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SHANK2 mutations associated with autism spectrum disorder cause hyperconnectivity of human neurons

Nature Neuroscience volume 22, pages 556–564 (2019)Cite this article

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Abstract

Heterozygous loss-of-function mutations in SHANK2 are associated with autism spectrum disorder (ASD). We generated cortical neurons from induced pluripotent stem cells derived from neurotypic and ASD-affected donors. We developed sparse coculture for connectivity assays where SHANK2 and control neurons were differentially labeled and sparsely seeded together on a lawn of unlabeled control neurons. We observed increases in dendrite length, dendrite complexity, synapse number, and frequency of spontaneous excitatory postsynaptic currents. These findings were phenocopied in gene-edited homozygous SHANK2 knockout cells and rescued by gene correction of an ASD SHANK2 mutation. Dendrite length increases were exacerbated by IGF1, TG003, or BDNF, and suppressed by DHPG treatment. The transcriptome in isogenic SHANK2 neurons was perturbed in synapse, plasticity, and neuronal morphogenesis gene sets and ASD gene modules, and activity-dependent dendrite extension was impaired. Our findings provide evidence for hyperconnectivity and altered transcriptome in SHANK2 neurons derived from ASD subjects.

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Fig. 1: SparCon assays of iPSC-derived SHANK2 ASD neurons compare marked mutant and control neurons seeded on the consistent synaptogenic environment of a lawn of unlabeled control or mutant neurons.
Fig. 2: Synapse numbers, dendrite length, and neuron complexity are enhanced in SHANK2 mutant neurons, and the length phenotype is exacerbated by treatment with IGF1 and BDNF.
Fig. 3: Excitatory synaptic function is enhanced in SHANK2 mutant neurons on control and mutant lawns.
Fig. 4: Deeply perturbed transcriptome and defective activity-dependent dendrite extension in R841X neurons.

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Code availability

The RNA-seq R scripts used to generate the figures in the manuscript are available in the Supplementary Software Zip file and at https://github.com/kzaslavsky/SparCon. The ‘GENERIC_SPARCON_ANALYSIS’ folder contains scripts for users to analyze their own coculture data. It performs within-well normalization, plotting, and statistical analysis. Sample data (excerpted from current study) are provided as outlined under Data availability.

Data availability

The whole genome sequence dataset used for off-target analysis can be accessed at EGA (EGAS00001003436). These raw data are associated with Fig. 1a, Supplementary Figs. 1–3, and Supplementary Tables 1–2. SparCon and dendrite extension datasets used to generate the figures in the manuscript are provided in the Supplementary Software Zip file and on GitHub (https://github.com/kzaslavsky/SparCon). These raw data are associated with Figs.1–4, Supplementary Figs. 7–15, and Supplementary Tables 3–9. The RNA-seq dataset can be accessed at GEO (GSE122550). These raw datasets are associated with Fig. 4a–d,f, Supplementary Fig. 16, and Supplementary Tables 10–12.

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Acknowledgements

This study was funded by grants from the National Institutes of Health (award no. R33MH087908 to J.E. and S.W.S.), the Ontario Brain Institute (J.E. and S.W.S.), the Canadian Institutes of Health Research (grant no. EPS-129129 to J.E.; nos. MOP-102649 and MOP-133423 to J.E. and M.W.S.), and the Simons Foundation/SFARI (grant no. 514918 to J.E.). We thank the MSSNG Open Science project for sharing data. Fellowship and studentship support: CIHR Canada Vanier Graduate Scholarship (K.Z.), MD/PhD studentships at the University of Toronto and McLaughlin Centre (K.Z.), CIHR Banting Fellowship (E.D.), Ontario Stem Cell Initiative Fellowship (P.J.R.), Ontario Ministry of Research & Innovation Fellowship (P.J.R.), and the International Rett Syndrome Foundation Fellowship (D.C.R.). S.W.S. is the GlaxoSmithKline–CIHR Endowed Chair in Genome Sciences at The Hospital for Sick Children. M.W.S. is the Northbridge Chair in Paediatric Research at the Hospital for Sick Children. We thank R. Yuen for comments regarding whole genome sequencing analysis of the two children in the study and J. Hicks for her technical help. We thank W. Roberts, R. Weksberg, B. Chung, and M. Carter for obtaining skin biopsies. We also thank the participants and their family members for their contributions to this study. We thank the Centre for Commercialization of Regenerative Medicine for in-kind access to equipment and project resources.

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Author notes

  1. P. Joel Ross

    Present address: Department of Biology, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada

  2. These authors contributed equally: Kirill Zaslavsky, Wen-Bo Zhang.

Authors and Affiliations

  1. Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada

    Kirill Zaslavsky, Fraser P. McCready, Deivid C. Rodrigues, Caitlin Loo, Melody Zhao, P. Joel Ross, Joelle El Hajjar, Asli Romm, Tadeo Thompson, Alina Piekna, Wei Wei, Marat Mufteev, Peter Pasceri & James Ellis

  2. Department of Molecular Genetics, The University of Toronto, Toronto, Ontario, Canada

    Kirill Zaslavsky, Fraser P. McCready, Marat Mufteev, Stephen W. Scherer & James Ellis

  3. Neuroscience & Mental Health Program, The Hospital for Sick Children, Toronto, Ontario, Canada

    Wen-Bo Zhang & Michael W. Salter

  4. Genetics & Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada

    Eric Deneault & Stephen W. Scherer

  5. The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada

    Eric Deneault, Zhuozhi Wang & Stephen W. Scherer

  6. The Centre for Commercialization of Regenerative Medicine, Toronto, Ontario, Canada

    Shahryar Khattak

  7. McLaughlin Centre, The University of Toronto, Toronto, Ontario, Canada

    Stephen W. Scherer

  8. Department of Physiology, University of Toronto, Toronto, Ontario, Canada

    Michael W. Salter

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Contributions

K.Z. and J.E. conceived the sparse-seeding coculture assay. T.T. generated iPS cells. P.P. performed teratoma assays. K.Z., A.R., A.P., and W.W. contributed to neuronal differentiation. E.D. and S.W.S. conceived the selection-free KO strategy and K.Z. isolated SHANK2 KO and R841X-C cells. P.J.R. cloned the CaMKII-mKO2 plasmid and characterized iPSC lines. K.Z. and D.C.R. performed western blots. K.Z., F.P.M., C.L., T.T., and M.Z. performed all immunocytochemical characterization of iPS cells, NPCs, and neurons. K.Z., F.P.M., C.L., M.Z., J.E.H., and S.K. performed synapse counting, morphological analyses, and live imaging. D.C.R., K.Z., F.P.M., and M.M. performed RNA-seq. W.Z. performed electrophysiological analyses. Z.W. performed WGS off-target analyses. K.Z., W.Z., F.P.M., M.W.S., and J.E. wrote the manuscript. P.J.R. helped edit the manuscript. K.Z., S.W.S., M.W.S., and J.E. supervised the project.

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Correspondence to Michael W. Salter or James Ellis.

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Journal peer review information: Nature Neuroscience thanks Kristen Brennand, Alysson Muotri, and Craig Powell for their contribution to the peer review of this work.

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Zaslavsky, K., Zhang, WB., McCready, F.P. et al. SHANK2 mutations associated with autism spectrum disorder cause hyperconnectivity of human neurons. Nat Neurosci 22, 556–564 (2019). https://doi.org/10.1038/s41593-019-0365-8

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