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（NRXN）是一个必不可少但知之甚少的家族，突触前细胞粘附分子是经常与神经精神科和神经发育障碍（例如自闭症谱系障碍）有关（ASDS），精神分裂症和智力残疾（ID）。三个进化保守的神经氧蛋白基因产生较长的α和较短的β神经蛋白mRNA，具有广泛的替代剪接。 α-和β-NRXN 具有共同的跨膜和细胞质序列，但其长度和复杂性不同细胞外结构域（ECD；9α-NRXN结构域与1β-NRXN结构域相比）。单个α-nrxn是与不同的神经精神疾病和与疾病相关的突变相关的为α-NRXN特异性细胞外序列编码的基因组区域，表明单个α Neurexin ECD可以控制突触功能的不同方面。尽管他们发现了二十年以前，关于单个α-nrxn ECD在突触中的基本作用的基本问题仍然存在未解决。作为理解α-nrxn特异性细胞外序列的重要第一步突触，我们的实验室已经确定了具有深度ID和癫痫。一个等位基因产生非功能蛋白质，第二个等位基因在所有α-NRXN共享的细胞外序列。有趣的是，有多个ASD相关的突变 NRXN1α的等效区域表明该区域在突触中起重要作用。初步数据从原发性神经元和离体急性切片中表明，错义NRXN3α突变体的表达在兴奋性和抑制性突触时产生了惊人的形态和功能表型。从生化上，NRXN3α的错义突变意外地与两种兴奋性的结合不同突触后配体。根据我们的初步数据，我们假设个人的细胞外序列 α神经氧素控制兴奋和抑制突触功能的不同方面。在这里，我们将测试我们的三个特定目的的中心假设：1。确定NRXN3α细胞外序列对突触形态和体外神经元培养的功能； 2。从生化评估NRXN3α如何错义突变会影响透射性结合；和3。在体内操纵NRXN3αECD并评估其影响在离体切片中的基本兴奋性和抑制性突触传递和活性依赖性可塑性上。到完成目标1和3，我们将使用分子替换，shRNA介导的内源性敲低 NRXN3α和用野生型或突变体NRXN3α替代，忠实地概括了疾病状态，合并具有免疫细胞化学，电生理学和电子显微镜。 AIM 2将使用体外生化和测量与已知NRXN配体的结合亲和力的结构/功能方法。这些目标将首先提供洞悉NRXN3α细胞外序列的形态，功能和生化特性和该地区的突变如何促进认知疾病。