Investigating Adenosine Deaminase and Purine Metabolism in C9orf72 Amyotrophic Lateral Sclerosis

Background: ALS is a neurodegenerative disorder characterised by the death of motor neurons (MNs) in the brain stem and spinal cord. The most common genetic mutation associated with the development of ALS is a hexanucleotide repeat expansion (HRE) in the C9orf72 gene. Previous data from our laboratory has revealed that induced astrocytes (iAstrocytes) from C9orf72 ALS patients exhibit a significant downregulation in the purine metabolism enzyme adenosine deaminase (ADA), that deaminates adenosine to form inosine, leading to increased sensitivity to adenosine-mediated toxicity. Inosine supplementation to bypass the defect was beneficial bioenergetically in iAstrocytes and decreased iAstrocyte-mediated MN toxicity in co-cultures. Our data raised questions as to what other effects the C9orf72 HRE has on purine metabolism, why and how ADA is downregulated, and led us to hypothesise that restoring ADA levels would be beneficial for C9orf72 iAstrocytes. Results: ADA activity and inosine levels were reduced in C9orf72 iAstrocytes. We also observed a downregulation of the purine enzymes ecto-5’-nucleotidase (CD73) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). ADA and CD73 downregulation were both recapitulated in C9orf72 HRE gain-of-function models. ADA gene therapy increased ADA activity and inosine output and restored ADA expression. p62 and NQO-1 were unaffected by ADA gene therapy. ADA gene therapy also did not alter CD73 or HGPRT levels and had no effect on adenosine-mediated toxicity or increase ATP levels. Conclusions: These data demonstrate that the C9orf72 HRE induces several aberrations in purine metabolism, which may in part be caused by gain-of-function mechanisms. HGPRT upregulation may be induced via a separate mechanism currently under investigation. ADA gene therapy can restore ADA expression and function, but further study is required to more robustly characterise the effect of ADA gene therapy on ALS iAstrocytes.