Epilepsy-Associated SCN2A-L1342P Mutation Drives Network Hyperexcitability and Widespread Transcriptomic Changes in Human Cortical Organoids.

Objective: pathogenic mutations, such as the recurrent heterozygous Nav1.2-L1342P, are monogenic causes of epilepsy. In this human-induced pluripotent stem cell model system, we aim to investigate the molecular and cellular mechanisms underlying the SCN2A-L1342P-associated pathology.

Methods: Using a human male iPSC reference line (KOLF) carrying the Nav1.2-L1342P mutation, we generated 3D cortical organoids for functional studies. Patch clamp and multi-electrode array (MEA) recordings, immunocytochemistry, and RNA sequencing were used to characterize the disease phenotypes.

Results: Nav1.2-L1342P organoid neurons displayed increased intrinsic excitability, and amplified excitatory post-synaptic currents, which are consistent with an increase in excitatory synapse formation revealed by PSD95/SYN1 immunostaining. Moreover, elevated network firing activity, as demonstrated by MEA, indicates a pronounced network hyperexcitability. Transcriptomic profiling of organoids carrying the Nav1.2-L1342P mutation further revealed significant alterations in synaptic, glutamatergic, and developmental pathways.

Significance: Our findings demonstrate that the Nav1.2-L1342P mutation drives a multifaceted disease phenotype, including network hyperexcitability and disruption of pathways related to neuronal and synaptic functions. These results advance our understanding of SCN2A-related Developmental and Epileptic Encephalopathy (DEE), laying a foundation for personalized interventions.

Key Points: : Human neurons carrying epilepsy-causing -L1342P display intrinsic hyperexcitability in cortical organoids. : Synaptic transmission is enhanced in organoids carrying the -L1324P mutation, consistent with the presence of elevated excitatory synapses, which leads to increased network hyperexcitability. Transcriptomics analysis reveals that the -L1342P mutation causes significant differential changes in forebrain developmental genes, synaptic and glutamatergic signaling pathways, and enhances cellular senescence and apoptosis.