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.

CNTNAP2 stabilizes interneuron dendritic arbors through CASK

Abstract

Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated-emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2’s C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching in particular cortical regions, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, Wu C. Interneurons of the neocortical inhibitory system. Nat Rev Neurosci. 2004;5:793–807.

    CAS  Article  Google Scholar 

  2. 2.

    Marin O. Interneuron dysfunction in psychiatric disorders. Nat Rev Neurosci. 2012;13:107–20.

    CAS  Article  Google Scholar 

  3. 3.

    Gao R, Penzes P. Common mechanisms of excitatory and inhibitory imbalance in schizophrenia and autism spectrum disorders. Curr Mol Med. 2015;15:146–67.

    CAS  Article  Google Scholar 

  4. 4.

    Srivastava DP, Copits BA, Xie Z, Huda R, Jones KA, Mukherji S, et al. Afadin is required for maintenance of dendritic structure and excitatory tone. J Biol Chem. 2012;287:35964–74.

    CAS  Article  Google Scholar 

  5. 5.

    Kulkarni VA, Firestein BL. The dendritic tree and brain disorders. Mol Cell Neurosci. 2012;50:10–20.

    CAS  Article  Google Scholar 

  6. 6.

    Kalus P, Bondzio J, Federspiel A, Muller TJ, Zuschratter W. Cell-type specific alterations of cortical interneurons in schizophrenic patients. Neuroreport. 2002;13:713–7.

    Article  Google Scholar 

  7. 7.

    Magloczky Z, Wittner L, Borhegyi Z, Halasz P, Vajda J, Czirjak S, et al. Changes in the distribution and connectivity of interneurons in the epileptic human dentate gyrus. Neuroscience. 2000;96:7–25.

    CAS  Article  Google Scholar 

  8. 8.

    Redolfi N, Galla L, Maset A, Murru L, Savoia E, Zamparo I, et al. Oligophrenin-1 regulates number, morphology and synaptic properties of adult-born inhibitory interneurons in the olfactory bulb. Hum Mol Genet. 2016;25:5198–211.

    CAS  PubMed  Google Scholar 

  9. 9.

    Cahill ME, Jones KA, Rafalovich I, Xie Z, Barros CS, Muller U, et al. Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition. Mol Psychiatry. 2012;17:99–107.

    CAS  Article  Google Scholar 

  10. 10.

    Cobos I, Calcagnotto ME, Vilaythong AJ, Thwin MT, Noebels JL, Baraban SC, et al. Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy. Nat Neurosci. 2005;8:1059–68.

    CAS  Article  Google Scholar 

  11. 11.

    Gomez-Climent MA, Guirado R, Castillo-Gomez E, Varea E, Gutierrez-Mecinas M, Gilabert-Juan J, et al. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is expressed in a subpopulation of mature cortical interneurons characterized by reduced structural features and connectivity. Cereb Cortex. 2011;21:1028–41.

    Article  Google Scholar 

  12. 12.

    Wirth MJ, Brun A, Grabert J, Patz S, Wahle P. Accelerated dendritic development of rat cortical pyramidal cells and interneurons after biolistic transfection with BDNF and NT4/5. Development. 2003;130:5827–38.

    CAS  Article  Google Scholar 

  13. 13.

    Zweier C, de Jong EK, Zweier M, Orrico A, Ousager LB, Collins AL, et al. CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila. Am J Hum Genet. 2009;85:655–66.

    CAS  Article  Google Scholar 

  14. 14.

    Sehested LT, Moller RS, Bache I, Andersen NB, Ullmann R, Tommerup N, et al. Deletion of 7q34-q36.2 in two siblings with mental retardation, language delay, primary amenorrhea, and dysmorphic features. Am J Med Genet A. 2010;152A:3115–9.

    Article  Google Scholar 

  15. 15.

    Bakkaloglu B, O’Roak BJ, Louvi A, Gupta AR, Abelson JF, Morgan TM, et al. Molecular cytogenetic analysis and resequencing of contactin associated protein-like 2 in autism spectrum disorders. Am J Hum Genet. 2008;82:165–73.

    CAS  Article  Google Scholar 

  16. 16.

    Rossi E, Verri AP, Patricelli MG, Destefani V, Ricca I, Vetro A, et al. A 12Mb deletion at 7q33-q35 associated with autism spectrum disorders and primary amenorrhea. Eur J Med Genet. 2008;51:631–8.

    Article  Google Scholar 

  17. 17.

    Alarcon M, Abrahams BS, Stone JL, Duvall JA, Perederiy JV, Bomar JM, et al. Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am J Hum Genet. 2008;82:150–9.

    CAS  Article  Google Scholar 

  18. 18.

    O’Roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, Girirajan S, et al. Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet. 2011;43:585–9.

    Article  Google Scholar 

  19. 19.

    Anney R, Klei L, Pinto D, Almeida J, Bacchelli E, Baird G, et al. Individual common variants exert weak effects on the risk for autism spectrum disorders. Hum Mol Genet. 2012;21:4781–92.

    CAS  Article  Google Scholar 

  20. 20.

    Ji W, Li T, Pan Y, Tao H, Ju K, Wen Z, et al. CNTNAP2 is significantly associated with schizophrenia and major depression in the Han Chinese population. Psychiatry Res. 2013;207:225–8.

    CAS  Article  Google Scholar 

  21. 21.

    Friedman JI, Vrijenhoek T, Markx S, Janssen IM, van der Vliet WA, Faas BH, et al. CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy. Mol Psychiatry. 2008;13:261–6.

    CAS  Article  Google Scholar 

  22. 22.

    Lee IS, Carvalho CM, Douvaras P, Ho SM, Hartley BJ, Zuccherato LW, et al. Characterization of molecular and cellular phenotypes associated with a heterozygous CNTNAP2 deletion using patient-derived hiPSC neural cells. NPJ Schizophr. 2015;1:pii: 15019.

    Article  Google Scholar 

  23. 23.

    Rodenas-Cuadrado P, Ho J, Vernes SC. Shining a light on CNTNAP2: complex functions to complex disorders. Eur J Hum Genet. 2014;22:171–8.

    CAS  Article  Google Scholar 

  24. 24.

    Rodenas-Cuadrado P, Pietrafusa N, Francavilla T, La Neve A, Striano P, Vernes SC. Characterisation of CASPR2 deficiency disorder--a syndrome involving autism, epilepsy and language impairment. BMC Med Genet. 2016;17:8.

    Article  Google Scholar 

  25. 25.

    Strauss KA, Puffenberger EG, Huentelman MJ, Gottlieb S, Dobrin SE, Parod JM, et al. Recessive symptomatic focal epilepsy and mutant contactin-associated protein-like 2. N Engl J Med. 2006;354:1370–7.

    CAS  Article  Google Scholar 

  26. 26.

    Smogavec M, Cleall A, Hoyer J, Lederer D, Nassogne MC, Palmer EE, et al. Eight further individuals with intellectual disability and epilepsy carrying bi-allelic CNTNAP2 aberrations allow delineation of the mutational and phenotypic spectrum. J Med Genet. 2016;53:820–7.

    CAS  Article  Google Scholar 

  27. 27.

    Penagarikano O, Abrahams BS, Herman EI, Winden KD, Gdalyahu A, Dong H, et al. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits. Cell. 2011;147:235–46.

    CAS  Article  Google Scholar 

  28. 28.

    Jurgensen S, Castillo PE. Selective dysregulation of hippocampal inhibition in the mouse lacking autism candidate gene CNTNAP2. J Neurosci. 2015;35:14681–7.

    CAS  Article  Google Scholar 

  29. 29.

    Bridi MS, Park SM, Huang S. Developmental disruption of GABAAR-meditated inhibition in Cntnap2 KO mice. eNeuro. 2017;4:5.

    Article  Google Scholar 

  30. 30.

    Vogt D, Cho KKA, Shelton SM, Paul A, Huang ZJ, Sohal VS, et al. Mouse Cntnap2 and human CNTNAP2 ASD alleles cell autonomously regulate PV+ cortical interneurons. Cereb Cortex 2017;1–12.

  31. 31.

    Ramanathan S, Wong CH, Rahman Z, Dale RC, Fulcher D, Bleasel AF. Myoclonic status epilepticus as a presentation of caspr2 antibody-associated autoimmune encephalitis. Epileptic Disord. 2014;16:477–81.

    PubMed  Google Scholar 

  32. 32.

    van Sonderen A, Arino H, Petit-Pedrol M, Leypoldt F, Kortvelyessy P, Wandinger KP, et al. The clinical spectrum of Caspr2 antibody-associated disease. Neurology. 2016;87:521–8.

    Article  Google Scholar 

  33. 33.

    Pinatel D, Hivert B, Boucraut J, Saint-Martin M, Rogemond V, Zoupi L, et al. Inhibitory axons are targeted in hippocampal cell culture by anti-Caspr2 autoantibodies associated with limbic encephalitis. Front Cell Neurosci. 2015;9:265.

    Article  Google Scholar 

  34. 34.

    Poliak S, Salomon D, Elhanany H, Sabanay H, Kiernan B, Pevny L, et al. Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1. J Cell Biol. 2003;162:1149–60.

    CAS  Article  Google Scholar 

  35. 35.

    Fazzari P, Paternain AV, Valiente M, Pla R, Lujan R, Lloyd K, et al. Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature. 2010;464:1376–80.

    CAS  Article  Google Scholar 

  36. 36.

    Chattopadhyaya B, Di Cristo G, Higashiyama H, Knott GW, Kuhlman SJ, Welker E, et al. Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period. J Neurosci. 2004;24:9598–611.

    CAS  Article  Google Scholar 

  37. 37.

    Srivastava DP, Woolfrey KM, Penzes P. Analysis of dendritic spine morphology in cultured CNS neurons. J Vis Exp. 2011;53:e2794.

    Google Scholar 

  38. 38.

    Nakagawa T, Cheng Y, Ramm E, Sheng M, Walz T. Structure and different conformational states of native AMPA receptor complexes. Nature. 2005;433:545–9.

    CAS  Article  Google Scholar 

  39. 39.

    Varea O, Martin-de-Saavedra MD, Kopeikina KJ, Schurmann B, Fleming HJ, Fawcett-Patel JM, et al. Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons. Proc Natl Acad Sci USA. 2015;112:6176–181.

    CAS  Article  Google Scholar 

  40. 40.

    Scorcioni R, Polavaram S, Ascoli GA. L-Measure: a web-accessible tool for the analysis, comparison and search of digital reconstructions of neuronal morphologies. Nat Protoc. 2008;3:866–76.

    CAS  Article  Google Scholar 

  41. 41.

    Smith KR, Kopeikina KJ, Fawcett-Patel JM, Leaderbrand K, Gao R, Schurmann B, et al. Psychiatric risk factor ANK3/ankyrin-G nanodomains regulate the structure and function of glutamatergic synapses. Neuron. 2014;84:399–415.

    CAS  Article  Google Scholar 

  42. 42.

    Chen N, Koopmans F, Gordon A, Paliukhovich I, Klaassen RV, van der Schors RC, et al. Interaction proteomics of canonical Caspr2 (CNTNAP2) reveals the presence of two Caspr2 isoforms with overlapping interactomes. Biochim Biophys Acta. 2015;1854:827–33.

    CAS  Article  Google Scholar 

  43. 43.

    Mo A, Mukamel EA, Davis FP, Luo C, Henry GL, Picard S, et al. Epigenomic signatures of neuronal diversity in the mammalian brain. Neuron. 2015;86:1369–84.

    CAS  Article  Google Scholar 

  44. 44.

    Scott EK, Luo L. How do dendrites take their shape? Nat Neurosci. 2001;4:359–65.

    CAS  Article  Google Scholar 

  45. 45.

    Gustafsson MG. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA. 2005;102:13081–6.

    CAS  Article  Google Scholar 

  46. 46.

    Hell SW, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 1994;19:780–2.

    CAS  Article  Google Scholar 

  47. 47.

    Cavallaro U, Dejana E. Adhesion molecule signalling: not always a sticky business. Nat Rev Mol Cell Biol. 2011;12:189–97.

    CAS  Article  Google Scholar 

  48. 48.

    Hata Y, Butz S, Sudhof TC. CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J Neurosci. 1996;16:2488–94.

    CAS  Article  Google Scholar 

  49. 49.

    Hsueh YP, Yang FC, Kharazia V, Naisbitt S, Cohen AR, Weinberg RJ, et al. Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses. J Cell Biol. 1998;142:139–51.

    CAS  Article  Google Scholar 

  50. 50.

    Frese CK, Mikhaylova M, Stucchi R, Gautier V, Liu Q, Mohammed S, et al. Quantitative map of proteome dynamics during neuronal differentiation. Cell Rep. 2017;18:1527–42.

    CAS  Article  Google Scholar 

  51. 51.

    Chao HW, Hong CJ, Huang TN, Lin YL, Hsueh YP. SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis. J Cell Biol. 2008;182:141–55.

    CAS  Article  Google Scholar 

  52. 52.

    Liska A, Bertero A, Gomolka R, Sabbioni M, Galbusera A, Barsotti N, et al. Homozygous loss of autism-risk gene CNTNAP2 results in reduced local and long-range prefrontal functional connectivity. Cereb Cortex 2017;1–13.

  53. 53.

    Selimbeyoglu A, Kim CK, Inoue M, Lee SY, Hong ASO, Kauvar I, et al. Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice. Sci Transl Med. 2017;9:401.

    Article  Google Scholar 

  54. 54.

    Jan YN, Jan LY. Branching out: mechanisms of dendritic arborization. Nat Rev Neurosci. 2010;11:316–28.

    CAS  Article  Google Scholar 

  55. 55.

    Anderson GR, Galfin T, Xu W, Aoto J, Malenka RC, Sudhof TC. Candidate autism gene screen identifies critical role for cell-adhesion molecule CASPR2 in dendritic arborization and spine development. Proc Natl Acad Sci USA. 2012;109:18120–5.

    CAS  Article  Google Scholar 

  56. 56.

    Yam PT, Pincus Z, Gupta GD, Bashkurov M, Charron F, Pelletier L, et al. N-cadherin relocalizes from the periphery to the center of the synapse after transient synaptic stimulation in hippocampal neurons. PLoS ONE. 2013;8:e79679.

    CAS  Article  Google Scholar 

  57. 57.

    Gregor A, Albrecht B, Bader I, Bijlsma EK, Ekici AB, Engels H, et al. Expanding the clinical spectrum associated with defects in CNTNAP2 and NRXN1. BMC Med Genet. 2011;12:106.

    CAS  Article  Google Scholar 

  58. 58.

    Bel C, Oguievetskaia K, Pitaval C, Goutebroze L, Faivre-Sarrailh C. Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis. J Cell Sci. 2009;122:3403–13.

    CAS  Article  Google Scholar 

  59. 59.

    Denisenko-Nehrbass N, Oguievetskaia K, Goutebroze L, Galvez T, Yamakawa H, Ohara O, et al. Protein 4.1B associates with both Caspr/paranodin and Caspr2 at paranodes and juxtaparanodes of myelinated fibres. Eur J Neurosci. 2003;17:411–6.

    Article  Google Scholar 

  60. 60.

    Horresh I, Bar V, Kissil JL, Peles E. Organization of myelinated axons by Caspr and Caspr2 requires the cytoskeletal adapter protein 4.1B. J Neurosci. 2010;30:2480–9.

    CAS  Article  Google Scholar 

  61. 61.

    Samuels BA, Hsueh YP, Shu T, Liang H, Tseng HC, Hong CJ, et al. Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK. Neuron. 2007;56:823–37.

    CAS  Article  Google Scholar 

  62. 62.

    Jeyifous O, Waites CL, Specht CG, Fujisawa S, Schubert M, Lin EI, et al. SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway. Nat Neurosci. 2009;12:1011–9.

    CAS  Article  Google Scholar 

  63. 63.

    Wang GS, Hong CJ, Yen TY, Huang HY, Ou Y, Huang TN, et al. Transcriptional modification by a CASK-interacting nucleosome assembly protein. Neuron. 2004;42:113–28.

    CAS  Article  Google Scholar 

  64. 64.

    Horresh I, Poliak S, Grant S, Bredt D, Rasband MN, Peles E. Multiple molecular interactions determine the clustering of Caspr2 and Kv1 channels in myelinated axons. J Neurosci. 2008;28:14213–22.

    CAS  Article  Google Scholar 

  65. 65.

    Tabuchi K, Biederer T, Butz S, Sudhof TC. CASK participates in alternative tripartite complexes in which Mint 1 competes for binding with caskin 1, a novel CASK-binding protein. J Neurosci. 2002;22:4264–73.

    CAS  Article  Google Scholar 

  66. 66.

    Mukherjee K, Sharma M, Urlaub H, Bourenkov GP, Jahn R, Sudhof TC, et al. CASK Functions as a Mg2+ -independent neurexin kinase. Cell. 2008;133:328–39.

    CAS  Article  Google Scholar 

  67. 67.

    LaConte LE, Chavan V, Liang C, Willis J, Schonhense EM, Schoch S, et al. CASK stabilizes neurexin and links it to liprin-alpha in a neuronal activity-dependent manner. Cell Mol Life Sci. 2016;73:3599–621.

    CAS  Article  Google Scholar 

  68. 68.

    Zhou W, Zhang L, Guoxiang X, Mojsilovic-Petrovic J, Takamaya K, Sattler R, et al. GluR1 controls dendrite growth through its binding partner, SAP97. J Neurosci. 2008;28:10220–33.

    CAS  Article  Google Scholar 

  69. 69.

    Lee S, Fan S, Makarova O, Straight S, Margolis B. A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia. Mol Cell Biol. 2002;22:1778–91.

    CAS  Article  Google Scholar 

  70. 70.

    Hsueh YP, Wang TF, Yang FC, Sheng M. Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature. 2000;404:298–302.

    CAS  Article  Google Scholar 

  71. 71.

    Moog U, Bierhals T, Brand K, Bautsch J, Biskup S, Brune T, et al. Phenotypic and molecular insights into CASK-related disorders in males. Orphanet J Rare Dis. 2015;10:44.

    Article  Google Scholar 

  72. 72.

    Hackett A, Tarpey PS, Licata A, Cox J, Whibley A, Boyle J, et al. CASK mutations are frequent in males and cause X-linked nystagmus and variable XLMR phenotypes. Eur J Hum Genet. 2010;18:544–52.

    CAS  Article  Google Scholar 

  73. 73.

    Lee BH, Smith T, Paciorkowski AR. Autism spectrum disorder and epilepsy: Disorders with a shared biology. Epilepsy Behav. 2015;47:191–201.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the grants NS100785 and MH097216 from the NIH-NIMH to P.P and F30MH096457 to R.G. SIM imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by the NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Structured illumination microscopy was performed on a Nikon N-SIM system, purchased through the support of NIH 1S10OD016342–01. We thank Dr. Joshua Zachary Rappoport for help with SIM imaging, Dr. Daniel Vogt for his consultation, and Xi Chao for help with figure illustrations.

Author contributions

R.G. led the project and performed all confocal and SIM imaging experiments, K.M. performed STED imaging experiments, R.G., A.E.M.-Z, S.Y., and T.A.R. performed in vivo experiments, R.G., N.H.P., M.P.F., and M.D.M.-de.S. performed biochemistry experiments, G.Z. assisted with data analysis. P.P. supervised the project while J.G.C. and D.J.S. advised. R.G. and P.P. wrote the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Peter Penzes.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gao, R., Piguel, N.H., Melendez-Zaidi, A.E. et al. CNTNAP2 stabilizes interneuron dendritic arbors through CASK. Mol Psychiatry 23, 1832–1850 (2018). https://doi.org/10.1038/s41380-018-0027-3

Download citation

Further reading

Search

Quick links