Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis

Nature Neuroscience volume 26, pages 2090–2103 (2023)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: SYNGAP1 is expressed in human radial glia progenitors and colocalizes with the tight junction protein TJP1.
Fig. 2: SYNGAP1 plays a role in the cytoskeletal organization of human radial glia.
Fig. 3: SYNGAP1 haploinsufficiency disrupts the organization of the developing cortical plate.
Fig. 4: SYNGAP1 haploinsufficiency affects the division mode of human radial glial progenitors.
Fig. 5: Decrease in SYNGAP1 levels leads to asynchronous cortical neurogenesis.
Fig. 6: SYNGAP1haploinsufficient organoids exhibit accelerated maturation of cortical projection neurons.

Similar content being viewed by others

Data availability

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).

References

  1. De Rubeis, S. et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature 515, 209–215 (2014).

    PubMed  PubMed Central  Google Scholar 

  2. Paulsen, B. et al. Autism genes converge on asynchronous development of shared neuron classes. Nature 602, 268–273 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Jourdon, A. et al. Modeling idiopathic autism in forebrain organoids reveals an imbalance of excitatory cortical neuron subtypes during early neurogenesis. Nat. Neurosci. https://doi.org/10.1038/s41593-023-01399-0 (2023).

  5. Villa, C. E. et al. CHD8 haploinsufficiency links autism to transient alterations in excitatory and inhibitory trajectories. Cell Rep. 39, 110615 (2022).

    CAS  PubMed  Google Scholar 

  6. Schafer, S. T. et al. Pathological priming causes developmental gene network heterochronicity in autistic subject-derived neurons. Nat. Neurosci. 22, 243–255 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. de Jong, J. O. et al. Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder. Nat. Commun. 12, 4087 (2021).

    PubMed  PubMed Central  Google Scholar 

  8. Urresti, J. et al. Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism. Mol. Psychiatry 26, 7560–7580 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Satterstrom, F. K. et al. Large-scale exome sequencing study implicates both developmental and functional changes in the neurobiology of autism. Cell 180, 568–584 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, H. J., Rojas-Soto, M., Oguni, A. & Kennedy, M. B. A synaptic Ras-GTPase activating protein (p135 SynGAP) inhibited by CaM kinase II. Neuron 20, 895–904 (1998).

    CAS  PubMed  Google Scholar 

  11. Kim, J. H., Liao, D., Lau, L. F. & Huganir, R. L. SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family. Neuron 20, 683–691 (1998).

    CAS  PubMed  Google Scholar 

  12. Komiyama, N. H. et al. SynGAP regulates ERK/MAPK signaling, synaptic plasticity, and learning in the complex with postsynaptic density 95 and NMDA receptor. J. Neurosci. 22, 9721–9732 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kim, J. H., Lee, H. K., Takamiya, K. & Huganir, R. L. The role of synaptic GTPase-activating protein in neuronal development and synaptic plasticity. J. Neurosci. 23, 1119–1124 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhu, J. J., Qin, Y., Zhao, M., Van Aelst, L. & Malinow, R. Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell 110, 443–455 (2002).

    CAS  PubMed  Google Scholar 

  15. Araki, Y., Zeng, M., Zhang, M. & Huganir, R. L. Rapid dispersion of SynGAP from synaptic spines triggers AMPA receptor insertion and spine enlargement during LTP. Neuron 85, 173–189 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Walkup, W. G. et al. A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain ‘slots’ in the postsynaptic density. eLife 5, e16813 (2016).

  17. Zeng, M. et al. Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity. Cell 166, 1163–1175 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Zeng, M., Bai, G. & Zhang, M. Anchoring high concentrations of SynGAP at postsynaptic densities via liquid–liquid phase separation. Small GTPases 10, 296–304 (2019).

    CAS  PubMed  Google Scholar 

  19. Kilinc, M. et al. Endogenous Syngap1 α splice forms promote cognitive function and seizure protection. eLife 11, e75707 (2022).

  20. Knuesel, I., Elliott, A., Chen, H. J., Mansuy, I. M. & Kennedy, M. B. A role for synGAP in regulating neuronal apoptosis. Eur. J. Neurosci. 21, 611–621 (2005).

    PubMed  Google Scholar 

  21. Willsey, H. R. et al. Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience. Neuron 109, 1409 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Su, P. et al. Disruption of SynGAP–dopamine D1 receptor complexes alters actin and microtubule dynamics and impairs GABAergic interneuron migration. Sci. Signal. 12, eaau9122 (2019).

  23. Berryer, M. H. et al. Mutations in SYNGAP1 cause intellectual disability, autism, and a specific form of epilepsy by inducing haploinsufficiency. Hum. Mutat. 34, 385–394 (2013).

    CAS  PubMed  Google Scholar 

  24. Kilinc, M. et al. Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders. Mol. Cell. Neurosci. 91, 140–150 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Gamache, T. R., Araki, Y. & Huganir, R. L. Twenty years of SynGAP research: from synapses to cognition. J. Neurosci. 40, 1596–1605 (2020).

    PubMed  PubMed Central  Google Scholar 

  26. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    CAS  PubMed  Google Scholar 

  27. Bershteyn, M. et al. Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell 20, 435–449 (2017).

    CAS  Google Scholar 

  28. Kanton, S. et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574, 418–422 (2019).

    CAS  PubMed  Google Scholar 

  29. Klaus, J. et al. Altered neuronal migratory trajectories in human cerebral organoids derived from individuals with neuronal heterotopia. Nat. Med. 25, 561–568 (2019).

    CAS  PubMed  Google Scholar 

  30. Esk, C. et al. A human tissue screen identifies a regulator of ER secretion as a brain-size determinant. Science 370, 935–941 (2020).

    CAS  PubMed  Google Scholar 

  31. Khan, T. A. et al. Neuronal defects in a human cellular model of 22q11.2 deletion syndrome. Nat. Med. 26, 1888–1898 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Samarasinghe, R. A. et al. Identification of neural oscillations and epileptiform changes in human brain organoids. Nat. Neurosci. 24, 1488–1500 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tidball, A. M. et al. Self-organizing single-rosette brain organoids from human pluripotent stem cells. Preprint at bioRxiv https://doi.org/10.1101/2022.02.28.482350 (2022).

  34. Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Velasco, S. et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 570, 523–527 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Camp, J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl Acad. Sci. USA 112, 15672–15677 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Trevino, A. E. et al. Chromatin accessibility dynamics in a model of human forebrain development. Science 367, eaay1645 (2020).

  38. Gordon, A. et al. Long-term maturation of human cortical organoids matches key early postnatal transitions. Nat. Neurosci. 24, 331–342 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Michaelson, S. D. et al. SYNGAP1 heterozygosity disrupts sensory processing by reducing touch-related activity within somatosensory cortex circuits. Nat. Neurosci. 21, 1–13 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Aceti, M. et al. Syngap1 haploinsufficiency damages a postnatal critical period of pyramidal cell structural maturation linked to cortical circuit assembly. Biol. Psychiatry 77, 805–815 (2015).

    CAS  PubMed  Google Scholar 

  41. Eze, U. C., Bhaduri, A., Haeussler, M., Nowakowski, T. J. & Kriegstein, A. R. Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia. Nat. Neurosci. 24, 584–594 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Li, J. et al. Spatiotemporal profile of postsynaptic interactomes integrates components of complex brain disorders. Nat. Neurosci. 20, 1150–1161 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Araki, Y. et al. SynGAP isoforms differentially regulate synaptic plasticity and dendritic development. eLife 9, e56273 (2020).

  44. Knight, G. T. et al. Engineering induction of singular neural rosette emergence within hPSC-derived tissues. eLife 7, e37549 (2018).

  45. Wilkinson, B. et al. Endogenous cell type-specific disrupted in schizophrenia 1 interactomes reveal protein networks associated with neurodevelopmental disorders. Biol. Psychiatry 85, 305–316 (2019).

    CAS  PubMed  Google Scholar 

  46. Carlisle, H. J., Manzerra, P., Marcora, E. & Kennedy, M. B. SynGAP regulates steady-state and activity-dependent phosphorylation of cofilin. J. Neurosci. 28, 13673–13683 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Tomoda, T., Kim, J. H., Zhan, C. & Hatten, M. E. Role of Unc51.1 and its binding partners in CNS axon outgrowth. Genes Dev. 18, 541–558 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Aaku-Saraste, E., Hellwig, A. & Huttner, W. B. Loss of occludin and functional tight junctions, but not ZO-1, during neural tube closure—remodeling of the neuroepithelium prior to neurogenesis. Dev. Biol. 180, 664–679 (1996).

    CAS  PubMed  Google Scholar 

  49. Edmondson, J. C. & Hatten, M. E. Glial-guided granule neuron migration in vitro: a high-resolution time-lapse video microscopic study. J. Neurosci. 7, 1928–1934 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Rakic, P. Guidance of neurons migrating to the fetal monkey neocortex. Brain Res. 33, 471–476 (1971).

    CAS  Google Scholar 

  51. Rakic, P. Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol. 145, 61–83 (1972).

    CAS  PubMed  Google Scholar 

  52. Rakic, P. Neuronal migration and contact guidance in the primate telencephalon. Postgrad. Med. J. 54, 25–40 (1978).

    PubMed  Google Scholar 

  53. Nowakowski, T. J., Pollen, A. A., Sandoval-Espinosa, C. & Kriegstein, A. R. Transformation of the radial glia scaffold demarcates two stages of human cerebral cortex development. Neuron 91, 1219–1227 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Chenn, A. & McConnell, S. K. Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 82, 631–641 (1995).

    CAS  PubMed  Google Scholar 

  55. Shitamukai, A., Konno, D. & Matsuzaki, F. Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors. J. Neurosci. 31, 3683–3695 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Nowakowski, T. J. et al. Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. Science 358, 1318–1323 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Llamosas, N. et al. SYNGAP1 controls the maturation of dendrites, synaptic function, and network activity in developing human neurons. J. Neurosci. 40, 7980–7994 (2020).

  58. Fromer, M. et al. De novo mutations in schizophrenia implicate synaptic networks. Nature 506, 179–184 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Genovese, G. et al. Increased burden of ultra-rare protein-altering variants among 4,877 individuals with schizophrenia. Nat. Neurosci. 19, 1433–1441 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Kirov, G. et al. De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia. Mol. Psychiatry 17, 142–153 (2012).

    CAS  PubMed  Google Scholar 

  61. O’Roak, B. J. et al. Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat. Genet. 43, 585–589 (2011).

    PubMed  PubMed Central  Google Scholar 

  62. O’Roak, B. J. et al. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485, 246–250 (2012).

    PubMed  PubMed Central  Google Scholar 

  63. Peça, J. & Feng, G. Cellular and synaptic network defects in autism. Curr. Opin. Neurobiol. 22, 866–872 (2012).

    PubMed  PubMed Central  Google Scholar 

  64. Kawaguchi, A. Neuronal delamination and outer radial glia generation in neocortical development. Front. Cell Dev. Biol. 8, 623573 (2021).

    Google Scholar 

  65. Kadowaki, M. et al. N-cadherin mediates cortical organization in the mouse brain. Dev. Biol. 304, 22–33 (2007).

    CAS  PubMed  Google Scholar 

  66. Cappello, S. et al. A radial glia-specific role of RhoA in double cortex formation. Neuron 73, 911–924 (2012).

    CAS  PubMed  Google Scholar 

  67. Gil-Sanz, C., Landeira, B., Ramos, C., Costa, M. R. & Müller, U. Proliferative defects and formation of a double cortex in mice lacking Mltt4 and Cdh2 in the dorsal telencephalon. J. Neurosci. 34, 10475–10487 (2014).

    PubMed  PubMed Central  Google Scholar 

  68. Yoon, K. J. et al. Modeling a genetic risk for schizophrenia in iPSCs and mice reveals neural stem cell deficits associated with adherens junctions and polarity. Cell Stem Cell 15, 79–91 (2014).

    CAS  Google Scholar 

  69. Hansen, D. V., Lui, J. H., Parker, P. R. & Kriegstein, A. R. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464, 554–561 (2010).

    CAS  PubMed  Google Scholar 

  70. Clement, J. P., Ozkan, E. D., Aceti, M., Miller, C. A. & Rumbaugh, G. SYNGAP1 links the maturation rate of excitatory synapses to the duration of critical-period synaptic plasticity. J. Neurosci. 33, 10447–10452 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Clement, J. P. et al. Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses. Cell 151, 709–723 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Ozkan, E. D. et al. Reduced cognition in Syngap1 mutants is caused by isolated damage within developing forebrain excitatory neurons. Neuron 82, 1317–1333 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Creson, T. K. et al. Re-expression of SynGAP protein in adulthood improves translatable measures of brain function and behavior. eLife 8, e46752 (2019).

  74. Noctor, S. C., Martínez-Cerdeño, V., Ivic, L. & Kriegstein, A. R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat. Neurosci. 7, 136–144 (2004).

    CAS  PubMed  Google Scholar 

  75. He, S., Li, Z., Ge, S., Yu, Y. C. & Shi, S. H. Inside–out radial migration facilitates lineage-dependent neocortical microcircuit assembly. Neuron 86, 1159–1166 (2015).

    CAS  PubMed  Google Scholar 

  76. Coba, M. P. et al. Dlgap1 knockout mice exhibit alterations of the postsynaptic density and selective reductions in sociability. Sci. Rep. 8, 2281 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Li, J. et al. Long-term potentiation modulates synaptic phosphorylation networks and reshapes the structure of the postsynaptic interactome. Sci. Signal. 9, rs8 (2016).

    PubMed  Google Scholar 

  78. Romero, D. M. et al. Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development. Nat. Commun. 13, 2746 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458–466 (2013).

    CAS  Google Scholar 

  80. Klose, A. et al. Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1. Hum. Mol. Genet. 7, 1261–1268 (1998).

    CAS  PubMed  Google Scholar 

  81. Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Lippmann, E. S., Estevez-Silva, M. C. & Ashton, R. S. Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors. Stem Cells 32, 1032–1042 (2014).

    CAS  Google Scholar 

  83. Hughes, C. S. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 14, 68–85 (2019).

    CAS  PubMed  Google Scholar 

  84. Uzquiano, A. et al. Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex. Cell https://doi.org/10.1016/j.cell.2022.09.010 (2023).

  85. Giovannucci, A. et al. CaImAn an open source tool for scalable calcium imaging data analysis. eLife 8, e38173 (2019).

Download references

Acknowledgements

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.

Author information

Author notes

  1. These authors contributed equally: Marcella Birtele, Ashley Del Dosso.

Authors and Affiliations

  1. Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Marcella Birtele, Ashley Del Dosso, Tiantian Xu, Tuan Nguyen, Negar Hosseini, Sarah Nguyen, Jean-Paul Urenda, Alexander Atamian & Giorgia Quadrato

  2. Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Marcella Birtele, Ashley Del Dosso, Tiantian Xu, Tuan Nguyen, Negar Hosseini, Sarah Nguyen, Jean-Paul Urenda, Alexander Atamian & Giorgia Quadrato

  3. Xiangya Hospital, Central South University, Changsha, China

    Tiantian Xu

  4. Department of Psychiatry and Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Brent Wilkinson, Ilse Flores & Marcelo P. Coba

  5. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Brent Wilkinson, Ilse Flores & Marcelo P. Coba

  6. Department of Biomedical Engineering, University of Wisconsin—Madison, Madison, WI, USA

    Gavin Knight & Randolph S. Ashton

  7. Wisconsin Institute for Discovery, University of Wisconsin—Madison, Madison, WI, USA

    Gavin Knight & Randolph S. Ashton

  8. Departments of Neuroscience and Molecular Medicine, University of Florida Scripps Biomedical Research Institute, Jupiter, FL, USA

    Camilo Rojas & Gavin Rumbaugh

  9. Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, Jupiter, FL, USA

    Camilo Rojas & Gavin Rumbaugh

  10. Integrated Mass Spectrometry Shared Resource, City of Hope Comprehensive Cancer Center, Duarte, CA, USA

    Roger Moore, Ritin Sharma & Patrick Pirrotte

  11. Cancer and Cell Biology Division, Translational Genomics Research Institute, Phoenix, AZ, USA

    Ritin Sharma & Patrick Pirrotte

  12. Department of Pathology, University of California, San Francisco, CA, USA

    Eric J. Huang

  13. Eli and Edythe Broad Institute for Stem Cell Research and Regeneration Medicine, University of California, San Francisco, CA, USA

    Eric J. Huang

  14. Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Marcelo P. Coba

Authors
  1. Marcella Birtele
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashley Del Dosso
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tiantian Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tuan Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brent Wilkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Negar Hosseini
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sarah Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-Paul Urenda
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gavin Knight
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Camilo Rojas
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ilse Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Alexander Atamian
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Roger Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Ritin Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Patrick Pirrotte
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Randolph S. Ashton
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Eric J. Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Gavin Rumbaugh
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Marcelo P. Coba
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Giorgia Quadrato
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.B., A.D.D., T.N., M.P.C. and G.Q. conceived the experiments. M.B. and A.D.D. generated, cultured and characterized single-rosette cultures; J.-P.U. performed live imaging of single rosettes; G.K. and R.S.A. prepared single-rosette culture substrates; M.B., A.D.D., T.X., N.H. and S.N. generated, cultured and characterized all organoids used in this study. A.D.D. and M.B. performed scRNA-seq and bulk RNA-seq experiments with help from T.X., A.A. and T.N.; T.N. performed scRNA-seq analysis and worked on cell type assignments and data analysis; T.X. performed bulk scRNA-seq analysis. B.W. performed sample preparation for proteomic analysis, and R.M., R.S. and P.P. performed analysis of the proteomic data under the supervision of M.P.C.; M.B. performed calcium imaging experiments and analysis; B.W. designed and generated the SYNGAP1p.Q503X, corrected, RGD and 03231 SYNGAP1p.Q503X-edited lines under the supervision of M.P.C. I.F. performed off-target site analysis and validation of cell lines under the supervision of M.P.C. E.J.H. prepared and provided human fetal tissue. C.R. prepared and provided mouse fetal tissue under the supervision of G.R. G.R. contributed to data interpretation. G.Q. supervised all aspects of the project; M.B., A.D.D. and G.Q. wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to Giorgia Quadrato.

Ethics declarations

Competing interests

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.

Peer review

Peer review information

Nature Neuroscience thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Validation of SYNGAP1 protein expression in RGCs in vitro and in vivo.

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.

Extended Data Fig. 2 Identification of SYNGAP1 protein isoform in hRGCs.

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.

Extended Data Fig. 3 Generation of patient derived and isogenic control cell lines.

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.

Extended Data Fig. 6 Organoids and Patient brain size.

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 information

Reporting Summary

Supplementary Tables 1–6

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.

Supplementary Table 7

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.

Supplementary Table 8

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.

Supplementary Table 9

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.

Supplementary Table 10

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.

Supplementary Video 1

Live imaging of corrected rosette formation from day 5 to day 7 after initial seeding.

Supplementary Video 2

Live imaging of SYNGAP1p.Q503X rosette formation from day 5 to day 7 after initial seeding.

Supplementary Video 3

Video recordings (10 min) of GCaMP6f corrected organoids.

Supplementary Video 4

Video recordings (10 min) of GCaMP6f SYNGAP1p.Q503X organoids.

Supplementary Video 5

Video recordings (10 min) of GCaMP6f 03231 organoids.

Supplementary Video 6

Video recordings (10 min) of GCaMP6f 03231p.Q503X organoids.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-023-01477-3

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing