Functional and mechanistic interrogation of alpha neurexin extracellular domains

神经毒素（Nrxns）是一个重要的但知之甚少的突触前细胞粘附分子家族， 通常与神经精神和神经发育障碍有关， （ASD），精神分裂症和智力残疾（ID）。三个进化上保守的neurexin基因产生 较长的α和较短的β neurexin mRNA经历广泛的选择性剪接。α-和β-Nrxns 共有共同的跨膜和胞质序列，但其长度和复杂性不同， 细胞外结构域（ECD; 9个α-Nrxn结构域与1个β-Nrxn结构域相比）。单个α-Nrxns为 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-Nrxn neurexin ECD可以控制突触功能的不同方面。尽管他们发现了二十多年 以前，关于单个α-Nrxn ECDs在突触中的重要作用的基本问题仍然存在 悬而未决作为了解α-Nrxn特异性细胞外序列在细胞内如何发挥作用的重要的第一步， 我们的实验室已经确定了一个Nrxn 3 α复合杂合子患者具有深刻的ID， 癫痫一个等位基因产生一种无功能的蛋白质，第二个等位基因在一个基因中含有一个错义突变。 所有α-Nrxns共有的胞外序列。有趣的是，有多个ASD相关的突变， Nrxn 1 α的等效区域表明该区域在突触中起重要作用。初步数据 从原代神经元和离体急性切片中发现，错义Nrxn 3 α突变体的表达 在兴奋性和抑制性突触上产生显著的形态和功能表型。 在生物化学上，Nrxn 3 α错义突变出乎意料地差异调节了与两种兴奋性受体的结合。 突触后配体基于我们的初步数据，我们假设个体的细胞外序列 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 中心假设在三个具体目标：1。确定Nrxn 3 α胞外序列对 体外神经元培养中突触形态和功能; 2.生物化学评估Nrxn 3 α 错义突变影响跨突触结合;和3.在体内操作Nrxn 3 α ECD并评估其影响 对离体切片中的基础兴奋性和抑制性突触传递以及活性依赖性可塑性的影响。到 为了实现目标1和3，我们将使用分子置换，shRNA介导的内源性敲低， Nrxn 3 α和用野生型或突变型Nrxn 3 α替换以忠实地再现疾病状态，组合 免疫细胞化学、电生理学和电子显微镜检查。目标2将使用体外生化和 结构/功能方法来测量与已知Nrxn配体的结合亲和力。这些目标将首先提供 深入了解Nrxn 3 α胞外序列的形态、功能和生化特性， 这个区域的突变是如何导致认知疾病的

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.