Heat stress-induced genomic instability in neural stem cells and its association with neuronal developmental deficits.

BACKGROUND: Maternal hyperthermia, an increasingly prevalent environmental stressor linked to global climate change, is epidemiologically associated with a higher risk of neurodevelopmental disorders, including autism and schizophrenia. However, the molecular mechanisms underlying this neurotoxicity, particularly those leading to long-term neurological deficits, remain poorly understood. This study tested the novel hypothesis that hyperthermia acts not only as an acute physiological stressor but also as a potent genotoxic agent, inducing persistent genomic alterations in neural progenitor cells.

METHODS: We established an in vitro model using mouse embryonic neural stem cells (eNSCs) differentiated into developing neurons. These cells were subjected to acute hyperthermic stress (40-43°C for 2 h). We employed a multi-omics approach to assess the consequences, including high-content neuromorphometrics, strand-specific RNA sequencing (RNA-seq) for transcriptomic and genomic variant analysis, and immunofluorescence for DNA double-strand breaks (DSBs) using the γH2AX marker.

RESULTS: Hyperthermia exposure at a clinically relevant temperature of 41°C induced a dose-dependent inhibition of neurite outgrowth and branching complexity. This morphological defect was underpinned by the induction of DNA DSBs, which triggered a robust p53-mediated DNA damage response, characterized by the upregulation of cell cycle arrest genes like Cdkn1a (p21). Critically, we uncovered evidence of lasting genomic damage. Hyperthermia-exposed neurons exhibited a significant increase in the frequency of single-nucleotide polymorphisms (SNPs), a lower transition-to-transversion (Ti/Tv) ratio indicative of genomic instability, and the formation of novel gene fusions. We identified a "heat stress signature" of unique mutations in key neurodevelopmental genes, including a missense variant in the inflammasome component Nlrp3 and structural rearrangements involving the axon guidance receptor Robo2 and the actin cytoskeleton regulator Cyth3.

CONCLUSION: Our findings reveal that hyperthermia impairs neuronal development through a "double-hit" mechanism. The "first hit" is an acute disruption of developmental programming via a p53-p21-mediated response to DNA damage. The "second hit" consists of permanent "genomic scars"-including SNPs and gene fusions-that can irreversibly alter the function of critical neurodevelopmental genes. By demonstrating that a transient environmental stressor can induce lasting genomic instability in neural progenitors, this study provides a compelling mechanistic framework linking maternal fever to the etiology of neurodevelopmental disorders and highlights a potentially crucial pathway for gene-environment interactions in brain development.