SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients

Abstract

Phelan–McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs)1. PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD)2,3. Rare mutations in SHANK3 have been associated with idiopathic ASDs4,5,6,7, non-syndromic intellectual disability8, and schizophrenia9. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients10, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-d-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.

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Figure 1: CaMKIIα-GFP labels functional iPS cell-derived forebrain neurons that express SHANK3.
Figure 2: PMDS neurons display impaired excitatory synaptic transmission.
Figure 3: PMDS neurons show reduced expression of glutamate receptors and decreased number of synapses.
Figure 4: Reduced SHANK3 expression contributes to synaptic defects in PMDS neurons.
Figure 5: IGF1 treatment restores excitatory synaptic transmission in PMDS neurons.

References

  1. 1

    Phelan, K. & McDermid, H. E. The 22q13.3 deletion syndrome (Phelan-McDermid syndrome). Mol. Syndromol. 2, 186–201 (2012)

    CAS  PubMed  Google Scholar 

  2. 2

    Boeckers, T. M., Bockmann, J., Kreutz, M. R. & Gundelfinger, E. D. ProSAP/Shank proteins – a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease. J. Neurochem. 81, 903–910 (2002)

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  PubMed  Google Scholar 

  4. 4

    Boccuto, L. et al. Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders. Eur. J. Hum. Genet. 21, 310–316 (2013)

    CAS  Article  Google Scholar 

  5. 5

    Durand, C. M. et al. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nature Genet. 39, 25–27 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Gauthier, J. et al. Novel de novo SHANK3 mutation in autistic patients. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 150B, 421–424 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Moessner, R. et al. Contribution of SHANK3 mutations to autism spectrum disorder. Am. J. Hum. Genet. 81, 1289–1297 (2007)

    CAS  Article  Google Scholar 

  8. 8

    Hamdan, F. F. et al. Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability. Am. J. Hum. Genet. 88, 306–316 (2011)

    CAS  Article  Google Scholar 

  9. 9

    Gauthier, J. et al. De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia. Proc. Natl Acad. Sci. USA 107, 7863–7868 (2010)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Wilson, H. L. et al. Molecular characterisation of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms. J. Med. Genet. 40, 575–584 (2003)

    CAS  Article  Google Scholar 

  11. 11

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)

    CAS  Article  Google Scholar 

  12. 12

    Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230–234 (2011)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Paşca, S. P. et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nature Med. 17, 1657–1662 (2011)

    Article  Google Scholar 

  14. 14

    Yoo, A. S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231 (2011)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl Acad. Sci. USA 101, 18206–18211 (2004)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Nathanson, J. L., Yanagawa, Y., Obata, K. & Callaway, E. M. Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors. Neuroscience 161, 441–450 (2009)

    CAS  Article  Google Scholar 

  17. 17

    Akhtar, M. W. et al. Histone deacetylases 1 and 2 form a developmental switch that controls excitatory synapse maturation and function. J. Neurosci. 29, 8288–8297 (2009)

    CAS  Article  Google Scholar 

  18. 18

    Maunakea, A. K. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Rinaldi, T., Kulangara, K., Antoniello, K. & Markram, H. Elevated NMDA receptor levels and enhanced postsynaptic long-term potentiation induced by prenatal exposure to valproic acid. Proc. Natl Acad. Sci. USA 104, 13501–13506 (2007)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Goold, C. P. & Nicoll, R. A. Single-cell optogenetic excitation drives homeostatic synaptic depression. Neuron 68, 512–528 (2010)

    CAS  Article  Google Scholar 

  21. 21

    Marchetto, M. C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539 (2010)

    CAS  Article  Google Scholar 

  22. 22

    O’Kusky, J. R., Ye, P. & D’Ercole, A. J. Insulin-like growth factor-I promotes neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development. J. Neurosci. 20, 8435–8442 (2000)

    Article  Google Scholar 

  23. 23

    Tropea, D. et al. Partial reversal of Rett syndrome-like symptoms in MeCP2 mutant mice. Proc. Natl Acad. Sci. USA 106, 2029–2034 (2009)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Chen, D. Y. et al. A critical role for IGF-II in memory consolidation and enhancement. Nature 469, 491–497 (2011)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Bozdagi, O. et al. Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication. Mol. Autism 1, 15 (2010)

    CAS  Article  Google Scholar 

  26. 26

    Peça, J. et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437–442 (2011)

    ADS  Article  Google Scholar 

  27. 27

    Wang, X. et al. Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum. Mol. Genet. 20, 3093–3108 (2011)

    CAS  Article  Google Scholar 

  28. 28

    Paoletti, P., Bellone, C. & Zhou, Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nature Rev. Neurosci. 14, 383–400 (2013)

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

    Gaspard, N. et al. Generation of cortical neurons from mouse embryonic stem cells. Nature Protocols 4, 1454–1463 (2009)

    CAS  Article  Google Scholar 

  31. 31

    Bellone, C. & Nicoll, R. A. Rapid bidirectional switching of synaptic NMDA receptors. Neuron 55, 779–785 (2007)

    CAS  Article  Google Scholar 

  32. 32

    Sommer, C. A. et al. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27, 543–549 (2009)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to participants and their families for their support; M. Adam for assistance with recruitment; to X. Jia, A. Cherry, C. Bangs, P. Jones, and J. Williams for assistance with tissue culture; P. Liao for help with multiplex ligation-dependent probe amplification (MLPA); M. Fabian for astrocyte preparations; H.N. Nguyen for consultations on the neural differentiation protocol and spectral karyotyping (SKY); V. Vu and G. Lin for help with data analysis; T. Sudhof, T. Boeckers, A. Grabruker, C. Garner and C. Sala for antibodies; R. Xavier for SHANK3 complementary DNA; R. Reijo-Pera and members of the Dolmetsch laboratory for commenting on the manuscript; E. Nigh for editing the manuscript. We also thank the Stanford Neuroscience Microscopy Service (supported by National Institutes of Health (NIH) NS069375). Support for this study came from the California Institute for Regenerative Medicine CIRM, the Autism Science Foundation and the Phelan-McDermid Syndrome Foundation (to A.S.), the Swiss National Science Foundation (to T.P.), the Japan Society for the Promotion of Research Abroad and American Heart Association (to M.Y.), the National Institute of Mental Health (NIMH) grant R33MH087898 (to J.F.H.); NIH Pioneer Award (5DP1OD3889), CIRM (grant RT2-01906) and Simons Foundation (to R.E.D.). We are also grateful for funding from the JDH research fund, N. Juaw, B. and F. Horowitz, M. McCafferey, B. and J. Packard, P. Kwan and K. Wang, and the Flora foundation.

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A.S. and R.E.D. designed experiments and wrote the manuscript; A.S. performed iPS cell maintenance, neural differentiation, electrophysiology, cloning and immunocytochemistry; O.S. maintained and characterized iPS cells, performed western blot and qRT–PCR; M.Y. generated and characterized iPS cells; T.P. performed multiplex single-cell qRT–PCR; V.S. performed teratoma assay; R.S. and A.K. performed qRT–PCR and data analysis; W.F., J.A.B. and J.F.H. recruited and characterized patients and performed the MLPA assay.

Corresponding author

Correspondence to Ricardo E. Dolmetsch.

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The authors declare no competing financial interests.

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Shcheglovitov, A., Shcheglovitova, O., Yazawa, M. et al. SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients. Nature 503, 267–271 (2013). https://doi.org/10.1038/nature12618

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