Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation

Neuronal hyperexcitability is a hallmark of seizures. It has been recently shown in rodent models of seizures that microglia, the brains resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interacts with human neurons to regulate hyperexcitability mediated by epilepsy-causing genetic mutation found in human patients remains unknown. The SCN2A genetic locus is responsible for encoding the voltage-gated sodium channel Nav1.2, recognized as one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation identified in patients with epilepsy leads to hyperexcitability in a hiPSC-derived cortical neuron model from a male donor. While microglia play an important role in the brain, these cells originate from a different lineage (yolk sac) and thus are not naturally present in hiPSCs-derived neuronal culture. To study how microglia respond to diseased neurons and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display altered morphology with increased branch length and enhanced calcium signal when co-cultured with neurons carrying the Nav1.2-L1342P mutation. Moreover, the presence of microglia significantly lowers the action potential firing of neurons carrying the mutation. Interestingly, we further demonstrated that the current density of sodium channels in neurons carrying the epilepsy-associated mutation was reduced in the presence of microglia. Taken together, our work reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation present in human neurons, highlighting the importance of neuron-microglia interactions in human pathophysiology. Significance StatementSeizure studies in mouse models have highlighted the role of microglia in modulating neuronal activity, particularly in the promotion or suppression of seizures. However, a gap persists in comprehending the influence of human microglia on intrinsically hyperexcitable neurons carrying epilepsy-associated pathogenic mutations. This research addresses this gap by investigating human microglia and their impact on neuronal functions. Our findings demonstrate that microglia exhibit dynamic morphological alterations and calcium fluctuations in the presence of neurons carrying an epilepsy-associated SCN2A mutation. Furthermore, microglia suppressed the excitability of diseased hyperexcitable neurons, suggesting a potential beneficial role. This study underscores the role of microglia in the regulation of abnormal neuronal activity, providing insights into therapeutic strategies for neurological conditions associated with hyperexcitability.