# Antisense Dipeptide Repeat Proteins Drive Widescale Purine Metabolism Aberration in C9orf72 Amyotrophic Lateral Sclerosis via ADA

## TL;DR

This study shows that dipeptide repeat proteins in C9orf72 ALS disrupt purine metabolism, contributing to disease progression.

## Contribution

The study identifies a novel mechanism by which DPRs impair purine metabolism through ADA loss in C9orf72 ALS.

## Key findings

- Loss of ADA and altered nucleotidase activity disrupts purine dNTP levels in C9-ALS astrocytes.
- Purine metabolism dysfunction is observed in patient CSF and white matter.
- Reducing DPR levels restores ADA activity in C9-ALS models.

## Abstract

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.