ROLE OF PRESENILIN 1 AT THE SYNAPSE

突触功能障碍是阿尔茨海默病（AD）的一个重要特征，与记忆密切相关 损伤Ap的活性依赖性产生和Ap寡聚体在突触处的靶向积累 已经报道，但是基本的分子机制仍然知之甚少。我们有 先前提出，早老素（PSI）可以采用两种构象之一-称为“开放”， 封闭的”，基于γ-分泌酶复合物的其它蛋白质成分（Serneels等，2009年），在 Gamma-分泌酶调节剂的作用（Uemura等人，2010），或由于家族性AD相关的PSI突变 （Berezovska等人，2005年）。然而，这些是否是不同的γ复合物， 另一个在细胞内，或PSI可以动态地改变其构象内的特定复合物，是不清楚的。 此外，可能参与调节PSI构象和功能的事件和/或蛋白质在细胞内表达。 细胞仍然未知。现在，我们建立了一个独特的构象敏感的FRET为基础的分析在生活中， 细胞，我们的新的初步数据表明，PSI的构象变化迅速，可逆的一致 突触活动。Aim 1将建立在这一观察的基础上，并将其扩展到PSI-APP的相互作用， 突触为了寻找潜在的PSI构象的活性依赖性调节剂，我们进行了 在存在或不存在钙的情况下，对小鼠脑裂解物进行蛋白质组学筛选，并鉴定了几种 新的PSI相互作用物，包括突触结合蛋白1和9（Sy1.9）和突触结合蛋白1（Svn 1）。由于Syt 1和 Synl显示出与PSI相互作用的强烈但相反的Ca依赖性概况，我们将指导我们的初步研究。 注意这些可能的候选人，以调节PSI/γ-分泌酶及其与APP的相互作用， 以活性-伽马控制的方式形成突触。有趣的是，Sytl和9也在一个独立的研究中被鉴定出来。 Kovacs博士进行的蛋白质组学筛选，作为APP相互作用蛋白（项目4）。目标2将测试 假设Ca 2+内流作为两个PSI构象状态之间的开关， 通过控制PSI与突触蛋白的相互作用来控制突触体隔室。目标3遵循这一理念 PSI可以经历快速且可逆的构象变化，以检查药理作用 干预PSI/γ-分泌酶的变构调节（与项目1和2合作），及其 在体外和体内与突触蛋白的相互作用。理解这个问题非常重要， 因为“闭合”PSI构象与增加的Ap 42（AD相关裂解）相关， 并且因为操纵PS1与Syt 1和Syn 1的相互作用可以转化为治疗 重点放在突触上

Synaptic dysfunction is a key feature of Alzheimer's disease (AD) and closely correlates with memory impairment. Activity-dependent production of Ap, and targeted accumulation of Ap oligomers at the synapse have been reported, however underlying molecular mechanisms remain poorly understood. We have previously suggested that presenilin (PSI) can adopt one of two conformations -termed "open" and closed", based on the other protein constituents of the gamma-secretase complex (Serneels et al., 2009,), on the effects of GAMMA-secretase modulators (Uemura et al., 2010), or due to familial AD-related mutations in PSI (Berezovska et al., 2005). However, whether these are different gamma-complexes that are in equilibrium with one another within a cell, or PSI can dynamically change its conformation within a specific complex, is unclear. Furthermore, events and/or protein(s) that may be involved in regulation of PSI conformation and function in the cell remain unknown. Now we established a unique conformation-sensitive FRET-based assay in living cells, and our new preliminary data show that conformation of PSI changes rapidly and reversibly in concert with synaptic activity. Aim1 will build on this observation and extend it to PSI-APP interactions at the synapse. To search for potential activity-dependent modulators of PSI conformation, we performed a proteomics screen of mouse brain lysates in the presence or absence of calcium, and identified several novel PSI interactors, including synaptotagminsi and 9 (Sytl .9), and synapsini (Svnl). Since Syt1 and Synl showed strong but opposing Ca-dependent profiles of interaction with PSI, we will direct our initial attention to these as likely candidates to modulate PSI/gamma-secretase and its interaction with APP at the synapse in an activity-gamma-controlled manner. Intriguingly, Sytl and 9 were also identified in an independent proteomics screen performed by Dr. Kovacs as APP-interacting proteins (Project 4). Aim 2 will test the hypothesis that Ca2+ influx serves as a switch between the two PSI conformational states in the synaptosomal compartment by controlling PSI interactions with synaptic proteins. Aim 3 follows on the idea that PSI can undergo rapid and reversible changes in conformation to examine the role of pharmacological interventions in allosteric modulation of PSI/gamma-secretase (collaboration with Projects 1 and 2), and its interactions with synaptic proteins in vitro and in vivo. Understanding this issue is of high importance both because the "closed" PSI conformation is associated with increased Ap42 (the AD-associated cleavage) and because manipulation of the PS1 interactions with Sytl and Synl may be translated into therapeutics with a focus on the synapse.