Investigating role of neurexin-1 mutation in autism using human induced neurons

描述（由申请人提供）：本提案的目的是加强我们对自闭症谱系障碍（asd）的病理生理学的有限理解，asd是一种破坏性的神经发育疾病，影响88名美国儿童中的1名。这将通过研究由人类胚胎干细胞（ESCs）诱导的人类神经元来实现，这些胚胎干细胞有条件地敲除神经素-1 (Nrxn1)，这是一种经常在asd患者中发生突变的基因。在人类神经元环境中，Nrxn1的突触作用及其对自闭症的功能贡献尚未被研究，这使得这项研究具有独特性。初步数据表明了这种方法的可行性，由此产生了Nrxn1位点的ESCs条件突变体。利用这些细胞，将产生人类兴奋性神经元，在神经元发育过程中，Nrxn1基因将被有条件地敲除。基于Nrxn1是人类神经元正常突触功能所必需的假设，我将比较控制和Nrxn1突变的人类神经元的突触发育和功能（突触传递）。Nrxn1在突触发育中的功能将通过形态学研究来监测突触密度和大小以及突触囊泡循环。使用电生理分析，我将评估Nrxn1在介导人类兴奋性突触突触传递中的突触前和突触后功能。总的来说，这些实验试图确定突变的Nrxn1影响人类突触传递的机制，从而导致自闭症，使用评估突触功能的实验方法相结合。！

DESCRIPTION (provided by applicant): The object of this proposal is to enhance our limited understanding of the pathophysiology of autism spectrum disorders (ASDs), which is a devastating neurodevelopmental condition affecting 1 in 88 US children. This will be accomplished by studying human neurons induced from human embryonic stem cells (ESCs) that are conditionally knock out for neurexin-1 (Nrxn1), a gene that is frequently mutated in patients with ASDs. The synaptic role of Nrxn1 and its functional contribution to autism in human neuronal context has not been studied, which makes this study unique. Preliminary data indicates the feasibility of this approach, whereby ESCs conditionally mutant for Nrxn1 locus has been generated. Using these cells, human excitatory neurons will be generated whereby Nrxn1 gene will be conditionally knocked out during neuronal development. Based on the hypothesis that Nrxn1 is required for proper synaptic function in human neurons, I will compare synapse development and function (synaptic transmission) in control and Nrxn1 mutant human neurons. Nrxn1 function in synapse development will be monitored by morphological studies examining synapse density and size as well as synaptic vesicle cycling. Using, electrophysiolgical assays, I will assess both pre and postsynaptic functions of Nrxn1 in mediating synaptic transmission at human excitatory synapses. In total, these experiments attempt to determine the mechanism by which mutated Nrxn1 influence human synaptic transmission and as a consequence, contribute to autism, using a combination of experimental approaches that assess synaptic function. !