# Aberrant CDK4/6-driven cell-cycle reentry drives neuronal loss and defines a therapeutic target in C9orf72 ALS/FTD

## TL;DR

This study identifies CDK4/6-driven cell-cycle reentry as a key mechanism in C9orf72-related ALS/FTD and suggests CDK4/6 inhibitors like palbociclib as a potential treatment.

## Contribution

The study reveals that arginine-rich dipeptide repeat proteins drive CDK4/6-mediated cell-cycle dysregulation in C9orf72 ALS/FTD neurons.

## Key findings

- C9orf72 neurons exhibit age-dependent cell-cycle reentry and increased S-phase entry.
- CDK4/6 inhibition with palbociclib reduces neuronal death and restores synaptic proteins.
- Single-nucleus RNA sequencing confirms cell-cycle activation in excitatory neurons from C9orf72 patients.

## Abstract

The C9orf72 hexanucleotide repeat expansion (G4C2) is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet targeted therapies remain unavailable. Here, we show that induced pluripotent stem cell (iPSC)-derived post-mitotic neurons from C9orf72 carriers exhibit age-dependent cell-cycle reentry, increased S-phase entry, and elevated cyclin and CDK expression. Mechanistically, arginine-containing dipeptide repeat proteins (poly-GR and poly-PR) translated from G4C2 repeats drive this aberrant activation through stimulation of the CDK4/6 pathway, whereas poly-GP and C9orf72 loss-of-function show no effect. Importantly, the FDA-approved CDK4/6 inhibitor palbociclib normalizes cell-cycle progression, reduces S-phase entry, decreases motor neuron death, and restores synaptic proteins PSD95 and synapsin-1. Single-nucleus RNA sequencing from C9orf72 patient cortex reveals cell-cycle activation within excitatory neuron subclusters and alterations in DNA repair and cell-cycle regulation pathways, supporting our in vitro findings. These findings establish cell-cycle dysregulation as a central pathogenic mechanism in C9orf72 ALS/FTD and highlight CDK4/6 signaling as a promising therapeutic target.

•C9orf72 neurons show age-dependent cell-cycle reentry and S-phase activation•Arginine-rich DPRs (poly-GR/PR) drive CDK4/6-mediated cell-cycle dysregulation•CDK4/6 inhibition with palbociclib restores neuronal survival and synaptic markers•snRNA-seq of C9orf72 cortex reveals excitatory neuron-specific cell-cycle activation

C9orf72 neurons show age-dependent cell-cycle reentry and S-phase activation

Arginine-rich DPRs (poly-GR/PR) drive CDK4/6-mediated cell-cycle dysregulation

CDK4/6 inhibition with palbociclib restores neuronal survival and synaptic markers

snRNA-seq of C9orf72 cortex reveals excitatory neuron-specific cell-cycle activation

Health sciences; Pharmaceutical science; Neuroscience; Clinical neuroscience; Cellular neuroscience