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

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.

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.

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.

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.