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.

  • Article
  • Published:

An in vitro model of lissencephaly: expanding the role of DCX during neurogenesis

Abstract

Lissencephaly comprises a spectrum of brain malformations due to impaired neuronal migration in the developing cerebral cortex. Classical lissencephaly is characterized by smooth cerebral surface and cortical thickening that result in seizures, severe neurological impairment and developmental delay. Mutations in the X-chromosomal gene DCX, encoding doublecortin, is the main cause of classical lissencephaly. Much of our knowledge about DCX-associated lissencephaly comes from post-mortem analyses of patient’s brains, mainly since animal models with DCX mutations do not mimic the disease. In the absence of relevant animal models and patient brain specimens, we took advantage of induced pluripotent stem cell (iPSC) technology to model the disease. We established human iPSCs from two males with mutated DCX and classical lissencephaly including smooth brain and abnormal cortical morphology. The disease was recapitulated by differentiation of iPSC into neural cells followed by expression profiling and dissection of DCX-associated functions. Here we show that neural stem cells, with absent or reduced DCX protein expression, exhibit impaired migration, delayed differentiation and deficient neurite formation. Hence, the patient-derived iPSCs and neural stem cells provide a system to further unravel the functions of DCX in normal development and disease.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Rakic P. Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 1972; 145: 61–83.

    Article  CAS  Google Scholar 

  2. Meyer G, Schaaps JP, Moreau L, Goffinet AM. Embryonic and early fetal development of the human neocortex. J Neurosci 2000; 20: 1858–1868.

    Article  CAS  Google Scholar 

  3. Gleeson JG, Allen KM, Fox JW, Lamperti ED, Berkovic S, Scheffer I et al. Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell 1998; 92: 63–72.

    Article  CAS  Google Scholar 

  4. des Portes V, Pinard JM, Billuart P, Vinet MC, Koulakoff A, Carrié A et al. A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome. Cell 1998; 92: 51–61.

    Article  CAS  Google Scholar 

  5. Fry AE, Cushion TD, Pilz DT. The genetics of lissencephaly. Am J Med Genet C Semin Med Genet 2014; 166C: 198–210.

    Article  Google Scholar 

  6. Gleeson JG, Lin PT, Flanagan LA, Walsh CA. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons Brigham and Women’s hospital. Neuron 1999; 23: 257–271.

    Article  CAS  Google Scholar 

  7. Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, Vinet MC et al. Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 1999; 23: 247–256.

    Article  CAS  Google Scholar 

  8. Moores Ca, Perderiset M, Francis F, Chelly J, Houdusse A, Milligan RA. Mechanism of microtubule stabilization by doublecortin. Mol Cell 2004; 14: 833–839.

    Article  CAS  Google Scholar 

  9. Sapir T, Horesh D, Caspi M, Atlas R, Burgess HA, Wolf SG et al. Doublecortin mutations cluster in evolutionarily conserved functional domains. Hum Mol Genet 2000; 9: 703–712.

    Article  CAS  Google Scholar 

  10. Ross ME, Allen KM, Srivastava AK, Featherstone T, Gleeson JG, Hirsch B et al. Linkage and physical mapping of X-linked lissencephaly/SBH (XLIS): a gene causing neuronal migration defects in human brain. Hum Mol Genet 1997; 6: 555–562.

    Article  CAS  Google Scholar 

  11. Cappello S, Böhringer CRJ, Bergami M, Conzelmann KK, Ghanem A, Tomassy GS et al. A radial glia-specific role of RhoA in double cortex formation. Neuron 2012; 73: 911–924.

    Article  CAS  Google Scholar 

  12. Gil-Sanz C, Landeira B, Ramos C, Costa MR, Muller U. Proliferative defects and formation of a double cortex in mice lacking Mltt4 and Cdh2 in the dorsal telencephalon. J Neurosci 2014; 34: 10475–10487.

    Article  Google Scholar 

  13. Corbo JC, Deuel TA, Long JM, LaPorte P, Tsai E, Wynshaw-Boris A et al. Doublecortin is required in mice for lamination of the hippocampus but not the neocortex. J Neurosci 2002; 22: 7548–7557.

    Article  CAS  Google Scholar 

  14. Bai J, Ramos RL, Ackman JB, Thomas AM, Lee RV, LoTurco JJ. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci 2003; 6: 1277–1283.

    Article  CAS  Google Scholar 

  15. Brose K, Tessier-Lavigne M. Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr Opin Neurobiol 2000; 10: 95–102.

    Article  CAS  Google Scholar 

  16. Wu S, Johansson J, Damdimopoulou P, Shahsavani M, Falk A, Hovatta O et al. Spider silk for xeno-free long-term self-renewal and differentiation of human pluripotent stem cells. Biomaterials 2014; 35: 8496–8502.

    Article  CAS  Google Scholar 

  17. Falk A, Koch P, Kesavan J, Takashima Y, Ladewig J, Alexander M et al. Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PLoS ONE 2012; 7: e29597.

    Article  CAS  Google Scholar 

  18. Nishimura K, Sano M, Ohtaka M, Furuta B, Umemura Y, Nakajima Y et al. Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming. J Biol Chem 2011; 286: 4760–4771.

    Article  CAS  Google Scholar 

  19. Müller F-J, Schuldt BM, Williams R, Mason D, Altun G, Papapetrou EP et al. A bioinformatic assay for pluripotency in human cells. Nat Methods 2011; 8: 315–317.

    Article  Google Scholar 

  20. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402–408.

    Article  CAS  Google Scholar 

  21. Thomas PD, Campbell MJ, Kejariwal A, Mi H, Karlak B, Daverman R et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res 2003; 13: 2129–2141.

    Article  CAS  Google Scholar 

  22. Mi H, Lazareva-Ulitsky B, Loo R, Kejariwal A, Vandergriff J, Rabkin S et al. The PANTHER database of protein families, subfamilies, functions and pathways. Nucleic Acids Res 2005; 33: 284–288.

    Article  Google Scholar 

  23. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 2015; 43: D447–D452.

    Article  CAS  Google Scholar 

  24. Parrini E, Conti V, Dobyns WB, Guerrini R. Genetic basis of brain malformations. Mol Syndromol 2016; 7: 220–233.

    Article  CAS  Google Scholar 

  25. Melo AS de O, Aguiar RS, Amorim MMR, Arruda MB, Melo F de O, Ribeiro STC et al. Congenital Zika Virus Infection. JAMA Neurol 2016; 73: 1407–1416.

    Article  Google Scholar 

  26. Filipovic R, Kumar SS, Fiondella C. Increasing doublecortin expression promotes migration of human embryonic stem cell-derived neurons. Stem Cells 2012; 30: 1852–1862.

    Article  CAS  Google Scholar 

  27. Leventer RJ, Cardoso C, Ledbetter DH, Dobyns WB. LIS1: from cortical malformation to essential protein of cellular dynamics. Trends Neurosci 2001; 24: 489–492.

    Article  CAS  Google Scholar 

  28. Tsai JW, Chen Y, Kriegstein AR, Vallee RB. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages. J Cell Biol 2005; 170: 935–945.

    Article  CAS  Google Scholar 

  29. Moon HM, Youn YH, Pemble H, Yingling J, Wittmann T, Wynshaw-Boris A. LIS1 controls mitosis and mitotic spindle organization via the LIS1-NDEL1-dynein complex. Hum Mol Genet 2014; 23: 449–466.

    Article  CAS  Google Scholar 

  30. Kato M, Dobyns WB. X-linked lissencephaly with abnormal genitalia as a tangential migration disorder causing intractable epilepsy: proposal for a new term, ‘interneuronopathy’. J Child Neurol 2005; 20: 392–397.

    Article  Google Scholar 

  31. Jin M, Pomp O, Shinoda T, Toba S, Torisawa T, Furuta K et al. Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics. Sci Rep 2017; 7: 39902.

    Article  CAS  Google Scholar 

  32. Bershteyn M, Nowakowski TJ, Pollen AA, Di Lullo E, Nene A, Wynshaw-Boris A et al. Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell 2017; 20: 435–449.e4.

    Article  CAS  Google Scholar 

  33. Ha S, Tripathi PP, Mihalas AB, Hevner RF, Beier DR. C-terminal region truncation of RELN disrupts an interaction with VLDLR, causing abnormal development of the cerebral cortex and hippocampus. J Neurosci 2017; 37: 960–971.

    Article  CAS  Google Scholar 

  34. Iefremova V, Manikakis G, Krefft O, Jabali A, Weynans K, Wilkens R et al. An organoid-based model of cortical development identifies non-cell-autonomous defects in Wnt signaling contributing to Miller-Dieker syndrome. Cell Rep 2017; 19: 50–59.

    Article  CAS  Google Scholar 

  35. Maness PF, Schachner M. Neural recognition molecules of the immunoglobulin superfamily: signaling transducers of axon guidance and neuronal migration. Nat Neurosci 2007; 10: 19–26.

    Article  CAS  Google Scholar 

  36. Kizhatil K, Wu Y-X, Sen A, Bennett V. A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin. J Neurosci 2002; 22: 7948–7958.

    Article  CAS  Google Scholar 

  37. Deuel TaS, Liu JS, Corbo JC, Yoo S-Y, Rorke-Adams LB, Walsh CA. Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth. Neuron 2006; 49: 41–53.

    Article  CAS  Google Scholar 

  38. Moores Ca, Perderiset M, Kappeler C, Kain S, Drummond D, Perkins SJ et al. Distinct roles of doublecortin modulating the microtubule cytoskeleton. EMBO J 2006; 25: 4448–4457.

    Article  CAS  Google Scholar 

  39. Barry J, Gu Y, Gu C. Polarized targeting of L1-CAM regulates axonal and dendritic bundling in vitro. Eur J Neurosci 2010; 32: 1618–1631.

    Article  Google Scholar 

  40. Flynn KC. The cytoskeleton and neurite initiation. Bioarchitecture 2013; 3: 86–109.

    Article  Google Scholar 

  41. Madl CM, Heilshorn SC. Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding. Neural Regen Res 2015; 10: 514–517.

    Article  Google Scholar 

  42. Bielas SL, Serneo FF, Chechlacz M, Deerinck TJ, Perkins Ga, Allen PB et al. Spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist. Cell 2007; 129: 579–591.

    Article  CAS  Google Scholar 

  43. Bamba Y, Shofuda T, Kato M, Pooh RK, Tateishi Y, Takanashi J-I et al. In vitro characterization of neurite extension using induced pluripotent stem cells derived from lissencephaly patients with TUBA1A missense mutations. Mol Brain 2016; 9: 1–14.

    Article  Google Scholar 

  44. Killeen MT, Sybingco SS. Netrin, Slit and Wnt receptors allow axons to choose the axis of migration. Dev Biol 2008; 323: 143–151.

    Article  CAS  Google Scholar 

  45. Friedlander DR, Milev P, Karthikeyan L, Margolis RK, Margolis RU, Grumet M. The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth. J Cell Biol 1994; 125: 669–680.

    Article  CAS  Google Scholar 

  46. Byun J, Kim BT, Kim YT, Jiao Z, Hur EM, Zhou FQ. Slit2 Inactivates GSK3beta to signal neurite outgrowth inhibition. PLoS ONE 2012; 7: e51895.

    Article  CAS  Google Scholar 

  47. Blockus H, Chédotal A. Slit-Robo signaling. Development 2016; 143: 3037–3044.

    Article  CAS  Google Scholar 

  48. Kim MH, Cierpicki T, Derewenda U, Krowarsch D, Feng Y, Devedjiev Y et al. The DCX-domain tandems of doublecortin and doublecortin-like kinase. Nat Struct Biol 2003; 10: 324–333.

    Article  CAS  Google Scholar 

  49. Tsukada M, Prokscha A, Eichele G. Neurabin II mediates doublecortin-dephosphorylation on actin filaments. Biochem Biophys Res Commun 2006; 343: 839–847.

    Article  CAS  Google Scholar 

  50. Toriyama M, Mizuno N, Fukami T, Iguchi T, Toriyama M, Tago K et al. Phosphorylation of doublecortin by protein kinase A orchestrates microtubule and actin dynamics to promote neuronal progenitor cell migration. J Biol Chem 2012; 287: 12691–12702.

    Article  CAS  Google Scholar 

  51. Tanaka T, Serneo FF, Higgins C, Gambello MJ, Wynshaw-Boris A, Gleeson JG. Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration. J Cell Biol 2004; 165: 709–721.

    Article  CAS  Google Scholar 

  52. Hitoshi S, Alexson T, Tropepe V, Donoviel D, Elia AJ, Nye JS et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002; 16: 846–858.

    Article  CAS  Google Scholar 

  53. Schuurmans C, Guillemot F. Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr Opin Neurobiol 2002; 12: 26–34.

    Article  CAS  Google Scholar 

  54. Imayoshi I, Sakamoto M, Yamaguchi M, Mori K, Kageyama R. Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci 2010; 30: 3489–3498.

    Article  CAS  Google Scholar 

  55. Chambers CB, Peng Y, Nguyen H, Gaiano N, Fishell G, Nye JS. Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors. Development 2001; 128: 689–702.

    CAS  PubMed  Google Scholar 

  56. Maness PF, Shores CG, Ignelzi M. Localization of the normal cellular src protein to the growth cone of differentiating neurons in brain and retina. Adv Exp Med Biol 1990; 265: 117–125.

    Article  CAS  Google Scholar 

  57. Maness PF. Nonreceptor protein tyrosine kinases associated with neuronal development. Dev Neurosci 1992; 14: 257–270.

    Article  CAS  Google Scholar 

  58. Brusés JL, Rutishauser U. Roles, regulation, and mechanism of polysialic acid function during neural development. Biochimie 2001; 83: 635–643.

    Article  Google Scholar 

  59. Hong SE, Shugart YY, Huang DT, Shahwan SAl, Grant PE, Hourihane JO et al. Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nat Genet 2000; 26: 93–96.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The patients and their families who participated in this study are deeply acknowledged and appreciated. This study was supported by Stiftelsen för strategisk forskning, SSF (IB13-0074) (to AF), the Åke Wiberg foundation (to AF), Tore Nilsson foundation (to AF), Jeansson foundation (to AF), Thuring foundation (to AF), KID (AF), SFOs (to AF) and Swedish Research Council 2015-02424_3 (to ND). This work was also supported by the Paul G Allen Family Foundation, Bob and Mary Jane Engman, The Leona M and Harry B Helmsley Charitable Trust, Annette C Merle-Smith, R01 MH095741 (to FHG), U19MH106434 (to FHG) and by The G Harold & Leila Y Mathers Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to A Falk.

Ethics declarations

Conflict of Interest

The authors declare no conflict of interest.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shahsavani, M., Pronk, R.J., Falk, R. et al. An in vitro model of lissencephaly: expanding the role of DCX during neurogenesis. Mol Psychiatry 23, 1674–1684 (2018). https://doi.org/10.1038/mp.2017.175

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/mp.2017.175

This article is cited by

Search

Quick links