Aberrant calcium channel splicing drives defects in cortical differentiation in Timothy Syndrome

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Abstract

The syndromic autism spectrum disorder (ASD) Timothy Syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Ca_v1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Ca_v1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Ca_v1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Ca_v1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally-regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.

Data availability

All data analyzed in this study have been included in the manuscript and supporting files and figures. Source data files have been provided for Figures 1 to 4, as well as Figure 1-figure supplement 1

Article and author information

Author details

Georgia Panagiotakos

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

For correspondence
georgia.panagiotakos@ucsf.edu

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-9444-2480
Christos Haveles

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

Competing interests
No competing interests declared.
Arpana Arjun

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

Competing interests
No competing interests declared.
Ralitsa Petrova

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-5586-6192
Anshul Rana

Graduate Program in Biochemistry, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.
Thomas Portmann

Department of Neurobiology, Stanford University School of Medicine, Stanford, United States

Competing interests
Thomas Portmann, is currently an employee of Neucyte, Inc.
Sergiu P Paşca

Department of Neurobiology, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-3216-3248
Theo D Palmer

Department of Neurosurgery, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.
Ricardo E Dolmetsch

Department of Neurobiology, Stanford University School of Medicine, Stanford, United States

Competing interests
Ricardo E Dolmetsch, is currently an employee of Novartis Institutes for Biomedical Research.

"This ORCID iD identifies the author of this article:" 0000-0002-2738-8338

Funding

National Institutes of Health (F31 MH090648 Predoctoral Fellowship)

Georgia Panagiotakos

National Institutes of Health (R01 MH096815)

Theo D Palmer

National Institutes of Health (Pioneer Award 5DP1OD3889)

Ricardo E Dolmetsch

Stanford University School of Medicine (Frances B Nelson Neuroscience Graduate Fellowship)

Georgia Panagiotakos

University of California, San Francisco (Program for Breakthrough Biomedical Research Sandler Foundation)

Georgia Panagiotakos

Howard Hughes Medical Institute (International Student Research Award)

Anshul Rana

Stanford University School of Medicine (Lucile P Markey Graduate Fellowship)

Anshul Rana

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Postdoctoral Fellowship PBSKP3-123434)

Thomas Portmann

Brain and Behavior Research Foundation (NARSAD Young Investigator Award)

Sergiu P Paşca

Simons Foundation (SFARI 206574)

Theo D Palmer

Blume Foundation

Theo D Palmer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal experiments performed in this study were done so in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and protocols approved by the Stanford University and University of California, San Francisco (UCSF) Institutional Animal Care and Use Committees (Stanford protocol #13705 granted to the lab of Dr. Ricardo Dolmetsch, and UCSF protocol #AN109792 granted to the lab of Dr. Georgia Panagiotakos).

Human subjects: Collection of dermal fibroblasts from TS patients and unaffected control subjects, as well as iPSC generation was previously described (Pasca, et al. 2011). Experiments involving primary dermal fibroblasts and iPSCs from Timothy Syndrome patients and healthy control subjects were conducted at Stanford University under study protocols (#12481, #232, and #327) approved by the Institutional Review Board and Stem Cell Research Oversight (SCRO) committees of Stanford University after obtaining informed consent.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.