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

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.

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. 13, and Supplementary Tables 12. 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.14, Supplementary Figs. 715, and Supplementary Tables 39. 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 1012.

References

  1. 1.

    Djuric, U. et al. MECP2e1 isoform mutation affects the form and function of neurons derived from Rett syndrome patient iPS cells. Neurobiol. Dis. 76, 37–45 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Pak, C. et al. Human neuropsychiatric disease modeling using conditional deletion reveals synaptic transmission defects caused by heterozygous mutations in NRXN1. Cell Stem Cell 17, 316–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Yi, F. et al. Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons. Science 352, aaf2669 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Shcheglovitov, A. et al. SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients. Nature 503, 267–271 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Griesi-Oliveira, K. et al. Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons. Mol. Psychiatry 20, 1350–1365 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Brennand, K. J. et al. Creating patient-specific neural cells for the in vitro study of brain disorders. Stem Cell Rep. 5, 933–945 (2015).

    Article  Google Scholar 

  7. 7.

    Sandoe, J. & Eggan, K. Opportunities and challenges of pluripotent stem cell neurodegenerative disease models. Nat. Neurosci. 16, 780–789 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Berkel, S. et al. Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation. Nat. Genet. 42, 489–491 (2010).

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    C Yuen, R. K. et al. Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder. Nat. Neurosci. 20, 602–611 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Deneault, E. et al. Complete disruption of autism-susceptibility genes by gene editing predominantly reduces functional connectivity of isogenic human neurons. Stem Cell Rep. 11, 1211–1225 (2018).

    Article  CAS  Google Scholar 

  11. 11.

    Rodrigues, D. C. et al. MECP2 is post-transcriptionally regulated during human neurodevelopment by combinatorial action of RNA-binding proteins and miRNAs. Cell Rep. 17, 720–734 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Johnson, M. A., Weick, J. P., Pearce, R. A. & Zhang, S.-C. Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture. J. Neurosci. 27, 3069–3077 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Gupta, K., Hardingham, G. E. & Chandran, S. NMDA receptor-dependent glutamate excitotoxicity in human embryonic stem cell-derived neurons. Neurosci. Lett. 543, 95–100 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Nehme, R. et al. Combining NGN2 programming with developmental patterning generates human excitatory neurons with NMDAR-mediated synaptic transmission. Cell Rep. 23, 2509–2523 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Micheva, K. D., Busse, B., Weiler, N. C., O’Rourke, N. & Smith, S. J. Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639–653 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Berkel, S. et al. Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology. Hum. Mol. Genet. 21, 344–357 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Bidinosti, M. et al. CLK2 inhibition ameliorates autistic features associated with SHANK3 deficiency. Science 351, 1199–1203 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    Tyler, W. J. & Pozzo-Miller, L. D. BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses. J. Neurosci. 21, 4249–4258 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Djuric, U. et al. Spatiotemporal proteomic profiling of human cerebral development. Mol. Cell. Proteomics 16, 1548–1562 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  Google Scholar 

  21. 21.

    Pruunsild, P., Bengtson, C. P. & Bading, H. Networks of cultured iPSC-derived neurons reveal the human synaptic activity-regulated adaptive gene program. Cell Rep. 18, 122–135 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Parikshak, N. N. et al. Integrative functional genomic analyses implicate specific molecular pathways and circuits in autism. Cell 155, 1008–1021 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Willsey, A. J. et al. Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism. Cell 155, 997–1007 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Du, Y., Weed, S. A., Xiong, W. C., Marshall, T. D. & Parsons, J. T. Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons. Mol. Cell. Biol. 18, 5838–5851 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Vessey, J. P. & Karra, D. More than just synaptic building blocks: scaffolding proteins of the post-synaptic density regulate dendritic patterning. J. Neurochem. 102, 324–332 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. 27.

    Kirwan, P. et al. Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro. Development 142, 3178–3187 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Zhang, Y. et al. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78, 785–798 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    DeRosa, B. A. et al. hVGAT-mCherry: a novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells. Mol. Cell. Neurosci. 68, 244–257 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Bardy, C. et al. Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology. Mol. Psychiatry 21, 1573–1588 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Schmeisser, M. J. et al. Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2. Nature 486, 256–260 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Won, H. et al. Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function. Nature 486, 261–265 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Wegener, S. et al. Defective synapse maturation and enhanced synaptic plasticity in Shank2 Δex7−/− mice. eNeuro 5, ENEURO.0398–17.2018 (2018).

  34. 34.

    Grabrucker, A. M. et al. Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation. EMBO J. 30, 569–581 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Modi, M. E. et al. Hyperactivity and hypermotivation associated with increased striatal mGluR1 signaling in a Shank2 rat model ofautism. Front. Mol. Neurosci. 11, 107 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Peixoto, R. T., Wang, W., Croney, D. M., Kozorovitskiy, Y. & Sabatini, B. L. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B(-/-) mice. Nat. Neurosci. 19, 716–724 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Eltokhi, A., Rappold, G. & Sprengel, R. Distinct phenotypes of Shank2 mouse models reflect neuropsychiatric spectrum disorders of human patients with SHANK2 variants. Front. Mol. Neurosci. 11, 240 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Leblond, C. S. et al. Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders. PLoS Genet. 8, e1002521 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Leblond, C. S. et al. Meta-analysis of SHANK mutations in autism spectrum disorders: a gradient of severity in cognitive impairments. PLoS Genet. 10, e1004580 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Sheng, M. & Kim, E. The Shank family of scaffold proteins. J. Cell Sci. 113, 1851–1856 (2000).

    CAS  PubMed  Google Scholar 

  41. 41.

    Santini, E. & Klann, E. Reciprocal signaling between translational control pathways and synaptic proteins in autism spectrum disorders. Sci. Signal. 7, re10 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Testa-Silva, G. et al. Hyperconnectivity and slow synapses during early development of medial prefrontal cortex in a mouse model for mental retardation and autism. Cereb. Cortex 22, 1333–1342 (2012).

    Article  PubMed  Google Scholar 

  43. 43.

    Cline, H. & Haas, K. The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J. Physiol. (Lond.) 586, 1509–1517 (2008).

    Article  CAS  Google Scholar 

  44. 44.

    Hasegawa, S., Sakuragi, S., Tominaga-Yoshino, K. & Ogura, A. Dendritic spine dynamics leading to spine elimination after repeated inductions of LTD. Sci. Rep. 5, 7707 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Sugathan, A. et al. CHD8 regulates neurodevelopmental pathways associated with autism spectrum disorder in neural progenitors. Proc. Natl Acad. Sci. USA 111, E4468–E4477 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. 46.

    Tang, G. et al. Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits. Neuron 83, 1131–1143 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Luo, T. et al. Effect of the autism-associated lncRNA Shank2-AS on architecture and growth of neurons. J. Cell. Biochem. 57, 19 (2018).

    Google Scholar 

  48. 48.

    Deneault, E. et al. CNTN5 -/+ or EHMT2 -/+ iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks. eLife 8, e40092 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Hotta, A. et al. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nat. Meth. 6, 370–376 (2009).

    Article  CAS  Google Scholar 

  50. 50.

    Cheung, A. Y. L. et al. Isolation of MECP2-null Rett syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum. Mol. Genet. 20, 2103–2115 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Miyaoka, Y. et al. Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat. Meth.11, 291–293 (2014).

    Article  CAS  Google Scholar 

  52. 52.

    Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    McKenna, A. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Maroof, A. M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Brennand, K. J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473, 221–225 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Kim, H. J. & Magrané, J. Isolation and culture of neurons and astrocytes from the mouse brain cortex. Methods Mol. Biol. 793, 63–75 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. 58.

    Mali, P. gRNA synthesis protocol. Addgene.org https://www.addgene.org/static/data/93/40/adf4a4fe-5e77-11e2-9c30-003048dd6500.pdf (2013)..

  59. 59.

    Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Otto, E., Zalewski, C., Kaloss, M. & Del Giudice, R. A. Quantitative detection of cell culture Mycoplasmas by a one step polymerase chain reaction method. Methods Cell Sci. 18, 261–268 (1996).

    Article  Google Scholar 

  61. 61.

    Kwiatkowski, A. V. et al. Ena/VASP is required for neuritogenesis in the developing cortex. Neuron 56, 441–455 (2007).

    Article  CAS  PubMed  Google Scholar 

  62. 62.

    Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Meth. 12, 357–360 (2015).

    Article  CAS  Google Scholar 

  63. 63.

    Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  Google Scholar 

  64. 64.

    Patro, R., Mount, S. M. & Kingsford, C. Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms. Nat. Biotechnol. 32, 462–464 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Bader, G. et al. EM Genesets. BaderLab http://download.baderlab.org/EM_Genesets (2018).

  67. 67.

    Cline, M. S. et al. Integration of biological networks and gene expression data using Cytoscape. Nat. Protoc. 2, 2366–2382 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Merico, D., Isserlin, R., Stueker, O., Emili, A. & Bader, G. D. Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One 5, e13984 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Kucera, M., Isserlin, R., Arkhangorodsky, A. & Bader, G. D. AutoAnnotate: a Cytoscape app for summarizing networks with semantic annotations. F1000Res. 5, 1717 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Zhang, W.-B. et al. Fyn kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons. Sci. Rep. 6, 23837 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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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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Zaslavsky, K., Zhang, W., 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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