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ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington’s disease: a novel approach for cell clock research


Calcium, the most versatile second messenger, regulates essential biology including crucial cellular events in embryogenesis. We investigated impacts of calcium channels and purinoceptors on neuronal differentiation of normal mouse embryonic stem cells (ESCs), with outcomes being compared to those of in vitro models of Huntington’s disease (HD). Intracellular calcium oscillations tracked via real-time fluorescence and luminescence microscopy revealed a significant correlation between calcium transient activity and rhythmic proneuronal transcription factor expression in ESCs stably expressing ASCL-1 or neurogenin-2 promoters fused to luciferase reporter genes. We uncovered that pharmacological manipulation of L-type voltage-gated calcium channels (VGCCs) and purinoceptors induced a two-step process of neuronal differentiation. Specifically, L-type calcium channel-mediated augmentation of spike-like calcium oscillations first promoted stable expression of ASCL-1 in differentiating ESCs, which following P2Y2 purinoceptor activation matured into GABAergic neurons. By contrast, there was neither spike-like calcium oscillations nor responsive P2Y2 receptors in HD-modeling stem cells in vitro. The data shed new light on mechanisms underlying neurogenesis of inhibitory neurons. Moreover, our approach may be tailored to identify pathogenic triggers of other developmental neurological disorders for devising targeted therapies.

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Fig. 1: Roles of extracellular nucleotide receptors and voltage-gated calcium channels in RA-induced neural differentiation of ESCs.
Fig. 2: Effects of VGCCs and P2Y2 and P2X7 receptor-induced signaling on NPC differentiation.
Fig. 3: Effects of P2X7 and P2Y2 receptors on spontaneous calcium oscillations and proneural ASCL-1 and Ngn2 bHLH transcription factor expression during neurogenesis, as determined by time-lapse recording.
Fig. 4: human NPCs from HD-patient iPSCs do not show spike-like [Ca2+]i oscillations.
Fig. 5: Influence of mHtt overexpression on ESC neuronal differentiation and P2Y2 receptor activity.


  1. 1.

    Rossant J, Tam PP. Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development. 2009;136:701–13.

    CAS  PubMed  Google Scholar 

  2. 2.

    Huang G, Ye S, Zhou X, Liu D, Ying QL. Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network. Cell Mol Life Sci. 2015;72:1741–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    ten Berge D, Koole W, Fuerer C, Fish M, Eroglu E, Nusse R. Wnt Signaling Mediates Self-Organization and Axis Formation in Embryoid Bodies. Cell Stem Cell. 2008.

  4. 4.

    Dhara SK, Stice SL. Neural differentiation of human embryonic stem cells. J Cell Biochem. 2008;105:633–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Ulrich H, Majumder P. Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications. Cell Prolif. 2006;39:281–300.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA. 2004.

  7. 7.

    Chuang JH, Tung LC, Lin Y. Neural differentiation from embryonic stem cells in vitro: an overview of the signaling pathways. World J Stem Cells. 2015;7:437–47.

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Resende RR, Britto LRG, Ulrich H Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells. Int J Dev Neurosci. 2008.

  9. 9.

    Resende RR, Adhikari A, da Costa JL, Lorencon E, Ladeira MS, Guatimosim S, et al. Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells. Biochim Biophys Acta. 2009;1803:246–60.

    PubMed  Google Scholar 

  10. 10.

    Zimmermann H. Purinergic signaling in neural development. Semin Cell Dev Biol. 2011;22:194–204.

    CAS  PubMed  Google Scholar 

  11. 11.

    Neary JT, Zimmermann H. Trophic functions of nucleotides in the central nervous system. Trends Neurosci. 2009;32:189–98.

    CAS  PubMed  Google Scholar 

  12. 12.

    Oliveira A, Illes P, Ulrich H. Purinergic receptors in embryonic and adult neurogenesis. Neuropharmacology. 2016;104:272–81.

    CAS  PubMed  Google Scholar 

  13. 13.

    Illes P, Rubini P. Regulation of neural stem/progenitor cell functions by P2X and P2Y receptors. Neural Regen Res. 2017;12:395–6.

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Tang Y, Illes P. Regulation of adult neural progenitor cell functions by purinergic signaling. Glia. 2017;65:213–30.

    PubMed  Google Scholar 

  15. 15.

    Burnstock G, Knight GE. Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol. 2004;240:31–304.

    CAS  PubMed  Google Scholar 

  16. 16.

    Burnstock G. Purine and pyrimidine receptors. Cell Mol Life Sci. 2007;64:1471–83.

    CAS  PubMed  Google Scholar 

  17. 17.

    Verkhratsky A, Krishtal OA, Burnstock G. Purinoceptors on neuroglia. Mol Neurobiol. 2009;39:190–208.

    CAS  PubMed  Google Scholar 

  18. 18.

    Dubyak GR, el-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol. 1993;265:C577–606.

    CAS  PubMed  Google Scholar 

  19. 19.

    Spitzer NC, Lautermilch NJ, Smith RD, Gomez TM. Coding of neuronal differentiation by calcium transients. Bioessays. 2000;22:811–7.

    CAS  PubMed  Google Scholar 

  20. 20.

    Ulrich H, Abbracchio MP, Burnstock G. Extrinsic purinergic regulation of neural stem/progenitor cells: implications for CNS development and repair. Stem Cell Rev. 2012;8:755–67.

    CAS  Google Scholar 

  21. 21.

    Spitzer NC, Root CM, Borodinsky LN. Orchestrating neuronal differentiation: patterns of Ca2+spikes specify transmitter choice. Trends Neurosci. 2004;27:415–21.

    CAS  PubMed  Google Scholar 

  22. 22.

    Resende RR, da Costa JL, Kihara AH, Adhikari A, Lorençon E. Intracellular Ca2+ regulation during neuronal differentiation of murine embryonal carcinoma and mesenchymal stem cells. Stem Cells Dev. 2010.

  23. 23.

    Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 2000;1:11–21.

    CAS  PubMed  Google Scholar 

  24. 24.

    Lipp P, Thomas D, Berridge MJ, Bootman MD. Nuclear calcium signalling by individual cytoplasmic calcium puffs. EMBO J. 1997;16:7166–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Spitzer NC. Activity-dependent neuronal differentiation prior to synapse formation: the functions of calcium transients. J Physiol Paris. 2002;96:73–80.

    CAS  PubMed  Google Scholar 

  26. 26.

    Tonelli FM, Santos AK, Gomes DA, da Silva SL, Gomes KN, Ladeira LO, et al. Stem cells and calcium signaling. Adv Exp Med Biol. 2012;740:891–916.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Leclerc C, Néant I, Webb SE, Miller AL, Moreau M. Calcium transients and calcium signalling during early neurogenesis in the amphibian embryo Xenopus laevis. 2006 Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1763:1184–91.

  28. 28.

    Weber AM, Wong FK, Tufford AR, Schlichter LC, Matveev V, Stanley EF. N-type Ca2+ channels carry the largest current: Implications for nanodomains and transmitter release. Nat Neurosci. 2010;13:1348–50.

    CAS  PubMed  Google Scholar 

  29. 29.

    Weiss N. The N-type voltage-gated calcium channel: when a neuron reads a map. J Neurosci. 2008;28:5621–2.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Gray EE, Murphy JG, Liu Y, Trang I, Tabor GT, Lin L, et al. Disruption of GpI mGluR-dependent Cav2.3 translation in a mouse model of fragile X syndrome. J Neurosci. 2019;39:7453–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Moody WJ. 5 The development of voltage-gated Ion channels and its relation to activity-dependent developmental events. Curr Top Dev Biol. 1998;39:159–85.

    CAS  PubMed  Google Scholar 

  32. 32.

    Snutch TP. Voltage-gated calcium channels. Encycl Neurosci. 2009;1:427–41.

    Google Scholar 

  33. 33.

    Cao YQ, Tsien RW. Different relationship of N- and P/Q-type Ca2+ channels to channel-interacting slots in controlling neurotransmission at cultured hippocampal synapses. J Neurosci. 2010;30:4536–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Vonsattel JP, DiFiglia M. Huntington disease. J Neuropathol Exp Neurol. 1998;57:369–84.

    CAS  PubMed  Google Scholar 

  35. 35.

    Pchitskaya E, Popugaeva E, Bezprozvanny I. Calcium signaling and molecular mechanisms underlying neurodegenerative diseases. Cell Calcium. 2017;70:87–94.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    MacDonald Gillian P. Buckler, Altherr Alan J, Michael Tagle Danilo Snell. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell. 1993;72:971–83.

    Google Scholar 

  37. 37.

    Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR. A new model for prediction of the age of onset and penetrance for Huntington’s disease based on CAG length. Clin Genet. 2004;65:267–77.

    CAS  PubMed  Google Scholar 

  38. 38.

    Hooper M, Hardy K, Handyside A, Hunter S, Monk M. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 1987;326:292–5.

    CAS  PubMed  Google Scholar 

  39. 39.

    Magin TM, Mcwhir J, Melton DW A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res. 1992.

  40. 40.

    Fornazari M, Nascimento IC, Nery AA, da Silva CC, Kowaltowski AJ, Ulrich H. Neuronal differentiation involves a shift from glucose oxidation to fermentation. J Bioenerg Biomembr. 2011;43:531–9.

    CAS  PubMed  Google Scholar 

  41. 41.

    Young MT, Pelegrin P, Surprenant A. Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP. Mol Pharmacol. 2007;71:92–100.

    CAS  PubMed  Google Scholar 

  42. 42.

    Glaser T, De Oliveira SLB, Cheffer A, Beco R, Martins P, Fornazari M, et al. Modulation of mouse embryonic stem cell proliferation and neural differentiation by the P2X7 receptor. PLoS ONE. 2014;9:e96281.

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Negraes PD, Lameu C, Hayashi MA, Melo RL, Camargo AC, Ulrich H. The snake venom peptide Bj-PRO-7a is a M1 muscarinic acetylcholine receptor agonist. Cytom A. 2011;79:77–83.

    Google Scholar 

  44. 44.

    Sykes DA, Dowling MR, Charlton SJ. Exploring the mechanism of agonist efficacy: a relationship between efficacy and agonist dissociation rate at the muscarinic M3 receptor. Mol Pharmacol. 2009;76:543–51.

    CAS  PubMed  Google Scholar 

  45. 45.

    Pal R, Mamidi MK, Das AK, Rao M, Bhonde R. Development of a multiplex PCR assay for characterization of embryonic stem cells. Methods Mol Biol. 2013;1006:147–66.

    CAS  PubMed  Google Scholar 

  46. 46.

    Avelar GM, Glaser T, Leonard G, Richards TA, Ulrich H, Gomes SL A cyclic GMP-dependent K+ channel in the blastocladiomycete fungus Blastocladiella emersonii. Eukaryot Cell. 2015.

  47. 47.

    Imayoshi I, Isomura A, Harima Y, Kawaguchi K, Kori H, Miyachi H, et al. Oscillatory control of factors determining multipotency and fate in mouse neural progenitors. Science. 2013;342:1203–8.

    CAS  PubMed  Google Scholar 

  48. 48.

    Liu Y, Liu H, Sauvey C, Yao L, Zarnowska ED, Zhang SC. Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells. Nat Protoc. 2013;8:1670–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Roy NS, Wang S, Jiang L, Kang J, Benraiss A, Harrison-Restelli C, et al. In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat Med. 2000.

  50. 50.

    Grimm I, Ullsperger SN, Zimmermann H. Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cells. Acta Physiol. 2010;199:181–9.

    CAS  Google Scholar 

  51. 51.

    D’Ascenzo M, Piacentini R, Casalbore P, Budoni M, Pallini R, Azzena GB, et al. Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation. Eur J Neurosci. 2006;23:935–44.

    PubMed  Google Scholar 

  52. 52.

    Cheung KK, Ryten M, Burnstock G. Abundant and dynamic expression of G protein-coupled P2Y receptors in mammalian development. Dev Dyn. 2003;228:254–66.

    CAS  PubMed  Google Scholar 

  53. 53.

    Resende RR, Majumder P, Gomes KN, Britto LR, Ulrich H. P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-D-aspartate-glutamate and acetylcholine receptors during neuronal differentiation. Neuroscience. 2007;146:1169–81.

    CAS  PubMed  Google Scholar 

  54. 54.

    Malmersjo S, Liste I, Dyachok O, Tengholm A, Arenas E, Uhlen P. Ca2+ and cAMP signaling in human embryonic stem cell-derived dopamine neurons. Stem Cells Dev. 2013;19:1355–64.

    Google Scholar 

  55. 55.

    Lautermilch NJ, Spitzer NC. Regulation of calcineurin by growth cone calcium waves controls neurite extension. J Neurosci. 2000;20:315–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Liu YP, Yang CS, Chen MC, Sun SH, Tzeng SF. Ca2+-dependent reduction of glutamate aspartate transporter GLAST expression in astrocytes by P2X7 receptor-mediated phosphoinositide 3-kinase signaling. J Neurochem. 2010.

  57. 57.

    Naranjo JR, Mellström B. Ca2+-dependent transcriptional control of Ca2+ homeostasis. J Biol Chem. 2012;287:31674–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Nieto M, Schuurmans C, Britz O, Guillemot F. Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors. Neuron. 2001;29:401–13.

    CAS  PubMed  Google Scholar 

  59. 59.

    Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neurosci. 2002;3:517–30.

    CAS  PubMed  Google Scholar 

  60. 60.

    Sung MH, McNally JG. Live cell imaging and systems biology. Wiley Interdiscip Rev Syst Biol Med. 2011;3:167–82.

    CAS  PubMed  Google Scholar 

  61. 61.

    Wheeler DG, Barrett CF, Groth RD, Safa P, Tsien RW. CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation-transcription coupling. J Cell Biol. 2008;183:849–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Bito H, Deisseroth K, Tsien RW. CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression. Cell. 1996;87:1203–14.

    CAS  PubMed  Google Scholar 

  63. 63.

    Ciccolini F, Collins TJ, Sudhoelter J, Lipp P, Berridge MJ, Bootman MD. Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation. J Neurosci. 2018.

  64. 64.

    Poulter MO, Barker JL, O’Carroll AM, Lolait SJ, Mahan LC. Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS. J Neurosci. 1992;12:2888–900.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Poulter MO, Barker JL, O’Carroll AM, Lolait SJ, Mahan LC. Co-existent expression of GABAA receptor beta 2, beta 3 and gamma 2 subunit messenger RNAs during embryogenesis and early postnatal development of the rat central nervous system. Neuroscience. 1993;53:1019–33.

    CAS  PubMed  Google Scholar 

  66. 66.

    Ma W, Barker JL. GABA, GAD, and GABA(A) receptor α4, β1, and γ1 subunits are expressed in the late embryonic and early postnatal neocortical germinal matrix and coincide with gliogenesis. Microsc Res Tech. 1998.<398::AID-JEMT6>3.0.CO;2-N.

  67. 67.

    Ma W, Barker JL. Complementary expressions of transcripts encoding GAD67 and GABAA receptor alpha 4, beta 1, and gamma 1 subunits in the proliferative zone of the embryonic rat central nervous system. J Neurosci. 1995;15:2547–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Owens DF, Boyce LH, Davis MB, Kriegstein AR. Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci. 1996;16:6414–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    LoTurco JJ, Owens DF, Heath MJS, Davis MBE, Kriegstein AR GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron. 1995.

  70. 70.

    Behar TN, Schaffner AE, Scott CA, O’Connell C, Barker JL Differential Response of Cortical Plate and Ventricular Zone Cells to GABA as a Migration Stimulus. J Neurosci. 2018.

  71. 71.

    Maric D, Liu QY, Maric I, Chaudry S, Chang YH, Smith SV, et al. GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels. J Neurosci. 2001;21:2343–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Ben-Ari Y. Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci. 2002;3:728–39.

    CAS  PubMed  Google Scholar 

  73. 73.

    Kleppner SR, Tobin AJ. GABA signalling: therapeutic targets for epilepsy, Parkinson’s disease and Huntington’s disease. Expert Opin Ther Targets. 2001;5:219–39.

    CAS  PubMed  Google Scholar 

  74. 74.

    Hodges A, Strand AD, Aragaki AK, Kuhn A, Sengstag T, Hughes G, et al. Regional and cellular gene expression changes in human Huntington’s disease brain. Hum Mol Genet. 2006.

  75. 75.

    Anacker C, Hen R. Adult hippocampal neurogenesis and cognitive flexibility-linking memory and mood. Nat Rev Neurosci. 2017;18:335–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Shin E, Palmer MJ, Li M, Fricker RA. GABAergic neurons from mouse embryonic stem cells possess functional properties of striatal neurons in vitro, and develop into striatal neurons in vivo in a mouse model of Huntington’s disease. Stem Cell Rev Reports. 2012.

  77. 77.

    Sailor KA, Lledo PM. Youth comes but once in a lifetime for adult-born neurons. Trends Neurosci. 2018;41:563–6.

    CAS  PubMed  Google Scholar 

  78. 78.

    Kempermann G, Gage FH, Aigner L, Song H, Curtis MA, Thuret S, et al. Human adult neurogenesis: evidence and remaining questions. Cell Stem Cell. 2018;23:25–30.

    CAS  PubMed  PubMed Central  Google Scholar 

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We thank Adriana Yamaguti Matsukuma, Denise Yamamoto, Wilton José da Rocha Lima and Zilda Mendonça Izzo for technical assistance, Prof. Deborah Schechtman, Department of Biochemistry, Institute of Chemistry, University of São Paulo, for donating the E14Tg2A cell line and Prof. Dr. Soraya Smaili, Federal University of São Paulo for donating the Q23 and Q74 plasmids. This work was supported by the Brazilian funding agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 467465/2014–2), INCT-REGENERA (National Institute of Science and Technology) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant numbers 2012/50880–4, 2015/13345–1, 2010/50554–4 and 2018/07366–4). Japanese grants included a Grant-in-Aid for Scientific Research on Innovative Areas (Ministry of Education, Culture, Sports, Science, and Technology 16H06480) (RK), a Scientific Research (A) grant (Japan Society for Promotion of Science [JSPS] 24240049) (RK), a Young Scientists (A) grant (JSPS 24680035) (II), and a Takeda Foundation grant (RK). US grants that facilitated the project were W81XWH-15-1-0621 (DoD), 1-I01-RX000308-01A1 (VA), and funds from The Gordon Project to Cure Clinical Paralysis and Cele H. and William B. Rubin Family Fund, Incorporated (YDT). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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HU receives consulting fees and support from TissueGnostics GmbH, Vienna, Austria.

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Glaser, T., Shimojo, H., Ribeiro, D.E. et al. ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington’s disease: a novel approach for cell clock research. Mol Psychiatry (2020).

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