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Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment

Abstract

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAF Q257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

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References

  1. World Health Organization. The global burden of disease: 2004 update [Internet]. 2008 [cited 2017 Nov 06]; Available from: http://www.who.int/entity/healthinfo/global_burden_disease/GBD_report_2004update_full.pdf?ua=1.

  2. Cross-Disorder Group of the Psychiatric Genomics C, Lee SH, Ripke S, Neale BM, Faraone SV, Purcell SM et al. Genetic relationship between five psychiatric disorders estimated from genome-wide SNPs. Nat Genet 2013; 45: 984–994.

    Article  Google Scholar 

  3. Network, Pathway Analysis Subgroup of the Psychiatric, Genomic C, International Inflammatory Bowel Disease Genetics C, International Inflammatory Bowel Disease Genetics Consortium I. Psychiatric genome-wide association study analyses implicate neuronal, immune and histone pathways. Nat Neurosci 2015; 18: 199–209.

    Article  Google Scholar 

  4. Krencik R, Zhang SC. Directed differentiation of functional astroglial subtypes from human pluripotent stem cells. Nat Protoc 2011; 6: 1710–1717.

    Article  CAS  Google Scholar 

  5. Tidyman WE, Rauen Ka. The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 2009; 19: 230–236.

    Article  CAS  Google Scholar 

  6. Adviento B, Corbin IL, Widjaja F, Desachy G, Enrique N, Rosser T et al. Autism traits in the RASopathies. J Med Genet 2014; 51: 10–20.

    Article  CAS  Google Scholar 

  7. Adachi M, Abe Y, Aoki Y, Matsubara Y. Epilepsy in RAS/MAPK syndrome: two cases of cardio-facio-cutaneous syndrome with epileptic encephalopathy and a literature review. Seizure 2012; 21: 55–60.

    Article  Google Scholar 

  8. Miller JA, Ding SL, Sunkin SM, Smith KA, Ng L, Szafer A et al. Transcriptional landscape of the prenatal human brain. Nature 2014; 508: 199–206.

    Article  CAS  Google Scholar 

  9. Armour CM, Allanson JE. Further delineation of cardio-facio-cutaneous syndrome: clinical features of 38 individuals with proven mutations. J Med Genet 2008; 45: 249–254.

    Article  CAS  Google Scholar 

  10. Sabatino G, Verrotti A, Domizio S, Angeiozzi B, Chiarelli F, Neri G. The cardio-facio-cutaneous syndrome: a long-term follow-up of two patients, with special reference to the neurological features. Childs Nerv Syst 1997; 13: 238–241.

    Article  CAS  Google Scholar 

  11. Alfieri P, Piccini G, Caciolo C, Perrino F, Gambardella ML, Mallardi M et al. Behavioral profile in RASopathies. Am J Med Genet A 2014; 164A: 934–942.

    Article  Google Scholar 

  12. Cesarini L, Alfieri P, Pantaleoni F, Vasta I, Cerutti M, Petrangeli V et al. Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade. Am J Med Genet A 2009; 149A: 140–146.

    Article  CAS  Google Scholar 

  13. Pierpont EI, Pierpont ME, Mendelsohn NJ, Roberts AE, Tworog-Dube E, Rauen KA et al. Effects of germline mutations in the Ras/MAPK signaling pathway on adaptive behavior: cardiofaciocutaneous syndrome and Noonan syndrome. Am J Med Genet A 2010; 152A: 591–600.

    Article  CAS  Google Scholar 

  14. Yoon G, Rosenberg J, Blaser S, Rauen KA. Neurological complications of cardio-facio-cutaneous syndrome. Dev Med Child Neurol 2007; 49: 894–899.

    Article  Google Scholar 

  15. van Erp TG, Hibar DP, Rasmussen JM, Glahn DC, Pearlson GD, Andreassen OA et al. Subcortical brain volume abnormalities in 2028 individuals with schizophrenia and 2540 healthy controls via the ENIGMA consortium. Mol Psychiatry 2016; 21: 547–553.

    Article  CAS  Google Scholar 

  16. Haijma SV, Van Haren N, Cahn W, Koolschijn PC, Hulshoff Pol HE, Kahn RS. Brain volumes in schizophrenia: a meta-analysis in over 18000 subjects. Schizophr Bull 2013; 39: 1129–1138.

    Article  Google Scholar 

  17. Frazier TW, Hardan AY. A meta-analysis of the corpus callosum in autism. Biol Psychiatry 2009; 66: 935–941.

    Article  Google Scholar 

  18. Haar S, Berman S, Behrmann M, Dinstein I. Anatomical abnormalities in autism? Cereb Cortex 2014; 26: 1440–1452.

    Article  Google Scholar 

  19. Palmen SJ, Hulshoff Pol HE, Kemner C, Schnack HG, Janssen J, Kahn RS et al. Larger brains in medication naive high-functioning subjects with pervasive developmental disorder. J Autism Dev Disord 2004; 34: 603–613.

    Article  Google Scholar 

  20. Chen AP, Ohno M, Giese KP, Kuhn R, Chen RL, Silva AJ. Forebrain-specific knockout of B-raf kinase leads to deficits in hippocampal long-term potentiation, learning, and memory. J Neurosci Res 2006; 83: 28–38.

    Article  CAS  Google Scholar 

  21. Pfeiffer V, Gotz R, Xiang C, Camarero G, Braun A, Zhang Y et al. Ablation of BRaf impairs neuronal differentiation in the postnatal hippocampus and cerebellum. PLoS ONE 2013; 8: e58259.

    Article  CAS  Google Scholar 

  22. Zhong J, Li X, McNamee C, Chen AP, Baccarini M, Snider WD. Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo. Nat Neurosci 2007; 10: 598–607.

    Article  CAS  Google Scholar 

  23. Inoue S, Moriya M, Watanabe Y, Miyagawa-Tomita S, Niihori T, Oba D et al. New BRAF knockin mice provide a pathogenetic mechanism of developmental defects and a therapeutic approach in cardio-facio-cutaneous syndrome. Hum Mol Genet 2014; 23: 6553–6566.

    Article  CAS  Google Scholar 

  24. Urosevic J, Sauzeau V, Soto-Montenegro ML, Reig S, Desco M, Wright EM et al. Constitutive activation of B-Raf in the mouse germ line provides a model for human cardio-facio-cutaneous syndrome. Proc Natl Acad Sci USA 2011; 108: 5015–5020.

    Article  CAS  Google Scholar 

  25. Gulsuner S, Walsh T, Watts AC, Lee MK, Thornton AM, Casadei S et al. Spatial and temporal mapping of de novo mutations in schizophrenia to a fetal prefrontal cortical network. Cell 2013; 154: 518–529.

    Article  CAS  Google Scholar 

  26. Krencik R, Zhang SC. Stem cell neural differentiation: a model for chemical biology. Curr Opin Chem Biol 2006; 10: 592–597.

    Article  CAS  Google Scholar 

  27. Muotri AR. The human model: changing focus on autism research. Biol Psychiatry 2016; 79: 642–649.

    Article  Google Scholar 

  28. Brennand KJ, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 2011; 473: 221–225.

    Article  CAS  Google Scholar 

  29. Yagi T, Ito D, Okada Y, Akamatsu W, Nihei Y, Yoshizaki T et al. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum Mol Genet 2011; 20: 4530–4539.

    Article  CAS  Google Scholar 

  30. Mariani J, Coppola G, Zhang P, Abyzov A, Provini L, Tomasini L et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 2015; 162: 375–390.

    Article  CAS  Google Scholar 

  31. Khalilov I, Le Van Quyen M, Gozlan H, Ben-Ari Y. Epileptogenic actions of GABA and fast oscillations in the developing hippocampus. Neuron 2005; 48: 787–796.

    Article  CAS  Google Scholar 

  32. Krencik R, Hokanson KC, Narayan AR, Dvornik J, Rooney GE, Rauen KA et al. Dysregulation of astrocyte extracellular signaling in Costello syndrome. Sci Transl Med 2015; 7: 286ra266.

    Article  Google Scholar 

  33. Rooney GE, Goodwin AF, Depeille P, Sharir A, Schofield CM, Yeh E et al. Human iPS cell-derived neurons uncover the impact of increased Ras signaling in Costello syndrome. J Neurosci 2016; 36: 142–152.

    Article  CAS  Google Scholar 

  34. Gripp KW, Hopkins E, Doyle D, Dobyns WB. High incidence of progressive postnatal cerebellar enlargement in Costello syndrome: brain overgrowth associated with HRAS mutations as the likely cause of structural brain and spinal cord abnormalities. Am J Med Genet A 2010; 152A: 1161–1168.

    Article  Google Scholar 

  35. Villegas J, McPhaul M (2005) Establishment and culture of human skin fibroblasts. In: Frederick M, Ausubel (eds). Current Protocols in Molecular Biology. John Wiley & Sons, Inc: San Francisco, 2009, p 28.3.1-3.

  36. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S et al. A more efficient method to generate integration-free human iPS cells. Nat Methods 2011; 8: 409–412.

    Article  CAS  Google Scholar 

  37. Mali P, Ye Z, Chou BK, Yen J, Cheng L. An improved method for generating and identifying human induced pluripotent stem cells. Methods Mol Biol 2010; 636: 191–205.

    Article  CAS  Google Scholar 

  38. Mitra I, Lavillaureix A, Yeh E, Traglia M, Tsang K, Bearden CE et al. Reverse pathway genetic approach identifies epistasis in autism spectrum disorders. PLoS Genet 2017; 13: e1006516.

    Article  Google Scholar 

  39. 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 

  40. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001; 19: 1129–1133.

    Article  CAS  Google Scholar 

  41. Liu Y, Liu HS, Sauvey C, Yao L, Zarnowska ED, Zhang SC. Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells. Nat Protoc 2013; 8: 1670–1679.

    Article  CAS  Google Scholar 

  42. Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods 2004; 33: 95–103.

    Article  CAS  Google Scholar 

  43. von Bohlen Und, Halbach O. Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res 2007; 329: 409–420.

    Article  Google Scholar 

  44. Izant JG, McIntosh JR. Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons. Proc Natl Acad Sci USA 1980; 77: 4741–4745.

    Article  CAS  Google Scholar 

  45. Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development 1992; 116: 201–211.

    CAS  PubMed  Google Scholar 

  46. Leach MK, Naim YI, Feng ZQ, Gertz CC, Corey JM. Stages of neuronal morphological development in vitro—an automated assay. J Neurosci Methods 2011; 199: 192–198.

    Article  Google Scholar 

  47. Dehay C, Kennedy H, Kosik KS. The outer subventricular zone and primate-specific cortical complexification. Neuron 2015; 85: 683–694.

    Article  CAS  Google Scholar 

  48. Song M, Mohamad O, Chen D, Yu SP. Coordinated development of voltage-gated Na+ and K+ currents regulates functional maturation of forebrain neurons derived from human induced pluripotent stem cells. Stem Cells Dev 2013; 22: 1551–1563.

    Article  CAS  Google Scholar 

  49. Jo H, Mondal S, Tan D, Nagata E, Takizawa S, Sharma AK et al. Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proc Natl Acad Sci USA 2012; 109: 10581–10586.

    Article  CAS  Google Scholar 

  50. Perea G, Sur M, Araque A. Neuron-glia networks: integral gear of brain function. Front Cell Neurosci 2014; 8: 378.

    Article  Google Scholar 

  51. Letinic K, Zoncu R, Rakic P. Origin of GABAergic neurons in the human neocortex. Nature 2002; 417: 645–649.

    Article  CAS  Google Scholar 

  52. Papadopoulou E, Sifakis S, Sol-Church K, Klein-Zighelboim E, Stabley DL, Raissaki M et al. CNS imaging is a key diagnostic tool in the evaluation of patients with CFC syndrome: two cases and literature review. Am J Med Genet A 2011; 155A: 605–611.

    Article  Google Scholar 

  53. Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H, Macklis JD. Molecular logic of neocortical projection neuron specification, development and diversity. Nat Rev Neurosci 2013; 14: 755–769.

    Article  CAS  Google Scholar 

  54. Maddodi N, Huang W, Havighurst T, Kim K, Longley BJ, Setaluri V. Induction of autophagy and inhibition of melanoma growth in vitro and in vivo by hyperactivation of oncogenic BRAF. J Invest Dermatol 2010; 130: 1657–1667.

    Article  CAS  Google Scholar 

  55. Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, Cruz MS et al. Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006; 311: 1287–1290.

    Article  CAS  Google Scholar 

  56. Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, Verloes A et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006; 38: 294–296.

    Article  CAS  Google Scholar 

  57. Ritt DA, Monson DM, Specht SI, Morrison DK. Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling. Mol Cell Biol 2010; 30: 806–819.

    Article  CAS  Google Scholar 

  58. Lavoie H, Therrien M. Regulation of RAF protein kinases in ERK signalling. Nat Rev Mol Cell Biol 2015; 16: 281–298.

    Article  CAS  Google Scholar 

  59. Goyal Y, Jindal GA, Pelliccia JL, Yamaya K, Yeung E, Futran AS et al. Divergent effects of intrinsically active MEK variants on developmental Ras signaling. Nat Genet 2017; 49: 465–469.

    Article  CAS  Google Scholar 

  60. Sun T, Hevner RF. Growth and folding of the mammalian cerebral cortex: from molecules to malformations. Nat Rev Neurosci 2014; 15: 217–232.

    Article  CAS  Google Scholar 

  61. Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci 2011; 36: 320–328.

    Article  CAS  Google Scholar 

  62. Zhou Y, Kaiser T, Monteiro P, Zhang X, Van der Goes MS, Wang D et al. Mice with Shank3 mutations associated with ASD and schizophrenia display both shared and distinct defects. Neuron 2016; 89: 147–162.

    Article  CAS  Google Scholar 

  63. Seeburg PH, Colby WW, Capon DJ, Goeddel DV, Levinson AD. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature 1984; 312: 71–75.

    Article  CAS  Google Scholar 

  64. Chappell WH, Steelman LS, Long JM, Kempf RC, Abrams SL, Franklin RA et al. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: rationale and importance to inhibiting these pathways in human health. Oncotarget 2011; 2: 135–164.

    Article  Google Scholar 

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Acknowledgments

This work was supported by National Institutes of Health New Innovator (1DP2OD007449 to LAW), Simons Foundation Autism Research Initiative (to LAW and EMU), National Alliance for Research on Schizophrenia and Depression Young Investigator Grant from the Brain & Behavior Research Foundation (to EY), Staglin Family/ International Mental Health Research Organization Assistant Professorship (to LAW), University of California San Francisco Resource Allocation Program (to LAW and WZ), the LeJeune Foundation (to LAW and EY), the City College of San Francisco Bridges to Stem Cell Program (to ZYW, FMC and CT), National Institutes of Health (T32 EY007120 to DQD), Research to Prevent Blindness—Walt and Lilly Disney Award for Amblyopia Research (to DQD) and National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (5RO1AR062165 to KAR). We thank all of the participants in our study and their families; Dina Bseiso and Dr Alinoë Lavillaureix for collecting and reviewing the patients’ clinical data; Brigid Adviento and Dr Keren Messing-Guy for preparing and shipping the skin biopsies; Dr Michela Traglia for help with repeated measures ANOVA; Dr Arnold Kriegstein’s lab (UCSF), and, specifically, Dr Alex Pollen for kindly providing the control iPSC line HS1-11; Dr Jody Baron (UCSF) for allowing us to use the QuantStudio 6 Flex Real-Time PCR System; Dr Susan M. Voglmaier’s lab (UCSF) and, specifically Dr Magda Santos for generously providing anti-GAD65 and anti-GAD67 antibodies; Liorimar R Medina and John Paul Kwak for technical assistance; and Jody Williams, MA, for revision of the manuscript and department assistance. We also thank NF, Children's Tumor Foundation, Noonan Foundation, CFC International, Costello Syndrome Family Support Network, Costello Kids and RASopathy Network for their contribution to our recruitment efforts.

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Yeh, E., Dao, D.Q., Wu, Z.Y. et al. Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment. Mol Psychiatry 23, 1687–1698 (2018). https://doi.org/10.1038/mp.2017.238

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