Nectin-1: Synaptic processing and functions

描述（申请人提供）：Nectin-1是一种细胞粘附分子，位于突触的粘附点连接处。Nectin-1与其他蛋白质结合，共同参与神经元突触的形成。我们假设连接蛋白-1以一种依赖于活性的方式，经历了一系列受调节的多步骤内源性蛋白水解裂解事件，这些事件调节突触发生并有助于突触可塑性。我们的初步数据表明，至少有两个脱落酶会导致连接蛋白-1的外域脱落，从而产生两个c端片段（CTFs）。这些CTFs在膜内被y分泌酶进一步裂解，并从质膜上释放出NE-ICD。释放的NEICD易位到细胞核中，我们假设它诱导基因表达。在Specific Aim 1中，我们将通过免疫亲和纯化确定nectin-1的分泌酶裂解位点，然后进行Edman降解测序。我们还将通过CodeLink生物阵列研究海马神经元中NE-ICD调控的基因。然后，使用定量RT-PCR， ICC和Western blotting，我们将确认神经元中差异表达的基因。与NE-ICD相互作用的分子将通过酵母双杂交筛选来鉴定。一旦相互作用物被确定，它们的生物学功能将通过细胞和分子方法进行分析。在Specific Aim 2中，我们将研究BACE1在连接素-1加工中的生物学作用。初步实验表明，BACE1与nectin-1的脱落相关并参与脱落。我们将通过ICC、Western blotting和囊泡循环试验研究连接素-1脱落的破坏如何通过BACE1功能的丧失影响突触形成和突触可塑性。我们的初步数据表明，两个连接素-1点突变T310A和Y311A对BACE1的切割和can /ra”具有抗性。y主要干扰内源性nectin-1的加工。我们将研究这些点突变如何通过重组腺相关病毒载体转导海马神经元和体内成年海马来影响突触形成和突触功能。我们将通过ICC、活细胞成像和突触活性定量测量突触标记物、突触形态和大小的变化。随后，我们研究了跨显性连接蛋白-1突变体的表达是否会影响海马依赖性学习

DESCRIPTION (provided by applicant): Nectin-1 is a cell adhesion molecule localized the puncta adherentia junctions in synapses. Nectin-1 associates with other proteins, which collectively participate in the formation of neuronal synapses. We hypothesize that nectin-1-undergoes a regulated multi-step set of endoproteolytic cleavage events by several sheddases in an activity-dependent manner and that these events regulate synaptogenesis and contribute to synaptic plasticity. Our preliminary data indicate that nectin-1 undergoes ectodomain shedding by at least two sheddases that result in the production of two C-terminal fragments (CTFs). These CTFs are further cleaved intramembraneously by y-secretase and liberate the NE-ICD from the plasma membrane. The released NEICD translocates into the nucleus and where we postulate it induces gene expression. In Specific Aim 1, we will determine the secretase cleavage sites of nectin-1 by immunoaffinity purification, followed by Edman degradation sequencing. We will also investigate which genes are regulated by NE-ICD in hippocampal neurons by CodeLink Bioarrays. Then, using quantitative RT-PCR, ICC, and Western blotting we will confirm differentially expressed genes in neurons. The molecules that interact with NE-ICD will be identified by a yeast two-hybrid screen. Once interactors are identified, their biological function will be assayed by cellular and molecular approaches. In Specific Aim 2, we will investigate the biological role of BACE1 in nectin-1 processing. Initial experiments indicate that BACE1 associates with and participates in the shedding of nectin-1. We will investigate how disruption of nectin-1 shedding, through loss of BACE1 function, affects synapse formation and synaptic plasticity by ICC, Western blotting and-vesicle recycling assays. Our preliminary data indicate that two nectin-1 point mutations, T310A and Y311A, are refractory to BACE1 cleavage and can /ra".y-dominantly interfere with processing of endogenous nectin-1. We will examine how these point mutants affect the synapse formation and synaptic function by transduction of hippocampal neurons and in vivo adult hippocampus with recombinant adeno-associated viral vectors. We will quantitatively measure the changes in synaptic markers, synapse morphology, and size by ICC, live cell imaging and synaptic activity. Subsequently, we examine whether expression of trans-dominant nectin-1 mutants will affect hippocampal dependent learning