These 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, which advances understanding of SCN2A-related developmental and epileptic encephalopathy (DEE).
Abstract
Objective
SCN2A pathogenic mutations, such as the recurrent heterozygous Nav1.2-L1342P, are monogenic causes of epilepsy. In this human-induced pluripotent stem cell-derived model system, we aim to investigate the molecular and cellular mechanisms underlying SCN2A-L1342P-associated pathology.
Methods
Using a human male induced pluripotent stem cell (iPSC) reference line (KOLF) carrying the Nav1.2-L1342P mutation, we generated three-dimensional (3D) cortical organoids for functional studies. Patch-clamp, 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 SYN1/PSD95 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, developmental, and senescence/apoptotic 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.
BACKGROUND
Variants in STX1B/syntaxin-1B are linked to a spectrum of fever-associated epilepsy syndromes. While studies in murine models have provided mechanistic insights, their relevance to human disease in a heterozygous context may be limited.
METHODS
We investigated two pathogenic STX1B variants using isolated single neurons and neuronal network cultures derived from patient-specific induced pluripotent stem cells. These carried either a de novo p.G226R variant, associated with severe developmental epilepsy, or an InDel variant (p.K45delinsRCMIE/p.L46M) linked to a transient familial seizure syndrome. Synaptic function and network excitability were assessed using patch-clamp and multi-electrode array recordings, alongside morphological and transcriptomic profiling.
FINDINGS
G226R exhibited both gain- and loss-of-function characteristics, with increased miniature excitatory postsynaptic current frequency in networks but not in autapses, and synaptic failure during sustained high-frequency stimulation. For the InDel variant, the predicted loss-of-function phenotype based on reduced syntaxin-1B levels was not detectable at the single-cell level, likely masked by compensatory synaptic upregulation. At the network level, however, both variants were associated with neuronal hyperexcitability, characterised by more frequent and prolonged bursting activity, with a much stronger phenotype in G226R-containing networks. Transcriptomic profiling revealed a differential dysregulation of synaptic and other neuronal genes.
INTERPRETATION
The divergence between morphological, electrophysiological and transcriptomic findings suggests that compensatory mechanisms may contribute to network hyperexcitability. Initially engaged to maintain homoeostasis, they may ultimately contribute to a pathological network state. The graded severity of network alterations across STX1B variants correlates with the clinical phenotypes.
FUNDING
BMBF (Treat ION-01GM2210A, SNAREopathies-01EW1809A), 2023 FEBS Summer Fellowship, Fortüne programme (2610-0-0), EKFS college precise.net, Open Access Publishing Fund of University of Tübingen.
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