Highlights
- C9orf72 loss-of-function in vivo triggers cerebellar atrophy and depletion of Purkinje and Granule cells, preceding the onset of overt motor defects.
- Single-cell transcriptomics identifies the downregulation of paics, a gene essential for purine biosynthesis, as a primary driver of neuronal loss in the cerebellum.
- PAICS deficiency is validated in human post-mortem cerebellar tissue and C9orf72 iPSC-derived motor neurons, suggesting a conserved mechanism across species.
- Knockout of paics leads to catastrophic DNA damage and repair (DDR) defects; conversely, restoring PAICS expression rescues the neurodegenerative phenotype and motor function.
Background
The hexanucleotide (GGGGCC) repeat expansion within the C9orf72 gene is recognized as the most prevalent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While research has historically focused on gain-of-function mechanisms—specifically the formation of toxic dipeptide repeat proteins (DPRs) and RNA foci—the contribution of C9orf72 haploinsufficiency and subsequent loss-of-function (LoF) remains a subject of intense investigation. C9orf72 is highly expressed in the cerebellum, an area of the brain increasingly implicated in the non-motor and cognitive manifestations of ALS/FTD. Despite this, the precise molecular pathways through which C9orf72 deficiency leads to specific neuronal loss in the cerebellum have remained elusive. Recent evidence, spearheaded by the work of Singh et al. (2026), suggests that the disruption of purine metabolism, specifically via the enzyme PAICS, is a critical mediator of genomic instability and cerebellar degeneration in this context.
Key Content
The Cerebellum as an Early Pathological Site in C9orf72-ALS
While ALS is traditionally viewed as a disease of the upper and lower motor neurons, clinical and pathological studies have consistently shown cerebellar involvement in C9orf72 carriers. The cerebellum’s role extends beyond motor coordination into cognitive and affective domains, which are often impaired in FTD. Singh et al. (2026) utilized a zebrafish model to demonstrate that C9orf72 loss-of-function leads to significant cerebellar atrophy. Specifically, there is a marked loss of GABAergic interneurons and a depletion of both Purkinje and Granule cells. Crucially, these cerebellar anomalies were observed to precede the development of motor defects, suggesting that cerebellar dysfunction may be one of the earliest stages of the C9orf72 pathological cascade.
Transcriptomic Discovery of PAICS Downregulation
To identify the molecular drivers of this cerebellar degeneration, researchers performed single-cell transcriptomics on the brains of C9orf72-deficient zebrafish. This high-resolution analysis revealed a significant downregulation of paics (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase) specifically within Purkinje cells. PAICS is a bifunctional enzyme that catalyzes the sixth and seventh steps of the de novo purine biosynthetic pathway. This metabolic pathway is essential for providing the precursors required for DNA and RNA synthesis, as well as for cellular energy (ATP/GTP) and signaling.
Mechanistic Insights: Purine Biosynthesis and DNA Damage
The link between PAICS and neurodegeneration was further elucidated through paics knockout experiments in zebrafish. The depletion of paics recapitulated the C9orf72 LoF phenotype, including cerebellar neuronal loss, neuromuscular junction (NMJ) disruption, and motor impairment. Mechanistically, PAICS deficiency resulted in widespread DNA damage and repair (DDR) defects. The researchers noted a suppression of key DNA repair pathways, leading to the accumulation of double-strand breaks and genomic instability. This suggests that the metabolic stress caused by impaired purine synthesis directly compromises the cell’s ability to maintain genomic integrity, particularly in highly metabolic cells like Purkinje neurons.
Translational Validation in Human Models
The relevance of these findings to human disease was confirmed through the examination of human post-mortem cerebellar sections and induced pluripotent stem cell (iPSC)-derived motor neurons. A consistent reduction in PAICS expression was observed in samples from patients with C9orf72-associated ALS and, notably, in some sporadic ALS cases as well. This indicates that the PAICS-DDR axis may be a broader feature of ALS pathology, extending beyond the C9orf72 mutation. The study also demonstrated that restoring paics expression in C9orf72-deficient zebrafish was sufficient to resolve DNA damage and preserve cerebellar architecture, providing a strong proof-of-concept for therapeutic intervention.
Expert Commentary
The discovery of the PAICS-mediated pathway provides a compelling mechanistic bridge between metabolic dysfunction and DNA damage in ALS. For years, the field has debated whether C9orf72 pathology is driven primarily by toxic gain-of-function or loss-of-function. These findings reinforce the importance of the LoF component, showing that reduced C9orf72 levels directly impair metabolic enzymes vital for neuronal survival.
From a clinical perspective, the observation that cerebellar changes precede motor symptoms is particularly significant. It suggests that cerebellar imaging or biomarkers related to purine metabolism could potentially serve as early diagnostic tools. Furthermore, the ability of PAICS restoration to rescue the phenotype in vivo highlights a metabolic therapeutic window that has been largely overlooked in neurodegeneration research. However, a major challenge remains: how to selectively upregulate purine biosynthetic enzymes in the CNS without inducing systemic metabolic imbalances or promoting oncogenic pathways, as PAICS is also known to be overexpressed in various cancers.
Conclusion
In summary, the identification of PAICS as a mediator of DNA damage and cerebellar loss represents a significant advance in our understanding of C9orf72-associated ALS and FTD. By linking the C9orf72 protein to the regulation of de novo purine biosynthesis, this research underscores the vulnerability of specific cerebellar populations to metabolic and genomic stress. Future studies should focus on the upstream regulatory mechanisms of PAICS by C9orf72 and explore small-molecule or gene therapy approaches to stabilize purine metabolism in at-risk neurons. Understanding the full scope of the PAICS-DDR axis may ultimately lead to disease-modifying therapies that can halt the progression of neurodegeneration long before the first motor symptoms appear.
References
- Singh J, Lescouzères L, Zaouter C, Chaineau M, Haghi G, Durcan TM, Patten SA. PAICS mediates DNA damage and cerebellar neuronal loss in C9orf72 amyotrophic lateral sclerosis. Brain. 2026. PMID: 41810938.
- DeJesus-Hernandez M, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p21-linked ALS-FTD. Neuron. 2011;72(2):245-56. PMID: 21944778.
- Renton AE, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72(2):257-68. PMID: 21944779.
- Farg MA, et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Hum Mol Genet. 2014;23(13):3579-95. PMID: 24549040.
