Autism Spectrum Disorder Induced Pluripotent Stem Cells Display Dysregulated Calcium Signaling During Neural Differentiation.

Abstract

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication, social interaction, and behavior. Calcium (Ca²⁺) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein, we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca²⁺ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing, spanning the following sequential stages: the iPSC stage, neural induction (NI) stage, neurosphere (NSP) stage, and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca²⁺ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly, samples from the iPSC and Diff stages were processed for Ca²⁺ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca²⁺ levels in response to ATP compared to controls. By contrast, in the Diff stage, ASD neurons showed reduced maximum Ca²⁺ levels in response to ATP but increased maximum Ca²⁺ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli, this differential profile of receptor- and ionophore-mediated Ca²⁺ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca²⁺ signaling dysregulation during neurogenesis in idiopathic ASD.