Neuronal impact of patient-specific aberrant NRXN1α splicing

Abstract

NRXN1 undergoes extensive alternative splicing, and non-recurrent heterozygous deletions in NRXN1 are strongly associated with neuropsychiatric disorders. We establish that human induced pluripotent stem cell (hiPSC)-derived neurons well represent the diversity of NRXN1α alternative splicing observed in the human brain, cataloguing 123 high-confidence in-frame human NRXN1α isoforms. Patient-derived NRXN1^+/- hiPSC-neurons show a greater than twofold reduction in half of the wild-type NRXN1α isoforms and express dozens of novel isoforms from the mutant allele. Reduced neuronal activity in patient-derived NRXN1^+/- hiPSC-neurons is ameliorated by overexpression of individual control isoforms in a genotype-dependent manner, whereas individual mutant isoforms decrease neuronal activity levels in control hiPSC-neurons. In a genotype-dependent manner, the phenotypic impact of patient-specific NRXN1^+/- mutations can occur through a reduction in wild-type NRXN1α isoform levels as well as the presence of mutant NRXN1α isoforms.

Extended Data Fig. 1. Validation of deletions in hiPSC cohort and whole transcriptome RNA-seq analysis

a, Schematic showing the structure of the NRXN1 gene and location of 5’-((blue) and 3’-deletions (red). b,c, Mean and s.e. of Taqman CNV assay confirming 3’-CNV in fibroblasts from one 3’-NRXN1^+/− case (b) and 5’-CNV in fibroblasts from both 5’-NRXN1^+/− cases (c); two replicates per sample per probe. d, PCR of cDNA across exons encompassed by the 3’-deletion in controls and 3’-NRXN1^+/− hiPSC-NPCs, two independent validations. e, Mean and s.e. of the log₂FoldChange by qPCR across the novel junction (exon 20–24) created by 3’-deletion across 2 controls and 2 3’-NRXN1^+/− hiPSC-NPCs compared using a two-sided t-test. f, Sanger sequencing result from a TOPO cloned NRXN1α isoform from 3’-NRXN1^+/− hiPSC-neurons (n = 1) confirming presence of novel exon junction (exon 20–24). g, Confirmation of the sex of each sample. h, PCA plot of combined hiPSC-NPC (19 samples, 8 donors) and hiPSC-neuron (18 samples, 8 donors) RNA-seq dataset showing separation by cell type on PC1. i,j, Volcano plot showing differentially expressed genes within hiPSC-NPCs (i) and hiPSC-neurons (j) individually. k, FPKM of NRXN1 expression across fetal PFC (1), adult dlPFC (3) and control hiPSC-neurons (3). l, Circle plot showing hierarchical clustering of samples by cell type and by donor. m,n Pearson correlation of gene expression within and between donors in hiPSC-NPCs (19 samples, 8 donors) (m) and hiPSC-neurons (18 samples, 8 donors) (n) by one-sided Wilcoxon test. o, Gene set enrichments for genes correlated with NRXN1 spicing in the Common Mind Consortium dlPFC dataset.

Extended Data Fig. 2. Cell type composition

a, Heatmap showing log₂FPKM values of sub-type marker genes in hiPSC-neurons across genotypes. b, Cell type composition scores obtained using Cibersort in hiPSC-neurons (9 controls, 4 donors; 9 cases, 4 donors), by diagnosis. c, Images of GABA immunostaining overlaid with MAP2. d, Mean percent of GABA⁺ cells from control (8 images, 2 coverslips), 5’-NRXN1^+/− (8 images, 2 coverslips) and 3’-NRXN1^+/− (11 images, 3 coverslips). Error bars are s.e. e, Representative images of SYN1 immunostaining alone and overlaid with MAP2 immunostaining and DAPI. f, Mean intensity of SYN1⁺ puncta normalized to MAP2 intensity (3 images per coverslip, 8 coverslips per donor, 2 donors per genotype). Error bars are s.e.

Extended Data Fig. 3. Pipeline Schematic and quality control of Iso-seq data

a, Schematic of the sample preparation for the hybrid sequencing approach. b, Schematic of the computational pipeline developed for the hybrid sequencing approach. c, UCSC genome browser view of whole transcriptome Iso-seq data across the NRXN1 locus from five hiPSC-neuron samples. d,e, Representative bioanalyzer traces from Iso-seq library prep passing QC (d) compared to library prep failed QC (e). f, Targeted short-read sequencing counts per million for 3’-NRXN1^+/− specific junction site showing 3’-NRXN1^+/− hiPSC-neurons passing QC in green and failing QC in red. g,h, Pearson’s correlation of junction site expression from targeted long read vs. targeted short read data showing one of the samples passing QC (g) and one sample failing QC (h).

Extended Data Fig. 4. Comparison of long and short read data for quantification and threshold testing

a, Correlation of NRXN1α junction expression from long read and short read sequencing across control (control 1 n = 39, control 2 n = 37) and 3’-NRXN1^+/− (3’-NRXN1^+/− 1 n = 36, 3’-NRXN1^+/− 2 n = 45) hiPSC-neuron samples. Red triangles represent canonical junctions while black represent non-canonical. b, Correlation of NRXN1α isoform expression from long read quantification and short read quantification across control (Control 1 n = 90, Control 2 n = 88) and 3’-NRXN1^+/− (3’-NRXN1^+/− 1 n = 89, 3’-NRXN1^+/− 2 n = 96) hiPSC-neuron samples. Colored triangles represent in-frame isoforms, predicted to be translated (red), untranslated (black) and TOPO cloned (green). c, Correlation of mouse and human NRXN1α isoform expression and corresponding Venn diagrams for the number of isoforms across expression thresholds (≥2 n = 112, ≥3 n = 88, ≥4 n = 75, ≥5 n = 63, ≥6 n = 60, ≥7 n = 57, ≥8 n = 54, ≥9 n = 52, ≥10 n = 50).

Extended Data Fig. 5. Cell type specific NRXN1 expression

a,b, PCA of isogenic samples differentiated into three hiPSC-neuronal cell types colored by cell type (ASCL1/DLX2 (5), hiPSC-neuron (6), and NGN2 (6) (a) or by NRXN1 genotype (control (8), case (9) from 3 donors each) (b). c, Representative confocal images from 2 independent differentiations of control and NRXN1^+/− ASCL1/DLX2-GABAergic hiPSC-neurons. d, Expression of glutamatergic, GABAergic and pan-neuronal marker genes across NRXN1^+/− (9) and control (8) NGN2-glutamatergic and ASCL1/DLX2-GABAergic hiPSC-neurons. Boxplot shows median and IQR. e, Sum of all NRXN1 transcripts expressed across control (2 donors), 3’-NRXN1^+/− (2 donors) and 5’-NRXN1^+/− (2 donors) in NGN2-glutamatergic and ASCL1/DLX2-GABAergic hiPSC-neurons. f,g, NRXN1α (f) and NRXN1β (g) isoform usage expressed across control, 3’-NRXN1^+/− and 5’-NRXN1^+/− donors in NGN2-glutamatergic and ASCL1/DLX2-GABAergic hiPSC-neurons. Boxplot displays median and range with P < 0.01 indicated by “**” from two way ANOVA with Holm-Sidak’s test.

Extended Data Fig. 6. Examination of NRXN1α canonical splice sites and total NRXN1 isoform expression

a, Pearson’s correlation of NRXN1α isoform expression across control and 3’-NRXN1^+/− hiPSC-neurons (n = 99 isoforms, r computed by t-statistics). b,c, Bar plot of the total read count for each NRXN1α exon along with the fraction that each NRXN1α junction is included in control hiPSC-neurons (b) and 3’-NRXN1^+/− hiPSC-neurons. Red circle indicates the novel junction created by the 3’-NRXN1^+/− deletion (c). d, Schematic of the experimental design to test activity induced regulation at NRXN1α canonical splice sites. e, Fold change of canonical splice site exclusion in controls (gray) and a 3’-NRXN1^+/− hiPSC-neuron (red) plus KCl compared to PBS control (dotted line). f, Bar plot showing fold change of SS4 in KCl treated control and 3’-NRXN1^+/- hiPSC-neurons (compared to PBS). g, Schematic of the experimental design to test developmental regulation at NRXN1α canonical splice sites. h, Fold change of canonical splice sites in control hiPSC-neurons at 2-weeks (light gray), 4-weeks (gray) and 6-weeks (dark gray) post-differentiation compared to NPCs (dotted line). i, Specific examination of developmental exclusion of SS4. Error bars are s.e. j-m, Expression of levels of all NRXN1 isoforms (j), NRXN1α (k), NRXN1β (l), NRXN1γ (m) across NRXN1 genotypes (8 control, 3 donors; 5 3’-NRXN1^+/−, 2 donors; 5 5’-NRXN1^+/−, 2 donors) in hiPSC-neurons. Violin plot displays density and range with P < 0.05 indicated by “*” from Wilcoxon Signed Rank Test. n, Pearson’s correlation of all NRXN1 isoforms (18 samples, 6 donors) with NRXN1α, NRXN1β and NRXN1γ (r values calculated using t-statistics).

Extended Data Fig. 7. Single cell expression of synaptic genes

a, Violin plot displaying density of the expression of NRXN and multiple synaptic marker genes in single cells across control (2 donors) and NRXN1^+/− (3 donors) hiPSC-neurons. b, Violin plot displaying density and range of the expression of multiple synaptic genes identified as marker genes in immature neuronal clusters from scRNA-seq data across control (2 donors) and NRXN1^+/− (3 donors) hiPSC-neurons.

Extended Data Fig. 8. Investigation of hiPSC-neuron morphology, cellular signaling, and NRXN1 overexpression

a, Strategy to label individual hiPSC-neurons. b-g, Mean neurite number across genotypes (control 71 neurons, 2 donors; 3’-NRXN1^+/− 72 neurons, 2 donors; 5’-NRXN1^+/− 50 neurons 2 donors) “****” indicates P < 0.00001 and “***”indicates P < 0.0001 by one-way ANOVA with Holm-Sidak’s test; (b) or by coverslip (2 donors, 12 coverslips, 3 regions each) (c,d) mean neurite length across genotypes (control 71 neurons, 2 donors; 3’-NRXN1^+/− 72 neurons, 2 donors; 5’-NRXN1^+/- 50 neurons, 2 donors); (e) or by coverslip (2 donors, 12 coverslips, 3 regions each) (f,g). Two donors per genotype indicated by different shading within each plot. h, Differentially active kinases (3’- NRXN1^+/− hiPSC-neurons: 6 samples, 2 donors; controls: 5 samples, 2 donors). i, Volcano plot of –log₁₀(P-value) and log₂(FoldChange) from linear model of RNA-seq (3’-NRXN1^+/− hiPSC-neurons: 5 samples, 2 donors; controls 6 samples, 2 donors); DE kinase associated genes labeled. j, Violin plot of median and quartiles of RPKM for kinase hits with largest fold-change in RNA-seq (3’-NRXN1^+/− hiPSC-neurons: 5 samples, 2 donors; controls: 6 samples, 2 donors). k, Differentially active kinases in (5’- NRXN1^+/− hiPSC-neurons: 3 samples, 1 donors; controls 5 samples, 2 donors). l, Volcano plot of –log₁₀(P-value) and log₂(FoldChange) from linear model of RNA-seq (5’-NRXN1^+/− hiPSC-neurons: 3 samples, 1 donors; controls 6 samples, 2 donors); DE kinase associated genes labeled. m, Violin plot of median and quartiles of RPKM for kinase hits with largest fold change values in RNA-seq (5’-NRXN1^+/− hiPSC-neurons: 3 samples, 1 donors; controls 6 samples, 2 donors). n, Isoform constructs for overexpression with log₂(FoldChange) from hybrid sequencing dataset. o, Mean fold-change from qPCR of NRXN1 expression (3 replicates per condition. p, Representative western blot (2 replicates) for anti-FLAG (48hr expression of control-enriched NRXN1α-FLAG). q, Representative western blot (2 replicates) for anti-FLAG (48hr expression of 3’-NRXN1^+/− specific NRXN1α-FLAG). All error bars are s.e.

Extended Data Fig. 9. Predicted protein models for wild-type and mutant NRXN1α isoforms

a, Superimposed image of the top ten most abundant wildtype NRXN1α isoforms in hiPSC-neurons. b, Superimposed predicted protein model of the top ten most abundant mutant isoforms. c, Superimposed predicted protein model of the top ten most abundant mutant isoforms compared to the most abundant wildtype isoform (grey). d, Individual predicted protein models of the top ten most abundant wild type isoforms. e, Individual predicted protein models of the top ten most abundant mutant isoforms. Insets in each panel highlight C-terminal region of NRXN1α isoforms where 3’-NRXN1^+/− deletion is located.

Extended Data Fig. 10. Investigation of NRXN1α isoform changes across development

a,b, Bar plot of the total read count for each NRXN1α exon along with the fraction that each NRXN1α junction is included in adult dlPFC samples (a) and fetal PFC samples (b); pink boxes represent potential developmentally regulated exons. c, Schematic of NRXN1α isoform structure, with each row representing a unique NRXN1α isoform and each column representing a NRXN1 exon. Colored exons (blue, fetal PFC specific; green, adult dlPFC specific; orange, shared) are spliced into the transcript while blank exons are spliced out. Abundance of each NRXN1α isoform across fetal PFC and adult dlPFC samples.

Figure 1 |. Cohort description and transcriptomic analysis.

a, Representation of individuals in the hiPSC cohort used for this study (5’-NRXN1^+/− deletions in blue; 3’-NRXN1^+/− deletions in red; controls in gray) with schematic of the NRXN1 gene structure highlighting the exons encompassed by 5’-(blue) and 3’-(red) NRXN1^+/− deletions. b, Validation of hiPSC-derived neural cells by immunostaining for SOX2 and NESTIN (NPCs) and MAP2 (6-week hiPSC-neurons) with DAPI stained nuclei, 3 independent differentiations per line. c, Volcano plot showing log₂(foldchange) between NRXN1^+/− (18 samples, 4 donors) and controls (19 samples, 4 donors) and the –log₁₀(P-value) by linear model for each gene with FDR < 20% in red, FDR < 10% in orange and NRXN1 in blue. d, Gene set enrichment analysis, with –log₁₀(P-value) computed by Fisher Exact test using mSigDB. e, Concordance computed using Spearman correlation of the t-statistics between two datasets. Comparisons were made between this study and postmortem RNA-seq datasets of schizophrenia (SZ), major depressive disorder (MDD), bipolar disorder (BD), and autism spectrum disorder (ASD) from CommonMind, NIMH HBCC and UCLA. f, Sum of all NRXN1 transcript expression by cell type (19 samples, 8 donors hiPSC-NPC; 18 samples, 8 donors hiPSC-neurons) and genotype (19 samples, 4 donors controls; 10 samples, 2 donors 5’-NRXN1^+/−; 8 samples, 3 donors 3’-NRXN1^+/−). g,h, Differential isoform usage in hiPSC-neurons across genotypes (9 samples, 3 donors controls; 5 samples, 2 donors 5’-NRXN1^+/−; 5 samples, 2 donors 3’-NRXN1^+/−) when sub-setting for NRXN1α isoforms (g) or NRXN1β isoforms (h). Violin plots display median and quartiles (*P < 0.05, **P < 0.01 by one-way ANOVA with Dunnett’s Test). i, Gene set enrichments for genes correlated with NRXN1 spicing in hiPSC-neurons.

Figure 2 |. Conservation of NRXN1α isoforms across mouse PFC, postmortem PFC and hiPSC-neurons.

a, Overlap of NRXN1α isoforms identified in mouse PFC (1 sample), adult dlPFC (3 donors) and fetal PFC (3 donors). b, Pearson’s correlation of the abundance of 57 NRXN1α isoforms shared between mouse (1 sample) and human (7 samples) with two-sized t-test. c, Schematic of NRXN1α isoform structure, with each row representing a unique NRXN1α isoform and each column representing a NRXN1α exon. Colored exons (green, shared; purple, mouse specific) are spliced into the transcript while blank exons are spliced out. d, Read count for each NRXN1α isoform in mouse PFC. e, Overlap of NRXN1α isoforms identified in adult dlPFC (3 donors), fetal PFC (3 donors) and control hiPSC-neurons (2 donors). f, PCA of NRXN1α isoforms across adult postmortem (3 donors), fetal postmortem (3 donors) and hiPSC-neuron samples (3 donors). g, Schematic of NRXN1α isoform structure, with each row representing a unique NRXN1α isoform and each column representing a NRXN1 exon. Colored exons (orange, shared; green, postmortem specific; gray, hiPSC-neuron specific) are spliced into the transcript while blank exons are spliced out. h, Abundance of each NRXN1α isoform by sample.

Figure 3 |. Identification of cell type specific NRXN1 isoforms from control isogenic hiPSC-neuronal subtypes.

a, Overlap of NRXN1α isoforms identified in forebrain hiPSC-neurons (3 donors), NGN2-glutamatergic hiPSC-neurons (1 donor) and ASCL1/DLX2-GABAergic hiPSC-neurons (1 donor). b, Bar plot of the total read count for each NRXN1α exon along with the fraction that each NRXN1α junction (row, 5’ end; column, 3’ end) is included in control NGN2-glutamatergic hiPSC-neurons (green, left) and control ASCL1/DLX2-GABAergic hiPSC-neurons (blue, right). c, Schematic of NRXN1α isoform structure, with each row representing a unique NRXN1α isoform and each column representing a NRXN1 exon. Colored exons (blue, ASCL1/DLX2-GABAergic specific, green, NGN2-glutamatergic specific; orange, shared) are spliced into the transcript while blank exons are spliced out. d, Abundance of each NRXN1α isoform across control isogenic hiPSC-neuronal subtypes. e, Validation of each isoform in control forebrain hiPSC-neurons (black, expressed).

Figure 4 |. Identification of mutant NRXN1α isoforms.

a, Schematic of NRXN1α isoform structure, with each row representing a unique NRXN1α isoform and each column representing a NRXN1 exon. Colored exons (red, 3’-NRXN1^+/− specific; gray, control specific; orange, shared) are spliced into the transcript while blank exons are spliced out. b, log₂(foldchange) of each NRXN1α isoform in 3’-NRXN1^+/− hiPSC-neurons (2 donors) compared to control hiPSC-neurons (2 donors). c, Abundance of each NRXN1α isoform across 3’-NRXN1^+/− and control hiPSC-neurons. d, Validation of each isoform in postmortem samples (black, expressed in postmortem PFC). e, Pearson’s correlation of 47 NRXN1α isoforms between hiPSC-neurons (forebrain) and ASCL1/DLX2-GABAergic neurons from 3’-NRXN1^+/− (1 donor) and controls (1 donor) with two-sized t-test. f, Pearson’s correlation of 57 NRXN1α isoforms between hiPSC-neurons (forebrain) and NGN2-glutamatergic neurons from 3’-NRXN1^+/− (1 donor) and controls (1 donor) with two-sized t-test.

Figure 5 |. Single-cell sequencing of hiPSC-neurons.

a, tSNE plot of 15 clusters identified in combined Seurat analysis of hiPSC-neuron samples from 5 donors (control (2), 5’-NRXN1^+/− (2) and 3’-NRXN1^+/−(1)) and marker genes of relevant clusters. b, tSNE plot colored by NRXN1 genotype (control (2), 5’-NRXN1^+/− (2) and 3’-NRXN1^+/−(1)). c-e, Percent cells from each genotype (control (2), 5’-NRXN1^+/− (2) and 3’-NRXN1^+/−(1)) across 2 glial clusters (c), 3 immature neuronal clusters (d) and 4 neuron clusters (e). f, Differentially expressed genes by linear model between NRXN1^+/− hiPSC-neurons and controls within C4, the most mature neuronal cluster.

Figure 6 |. Impact of specific NRXN1α isoforms on neuronal activity.

a-c, Results of hiPSC-neuron (forebrain) multi-electrode array (MEA) showing representative raster plot for each genotype (a), a time-course of spontaneous activity over two independent 6-week differentiations (b), and quantification of the number of spontaneous spikes at 6-weeks across controls (30 wells, 2 donors), related non-carrier (3 wells, 1 donor), 5’-NRXN1^+/− (30 wells, 2 donors), 3’-NRXN1^+/− (30 wells, 2 donors) (c). d-f, Results of NGN2-neuron MEA showing representative raster plots for each genotype (d), a time-course of spontaneous activity over 7-weeks of two independent differentiations (e) and quantification of number of spontaneous spikes at 6-weeks across controls (22 wells, 2 donors), 5’-NRXN1^+/− (22 wells, 2 donors), 3’-NRXN1^+/− (17 wells, 2 donors) (f). Two unique donors per genotype indicated by different shading within each plot. Plots display mean with s.e.; *P < 0.05 by one way ANOVA with Dunnett’s test. g, Schematic of timeline used for over-expression of NRXN1α isoforms. h-j, Quantification of spontaneous spikes after over-expression of individual NRXN1α isoforms in control hiPSC-neurons (1 donor) (h), 5’-NRXN1^+/− hiPSC-neurons (1 donor) (i) or 3’-NRXN1^+/− hiPSC-neurons (1 donor) (j). k, A model for the contribution of both NRXN1^+/− genotype and NRXN1α isoform expression on changes in neuronal activity. g-j, One donor per genotype. Plots display mean with s.e.; *P < 0.05 by one way ANOVA after log-transformation with Dunnett’s test.