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.
Access options
Subscribe to Journal
Get full journal access for 1 year
$225.00
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
from$8.99
All prices are NET prices.
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).
- 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).
- 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).
- 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).
- 5.
Wang, K. et al. Common genetic variants on 5p14.1 associate with autism spectrum disorders. Nature 459, 528–533 (2009).
- 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).
- 7.
Nyegaard, M. et al. CACNA1C (rs1006737) is associated with schizophrenia. Mol. Psychiatry 15, 119–121 (2010).
- 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).
- 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).
- 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).
- 11.
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
- 12.
Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230–234 (2011).
- 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).
- 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).
- 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).
- 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).
- 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).
- 18.
Garbelli, R. et al. Layer-specific genes reveal a rudimentary laminar pattern in human nodular heterotopia. Neurology 73, 746–753 (2009).
- 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).
- 20.
Alcamo, E.A. et al. Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57, 364–377 (2008).
- 21.
Britanova, O. et al. Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57, 378–392 (2008).
- 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).
- 23.
Pinto, D. et al. Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466, 368–372 (2010).
- 24.
Garbett, K. et al. Immune transcriptome alterations in the temporal cortex of subjects with autism. Neurobiol. Dis. 30, 303–311 (2008).
- 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).
- 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).
- 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).
- 28.
Dulcis, D. & Spitzer, N.C. Illumination controls differentiation of dopamine neurons regulating behaviour. Nature 456, 195–201 (2008).
- 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).
- 30.
Geschwind, D.H. & Levitt, P. Autism spectrum disorders: developmental disconnection syndromes. Curr. Opin. Neurobiol. 17, 103–111 (2007).
- 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).
- 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).
- 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).
- 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).
- 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).
- 36.
Du, P., Kibbe, W.A. & Lin, S.M. lumi: a pipeline for processing Illumina microarray. Bioinformatics 24, 1547–1548 (2008).
- 37.
Coppola, G. et al. Gene expression study on peripheral blood identifies progranulin mutations. Ann. Neurol. 64, 92–96 (2008).
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
Author notes
- Thomas Portmann
- , Irina Voineagu
- & Masayuki Yazawa
These authors contributed equally to this work.
Affiliations
Department of Neurobiology, Stanford University School of Medicine, Stanford, California, USA.
- Sergiu P Paşca
- , Thomas Portmann
- , Masayuki Yazawa
- , Aleksandr Shcheglovitov
- , Anca M Paşca
- & Ricardo E Dolmetsch
Department of Neurology and Program in Neurogenetics, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
- Irina Voineagu
- & Daniel H Geschwind
Stanford Institute for Stem Cell Biology and Regenerative Medicine and Department of Neurosurgery, Stanford School of Medicine, Stanford, California, USA.
- Branden Cord
- & Theo D Palmer
Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, Stanford, California, USA.
- Sachiko Chikahisa
- , Seiji Nishino
- & Joachim Hallmayer
Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
- Sachiko Chikahisa
Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
- Jonathan A Bernstein
Authors
Search for Sergiu P Paşca in:
Search for Thomas Portmann in:
Search for Irina Voineagu in:
Search for Masayuki Yazawa in:
Search for Aleksandr Shcheglovitov in:
Search for Anca M Paşca in:
Search for Branden Cord in:
Search for Theo D Palmer in:
Search for Sachiko Chikahisa in:
Search for Seiji Nishino in:
Search for Jonathan A Bernstein in:
Search for Joachim Hallmayer in:
Search for Daniel H Geschwind in:
Search for Ricardo E Dolmetsch in:
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.
Competing interests
The authors declare no competing financial interests.
Corresponding author
Correspondence to Ricardo E Dolmetsch.
Supplementary information
PDF files
- 1.
Supplementary Text and Figures
Supplementary Methods, Supplementary Figures 1–9 and Supplementary Tables 1–3 and 5
Excel files
- 1.
Supplementary Table 4
Genes differentially expressed between TS and controls
Rights and permissions
To obtain permission to re-use content from this article visit RightsLink.
About this article
Further reading
-
Induced pluripotent stem cells as a tool to study brain circuits in autism-related disorders
Stem Cell Research & Therapy (2018)
-
Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders
Molecular Autism (2018)
-
Generation and assembly of human brain region–specific three-dimensional cultures
Nature Protocols (2018)
-
Genomics in neurodevelopmental disorders: an avenue to personalized medicine
Experimental & Molecular Medicine (2018)
-
The state of the art in stem cell biology and regenerative medicine: the end of the beginning
Pediatric Research (2018)