Functional and mechanistic interrogation of alpha neurexin extracellular domains

α神经毒素细胞外结构域的功能和机制研究

基本信息

批准号：
10377418
负责人：
Jason Aoto
金额：
$ 37.98万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10377418
关键词：
Acute Address Affect Affinity Alleles Alternative Splicing Amino Acids Binding Biochemical Biological Biological Assay Cell Adhesion Molecules Cellular Assay Code Cognition Disorders Communication Data Diagnosis Disease EGF gene Electron Microscopy Electrophysiology (science)Epilepsy Etiology Excitatory Synapse Exons Extracellular Domain Family Functional disorder Genes Genomic Segment Gonadal Steroid Hormones Hippocampus (Brain)Impairment In Vitro Individual Inhibitory Synapse Injections Intellectual functioning disability Laboratories Laminin Length Ligand Binding Ligands Light Link Masks Measures Mediating Mental disorders Messenger RNA Missense Mutation Molecular Morphology Mus Mutation Neurodevelopmental Disorder Neurons Patients Phenotype Play Point Mutation Property Protein Family Protein Isoforms Proteins RNA Splicing Role Schizophrenia Shapes Site Slice Specificity Structure Surface Synapses Synaptic Transmission Synaptic plasticity Testing autism spectrum disorder base combinatorial extracellular immunocytochemistry in vivo insight interdisciplinary approach knock-down mutant neuroligin 1 neuropsychiatric disorder overexpression postsynaptic presynaptic small hairpin RNA stem synaptic function trafficking transmission process

项目摘要

Neurexins (Nrxns) are a family of essential but poorly understood presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders such as autism spectrum disorders (ASDs), schizophrenia and intellectual disability (ID). Three evolutionarily conserved neurexin genes produce longer alpha and shorter beta neurexin mRNAs that undergo extensive alternative splicing. α- and β-Nrxns share common transmembrane and cytoplasmic sequences but differ in the length and complexity of their extracellular domains (ECDs; 9 α-Nrxn domains compared to 1 β-Nrxn domain). Individual α-Nrxns are associated with distinct neuropsychiatric disorders and disease-relevant mutations are commonly located in genomic regions that code for α-Nrxn-specific extracellular sequences, suggesting that individual alpha neurexin ECDs may control distinct aspects of synapse function. Despite their discovery over twenty years ago, the fundamental question regarding the essential role of individual α-Nrxn ECDs at the synapse remains unresolved. As an important first step in understanding how α-Nrxn-specific extracellular sequences function at the synapse, our laboratory has identified an Nrxn3α compound heterozygous patient with profound ID and epilepsy. One allele produces a non-functional protein and the second harbors a missense mutation in an extracellular sequence shared by all α-Nrxns. Intriguingly, there are multiple ASD associated mutations in the equivalent region of Nrxn1α indicating that this region plays an important role at the synapse. Preliminary data from primary neurons and ex vivo acute slices revealed that expression of the missense Nrxn3α mutant produced striking morphological and functional phenotypes at excitatory and inhibitory synapses. Biochemically, the Nrxn3α missense mutation unexpectedly differentially modulated binding to two excitatory postsynaptic ligands. Based on our preliminary data, we hypothesize that extracellular sequences of individual alpha neurexins control distinct aspects of excitatory and inhibitory synapse function. Here, we will test our central hypothesis in three specific aims: 1. Determine the impact of Nrxn3α extracellular sequences on synaptic morphology and function in in vitro neuron cultures; 2. Biochemically assess how the Nrxn3α missense mutation affects transsynaptic binding; and 3. Manipulate Nrxn3α ECD in vivo and assess its impact on basal excitatory and inhibitory synaptic transmission and activity-dependent plasticity in ex vivo slices. To accomplish aims 1 and 3, we will use molecular replacement, shRNA-mediated knockdown of endogenous Nrxn3α and replacement with wild-type or mutant Nrxn3α to faithfully recapitulate the disease state, combined with immunocytochemistry, electrophysiology and electron microscopy. Aim 2 will use in vitro biochemical and structure/function approaches to measure binding affinities to known Nrxn ligands. These aims will provide first insight into the morphological, functional and biochemical properties of Nrxn3α extracellular sequences and how mutations in this region contribute to cognitive disease.

神经毒素 (Nrxns) 是一类重要但知之甚少的突触前细胞粘附分子，经常与神经精神和神经发育障碍（例如自闭症谱系障碍）有关（ASD）、精神分裂症和智力障碍（ID）。三个进化上保守的神经素基因。较长的 α 和较短的 β 神经素 mRNA 进行广泛的 α- 和 β-Nrxns 剪接。共享共同的跨膜和细胞质序列，但其长度和复杂性不同胞外结构域（ECD；9 个 α-Nrxn 结构域与 1 个 β-Nrxn 结构域相比）。与不同的神经精神疾病和疾病相关的突变通常位于编码 α-Nrxn 特异性细胞外序列的基因组区域，表明个体 α 尽管神经素 ECD 的发现已经有二十多年了，但它们可能控制着突触功能的不同方面。以前，关于单个 α-Nrxn ECD 在突触中的重要作用的基本问题仍然存在作为了解 α-Nrxn 特异性细胞外序列如何发挥作用的重要第一步。突触方面，我们的实验室已经鉴定出一名 Nrxn3α 复合杂合子患者，具有深度 ID 和一个等位基因产生一种无功能的蛋白质，而第二个等位基因则含有错义突变。所有 α-Nrxns 共有的细胞外序列有趣的是，存在多种 ASD 相关突变。 Nrxn1α 的等效区域表明该区域在突触中起着重要作用。来自原代神经元和离体急性切片的结果表明，错义 Nrxn3α 突变体的表达在兴奋性和抑制性突触处产生惊人的形态和功能表型。从生化角度来看，Nrxn3α 错义突变出乎意料地差异调节了与两种兴奋剂的结合。根据我们的初步数据，我们捕获了个体的细胞外序列。 α神经毒素控制兴奋性和抑制性突触功能的不同方面。在这里，我们将测试我们的神经突触功能。三个具体目标的中心假设： 1. 确定 Nrxn3α 细胞外序列对 2. 体外神经元培养中的突触形态和功能；错义突变影响跨突触结合；3. 体内操作 Nrxn3α ECD 并评估其影响对离体切片中基础兴奋性和抑制性突触传递以及活动依赖性可塑性的影响。为了实现目标 1 和 3，我们将使用分子替代、shRNA 介导的内源性敲低 Nrxn3α 和用野生型或突变型 Nrxn3α 替换以忠实地再现疾病状态，结合目标 2 将使用免疫细胞化学、电生理学和电子显微镜。这些目标将首先提供测量与已知 Nrxn 配体的结合亲和力的结构/功能方法。深入了解 Nrxn3α 胞外序列的形态、功能和生化特性，该区域的突变如何导致认知疾病。