FUNCTIONAL ANALYSIS OF AD-LINKED PRESENILIN 2 MUTATIONS

大多数早发性家族性阿尔茨海默病（FAD）最近 被证明是由两个新基因突变引起的， 早老素1（PS 1）和早老素2（PS 2）。虽然正常的生物学 这两种蛋白质的作用以及 FAD相关突变发挥其影响仍然未知，最近 已经证明，来自受影响个体的成纤维细胞携带 这些突变中的某些具有增加的A β 42水平。到目前为止， 对这种生物的基本生物学特性知之甚少。 早老素显然，未来的研究必须旨在确定正常的 这两种蛋白质的作用以及它们如何与淀粉样蛋白连接 前体蛋白（APP）和Abeta的产生。 在目前的提案中，我们选择将重点放在 早老素家族，PS2.我们建议进行一系列实验， 旨在提供有关PS2的基本但关键的信息， FAD相关突变对其基本功能的影响 特性.在前两个目标中，我们将扩展我们的初步目标。 调查结果，并继续分析表达，处理， PS2的亚细胞定位，同时检查影响 N141 I突变的基因。由于明显的 早老素突变与Abeta产生之间的关联， 后两个目标的目标是确定是否存在 PS2和APP之间的可检测的相互作用，并检查是否 PS2的质或量的变化对加工有影响 我们的总体目标是为研究提供一阶信息， 旨在确定PS2的正常生物学作用和 这种蛋白质突变导致神经变性的机制 和早发性阿尔茨海默病中的突触丧失。的结果 提出的实验不仅有助于阐明PS2在 阿尔茨海默病的病理生理学，而且还提供了一个坚实的 最终有效治疗策略的基础框架， 未来的实验具体而言，我们计划：l）继续分析 突变对PS2加工和定位的影响 2）定义PS2的哪些结构域决定其细胞内 定位到高尔基体/ER，并评估PS2的特定区域是否 在PS1中不保守的基因导致了细胞内定位模式 3）确定PS2是否与PS1略有不同; N1411 PS2突变或PS2水平的变化改变了细胞的加工或表达。 APP的亚细胞定位; 4）评估是否存在可检测的 APP和PS2之间的相互作用，如果是这样，N141 I突变是否 改变了这种互动。虽然我们将首先集中分析 N141 I突变，我们还将评估其他PS2突变的影响 正如他们所描述的。

The majority of early-onset familial Alzheimer's disease (FAD) has recently been demonstrated to be caused by mutations in two novel genes termed presenilin 1 (PS1) and presenilin 2 (PS2). Although the normal biological role(s) of these two proteins and the details of the mechanism by which the FAD-associated mutations exert their effect remain unknown, it has recently been demonstrated that fibroblasts from affected individuals harboring certain of these mutations have increased levels of Abeta42. To date, little is known about the basic biological characteristics of the presenilins. Clearly future studies must be aimed at determining the normal roles of these two proteins and how they are linked to the amyloid precursor protein (APP) and the production of Abeta. In the current proposal we have elected to focus on one member of the presenilin family, PS2. We propose to perform a series of experiments which are aimed at providing rudimentary but critical information about PS2 and the effects that FAD-associated mutation(s) have on its fundamental properties. In the first two aims we will expand upon our preliminary findings and continue the analysis of the expression, processing, and subcellular localization of PS2, while concurrently examining the effects of the N141I mutation on these characteristics. Because of the apparent association between the presenilin mutations and the generation of Abeta, the goals of the second two aims are to determine whether there is a detectable interaction between PS2 and APP, and to examine whether quantitative or qualitative changes in PS2 have an effect on the processing of APP. Our overall goal is to provide first order information for studies aimed at determining both the normal biological role of PS2 and the mechanism by which mutations in this protein lead to the neurodegeneration and synaptic loss in early-onset Alzheimer's disease. The results of the proposed experiments should help to not only elucidate the role of PS2 in the pathophysiology of Alzheimer's disease, but also to provide a solid framework on which to ultimately base effective therapeutic strategies and future experiments. Specifically, we plan to: l) Continue our analysis of the effects that mutations have on the processing & localization of PS2 itself; 2) Define which domains of PS2 dictate its intracellular localization to the Golgi/ER, and assess whether particular regions of PS2 that are not conserved in PS1 cause the intracellular localization pattern of PS2 to be slightly different from that of PS1; 3) Determine whether the N141l PS2 mutation or changes in the level of PS2 alter the processing or subcellular localization of APP; 4) Assess whether there is a detectable interaction between APP and PS2 and, if so whether the N141I mutation alters this interaction. Although we will first concentrate on analysis of the N141I mutation, we will also assess the effects of other PS2 mutations as they are described.