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

内质网应激作为自闭症相关 NLGN3 R451C 人类神经元突触功能障碍的新机制

• 批准号: 9257197
• 负责人: Vincent Mirabella
• 金额: $ 3.83万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257197
• 关键词: ATF6 gene; Adhesions; Amino Acid Substitution; Animals; Arginine; Attenuated; Autistic Disorder; Biochemical; Biochemistry; Biological; Brain; Cell Adhesion Molecules; Cell Line; Cell surface; Cells; Chemicals; Complex; Cysteine; Data; Defect; Development; Disease; Dose; ES Cell Line; Effectiveness; Electrophysiology (science); Endoplasmic Reticulum; Equilibrium; Etiology; Fellowship; Functional disorder; GRP94; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic study; Goals; Human; Human Genetics; Image; Impairment; Individual; Knock-in; Knock-in Mouse; Knockout Mice; Link; Luciferases; Maintenance; Measures; Mediating; Methods; Missense Mutation; Molecular; Molecular Abnormality; Molecular Chaperones; Morphology; Mutation; Neurodevelopmental Disorder; Neuronal Dysfunction; Neurons; Noise; PC12 Cells; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Pharmacology; Phenotype; Phosphorylation; Physicians; Physiology; Positioning Attribute; Property; Proteins; RNA Splicing; Reporter; Rett Syndrome; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Schizophrenia; Scientist; Social Interaction; Source; Stabilizing Agents; Stem Cell Research; Surface; Synapses; Synapsins; Synaptic Transmission; System; Techniques; Testing; Translating; Tunicamycin; United States National Institutes of Health; Variant; Western Blotting; arm; autism spectrum disorder; embryonic stem cell; endoplasmic reticulum stress; experimental study; gephyrin; immunocytochemistry; inhibitor/antagonist; innovation; insight; mouse model; mutant; neuroligin 3; neuropsychiatric disorder; neurotransmission; novel; overexpression; postsynaptic; protein transport; public health relevance; repetitive behavior; response; small molecule; stem cell biology; synaptic function; synaptogenesis; tauroursodeoxycholic acid; trafficking; transcription factor CHOP

 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.

 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多种神经发育障碍的生物学基础，包括自闭症谱系障碍（ASD）、雷特综合征和精神分裂症等神经精神疾病。人类遗传学研究表明，编码突触粘附蛋白（例如神经连接蛋白（NLGN））的基因中越来越多的突变与自闭症谱系障碍（ASD）有关。尽管进行了大量的动物研究，但由于缺乏自闭症患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。利用异源细胞系统，我们最近发现一种与自闭症相关的错义突变，即人类 NLGN3 (NLGN3 R451C) 451 位的精氨酸 (R) 变为半胱氨酸 (C)，会破坏蛋白质运输并通过激活未折叠蛋白质反应 (UPR) 引起内质网 (ER) 应激，这提出了一种可能的自闭症病因学新机制。这一假设以前从未在神经元中进行过测试，更重要的是，它是否适用于人类尚不清楚。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，现在可以在同基因背景下创建携带基因突变的人类神经元，从而消除了先前使用多个个体样本进行干细胞研究的遗传背景“噪音”。因此，我建议检查 ER 应激和 UPR 是否与在同基因背景下携带 R451C 基因突变的人类神经元的突触功能障碍存在机械联系。我将研究与 UPR 和 ER 应激相关的特定蛋白质的亚细胞定位，并与推定的相关途径并行。通过形态测量和电生理学分析将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潜力：1）揭示一种新的致病机制，通过该机制，与自闭症相关的关键突变导致神经元功能障碍；2）确定内质网应激和 UPR 在调节人类神经元突触传递中的作用。