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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.

Neurexins （Nrxns）是一类重要的但鲜为人知的突触前细胞粘附分子