Loss of schizophrenia risk gene XPO7 disrupts neuronal excitability and network regularity via altered Na+ channel dynamics in human neurons

Figures

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  Fig. 1. XPO7 LoF iNs exhibit distinct electrophysiological properties with elevated Na+ channel conductance compared to parental controls. A Representative traces of whole-cell voltage-gated Na+ and K+ currents in response to voltage steps recorded from iNs at day 56 using the automated patch-clamp instrument, SyncroPatch384. B Quantification of current-voltage (I/V) relationships for K+ (top) and Na+ (bottom) currents in iNs. C Quantification of maximum Na+ (left) and K+ (right) channel current densities in XPO7+/+ (n = 97), XPO7+/- (n = 79), and XPO7-/- (n = 93) neurons. D Summary data showing a significant hyperpolarizing shift in Na+ channel activation in XPO7 LoF (XPO7+/- and XPO7-/-) neurons compared to parental controls. E Representative traces of whole-cell voltage-gated Na+ currents in response to a steady-state inactivation protocol used to measure channel inactivation. F Quantification of maximum Na+ inactivation current densities in XPO7+/+ (n = 54), XPO7+/- (n = 49), and XPO7-/- (n = 47) neurons. G Summary data showing a significant hyperpolarizing shift in Na+ channel inactivation in XPO7-/- neurons compared to parental controls. H Representative traces of Na+ currents in…

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  Fig. 2. XPO7 LoF alters neuronal excitability by disrupting action potential regularities and dynamics. A Schematic workflow illustrating HD-MEA data processing, including spike sorting, extraction of activity and waveform features, and analysis of action potential propagation dynamics. B Principal component analysis of excitability features from control and XPO7 LoF neurons across five independent differentiations. Genotype-specific clustering demonstrates strong reproducibility across batches, while clear separation between genotypes indicates robust discrimination of distinct excitability profiles. C Heatmap illustrating key electrophysiological measurements from HD-MEA assessments. Color scale indicates the relative change of each measurement, as annotated on the left. Outlined cells represent features significantly altered in XPO7+/- or XPO7-/- neurons compared to parental controls (XPO7+/+). Asterisks denote levels of statistical significance. D, E Quantification of single-unit analysis results in XPO7+/+ (n = 23/5), XPO7+/- (n = 30/5), and XPO7-/- neurons (n = 31/5), where n indicates the number of MEA cultures per genotype derived from 5 independent neuronal differentiation…

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  Fig. 3. XPO7 LoF alters transcriptomic profiles across neuronal maturation and upregulates pathways related to Na+ channel activity. A Principal component analysis of bulk RNA-seq data from control neurons at three different time points. B Heatmap showing the top 100 DEGs after hierarchical clustering of gene expression profiles in control neurons across three time points during neuronal maturation. Genes were clustered using fuzzy c-means clustering based on their expression trajectories across the three differentiation stages. C Gene Ontology (GO) enrichment analysis of RNA-seq data from control neurons. The size of each bubble indicates the number of gene counts. Bubbles filled with a gradient color represent pathways with significant enrichment (padj < 0.05), whereas bubbles filled with grey indicate pathways with nonsignificant enrichment (padj ≥ 0.05). D Principal component analysis of bulk RNA-seq data from XPO7 LoF neurons at day 56. E Gene set enrichment analysis of bulk RNA-seq data from XPO7 LoF neurons at day 56, using gene lists from the Molecular Signatures Database (MSigDB). The size of each bubble indicates the significance of pathway enrichment (-log10 converted).…

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  Fig. 4. XPO7 LoF reduces dendritic complexity, decreases synaptic density, and alters Nav channel subcellular localization. A Representative immunofluorescent images of day 42 XPO7+/+, XPO7+/-, XPO7-/- neurons stained with the dendritic marker MAP2. Black dashed boxes in the upper images indicate the regions that are cropped and magnified in the bottom images. Scale bar, 10 μm. B Morphological analysis of XPO7+/+ (n = 101), XPO7+/- (n = 99), and XPO7-/- (n = 91) neuronal cultures by quantifying the number of primary dendrites, the number of dendritic segments per primary dendrite, and the maximum dendrite length. Distribution plot showing Sholl intersections relative to distance from the soma. C Representative immunofluorescent images of day 42 XPO7+/+, XPO7+/-, and XPO7-/- neurons stained with presynaptic (Synapsin I/II), postsynaptic (PSD-95), and dendritic (MAP2) markers. Scale bar, 10 μm. D Quantification of synapse density in XPO7+/+ (n = 41), XPO7+/- (n = 26), and XPO7-/- (n = 36) neuronal cultures by calculating the total number of colocalized puncta along each dendritic segment, normalized to segment length. E Representative immunofluorescent images of day 42 XPO7+/+, XPO7+/-,…

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  Fig. 5. XPO7 LoF impairs synaptic composition and reduces Na+ channel abundance in the synaptic proteome. A GSEA of cytoplasmic proteomic data from XPO7 LoF neurons at day 42, using gene lists from MSigDB. The size of each bubble indicates the significance of pathway enrichment (-log10 of padj). Bubbles outlined in black indicate significantly enriched pathways (padj < 0.05). Bubbles are filled with a gradient color representing the GSEA normalized enrichment score of each pathway. B and C Volcano plots showing all synaptic proteins annotated by SynGO. Top significant DEPs are labeled with the gene symbol of the corresponding protein. The horizontal dashed line indicates padj = 0.05, and the vertical dashed lines indicate log2fold-change thresholds of 0.1 and -0.1. D Correlation of protein log2fold-changes between XPO7+/- and XPO7-/- cytoplasmic proteomes. Top significant DEPs are labeled with the gene symbol of the corresponding protein. E Heatmap showing log2fold-changes in the cytoplasmic proteomes of XPO7+/- and XPO7-/- neurons. F Cytoplasmic extracts were analyzed by Western blot to assess top DEPs identified by cytoplasmic proteomics, including NPTX2, PSD-95, Synaptophysin,…

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  Fig. 6. XPO7 LoF disrupts the temporal precision and regularity of neuronal network activity by altering firing rate dynamics and spike waveform properties. A Representative 1-minute raster plots of electrophysiological activity from XPO7+/+, XPO7+/-, and XPO7-/- neuronal networks at multiple developmental time points. A multielectrode array system with 9 electrodes per array and fixed 200 μm electrode spacing was used to monitor network activity across different stages of neural differentiation and maturation. B Network synchrony across developmental time. Each point represents the mean network burst rate (number of network bursts per minute) for a single neuronal network (one MEA chip) at the indicated time point in XPO7+/+ (n = 48/8), XPO7+/- (n = 69/8), and XPO7-/- (n = 60/8) neuronal cultures, where n indicates the number of MEA networks per genotype derived from 8 independent neuronal differentiation batches. Neuronal activity for each included network was recorded over a 59-day period, with two recordings performed per week. C Summary of the half-maximal network burst rate (NBR-50) values derived from sigmoid curve fitting of network burst rate trajectories. D Representative…