Cell cycle dysregulation contributes to neurodegeneration in human neurons and defines a druggable vulnerability in C9orf72 ALS/FTD

The C9orf72 hexanucleotide repeat expansion GGGGCC (G4C2) cause the most common genetic forms of ALS and frontotemporal dementia, affecting thousands of patients worldwide with uniformly fatal outcomes. C9orf72 ALS/FTD patients lack targeted treatments because druggable molecular vulnerabilities remain unidentified. Using iPSC-derived motor neurons from C9orf72 carriers and age-matched controls, we performed comprehensive cell cycle analysis, drug screening, and single-nucleus RNA sequencing validation in human brain tissue. C9orf72 neurons exhibit age-dependent cell cycle reentry with increased S-phase cells, elevated cyclin and CDK expression, and aberrant cell cycle gene signatures confirmed in patient brain excitatory neurons. Mechanistically, arginine-containing dipeptide repeat proteins (poly-GR, poly-PR) drive this cell cycle activation through CDK4/6 pathway stimulation, while C9orf72 loss-of-function alone shows no effect. Critically, the FDA-approved CDK4/6 inhibitor palbociclib normalizes cell cycle progression, reduces S-phase entry, and rescues neuronal survival with significant reduction in motor neuron death. Single-nucleus RNA-sequencing analyses from C9orf72 patient cortex reveals cell cycle-activated neuronal subclusters. Copy number variation, gene ontology and pathways analyses revealed alterations in DNA repair pathways, cell cycle regulation and cell cycle transition, validating our in vitro findings. These results identify cell cycle dysregulation as a therapeutic target in C9orf72 ALS/FTD with clinical translation potential using existing therapeutics.