Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the death of motor neurons leading to paralysis and death, generally 3–5 years post-symptom onset. The most frequent genetic cause of ALS is a hexanucleotide repeat expansion (HRE) in the chromosome 9 open reading frame 72 (C9orf72) gene, that has three major hypothesised pathological mechanisms including the production of dipeptide repeat proteins (DPRs). Our laboratory has previously identified purine metabolism dysfunction in induced neural progenitor cell-derived astrocytes (iAstrocytes) from C9orf72 ALS (C9-ALS) cases (C9-iAstrocytes), driven by loss of the enzyme adenosine deaminase (ADA). Here, we have demonstrated that loss of ADA along with changes to ecto-5′-nucleotidase and hypoxanthine-guanine phosphoribosyl transferase led to disruption in purine metabolite levels including purine dNTP output. These changes were recapitulated in patient CSF, whilst loss of ADA was recapitulated in patient white matter. Immunofluorescence also demonstrated purinosome formation dysfunction in C9-iAstrocytes. These changes are likely driven by DPRs as ADA loss was recapitulated in in vitro and in vivo DPR models. Finally, ADA levels could be recovered by reducing DPR levels either by inhibiting serine/arginine-rich splicing factor 1 or overexpressing RuvB-like 2. Our data demonstrate that DPR production negatively affects purine function in C9-ALS suggesting a potentially pivotal role for purine metabolism dysfunction in C9-ALS pathology.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the death of motor neurons leading to paralysis and death generally 3–5 years post-symptom onset. ALS is a cell- and non-cell-autonomous disease, with glia such as astrocytes influencing disease pathology and progression. Our laboratory has previously identified purine metabolism dysfunction in induced neural progenitor cell-derived astrocytes (iAstrocytes) from sporadic ALS (SALS) cases, driven by loss of the enzyme adenosine deaminase (ADA). Here, we have demonstrated that loss of ADA, along with changes to ecto-5′-nucleotidase and hypoxanthine-guanine phosphoribosyl transferase led to disruption in purine metabolite levels, linked to the level of the ADA enzyme. These alterations were recapitulated in SALS CSF and post-mortem tissue, with ageing and sex affecting purine metabolite levels downstream of ADA and positively correlating with disease progression. Loss of ADA led to reduced 53BP1-mediated DNA repair and increased P16 levels, which was recapitulated in control iAstrocytes via ADA inhibition. Our findings indicate that TDP43 dysfunction drives impairment of ADA-mediated purine metabolism in vitro, leading to downstream effects that include DNA damage, likely through inhibition of DNA repair mechanisms, and the induction of cellular senescence. Furthermore, these results suggest that therapeutic targeting of the ADA pathway may help slow ALS disease progression.