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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.

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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.

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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.

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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.

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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)

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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

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