Co-Expression Network Analysis of Activity-Dependent Gene Expression In Human Neurons Identifies Expression Changes Associated with the Timothy Syndrome CACNA1C Mutation

Background: Depolarization-induced gene expression is a fundamental process underlying synaptic activity, which involves several calcium-signaling pathways. Most of the current knowledge regarding gene expression changes in response to depolarization comes from experiments involving mouse neurons or human neuroblastoma cell lines. Timothy syndrome is caused by a mutation of the voltage-dependent Ca-channel CACNA1C and is manifested by cardiac abnormalities and high frequency of autism among affected individuals(>60%). 

Objectives: Here we aimed to (a) thoroughly characterize gene expression changes induced by depolarization  human neurons and (b) establish whether the Timothy syndrome CACNA1C mutation causes alteration in resting state gene transcription in human neurons and/or affects depolarization-induced gene expression

Methods: We obtained genome-wide transcription profiles iPSC-derived human neurons from normal individuals and Timothy Syndrome patients, in either a resting state or after 9h of depolarization by KCl treatment. Gene expression changes associated with depolarization as well as the TS mutation were characterized by weighted-gene co-expression network analysis. Motif-enrichment analysis was used to identify transcription factors driving specific network modules

Results: We identified modules of co-expressed genes corresponding to gene upregulation as well as downregulation in response to neuronal depolarization. We find that genes with significant depolarization-dependent expression changes are enriched for binding sites of several transcription factors, including known regulators of activity-dependent gene expression (CREB) and we find evidence for novel trascription factors regulating activity-dependent transcription. In addition we describe co-expression modules associated with the CACNA1C mutation and discuss their relationship to depolarization-induced changes.

Conclusions: The Timothy syndrome mutation affects gene expression in human neurons, and the dysregulated genes are primarily involved in inositol phosphate signaling pathway.