Abstract

Autism spectrum disorders (ASDs) are four times more common in males than in females, but the underlying mechanisms are poorly understood. We characterized sexually dimorphic changes in mice carrying a heterozygous mutation in Chd8 (Chd8+/N2373K) that was first identified in human CHD8 (Asn2373LysfsX2), a strong ASD-risk gene that encodes a chromatin remodeler. Notably, although male mutant mice displayed a range of abnormal behaviors during pup, juvenile, and adult stages, including enhanced mother-seeking ultrasonic vocalization, enhanced attachment to reunited mothers, and isolation-induced self-grooming, their female counterparts do not. This behavioral divergence was associated with sexually dimorphic changes in neuronal activity, synaptic transmission, and transcriptomic profiles. Specifically, female mice displayed suppressed baseline neuronal excitation, enhanced inhibitory synaptic transmission and neuronal firing, and increased expression of genes associated with extracellular vesicles and the extracellular matrix. Our results suggest that a human CHD8 mutation leads to sexually dimorphic changes ranging from transcription to behavior in mice.

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References

  1. 1.

    Elsabbagh, M. et al. Global prevalence of autism and other pervasive developmental disorders. Autism Res. 5, 160–179 (2012).

  2. 2.

    Südhof, T. C. Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455, 903–911 (2008).

  3. 3.

    Zoghbi, H. Y. & Bear, M. F. Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities. Cold Spring Harb. Perspect. Biol. 4, a009886 (2012).

  4. 4.

    Jiang, Y. H. & Ehlers, M. D. Modeling autism by SHANK gene mutations in mice. Neuron 78, 8–27 (2013).

  5. 5.

    Bourgeron, T. From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat. Rev. Neurosci. 16, 551–563 (2015).

  6. 6.

    de la Torre-Ubieta, L., Won, H., Stein, J. L. & Geschwind, D. H. Advancing the understanding of autism disease mechanisms through genetics. Nat. Med. 22, 345–361 (2016).

  7. 7.

    Krumm, N., O’Roak, B. J., Shendure, J. & Eichler, E. E. A de novo convergence of autism genetics and molecular neuroscience. Trends Neurosci. 37, 95–105 (2014).

  8. 8.

    Ronemus, M., Iossifov, I., Levy, D. & Wigler, M. The role of de novo mutations in the genetics of autism spectrum disorders. Nat. Rev. Genet. 15, 133–141 (2014).

  9. 9.

    Werling, D. M., Parikshak, N. N. & Geschwind, D. H. Gene expression in human brain implicates sexually dimorphic pathways in autism spectrum disorders. Nat. Commun. 7, 10717 (2016).

  10. 10.

    Werling, D. M. & Geschwind, D. H. Recurrence rates provide evidence for sex-differential, familial genetic liability for autism spectrum disorders in multiplex families and twins. Mol. Autism 6, 27 (2015).

  11. 11.

    Werling, D. M., Lowe, J. K., Luo, R., Cantor, R. M. & Geschwind, D. H. Replication of linkage at chromosome 20p13 and identification of suggestive sex-differential risk loci for autism spectrum disorder. Mol. Autism 5, 13 (2014).

  12. 12.

    Werling, D. M. & Geschwind, D. H. Sex differences in autism spectrum disorders. Curr. Opin. Neurol. 26, 146–153 (2013).

  13. 13.

    Werling, D. M. & Geschwind, D. H. Understanding sex bias in autism spectrum disorder. Proc. Natl Acad. Sci. USA 110, 4868–4869 (2013).

  14. 14.

    Robinson, E. B., Lichtenstein, P., Anckarsäter, H., Happé, F. & Ronald, A. Examining and interpreting the female protective effect against autistic behavior. Proc. Natl Acad. Sci. USA 110, 5258–5262 (2013).

  15. 15.

    Lu, A. T. & Cantor, R. M. Allowing for sex differences increases power in a GWAS of multiplex Autism families. Mol. Psychiatry 17, 215–222 (2012).

  16. 16.

    Sato, D. et al. SHANK1 deletions in males with autism spectrum disorder. Am. J. Hum. Genet. 90, 879–887 (2012).

  17. 17.

    Lo, S. C., Scearce-Levie, K. & Sheng, M. Characterization of social behaviors in caspase-3 deficient mice. Sci. Rep. 6, 18335 (2016).

  18. 18.

    Barak, B. & Feng, G. Neurobiology of social behavior abnormalities in autism and Williams syndrome. Nat. Neurosci. 19, 647–655 (2016).

  19. 19.

    Sakamoto, I. et al. A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation. J. Biol. Chem. 275, 32871–32878 (2000).

  20. 20.

    Barnard, R. A., Pomaville, M. B. & O’Roak, B. J. Mutations and modeling of the chromatin remodeler CHD8 define an emerging autism etiology. Front. Neurosci. 9, 477 (2015).

  21. 21.

    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).

  22. 22.

    Cotney, J. et al. The autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment. Nat. Commun. 6, 6404 (2015).

  23. 23.

    Wilkinson, B. et al. The autism-associated gene chromodomain helicase DNA-binding protein 8 (CHD8) regulates noncoding RNAs and autism-related genes. Transl. Psychiatry 5, e568 (2015).

  24. 24.

    Katayama, Y. et al. CHD8 haploinsufficiency results in autistic-like phenotypes in mice. Nature 537, 675–679 (2016).

  25. 25.

    Durak, O. et al. Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling. Nat. Neurosci. 19, 1477–1488 (2016).

  26. 26.

    Gompers, A. L. et al. Germline Chd8 haploinsufficiency alters brain development in mouse. Nat. Neurosci. 20, 1062–1073 (2017).

  27. 27.

    Platt, R. J. et al. Chd8 mutation leads to autistic-like behaviors and impaired striatal circuits. Cell Rep. 19, 335–350 (2017).

  28. 28.

    Stessman, H. A. et al. Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases. Nat. Genet. 49, 515–526 (2017).

  29. 29.

    Nishiyama, M. et al. Early embryonic death in mice lacking the beta-catenin-binding protein Duplin. Mol. Cell. Biol. 24, 8386–8394 (2004).

  30. 30.

    Suetterlin, P. et al. Altered neocortical gene expression, brain overgrowth and functional over-connectivity in Chd8 haploinsufficient mice. Cereb. Cortex 28, 2192–2206 (2018).

  31. 31.

    Bernier, R. et al. Disruptive CHD8 mutations define a subtype of autism early in development. Cell 158, 263–276 (2014).

  32. 32.

    O’Roak, B. J. et al. Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science 338, 1619–1622 (2012).

  33. 33.

    Merner, N. et al. A de novo frameshift mutation in chromodomain helicase DNA-binding domain 8 (CHD8): a case report and literature review. Am. J. Med. Genet. A. 170A, 1225–1235 (2016).

  34. 34.

    Colombo, M., Raposo, G. & Théry, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30, 255–289 (2014).

  35. 35.

    Porro, C., Trotta, T. & Panaro, M. A. Microvesicles in the brain: biomarker, messenger or mediator? J. Neuroimmunol. 288, 70–78 (2015).

  36. 36.

    Serra-Millàs, M. Are the changes in the peripheral brain-derived neurotrophic factor levels due to platelet activation? World J. Psychiatry 6, 84–101 (2016).

  37. 37.

    Song, I. & Dityatev, A. Crosstalk between glia, extracellular matrix and neurons. Brain Res. Bull. 136, 101–108 (2018).

  38. 38.

    Frischknecht, R., Chang, K. J., Rasband, M. N. & Seidenbecher, C. I. Neural ECM molecules in axonal and synaptic homeostatic plasticity. Prog. Brain Res. 214, 81–100 (2014).

  39. 39.

    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).

  40. 40.

    Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011).

  41. 41.

    White, S. W., Oswald, D., Ollendick, T. & Scahill, L. Anxiety in children and adolescents with autism spectrum disorders. Clin. Psychol. Rev. 29, 216–229 (2009).

  42. 42.

    Yizhar, O. et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011).

  43. 43.

    Selimbeyoglu, A. et al. Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice. Sci. Transl. Med. 9, eaah6733 (2017).

  44. 44.

    Cao, W. et al. Gamma oscillation dysfunction in mPFC leads to social deficits in Neuroligin 3 R451C knockin mice. Neuron 97, 1253–1260.e7 (2018).

  45. 45.

    Risher, W. C. & Eroglu, C. Thrombospondins as key regulators of synaptogenesis in the central nervous system. Matrix Biol. 31, 170–177 (2012).

  46. 46.

    Stryker, E. & Johnson, K. G. LAR, liprin alpha and the regulation of active zone morphogenesis. J. Cell Sci. 120, 3723–3728 (2007).

  47. 47.

    Zhao, X. et al. A unified genetic theory for sporadic and inherited autism. Proc. Natl Acad. Sci. USA 104, 12831–12836 (2007).

  48. 48.

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

  49. 49.

    Sanders, S. J. et al. Insights into autism spectrum disorder genomic architecture and biology from 71 risk loci. Neuron 87, 1215–1233 (2015).

  50. 50.

    Jacquemont, S. et al. A higher mutational burden in females supports a ‘female protective model’ in neurodevelopmental disorders. Am. J. Hum. Genet. 94, 415–425 (2014).

  51. 51.

    Mo, J. et al. Early growth response 1 (Egr-1) directly regulates GABAA receptor α2, α4, and θ subunits in the hippocampus. J. Neurochem. 133, 489–500 (2015).

  52. 52.

    Huttner, W. B., Schiebler, W., Greengard, P. & De Camilli, P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J. Cell Biol. 96, 1374–1388 (1983).

  53. 53.

    Kim, M. H. et al. Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation, and impaired learning and memory in mice lacking IRSp53. J. Neurosci. 29, 1586–1595 (2009).

  54. 54.

    Mok, H. et al. Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins. J. Neurosci. 22, 5253–5258 (2002).

  55. 55.

    Lerch, J. P., Sled, J. G. & Henkelman, R. M. MRI phenotyping of genetically altered mice. Methods Mol. Biol. 711, 349–361 (2011).

  56. 56.

    Dorr, A. E., Lerch, J. P., Spring, S., Kabani, N. & Henkelman, R. M. High resolution three-dimensional brain atlas using an average magnetic resonance image of 40 adult C57Bl/6J mice. Neuroimage 42, 60–69 (2008).

  57. 57.

    Steadman, P. E. et al. Genetic effects on cerebellar structure across mouse models of autism using a magnetic resonance imaging atlas. Autism Res. 7, 124–137 (2014).

  58. 58.

    Ullmann, J. F., Watson, C., Janke, A. L., Kurniawan, N. D. & Reutens, D. C. A segmentation protocol and MRI atlas of the C57BL/6J mouse neocortex. Neuroimage 78, 196–203 (2013).

  59. 59.

    Silverman, J. L., Yang, M., Lord, C. & Crawley, J. N. Behavioral phenotyping assays for mouse models of autism. Nat. Rev. Neurosci. 11, 490–502 (2010).

  60. 60.

    Chung, W. et al. Social deficits in IRSp53 mutant mice improved by NMDAR and mGluR5 suppression. Nat. Neurosci. 18, 435–443 (2015).

  61. 61.

    Jamain, S. et al. Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc. Natl Acad. Sci. USA 105, 1710–1715 (2008).

  62. 62.

    McFarlane, H. G. et al. Autism-like behavioral phenotypes in BTBR T+tf/J mice. Genes Brain Behav. 7, 152–163 (2008).

  63. 63.

    Long, J. M., LaPorte, P., Paylor, R. & Wynshaw-Boris, A. Expanded characterization of the social interaction abnormalities in mice lacking Dvl1. Genes Brain Behav. 3, 51–62 (2004).

  64. 64.

    Zhan, Y. et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nat. Neurosci. 17, 400–406 (2014).

  65. 65.

    Deacon, R. M. Digging and marble burying in mice: simple methods for in vivo identification of biological impacts. Nat. Protoc. 1, 122–124 (2006).

  66. 66.

    Yang, M. & Crawley, J. N. Simple behavioral assessment of mouse olfaction. Curr. Protoc. Neurosci. 48, 8.24 (2009).

  67. 67.

    Clapcote, S.J. & Roder, J.C. Simplex PCR assay for sex determination in mice. Biotechniques https://doi.org/10.2144/05385BM05 (2005).

  68. 68.

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

  69. 69.

    Anders, S., Pyl, P. T. & Huber, W. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

  70. 70.

    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).

  71. 71.

    Razick, S., Magklaras, G. & Donaldson, I. M. iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics 9, 405 (2008).

  72. 72.

    Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

  73. 73.

    Shah, H. How to calculate the sample size for animal studies? Natl J. Physiol. Pharm. Pharmacol 1, 35–39 (2011).

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Acknowledgements

This work was supported by the NRF Grant 2013M3C7A1056732 (to H.K.), the Future Systems Healthcare Project of KAIST (to S.P.), KISTI K-16-L03-C02-S01 (to H. Kang), and the Institute for Basic Science (IBS-R002-D1 to E.K.).

Author information

Author notes

  1. These authors contributed equally: Hwajin Jung, Haram Park.

Affiliations

  1. Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon, Korea

    • Hwajin Jung
    • , Eunee Lee
    • , Issac Rhim
    • , Su-Yeon Choi
    • , Mihyun Bae
    • , Sun-Gyun Kim
    • , Jiseok Lee
    •  & Eunjoon Kim
  2. Department of Biological Sciences, Korea Advanced Institute for Science and Technology (KAIST), Daejeon, Korea

    • Haram Park
    • , Yeonsoo Choi
    • , Hanseul Kweon
    • , Junyeop Daniel Roh
    • , Jaeseung Kang
    • , Changuk Chung
    • , Taesun Yoo
    • , Seungmin Ha
    • , Seung Min Um
    • , Yonghan Kwon
    •  & Eunjoon Kim
  3. Supercomputing Center, KISTI, Daejeon, Korea

    • Hyojin Kang
  4. Mouse Imaging Centre, Hospital for Sick Children, Toronto, Ontario, Canada

    • Jacob Ellegood
    •  & Jason P Lerch
  5. Program of Brain and Cognitive Engineering, Department of Bio and Brain Engineering, KAIST, Daejeon, Korea

    • Woochul Choi
    •  & Se-Bum Paik
  6. Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea

    • Hanwool Park
    •  & Yangsik Kim
  7. Department of Anatomy and Division of Brain Korea 21, Biomedical Science, College of Medicine, Korea University, Seoul, Korea

    • Seojung Mo
    •  & Hyun Kim
  8. Department of Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea

    • Won Mah
    •  & Yong Chul Bae
  9. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    • Jason P Lerch

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Contributions

W.M. designed the mouse knockout strategy. S.M. conducted in situ hybridization experiments. J.E. and J.P.L. performed MRI and DTI experiments and analysis. Y.C. and H. Kweon conducted immunohistochemistry experiments. Haram Park, H.J., Y.C., J.D.R., H. Kweon., C.C., S.H., T.Y., Hanwool Park, S.U., and S.K. conducted slice electrophysiology experiments. E.L., I.Y., W.C., and J.L. performed in vivo recording experiments and analysis. H.J., Haram Park, Y.C., J.K., M.B., S.C., S.K., Y. Kim., H. Kweon, and Y. Kwon conducted mouse behavioral experiments. Haram Park, H.J., H. Kweon, and Y.C. conducted all of the other experiments. H. Kang performed RNA transcript analysis. Y.C.B., H.K., S.P., and E.K. supervised the project and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Eunjoon Kim.

Supplementary Information

  1. Supplementary Text and Figures

    Supplementary Figures 1–21

  2. Reporting Summary

  3. Supplementary Table 1

    Summary of MRI and DTI analysis of brains from male and female, WT and Chd8+/N2373K mice at 4 weeks.

  4. Supplementary Table 2

    Summary of c-fos expression analyses of brains from male and female, WT and Chd8+/N2373K mice at P20.

  5. Supplementary Table 3

    All RNA-Seq data from male and female, WT and Chd8+/N2373K mice at P0 and P25 and details on all HT/WT DEGs.

  6. Supplementary Table 4

    Full GO annotations for DEGs.

  7. Supplementary Table 5

    GSEA results for C5, ASD-related, cell type-specific, and IEG-related, C2, and C3 gene sets.

  8. Supplementary Table 6

    Statistical analyses.

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DOI

https://doi.org/10.1038/s41593-018-0208-z

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