9 viral-vectors encoding 10,102 RREs (c) or 34, 69 DPRs (d) were processed with FISH-IF double staining (c) or with immunocytochemistry (d), as described for (a,b). Left, Representative images shown, scale bar 5μm. Right, S9.6 foci quantified from 3 seperate neuronal preperations, 20 neurons each, and data presented and analysed as described for (a). (e,g) MRC5 cells mock transfected or were transfected with 10,102 RREs (with GFP) (e) or 34, 69 DPRs (g). Cells were immunostained with anti-γH2AX antibodies ‘γH2AX’, with GFP (e) or anti-V5 antibodies ‘DPRs’ (g). Left, Representative images are shown, scale bar 5μm. Right, The percentage of cells with 10 or more foci was quantified, presented and analysed as described for (a). (f,h) HEK 293T cells mock transfected, transfected with 10,102 RREs (f), or 34, 69 DPRs (h). Neutral comet tail moments were quantified, 100 cells each, presented, and analysed as described for (a).(i-j) MRC5 cells mock transduced or transduced with adenoviral vectors encoding for SETX or RFP and then transfected with 10 or 102 RREs (with GFP) (i) or with 0, 69 DPRs (j). Left, Cells were immunostained with S9.6 antibodies ‘R-Loops’ alongside GFP (i) or alongside anti-V5 ‘DPRs’ antibodies (j). Representative images are shown, scale bar 5μm. Right, Cells were immunostained with anti-γH2AX antibodies as described for panels (e,f), and the average (± SEM) percentage of cells exhibiting 10 or more γH2AX foci was quantified, 25 cells each, and analysed using Student‘s t-test. (k,m) MRC5 cells transduced with adenoviral vector particles encoding for SETX or mock transduced and transfected with constructs encoding 10, 102 RREs (k) or 0 or 69 DPRs (m). Cells examined by immunocytochemistry using cleaved-PARP (Cell Signalling, 9548) ‘cle-PARP’ antibodies alongside GFP (k) or anti-V5 (Bethyl, A190-120A) ‘DPRs’ antibodies (m). Left, Representative images of cle-PARP-postive and -negative cells shown, scale bar 5μm. Right, the percentage of cells cleaved-PARP-positive was quantified, 50-100 cells each, presented and analysed as decribed for (i,j). (l,n) HEK 293T cells were mock transduced or transduced with adenoviral vector particles encoding for SETX and transfected with 10,102 RREs (l) or 0 or 69 DPRs (n). Left, Cells were analysed using Trypan blue exlusion assays, and the % of cells Trypan-permeable was quantified from 6 (l) and 4 (n) cell culture replicates, ~200 cells each, and was presented and analysed as decribed for (i,j). Right, Whole cell lysates from samples used in (l) and (n) were analysed by western blotting, using senataxin and anti-α-tubulin antibodies.

Figure 2. Expression of C9orf72 expansions leads to defective ATM activation.

(a,b) MRC5 cells mock transfected, transfected with 34, 69 DPRs (a), or 10, 102 RREs (b). Cells analysed using immunocytochemistry with anti-phosphorylated ATM ‘pATM’ and anti-V5 ‘DPRs’ antibodies (a) or with FISH-IF (b). Left, Representative images shown, scale bar 5μm. Right, The average (± SEM) number of pATM foci per cell was quantified from 3 cell culture replicates, 50 cells each, and analysed using a one-way ANOVA. (c) Left, HEK 293T cells were mock transfected ‘M’ or were transfected constructs encoding 10 or 102 RREs ’10, 102’ or 34, 69 DPRs ‘34, 69’. Whole cell lysates were analysed using anti-ATM and α-tubulin antibodies. Right, ATM protein expression (normalised to α-tubulin) is presented as average ± SEM from 3 cell culture replicates, and analysed using a one-way ANOVA. (d,e) MRC5 cells mock transfected, transfected with constructs encoding 34, 69 DPRs (d), or transfected with constructs encoding for 10, 102 RREs (e). Cells were incubated with 10μM CPT ,0.037% TBH, or DMSO for 1 hour, and analysed by immunocytochemistry as decribed for (a,b). Left, Representative images of 3 cell culture replicates are shown, scale bar 5μm. Right, pATM foci were quantified as decribed for (a,b).

Figure 3. Expression of C9orf72 expansions leads to defective ATM signalling.

(a,b) MRC5 cells mock transfected or transfected with 34 or 69 DPRs (a) or 10, 102 RREs (b), and immunostained with anti-53BP1 antibodies, alongside anti-V5 ‘DPRs’ antibodies or with FISH-IF double-staining ‘RNA’ (b). Left, Representative images are shown, scale bar 5μm. Right, The average (± SEM) number of 53BP1 foci per cell was quantified from 3 cell culture replicates, 50 cells each, and analysed using a one-way ANOVA. (c,d) MRC5 cells were mock transfected or transfected with constructs encoding 34 or 69 DPRs (c), or 10, 102 RREs (d). Cells were incubated with 10μM CPT or DMSO for 1hour, and immunostained as described for (a,b). Left, Representative images are shown, scale bar 5μm. Right, 53BP1 foci was quantified as decribed above and analysed using a Student’s t-test. (e,f) Rat cortical neurons mock transduced or transduced with AAV9 viral-vectors expressing 34 or 69 DPRs (e) or with 10 or 102 RREs (f). Neurons treated with 10μM CPT for 1 hour and analysed by immunocytochemistry as decribed for (a,b). Left, Representative images are shown, scale bar 5μm. Right, 53BP1 foci was quantified as decribed for (a,b), 20 neurons each, and analysed using a one-way ANOVA. (g,h) MRC5 cells were mock transfected or transfected with constructs encoding 34 or 69 DPRs (g), or 10, 102 RREs (h) and were then treated with DMSO or with 10μM CPT. Cells were then immunostained with anti-phosphorylated p53 antibodies, alongside anti-V5 ‘DPRs’ antibodies (g) or with FISH-IF double-staining ‘RNA’ (h). Left, Representative images are shown, scale bar 5μm. Right, The nuclear fluorescence value for 50 nuclei was quantified from 3 cell culture replicates, and presented as the average (± SEM) fold change in nuclear intensity (relative to control cells), and analysed using Student’s t-test. (i) Top, HEK 293T cells were mock transfected or were transfected with 69 DPRs or 102 RREs. Cells were treated 10μM CPT for 40 min, subjected to CsCl step gradients, and fractions slot blotted with anti-TOP1 antibodies. Bottom, The fold increase in TOP1-ccs, normalised to mock was calculated and presented as the average from 2 cell culture replicates ± range.

Figure 4. The expression of C9orf72 expansions in the murine CNS leads to DSBs, nuclear HDAC4, and neurodegeneration.

(a) Cerebellar sections from mice injected with AAV9-10 or -102 RREs subjected to immunohistochemistry using anti-HDAC4 antibodies. Left, Representative images shown, scale bar 10μm. Right, The average (± SEM) percentage of Purkinje cells displaying nuclear HDAC4 was calculated for 3 animals per group, 50 Purkinje cells per animal, and analysed using a Student’s t-test. (b) Cerebellar sections from mice injected with AAV9-10 or -102 RREs subjected to immunohistochemistry using anti-γH2AX antibodies. Left, Representative images shown, scale bar 10μm. Right, The average (± SEM) number of γH2AX-positive Purkinje cells was calculated from 3 animals per group, 10 images each, and analysed using a Student’s t-test. (c) Brainstem sections from mice injected with AAV9-0 or -69 poly-GA DPRs were subjected to immunohistochemistry using anti-HDAC4 and anti-V5 antibodies. Left, Representative images shown, scale bar 10μm. Right, The average (± SEM) percentage of brainstem cells displaying nuclear HDAC4 was calculated for 3 animals per group, 30 HDAC4-positive cells per animal, and analysed using a Student’s t-test. (d) Brainstem sections from mice injected with AAV9-0 or -69 poly-GA DPRs were subjected to immunohistochemistry using anti-γH2AX and anti-V5 antibodies. Left, Representative images shown, scale bar 10μm. Right, The average (± SEM) number of γH2AX foci per cell calculated from 3 animals per group, ~1000 cells per animal, and analysed using Student’s t-test.(e) Left, Brainstem tissue harvested from mice injected with AAV9-0 or -69 poly-GA DPRs were analysed using Western blotting, with anti-GAPDH and anti-cleaved PARP antibodies. Right, cleaved-PARP was quantified and normalised to GAPDH, presented as the average intensity ± SEM from 3 animals per group, and analysed using a Student’s t-test. (f) Left, Brainstem sections from mice injected with AAV9-0 or -69 poly-GA DPRs were subjected to immunohistochemistry using anti-NeuN and anti-V5 antibodies. The average (± SEM) number of NeuN-positive cells within the periaqueductal gray region of the brainstem was quantified from 3 animals per group, and analysed using Student’s t-test. (g) Catwalk analysis was performed in animals injected with AAV9-0 or -69 poly-GA DPRs, aged 6 months. Stand intensity, stride length, and swing speed were quantified (n=12/13 for 0-V5/69-V5), presented as average ± SEM, analysed using Student’s t-test.

Figure 5. Defective ATM-mediated DNA repair can be restored by RNF168 overexpression or p62 depletion.

(a) MRC5 cells transfected with 0 or 69 DPRs, and immunostained with anti-V5 ‘DPRs’ and anti-Nbs1 antibodies. Left, Representative images are shown, scale bar 5μm. Right, The average (± SEM) number of Nbs1 foci per cell was quantified and from 3 cell culture replicates, 50 cells each, and analysed using a Student’s t-test. (b) Left, Chromatin fractions from MRC5 cells transfected with 0 or 69 DPRs were seperated analysed with Western blotting using antibodies specific to H2A . Low exposure (LE) H2A and Nbs1 bands show equal loading. Right, The average (± SEM) percentage of H2A that was ubiqutinated was quantifed from 3 cell culture replicates and analysed using Student’s t-test. (c) Left, MRC5 cells transfected with constructs encoding 0 or 69 DPRs, and immunostained with anti-V5 ‘DPRs’ and anti-Ubiquinated-H2A ‘Ub-H2A’ antibodies. Representative images are shown, scale bar 5μm. Right, Ub-H2A foci was quantified, 25 cells each, presentaed and analysed as decribed for (a). (d) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, and with control-GFP or RNF168-GFP, were immunostained with anti-V5 and anti-53BP1 antibodies. Left, Reprentative images are shown. Right, the percentage of cells with 5 or more 53BP1 foci was quantified (25 cells each), presented, and analysed as decribed for (a).(e) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, and with control-GFP or RNF168-GFP plasmids, were immunostained with anti-V5 and anti-pATM antibodies. The percentage of cells with 5 or more pATM foci was quantified (25 cells each), presented and analysed as decribed for (a). (f) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, with either control siRNA particles or p62 siRNA particles, were analysed with Western blotting using antibodies specific to p62 and GAPDH. (g) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, and with control siRNA particles or p62 siRNA particles, were immunostained with anti-V5 and anti-53BP1 antibodies. The percentage of cells with 5 or more 53BP1 foci was quantified from 4 cell culture replications (25 cells each), presented and analysed as described for (a). (h) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, and with control siRNA particles or p62 siRNA particles, were immunostained with anti-V5 and anti-pATM antibodies. The percentage of cells with 5 or more pATM foci was quantified from 4 cell culture replicates (25 cells each), presented and analysed as decribed for (a). (i) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, with control siRNA particles or p62 siRNA particles. Cells were immunostained with anti-V5 ‘DPRs’ and anti--γH2AX antibodies. The percentage of cells with 10 or more γH2AX foci was quantified (25 cells each), presented, and analysed as decribed for (a). (j) MRC5 cells transfected with constructs encoding 0 or 69 DPRs, with control siRNA particles or p62 siRNA particles, were immunostained with anti-V5 ‘DPRs’ and anti—S9.6 antibodies. R-Loop foci were quantified, presented and analysed as described for (a).(k) MRC5 cells transduced with adenoviral vectors encoding for SETX or RFP, transfected with constructs encoding 0 or 69 DPRs, with control siRNA or p62 siRNA particles, were immunostained with anti-V5 and anti--γH2AX antibodies. Nuclei were counterstained with DAPI. The average (± SEM) percentage of cells with 10 or more γH2AX foci was quantified from 4 cell culture replicates (25 cells each), and analysed using a one-way ANOVA.

Figure 6. Spinal Cord Motor Neurons from C9orf72-ALS post-mortem show elevated levels of R-Loops, DSBs, and nuclear HDAC4.

(a) Human spinal cord sections were analysed by immunohistochemistry using S9.6 antibodies. Representative images are presented, scale bar 5μm. (b) The average (± SEM) percentage of R-Loop-positive motor neurons was quantified from 6 C9orf72 patient and 6 control sections, ~50 cells each, and analysed with Student’s t-test. (c) Human spinal cord sections were analysed by immunohistochemistry using anti-γH2AX antibodies. Representative images are presented, scale bar 5 μm. (d) The % of γH2AX-postive motor neurons was quantified, presented and analysed as described for (b). (e) Human spinal cord sections were analysed by immunohistochemistry using anti-HDAC4 antibodies. Representative images are presented, scale bar 5 μm. (f) The % of motor neurons displaying HDAC4 enrichment in the nucleus was quantified, presented and analysed as described for (b).