ALS-Linked FUS and SOD1 Mutations Elevate MCM2 in Human Motor Neurons
BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by progressive motor neuron loss. In typically post-mitotic neurons, abnormal reactivation of cell cycle regulators and DNA replication licensing factors is observed in ALS pathogenesis. Emerging evidence links components of the minichromosome maintenance (MCM) complex, notably MCM2, to replication stress and genomic instability, implying a mechanistic role in ALS pathogenesis. ObjectiveTo determine whether ALS-associated mutations in FUS and SOD1 influence MCM2 expression and localisation in human induced pluripotent stem cell (hiPSC)-derived spinal motor neurons (MNs). MethodsWe differentiated isogenic hiPSC lines carrying FUS P525L-GFP and R495QfsX527, a SOD1 mutant line, and matched wild-type controls into spinal MNs ([≥]28 days in vitro). QRT-PCR quantified MCM2 mRNA levels ({Delta}{Delta}Ct method; n=3 biological replicates, technical triplicates). Protein expression was assessed by Western blot densitometry (n=3). Subcellular distribution of MCM2 in FUS mutants was evaluated by immunofluorescence (pilot, N=1; [≥]50 cells quantified). ResultsFUS P525L MNs exhibited a modest, non-significant increase in MCM2 mRNA (1.3-fold vs. WT; p=0.1319) but a significant 1.8-fold elevation in MCM2 protein levels (p=0.034). The R495QfsX527 line showed a comparable trend at the transcript level (1.2-fold; p > 0.05) and a 1.6-fold increase in protein (p = 0.041). SOD1 mutant MNs demonstrated a pronounced 2.3-fold MCM2 protein upregulation (p=0.008). Immunofluorescence in FUS mutant MNs revealed no significant nuclear-to-cytoplasmic shift in MCM2 localisation, indicating that elevated MCM2 levels are not driven by subcellular mislocalization. ConclusionALS-linked FUS and SOD1 mutations upregulate MCM2 protein in human spinal MNs, suggesting post-transcriptional or stability-driven regulation. The absence of relocalisation in FUS mutants shows that this impact is caused by overexpression rather than mislocalisation. MCM2 may be a biomarker of disease-associated replication stress. Future studies will explore whether MCM2 overexpression exacerbates DNA damage or serves as a compensatory response, clarifying its role in ALS pathogenesis.