Endoplasmic Reticulum Stress as a Novel Mechanism of Synaptic Dysfunction in Autism-Associated NLGN3 R451C Human Neurons

 描述（由申请人提供）：突触传递控制大脑中的信息流，突触功能障碍可能是几种神经发育障碍的生物学基础，包括自闭症谱系障碍（ASD）、Rett综合征和神经精神障碍，如精神分裂症。人类遗传学研究表明，越来越多的编码突触粘附蛋白（如神经连接蛋白（NLGN））的基因突变与ASD有关。尽管有许多动物研究，但由于缺乏来自自闭症受试者的初级神经元的容易获得的来源，这些基因的突变如何导致人类的病理和突触功能障碍仍然是个谜。使用异源细胞系统，我们最近发现一个自闭症相关的错义突变，即人NLGN 3的451位精氨酸（R）至半胱氨酸（C）（NLGN 3 R451C），破坏蛋白质运输并导致内质网（ER）应激，激活未折叠蛋白反应（UPR），提出了自闭症病因学的一种可能的新机制。这一假设以前从未在神经元中进行过测试，更重要的是，它是否会转化为人类尚不清楚。幸运的是，最近开发的干细胞生物学新技术使这种分析成为可能。此外，现在有可能在等基因背景上创建携带基因突变的人类神经元，消除遗传背景“噪音”，这些噪音混淆了以前使用多个个体样本进行的干细胞研究。因此，我建议研究ER应激和UPR是否与携带R451C基因突变的人类神经元中的突触功能障碍有机械联系。我将研究亚细胞定位的特定蛋白质参与UPR和ER压力平行与pupillin相关的途径。将通过形态测量和电生理分析提供神经元表型的即时和功能读数。使用小分子伴侣和UPR抑制剂的机制研究将巩固ER应激，UPR和突触功能障碍之间的潜在联系。这项研究最重要的影响是，它有可能1）揭示一种新的致病机制，通过这种机制，一种关键的自闭症相关突变导致神经元功能障碍，2）确定ER应激和UPR在人类神经元环境中调节突触传递的作用。

 DESCRIPTION (provided by applicant): Synaptic transmission controls information flow in the brain, and synaptic dysfunction is likely the biological basis of several neurodevelopmental disorders including autism spectrum disorders (ASDs), Rett syndrome, and neuropsychiatric disorders such as schizophrenia. Human genetic studies revealed that an increasing number of mutations in genes encoding synaptic adhesion proteins, such as the neuroligins (NLGNs), are linked to ASDs. Despite numerous animal studies, given the lack of a readily accessible source of primary neurons from subjects with autism, how mutations in these genes cause pathology and synaptic dysfunction in humans remains enigmatic. Using heterologous cell systems, we recently discovered that one autism-linked missense mutation, arginine (R) to cysteine (C) at position 451 of the human NLGN3 (NLGN3 R451C), disrupts protein trafficking and causes endoplasmic reticulum (ER) stress with activation of the Unfolded Protein Response (UPR), presenting a possible novel mechanism of autism etiology. This hypothesis has never been tested before in neurons and, more importantly, whether it translates to humans is not known. Fortunately, recently developed novel techniques in stem cell biology have made this analysis possible. Furthermore, it is now possible to create human neurons carrying gene mutations on an isogenic background, eliminating the genetic background "noise" that confounded previous stem cell research using samples from multiple individuals. Therefore, I propose to examine if ER stress and UPR are mechanistically linked with the synaptic dysfunction in human neurons carrying the R451C gene mutation on an isogenic background. I will investigate the sub-cellular localization of specific proteins involved in UPR and ER stress in parallel with putatively correlating pathways. An immediate and functional readout of neuronal phenotypes will be provided by morphometric and electrophysiological analyses. Mechanistic studies using small molecule chaperones and inhibitors of the UPR will solidify the potential link between ER stress, UPR and synaptic dysfunction. The most significant impact of this study is that it has the potential to 1) uncover a novel pathogenic mechanism by which a key autism-linked mutation causes neuronal dysfunction and 2) determine the roles of ER stress and the UPR in regulating synaptic transmission in a human neuronal context.