Human induced pluripotent stem cells as an in vitro model for autism spectrum disorder
Ding, Yicheng
Ding, Yicheng
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Publication Date
2022-08-23
Type
Thesis
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Abstract
Autism spectrum disorders (ASD) are neurodevelopmental disorders which encompass a wide range of symptoms, including intellectual disability and delayed development, while the phenotype can range from mild to severe. Genetic studies revealed over 100 candidate genes associated with ASD. One of the major genetic risk factors of ASD, NRXN1, encodes pre-synaptic proteins with 3 major isoforms (NRXN1α, NRXN1β and NRXN1) which form synaptic complexes by binding with various pre- and post-synaptic partners to play a crucial role in both excitation and inhibition of neurons. The mutation of NRXN1 gene has been associated with various neurodevelopmental disorders including ASD. Human based-disease modelling study is limited due to difficulty in access to patient materials and primary brain cells. The induced pluripotent stem cell (iPSC) technology provides a great opportunity to investigate patient-derived neurons and/or engineered neurons with gene of interest. We hypothesize the deletion of NRNX1 will cause dysregulation on excitation and inhibition (E/I) balance which leads to aberrant network behaviour. We recruited ASD patients with or without NRXN1 deletion and healthy donors in this PhD project, who donated their skin biopsies for the iPSC research. The ASD patients included a rare family case of 3 patients of ASD with NRXN1α and 2 individuals from other families, and healthy controls include both familial and independent healthy volunteers. We derived dermal fibroblasts from the skin biopsies and reprogrammed them into iPSCs using the non-integration Sendai virus method. Stable iPSC clones were established which were shown to express pluripotency markers (OCT4, SOX2, NANOG, SSEA4 and TRA 1-81) and possess the capacity to differentiate into different cell types from all 3 germ layers. Different isoforms of NRXN1 are known to bind and interact differently with various binding partners, however, the function of NRXN1β is less well-studied. Due to the failure to recruit ASD patient with NRXN1β-specific deletion, we used CRISPR/Cas9 system to induce β-NRXN1-specific deletion in human iPSC lines from unrelated healthy controls. We successfully derived iPSC lines with either heterozygous or homozygous deletion of NRXN1β. The iPSCs were differentiated to mature neurons expressing MAP2 and SYN1 by Ngn2-forced neuronal differentiation. Neurons induced by Ngn2 (iNs) also transcribed pre- and post-synaptic interaction partners of NRXN1, including CASK, NLGN1, MUNC18 and SHANKs. The difference in neuronal maturity or synaptogenesis was not observed between controls and NRXN1 deletion lines. Neuronal network function was investigated using a multi-electrode array (MEA), and the deletion of NRXN1β displayed reduced spontaneous activity. Our results showed for the first time that the spontaneous firing of human neurons was affected by the deletion mutation of NRXN1β. The iPSCs established in this project are valuable resources for the disease modelling and further drug discovery of ASD.
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NUI Galway