Neuronal Cell-Cycle Re-entry Defines Divergent Outcomes Through Replication-Dependent DNA Damage in ALS

Abstract Cell-cycle dysregulation has emerged as a shared mechanism of neuronal loss across neurodegenerative diseases (NDDs), including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease. In post-mitotic neurons, aberrant reactivation of cell-cycle signaling precedes degeneration, yet the upstream triggers and functional consequences of this process remain poorly defined. Nucleocytoplasmic transport (NCT) dysfunction, a hallmark of ALS and related disorders, disrupts the spatial distribution of key regulatory proteins and may contribute to maladaptive cell-cycle activation. Our recent evidence suggests that impaired nuclear import may initiate, rather than merely accompany, neuronal cell-cycle re-entry. Here, we show that cell-cycle activation in motor neurons distinguishes molecular subtypes and outcomes in ALS. We analyzed the AnswerALS transcriptomic cohort and identified a patient cluster characterized by robust upregulation of cyclins B and D. Clusters with lower levels of cell-cycle gene expression exhibited accelerated ALSFRS-R decline, whereas the highest cyclin-expressing cluster demonstrated comparatively improved functional trajectories over time. To test whether NCT disruption can mechanistically drive aberrant cell-cycle activation, we pharmacologically inhibited importin-β in human iPSC-derived spinal motor neurons. NCT disruption induced widespread proteomic mislocalization, including TDP-43 pathology, and triggered a transient wave of cell-cycle activity preceding neuronal death. Mechanistically, we identified DNA-replication initiation as a pathological event driving degeneration and demonstrated that selective inhibition of G1/S-associated CDK4/6 activity confers neuroprotection. Together, these findings link impaired nuclear import to maladaptive cell-cycle reactivation in neurons and highlight stage-specific engagement of the cell-cycle machinery as a determinant of neuronal vulnerability in ALS. Competing Interest Statement The authors have declared no competing interest. Data Availability Data used in the preparation of this article were obtained from the AnswerALS Data Portal (AALS-01184). For up-to-date information on the study, visit https://dataportal.answerals.org. All data generated or analyzed during this study are included in the accompanying Source Data file. This file contains processed proteomic and quantitative datasets, as well as underlying numerical values for all main and supplementary figures.