The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease affecting motor neurons. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9orf72 are the major cause of familial ALS and frontotemporal dementia (FTD) worldwide. The C9orf72 repeat expansion undergoes repeat-associated non-ATG (RAN) translation to produce five dipeptide repeat proteins (DRPs), including poly(GR) and poly(PR). Whilst it remains unclear how mutations in C9orf72 lead to neurodegeneration in ALS/FTD, dysfunction to the nucleolus and R loop formation are implicated as pathogenic mechanisms. These events can damage DNA and hence genome integrity. Cells activate the DNA damage response (DDR) with the aim of repairing this damage. However, if the damage cannot be repaired, apoptosis is triggered. In lumbar motor neurons from C9orf72-positive ALS patients, we demonstrate significant up-regulation of markers of the DDR compared to controls: phosphorylated histone 2AX (c-H2AX), phosphorylated ataxia telangiectasia mutated (p-ATM), cleaved poly (ADP-Ribose) polymerase 1 (PARP-1) and tumour suppressor p53-binding protein (53BP1). Similarly, significant up-regulation of c-H2AX and p-ATM was detected in neuronal cells expressing poly(GR)(100) and poly(PR)(100) compared to controls, revealing that DNA damage is triggered by the DRPs. Nucleophosmin (NPM1) is a histone chaperone induced during the DDR, which interacts with APE1 to enhance DNA repair. We also demonstrate that more NPM1 precipitates with APE1 in C9orf72 patients compared to controls. Furthermore, overexpression of NPM1 inhibits apoptosis in cells expressing poly(GR)(100) and poly(PR)(100). This study therefore demonstrates that DNA damage is activated by the C9orf72 repeat expansion in ALS.