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

Olivero-Acosta, M. I.; Robinson, M.; Que, Z.; Zhang, Z.; Harlow, H. E.; Shankar, V.; Hong, S.; Wang, M.; Otterbacher, C. M.; Kadono, H.; Halurkar, M.; Kothandaraman, H.; Lanman, N.; Nguyen, T.; Wettschurack, K.; Zirkle, B.; Yunis, L.; Cui, N.; Chen, X.; Zhang, J.; Wu, J.; Skarnes, W. C.; Yuan, C.; Guo, F.; Yang, Y.

doi:10.1101/2025.08.18.670956

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bioRxiv · 10.1101/2025.08.18.670956

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

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Abstract

ObjectiveSCN2A 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. MethodsUsing 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. ResultsNav1.2-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. SignificanceOur 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 PointsO_LIKey Point 1: Human neurons carrying epilepsy-causing SCN2A-L1342P display intrinsic hyperexcitability in cortical organoids. C_LIO_LIKey Point 2: Synaptic transmission is enhanced in organoids carrying the SCN2A-L1324P mutation, consistent with the presence of elevated excitatory synapses, which leads to increased network hyperexcitability. C_LIO_LIKey Point 3: Transcriptomics analysis reveals that the SCN2A-L1342P mutation causes significant differential changes in forebrain developmental genes, synaptic and glutamatergic signaling pathways, and enhances cellular senescence and apoptosis. C_LI

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Olivero-Acosta, M. I., Robinson, M., Que, Z., Zhang, Z., Harlow, H. E., Shankar, V., Hong, S., Wang, M., Otterbacher, C. M., Kadono, H., Halurkar, M., Kothandaraman, H., Lanman, N., Nguyen, T., Wettschurack, K., Zirkle, B., Yunis, L., Cui, N., Chen, X., Zhang, J., Wu, J., Skarnes, W. C., Yuan, C., Guo, F., Yang, Y.. 2025-08-19. Epilepsy-Associated SCN2A-L1342P Mutation Drives Network Hyperexcitability and Widespread Transcriptomic Changes in Human Cortical Organoids. https://doi.org/10.1101/2025.08.18.670956

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