Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how ‘synaptic’ ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.
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Data are available through the following hyperlinks: bulk RNA-seq raw counts, https://figshare.com/s/fe1ab60f2a3ebcaae7ff; 2-month-old scRNA-seq data (PatientCorrected organoid 1, https://figshare.com/s/93d4d3b71ec8d9951eb4; PatientCorrected organoid 2, https://figshare.com/s/7603008aefc936fd53d7; PatientCorrected organoid 3, https://figshare.com/s/d19a8905e1296e2055bd; SYNGAP1p.Q503X organoid 1, https://figshare.com/s/794cfe68f8caedfbd440; SYNGAP1p.Q503X organoid 2, https://figshare.com/s/d5a9e7d3ab45b3c50a21; SYNGAP1p.Q503X organoid 3, https://figshare.com/s/05dc105003079568bfc3); 4-month-old scRNA-seq data (SYNGAP1p.Q503X organoids, matrix.mtx.gz, https://figshare.com/s/1cd0dc51bcf6f1028cd2; SYNGAP1p.Q503X organoids, features.tsv.gz, https://figshare.com/s/c08cfabef967271dcfbb; SYNGAP1p.Q503X organoids, barcodes.tsv.gz, https://figshare.com/s/2b493cc0108d5f2cbc35; PatientCorrected organoids, matrix.mtx.gz, https://figshare.com/s/5b8b4b0cd4818c9c2e7d; PatientCorrected organoids, features.tsv.gz, https://figshare.com/s/d6054204bd0514a87b5e; PatientCorrected organoids, barcodes.tsv.gz, https://figshare.com/s/c1684604c58b0b04ba05); Proteomics Identifications Database (project accession, PXD034090).
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We thank the SynGAP Research Fund and families participating in this study for their collaboration. We thank the Pediatric Neuropathology Research Lab of the UCSF and honor the families who generously donated the tissue samples used in this study. We thank P. Arlotta and F. Francis, current and former members of the Quadrato laboratory for insightful discussions and feedback on this project. We thank C. Lytal for editing the manuscript. We thank S.W. Ruffins and the Optical Imaging Facility at the USC for providing guidance and support for imaging analyses. We thank C. Taitano-Johnson, T. Rintoul and A. Albanese for outstanding technical support. This work was supported by the SynGAP Research Fund (SRF), the Donald D. and Delia B. Baxter Foundation, the Edward Mallinckrodt Jr. Foundation and the National Science Foundation (5351784498) and the Eli and Edythe Broad Foundation (G.Q.). This work was supported in part by NIH grants from the National Institute of Mental Health to M.P.C. (MH115005). This reported research includes work performed in the mass spectrometry core supported by the National Cancer Institute of the National Institutes of Health under grant number P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
G.K. and R.S.A. are inventors on US patent application no. 16/044236, which describes a platform for generating microarrayed single-rosette cultures, and they are cofounders of Neurosetta, which is focused on commercializing the culture platform. The remaining authors declare no competing interests.
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A. Expression of early forebrain marker genes of PAX6, HES5, EOMES (TBR2) and SYNGAP1 from post-conception day (PCD) 26 to 54 from single cell RNA-seq data. B. UMAP visualization of age-dependent clustering of fetal single cells. C. SYNGAP1 expression at PCD 56 grouped by cell types; intermediate progenitor cells (IPC), neuroepithelial cells (NE), radial glial cells (RGCs) and neurons. D. D.I.V. 7 cortical organoids are composed of cells positive for the neural stem cell marker SOX2, the radial glial progenitor marker PAX6, the nuclear marker DAPI and SYNGAP1. E. A coronal section from E13.5 mouse brain showing expression of the neural stem cell marker SOX2, the tight junction protein TJP1, and SYNGAP1. SYNGAP1 is highly expressed at the ventricular wall. White box indicates the Region of Interest selected for the merged images showing colocalization of DAPI, TJP1, and SYNGAP1. F. Peptide competition assay shows the specificity of the SYNGAP1 antibody used. 5X and 10X concentrations of the commercial antigenic peptide were evaluated, showing a strong reduction in specific signal in the apical wall of the ventricular zone. G. SynGAP1 expression in E18.5 wild type and SynGAP1 KO mouse showing the overall decrease in SynGAP1 levels. Decreased levels of SynGAP are most evident at the VZ.
A. Annotated spectra of the SYNGAP1 isoform alpha 1 specific peptide “GSFPPWQQTR” identified from MS analysis of immune-isolated SYNGAP1 protein from D.I.V. 7 organoids. B. Annotated spectra of the SYNGAP1 isoform alpha 1 specific peptide “LLDAQR” identified from MS analysis of immune-isolated SYNGAP1 protein from D.I.V. 7 organoids.
A. Selected GO terms for biological processes for SYNGAP1 immunoprecipitation data collected from D.I.V. 7 cortical organoids. B. Schematic of line generation details for isogenic control of Patientp.Q503X. C. Chromatogram of the generated corrected line (PatientC°rrected). The truncating “T” was substituted with the wild type “C” base pair. D. Representative Western blot for SYNGAP1 in the Patient p.Q503X(P), PatientC°rrected (C) and KO (K) iPSCs derived neurons showing a reduction of SYNGAP1 levels in P and complete loss in K iPSCs. E. Quantification of the western blot shows significant reduction in SYNGAP1 levels in Patient p.Q503X iPSCs compared to the PatientCorrected iPSCs in four biological replicates. Individual dots represent independent replicates. Data are presented as mean values± SD. Statistical analysis was performed using unpaired two-tailed t-test. P value <0.01. F. Quantification of the SYNGAP1 peptide. Graphical representation of SYNGAP1 protein levels quantified by timed parallel reaction monitoring (tRPM). Plot shows a decrease of SYNGAP1 total protein levels in Patientp.Q503X:64.83 (51.6%) as compared to its corresponding isogenic control (PatientCorrected): 125.6 expressed as fg peptide/ug digested protein. Individual dots represent 20 organoids pooled together. N=9 across three independent differentiations. Data are presented as mean values ± SD. Statistical analysis was performed using unpaired two-tailed t-test. P value <0.0001. G. Karyotypic analysis of PatientCorrected iPSCs revealed a normal karyotype. H. Chromatogram of the Patientp.Q503X iPSCs carrying the truncating mutation. I. PCR of RGD, Patientp.Q503X, 03231 cell line. J. Chromatogram of the 03231Control iPSCs carrying the wild type sequence. K. Chromatogram of the 03231RGD iPSCs carrying the homozygous mutation in the RGD domain. L. Karyotypic analysis of 03231RGD iPSCs revealed a normal karyotype. M. Chromatogram of the 03231p.Q503X iPSCs carrying the truncating mutation. N. PCR of 03231Control cell line and 03231p.Q503X cell line showing haploinsuffiency in the 03231p.Q503X line. O. Karyotypic analysis of 03231p.Q503X iPSCs revealed a normal karyotype.
Extended Data Fig. 4 Characterization of single rosettes and organoids modelling SYNGAP1 haploinsufficiency.
A. Single rosette from the PatientCorrected cell line expressing SOX2 and PAX6 markers. B. Single rosette from the Patientp.Q503X cell line expressing SOX2 and PAX6 markers. C. Single rosette from the 03231Control cell line expressing SOX2 and PAX6 markers. D. A single rosette was generated from PatientCorrected line. The rosette is composed of cells positive for the neural progenitor marker SOX2 and SYNGAP1. SYNGAP1 is also highly expressed at the apical wall of the lumen. The tight junction protein TJP1 labels the central luminal space of the rosette. Merged images show colocalization of DAPI, SYNGAP1, and TJP1. E. Single rosette from the Patientp.Q503X line. The rosette is composed of cells positive for the neural progenitor marker SOX2 and SYNGAP1. Merged images show colocalization of DAPI, SYNGAP1, and TJP1. The Patientp.Q503X single rosettes display a larger and more irregularly shaped central luminal space. F. Single rosette from the 03231Control line. The rosette is composed of cells positive for the neural progenitor marker SOX2 and SYNGAP1. SYNGAP1 is also highly expressed at the apical wall of the lumen. Merged images show colocalization of DAPI, SYNGAP1, and TJP1. G. Single rosette from the 03231p.Q503X cell line expressing SOX2 and PAX6 markers. H. Single rosette from the 03231RGD cell line expressing SOX2 and PAX6 markers. I. Single rosette from the 03231p.Q503X line. The rosette is composed of cells positive for the neural progenitor marker SOX2 and SYNGAP1. Merged images show colocalization of DAPI, SYNGAP1, and TJP1. The tight junction protein TJP1 is weakly expressed with little to no central luminal organization. J. Single rosette from the 03231RGD line. The rosette is composed of cells positive for the neural progenitor marker SOX2 and SYNGAP1. Merged images show colocalization of DAPI, SYNGAP1, and TJP1. The tight junction protein TJP1 is weakly expressed with no central luminal organization. K. Survival curve for organoids generated from the 03231RGD line. Data collected from 10 independent differentiations, each representing an average of 6 organoids for each time point. Single dots represent total averages for that time point. Two-tailed t-test was performed (Day 30 P=0.0134, Day 60 P<0.0001). Data represented as mean ± SEM.
Extended Data Fig. 5 Assessment of proliferation/differentiation ratio in 2-month-old SYNGAP1 haploinsufficient and control organoids.
A. Representative single channel images from BrdU pulse-chase experiments in 2-month-old PatientCorrected organoids. Images show the expression of the progenitor marker SOX2, the neuronal marker NeuN and the proliferative marker BrdU. B. Representative single channel images from BrdU pulse-chase experiments in 2-month-old Patientp.Q503X organoids. Images show the expression of the progenitor marker SOX2, the neuronal marker NeuN and the proliferative marker BrdU. C. Representative single channel images from BrdU pulse-chase experiments in 2-month-old 03231Control organoids. Images show the expression of the progenitor marker SOX2, the neuronal marker NeuN and the proliferative marker BrdU. D. Representative single channel images from BrdU pulse-chase experiments in 2-month-old 03231p.Q503X organoids. Images show the expression of the progenitor marker SOX2, the neuronal marker NeuN and the proliferative marker BrdU.
A. SOX2 and NeuN expression in 2-month-old 03231Control and 03231p.Q503X organoids. B. Total number of SOX2+ cells in 03231Control and 03231p.Q503X organoids. Dot represents an average value for all organoids from 1 differentiation. Two-tailed t-test on average values for 10 organoids from 4 differentiations. P Value =0.0112. Data shown as mean ± SD. C Total number of NeuN positive cells in 03231Control and 03231p.Q503X organoids. Two-tailed t-test on average values for 10 organoids from 4 differentiations. P Value =0.0217. Data shown as mean ± SD. D. TBR2 expression in 2-month-old PatientCorrected and Patientp.Q503X organoids. E. Total number of TBR2+ cells in PatientCorrected and Patientp.Q503X organoids. Two-tailed t-test on average values for 10 organoids from 4 differentiations. P= ns. Data shown as mean ± SD. F. SOX2 positive area in the dorsal cortex of E18.5 mouse brains. Data from WT=27, HET=24, KO =27 ventricles from 4 brains for each genotype. One Way ANOVA between the genotypes, showing a decrease in the SOX2 positive progenitor regions in Het and KO as compared to WT mice. P<0.0001. Data shown as mean ± SD. G. SOX2 positive area in the lateral cortex of E18.5 mouse brains. Data from WT=27, HET=24, KO =27 from 4 brains for each genotype. One Way ANOVA between the genotypes. P=ns. Data shown as mean ± SD. H. TBR2 expression in WT, Het and KO E18.5 mouse brains. I. TBR2 thickness in the cortical plate in in WT, Het and KO E18.5 mouse brain sections. One Way ANOVA on 8 ventricles from four animals for each genotype. P=ns. Data shown as mean ± SD. J. Number of TBR2+ cells in 100 um2 of the VZ area in WT, Het and KO E18.5 mouse brains. One Way ANOVA on 6 ventricles from four animals for each genotype. P=ns. Data shown as mean ± SD. K. Organoid area over time from the PatientCorrected and Patientp.Q503X lines. Data from 10 independent differentiations, each representing an average of 6 organoids for each time point. Data shown as mean ± SEM. L. Organoid perimeter over time from the PatientCorrected and Patientp.Q503X lines. Data from 10 independent differentiations, each representing an average of 6 organoids for each time point. Data shown as mean ± SEM. M. Head circumference measurements represented as dots from the Patientp.Q503X donor plotted against the WHO child growth standards.
Extended Data Fig. 7 scRNAseq and functional analysis of 4-months-old SYNGAP1 haploinsufficient organoids.
A. GO-Terms from single cell RNA sequencing preformed in 2-month-old Patientp.Q503X and PatientCorrected organoids. Graphical representation of upregulated terms for Patientp.Q503X Corticofugal Projection Neurons (CFuPN). Main biological process, cellular component, and molecular function GO-Terms are related to neuronal differentiation and synapse formation. Wilcoxon rank sum test was used for DEGs between control and mutant organoids for each cluster. B. GO-Terms from single cell RNA sequencing preformed in 4-month-old Patientp.Q503X and PatientCorrected organoids. Graphical representation of upregulated GO-Terms in Patientp.Q503X Callosal Projection Neurons (CPN). Main biological process and cellular component GO-Terms are related to neuronal differentiation and synapse formation. Wilcoxon rank sum test was used for DEGs between control and mutant organoids for each cluster. C. Combined t-distributed stochastic neighbor embedding (t-SNE) from single cell RNA sequencing analysis of pooled Patientp.Q503X and PatientCorrected organoids at 4 months. D. Individual t-SNE plot for pooled Patientcorrected organoids at 4 months (n=7540 cells). E. Individual t-SNE plot for pooled Patientp.Q503X organoids at 4 months (n=3123 cells). F. ΔF/F(t) from GCaMP6f2 recordings of PatientCorrected and Patientp.Q503X organoids. G. Calcium spike frequency analysis on 2-month-old Patientp.Q503X organoids before and during bath application of glutamate (Glu). Unpaired two-tailed t-test performed on 16 cells from Patientp.Q503X organoids, from 3 independent experiments. P value = 0.0005. Data shown as mean ± SD. H. ΔF/F(t) from GCaMP6f2 recordings of 03231Control and 03231p.Q503X organoids. I. Calcium spike frequency analysis on 2-month-old Patientp.Q503X organoids before and during bath application of tetrodotoxin (TTX). Unpaired two-tailed t-test performed on 9 cells from Patientp.Q503X organoids, from 3 independent experiments. P value =0.0001. Data shown as mean ± SD.
Supplementary Table 1. Proteome of 7-d.i.v. cortical organoids. Supplementary Table 2. SynGO analysis of the 7-d.i.v. cortical organoid proteome. Supplementary Table 3. Interactome of SYNGAP1 in 7-d.i.v. cortical organoids. Supplementary Table 4. Off-target sequences and sequencing. SYNGAP1p.Q503X-corrected guide RNA predicted off-target sequences and sequencing. Table contains the top predicted off-target regions of the guide RNA used to generate patient 1 corrected iPSCs along with sequencing results of each target that was amplified by PCR and then verified by Sanger sequencing. The predicted off-target sequence is highlighted along with 25 bp on either side. RDG guide RNA predicted off-target sequences and sequencing. Table contains the top ten predicted off-target regions of the guide RNA used to generate RDG iPSCs along with sequencing results of each target that was amplified by PCR and then verified by Sanger sequencing. The predicted off-target sequence is highlighted along with 25 bp on either side. Supplementary Table 5. SYNGAP1 quantitation. Table shows MS methods, acceptance criteria, peptide concentrations, calibration curve and statistical analysis for PRM quantitation of total protein levels of SYNGAP1. Supplementary Table 6. Differential expression analysis (DEGs) from bulk RNA-seq of 7-d.i.v. cortical organoids from SYNGAP1p.Q503X and corrected lines. DEGs were calculated using two-tailed Wilcoxon rank-sum test.
DEGs used to determine the identity of each cluster with the 2-month-old and 4-month-old scRNA-seq datasets. DEGs were calculated using two-tailed Wilcoxon rank-sum test.
Differential expression analysis (DEGs) from scRNA-seq of 2-month-old cortical organoids from SYNGAP1p.Q503X and corrected lines. DEGs were calculated using two-tailed Wilcoxon rank-sum test.
Raw counts and relative percentages of cells within each cluster from scRNA-seq of 2-month-old cortical organoids from SYNGAP1p.Q503X and corrected lines.
Differential expression analysis (DEGs) from scRNA-seq of 4-month-old cortical organoids from SYNGAP1p.Q503X and corrected lines. DEGs were calculated using two-tailed Wilcoxon rank-sum test.
Live imaging of corrected rosette formation from day 5 to day 7 after initial seeding.
Live imaging of SYNGAP1p.Q503X rosette formation from day 5 to day 7 after initial seeding.
Video recordings (10 min) of GCaMP6f corrected organoids.
Video recordings (10 min) of GCaMP6f SYNGAP1p.Q503X organoids.
Video recordings (10 min) of GCaMP6f 03231 organoids.
Video recordings (10 min) of GCaMP6f 03231p.Q503X organoids.
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Birtele, M., Del Dosso, A., Xu, T. et al. Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis. Nat Neurosci 26, 2090–2103 (2023). https://doi.org/10.1038/s41593-023-01477-3