Functional and mechanistic interrogation of alpha neurexin extracellular domains

Neurexins(Nrxns)是一个重要但知之甚少的突触前细胞黏附分子家族，它们是 经常与神经精神和神经发育障碍有关，如自闭症谱系障碍 (自闭症)、精神分裂症和智力残疾(ID)。三个进化上保守的神经毒素基因产生 经过广泛的选择性剪接的较长的α-和较短的β-神经氨酸mRNAs。α-和β-Nrxns 共享共同的跨膜和细胞质序列，但在长度和复杂性上有所不同 胞外结构域(ECDs；9个α-Nrxn结构域与1个β-Nrxn结构域)。单个α-Nrxn是 与不同的神经精神障碍和疾病相关突变相关的通常位于 编码α-Nrxn特异性胞外序列的基因组区域，表明单个阿尔法 Neuresin ECDs可能控制突触功能的不同方面。尽管他们的发现超过了二十年 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。 悬而未决。作为了解α-Nrxn特异性胞外序列如何发挥作用的重要第一步 突触，我们实验室已经鉴定出一例NRXN3α复合杂合子患者，具有深刻的ID和 癫痫。一个等位基因产生一种不起作用的蛋白质，第二个等位基因含有错义突变。 所有α-Nrxn共享的胞外序列。耐人寻味的是，在人类自闭症相关基因 NRXN1α的等价区，表明该区域在突触中起重要作用。初步数据 从原代神经元和体外急性切片中发现错义NRXN3α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。 生化方面，NRXN3α错义突变出人意料地差异调节与两种兴奋性物质的结合 突触后配体。根据我们的初步数据，我们假设个体的胞外序列 α-神经氨酸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 三个特定目的的中心假说：1.确定NRXN3胞外序列对α的影响 体外神经元培养中突触的形态和功能；2.生化评估NRXN3α是如何 错义突变影响跨突触结合；以及3.体内操纵NRXN3αECD并评估其影响 体外脑片基础兴奋性和抑制性突触传递及活性依赖性可塑性的研究。至 实现目标1和目标3，我们将使用分子置换、shRNA介导的内源敲除 NRXN3α和用野生型或突变型NRXN3α替换，以忠实地概括疾病状态，组合 用免疫细胞化学、电生理学和电子显微镜观察。AIM 2将使用体外生化和 测量已知Nrxn配体结合亲和力的结构/功能方法。这些目标将首先提供 深入了解NRXN3α胞外序列的形态、功能和生化性质 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.