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.

  • Letter
  • Published:

Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome

Abstract

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Cav1.2 that is associated with developmental delay and autism1. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca2+) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type–channel blocker2,3,4. These findings provide strong evidence that Cav1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Characterization of iPSC-derived NPCs and neurons.
Figure 2: Characterization of NPCs and neurons by genome-wide microarrays.
Figure 3: Characterization of neuronal subpopulations differentiated from Timothy syndrome and control iPSCs.
Figure 4: Abnormal expression of TH in iPSC-derived neurons from individuals with Timothy syndrome.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Splawski, I. et al. Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119, 19–31 (2004).

    Article  CAS  Google Scholar 

  2. Yarotskyy, V. et al. Roscovitine binds to novel L-channel (CaV1.2) sites that separately affect activation and inactivation. J. Biol. Chem. 285, 43–53 (2010).

    Article  CAS  Google Scholar 

  3. Yarotskyy, V. & Elmslie, K.S. Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels. Br. J. Pharmacol. 152, 386–395 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  4. Yarotskyy, V., Gao, G., Peterson, B.Z. & Elmslie, K.S. The Timothy syndrome mutation of cardiac CaV1.2 (L-type) channels: multiple altered gating mechanisms and pharmacological restoration of inactivation. J. Physiol. (Lond.) 587, 551–565 (2009).

    Article  CAS  Google Scholar 

  5. Wang, K. et al. Common genetic variants on 5p14.1 associate with autism spectrum disorders. Nature 459, 528–533 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  6. Moskvina, V. et al. Gene-wide analyses of genome-wide association data sets: evidence for multiple common risk alleles for schizophrenia and bipolar disorder and for overlap in genetic risk. Mol. Psychiatry 14, 252–260 (2009).

    Article  CAS  Google Scholar 

  7. Nyegaard, M. et al. CACNA1C (rs1006737) is associated with schizophrenia. Mol. Psychiatry 15, 119–121 (2010).

    Article  CAS  Google Scholar 

  8. Dolmetsch, R.E., Pajvani, U., Fife, K., Spotts, J.M. & Greenberg, M.E. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science 294, 333–339 (2001).

    Article  CAS  Google Scholar 

  9. Barrett, C.F. & Tsien, R.W. The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels. Proc. Natl. Acad. Sci. USA 105, 2157–2162 (2008).

    Article  CAS  Google Scholar 

  10. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  Google Scholar 

  11. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  12. Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230–234 (2011).

    Article  CAS  PubMed Central  Google Scholar 

  13. Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).

    Article  CAS  Google Scholar 

  14. Pankratz, M.T. et al. Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells 25, 1511–1520 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  15. Li, X.J. et al. Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells. Development 136, 4055–4063 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  16. Warren, L., Bryder, D., Weissman, I.L. & Quake, S.R. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc. Natl. Acad. Sci. USA 103, 17807–17812 (2006).

    Article  CAS  Google Scholar 

  17. Flatz, L. et al. Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines. Proc. Natl. Acad. Sci. USA 108, 5724–5729 (2011).

    Article  CAS  Google Scholar 

  18. Garbelli, R. et al. Layer-specific genes reveal a rudimentary laminar pattern in human nodular heterotopia. Neurology 73, 746–753 (2009).

    Article  CAS  Google Scholar 

  19. Saito, T. et al. Neocortical layer formation of human developing brains and lissencephalies: consideration of layer-specific marker expression. Cereb. Cortex 21, 588–596 (2010).

    Article  Google Scholar 

  20. Alcamo, E.A. et al. Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57, 364–377 (2008).

    Article  CAS  Google Scholar 

  21. Britanova, O. et al. Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57, 378–392 (2008).

    Article  CAS  Google Scholar 

  22. Leone, D.P., Srinivasan, K., Chen, B., Alcamo, E. & McConnell, S.K. The determination of projection neuron identity in the developing cerebral cortex. Curr. Opin. Neurobiol. 18, 28–35 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  23. Pinto, D. et al. Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466, 368–372 (2010).

    Article  CAS  PubMed Central  Google Scholar 

  24. Garbett, K. et al. Immune transcriptome alterations in the temporal cortex of subjects with autism. Neurobiol. Dis. 30, 303–311 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  25. Ip, B.K., Bayatti, N., Howard, N.J., Lindsay, S. & Clowry, G.J. The corticofugal neuron-associated genes ROBO1, SRGAP1 and CTIP2 exhibit an anterior to posterior gradient of expression in early fetal human neocortex development. Cereb. Cortex 21, 1395–1407 (2011).

    Article  Google Scholar 

  26. Romano, G., Suon, S., Jin, H., Donaldson, A.E. & Iacovitti, L. Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: implications for the engineering of a human TH minimal promoter assembled in a self-inactivating lentiviral vector system. J. Cell. Physiol. 204, 666–677 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  27. Raghanti, M.A. et al. Species-specific distributions of tyrosine hydroxylase–immunoreactive neurons in the prefrontal cortex of anthropoid primates. Neuroscience 158, 1551–1559 (2009).

    Article  CAS  Google Scholar 

  28. Dulcis, D. & Spitzer, N.C. Illumination controls differentiation of dopamine neurons regulating behaviour. Nature 456, 195–201 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  29. Barttfeld, P. et al. A big-world network in ASD: dynamical connectivity analysis reflects a deficit in long-range connections and an excess of short-range connections. Neuropsychologia 49, 254–263 (2011).

    Article  Google Scholar 

  30. Geschwind, D.H. & Levitt, P. Autism spectrum disorders: developmental disconnection syndromes. Curr. Opin. Neurobiol. 17, 103–111 (2007).

    Article  CAS  Google Scholar 

  31. Casanova, M.F. et al. Reduced gyral window and corpus callosum size in autism: possible macroscopic correlates of a minicolumnopathy. J. Autism Dev. Disord. 39, 751–764 (2009).

    Article  PubMed Central  Google Scholar 

  32. D'Souza, A., Onem, E., Patel, P., La Gamma, E.F. & Nankova, B.B. Valproic acid regulates catecholaminergic pathways by concentration-dependent threshold effects on TH mRNA synthesis and degradation. Brain Res. 1247, 1–10 (2009).

    Article  CAS  Google Scholar 

  33. Toru, M., Nishikawa, T., Mataga, N. & Takashima, M. Dopamine metabolism increases in post-mortem schizophrenic basal ganglia. J. Neural Transm. 54, 181–191 (1982).

    Article  CAS  Google Scholar 

  34. Elkabetz, Y. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22, 152–165 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  35. Tole, S. & Patterson, P.H. Regionalization of the developing forebrain: a comparison of FORSE-1, Dlx-2, and BF-1. J. Neurosci. 15, 970–980 (1995).

    Article  CAS  Google Scholar 

  36. Du, P., Kibbe, W.A. & Lin, S.M. lumi: a pipeline for processing Illumina microarray. Bioinformatics 24, 1547–1548 (2008).

    Article  CAS  Google Scholar 

  37. Coppola, G. et al. Gene expression study on peripheral blood identifies progranulin mutations. Ann. Neurol. 64, 92–96 (2008).

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank K. Timothy and the individuals with Timothy syndrome who participated in this study; E. Nigh for editing of the manuscript; U. Francke for karyotyping; A. Cherry and D. Bangs for help with fibroblast cultures; G. Panagiotakos and C. Young-Park for insightful discussions, and A. Krawisz, R. Schwemberger, D. Fu and R. Shu for help with data analysis. Antibodies to FORSE-1 were developed by P.H. Patterson and were obtained from the Developmental Studies Hybridoma Bank (University of Iowa). Financial support was provided by a US National Institutes of Health Director's Pioneer Award, and by grants to R.E.D. from the US National Institute of Mental Health, the California Institute for Regenerative Medicine and the Simons Foundation for Autism Research. S.P.P. was supported by awards from the International Brain Research Organization Outstanding Research Fellowship and the Tashia and John Morgridge Endowed Fellowship, M.Y. by a Japan Society of the Promotion for Science Postdoctoral Fellowship for Research Abroad and an American Heart Association Western States postdoctoral fellowship, T.P. by a Swiss National Science Foundation Postdoctoral Fellowship and A.S. by a California Institute for Regenerative Medicine Postdoctoral Fellowship. We are also grateful for funding from B. and F. Horowitz, M. McCafferey, B. and J. Packard, P. Kwan and K. Wang and the Flora foundation.

Author information

Authors and Affiliations

Authors

Contributions

R.E.D. and S.P.P. designed the experiments and wrote the manuscript. S.P.P. generated iPSC lines, differentiated the iPSC lines into neurons, performed the calcium imaging and immunocytochemistry studies and contributed to the mutant mouse characterization. T.P. designed and analyzed the Fluidigm microarray studies. M.Y. generated and characterized the iPSC lines, and generated and characterized the mutant mice. I.V. and D.H.G. performed and analyzed the microarray gene expression experiments. A.S. derived neurons and designed and performed the electrophysiological experiments. A.M.P. performed the karyotyping and immunocytochemistry. S.C. and N.S. performed and analyzed catecholamine concentrations by HPLC. B.C. and T.D.P. contributed to the Fluidigm studies. J.A.B. and J.H. recruited and characterized the subjects.

Corresponding author

Correspondence to Ricardo E Dolmetsch.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–9 and Supplementary Tables 1–3 and 5 (PDF 1779 kb)

Supplementary Table 4

Genes differentially expressed between TS and controls (XLS 4233 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paşca, S., Portmann, T., Voineagu, I. et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat Med 17, 1657–1662 (2011). https://doi.org/10.1038/nm.2576

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2576

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing