Uncovering the Functional Link Between SHANK3 Deletions and Deficiency in Neurodevelopment Using iPSC-Derived Human Neurons.

Jiandong Yu,Xiaokuang Ma,Siyi Gong,Libing Zhou,Shuting Chen,Jiajun Zheng,Lingling Shi,Shenfeng Qiu,Qingpei Chen,Zhuoran Xu,Abolfazl Doostparast Torshizi,Kai Wang,Xiaoxia Chen,Guanqun Huang

doi:10.3389/fnana.2019.00023

Abstract

SHANK3 mutations, including de novo deletions, have been associated with autism spectrum disorders (ASD). However, the effects of SHANK3 loss of function on neurodevelopment remain poorly understood. Here we generated human induced pluripotent stem cells (iPSC) in vitro, followed by neuro-differentiation and lentivirus-mediated shRNA expression to evaluate how SHANK3 knockdown affects the in vitro neurodevelopmental process at multiple time points (up to 4 weeks). We found that SHANK3 knockdown impaired both early stage of neuronal development and mature neuronal function, as demonstrated by a reduction in neuronal soma size, growth cone area, neurite length and branch numbers. Notably, electrophysiology analyses showed defects in excitatory and inhibitory synaptic transmission. Furthermore, transcriptome analyses revealed that multiple biological pathways related to neuron projection, motility and regulation of neurogenesis were disrupted in cells with SHANK3 knockdown. In conclusion, utilizing a human iPSC-based neural induction model, this study presented combined morphological, electrophysiological and transcription evidence that support that SHANK3 as an intrinsic, cell autonomous factor that controls cellular function development in human neurons.

Highlights

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impairments in social communication and interaction, and repetitive behaviors and restricted interests (Freitag et al, 2010; State and Levitt, 2011; Lyall et al, 2017; Rubeis et al, 2018)
The human induced pluripotent stem cells (iPSC) used in this experiment were stained positive with specific iPSC marker proteins (OCT4 and SSEA4) (Figure 1A) and specific neural stem cells marker proteins (SOX2, nestin) (Figure 1B), indicating that the cells have the potential of differentiating into neurons
The results showed that neurons derived from iPSC could be differentiated into multiple CNS neuron types, reflecting a highly induction efficiency using our method

Summary

Introduction

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impairments in social communication and interaction, and repetitive behaviors and restricted interests (Freitag et al, 2010; State and Levitt, 2011; Lyall et al, 2017; Rubeis et al, 2018). Abnormal cortico-striatal circuits, disrupted excitability and inhibitory (E/I) balance, and synaptic dysfunction have been identified to be associated with the mechanism of ASD-like behavior (Bozdagi et al, 2010; Peca et al, 2011; Wang et al, 2011; Mei et al, 2016; Vicidomini et al, 2017; Bey et al, 2018). These analysis of synaptic physiology and mice behavior revealed a strong causal connection between SHANK3 mutations and ASD-like endophenotypes

Methods

Results

Conclusion