Journal: bioRxiv
Article Title: Epilepsy-Associated SCN2A-L1342P Mutation Drives Network Hyperexcitability and Widespread Transcriptomic Changes in Human Cortical Organoids
doi: 10.1101/2025.08.18.670956
Figure Lengend Snippet: (A) Bulk RNA-seq experimental conditions. Control organoids (n = 6 organoids, three differentiations) and L1342P organoids (n = 6 organoids, two differentiations) were sequenced using the AVITI system. (B) Hierarchical clustering of cortical organoid sample replicates. Darker blue indicates higher similarity. (C) Principal Component Analysis (PCA) plot illustrating the clustering of replicates based on their variance in two dimensions. (D) DEG Heatmap showing the top 50 down (blue) and upregulated (red) genes across conditions. (E) Volcano plot highlighting significantly differentially expressed genes (DEGs, FDR < 0.05) in Nav1.2-L1342P hiPSC-derived cortical organoids compared to controls. Notable downregulated genes include SCN2A and potassium channel-related genes. In contrast, upregulated genes include: DLX2 (Distal-Less Homeobox 2), H2AX (histone family member X), PAX6 (Paired Box 6), and ASCL1 (Achaete-scute family bHLH transcription factor 1), which are important in neurodevelopment, neuronal function, and cellular function. (F) Gene Ontology (GO) analysis of biological processes reveals changes in synapse organization, microtubule-cytoskeleton arrangements, glutamate receptor signaling, and forebrain development (top). (G) GO molecular function analysis reveals enrichment in ion channel activity and alterations in the glutamatergic pathway. (H) GO cellular component identifies synaptic pathway alterations in the Nav1.2-L1342P cortical organoids. (I) Network analysis reveals numerous globally differentially expressed genes (DEGs) involved in synaptic signal regulation (magenta), calcium transport (dark green), synapse organization (orange), and forebrain development (lime). The colored edges connecting genes represent functional relationships or interactions within each cluster, highlighting how these DEGs collectively contribute to altered neuronal physiology. (J) REACTOME pathway analysis reveals disruptions in cell division, metabolism, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor trafficking, and neuronal synapses. (K) KEGG pathway enrichment analysis reveals alterations in MAPK and cAMP signaling pathways, as well as glutamatergic synapses. The circle size is represented by the number of DEGs highly ranked by p.adjust values.
Article Snippet: To this end, we extracted total RNA from the cortical organoids using the NucleoSpin miRNA Kit for the Isolation of Small and large RNA (Macherey-Nagel, Catalog No. 740971.50) according to the manufacturer’s instructions.
Techniques: RNA Sequencing, Control, Derivative Assay, Cell Function Assay, Activity Assay, Functional Assay, Protein-Protein interactions
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