Role of the g-secretase/PS1 complex in APP processing

描述(由申请人提供)：淀粉样贝塔多肽(ABeta)或特定亚型(Abeta42)的积累增加是所有形式的阿尔茨海默病(AD)神经变性的主要致病事件。在这个项目的前四年，我们专注于早老素(PS)FAD突变在细胞凋亡中的作用，以及细胞凋亡如何影响ABeta的产生。然而，越来越多的证据表明，虽然急性中枢神经系统损伤后可能发生凋亡诱导的ABeta产生，但由于APP和PS基因的家族性AD突变，ABeta的产生可能涉及其他致病机制。PS基因的克隆导致了被称为伽马分泌酶的蛋白酶的初步表征，该酶在ABeta的C末端裂解APP。伽马分泌酶是一种蛋白质的异构体复合体，迄今只鉴定出PS和尼古丁两种成分。PS1中的FAD突变增加了ABeta 42：Abeta40的比例，并可能涉及伽马分泌酶/PS1中一些尚未识别的蛋白质。因此，在接下来的资助期，我们建议在最初应用程序的特定目标3的基础上进行扩展，探索PS中的伽马分泌酶复合体/PS1和FAD突变如何导致APP成熟和加工的改变，并影响ABeta的生产。在我们的初步数据中，我们发现尼古丁和13个未知蛋白与来自碳酸钠洗涤裂解物的PS1 C-端和N-端片段进行免疫共沉淀，从而代表了潜在的新的膜相关成分和/或γ-分泌酶/PS1复合体的底物。我们还在含有该复合体的高尔基体/内体及其APP C末端底物中鉴定了一种亚细胞组分。我们已经初步确定了该复合体中的一条未知蛋白质带。为了跟进这些发现并扩大我们对FAD早老素突变对伽马分泌酶活性影响的研究，我们建议鉴定和表征伽玛分泌酶/PS1复合体的新成分，特别是那些调节ABeta产生和Abeta42/ABetattal比率的成分。我们还将测试编码伽马分泌酶/PS1复合体新成分的基因的多态，以确定与阿尔茨海默病的家族相关性。我们计划确定γ-分泌酶/PS1复合体的亚细胞定位并阐明其生理功能。最后，我们正在进行体外伽马分泌酶测定，并将分离的复合体重组为单层脂质体，以研究其体外活性。这些研究的总体目标是确定PS中100多个FAD突变影响ABeta生成的致病机制，并最终确定减少阿尔茨海默病ABeta生成的潜在靶点。

DESCRIPTION (provided by applicant): Increased accumulation of the amyloid-Beta peptide (ABeta) or specific isoforms (ABeta42) is a major pathogenic event underlying neurodegeneration in all forms of Alzheimer's disease (AD). In the first four years of this project, we have focused on the role of presenilin (PS) FAD mutations in apoptosis, and how apoptosis influences ABeta production. However, evidence has mounted to suggest that while apoptosis-induced ABeta generation may occur following acute injury to the CNS, other pathogenic mechanisms are likely involved in ABeta production owing to familial AD mutations in APP and the PS genes. Cloning of the PS genes has led to the initial characterization of the protease called gamma-secretase that cleaves APP at the C-terminal end of ABeta. Gamma-secretase is a heteromeric complex of proteins, in which only two components have been identified to date, PS and nicastrin. FAD mutations in PS1 increase the ratio of ABeta 42:ABeta40 and are likely to involve a number of as of yet unidentified proteins in the gamma-secretase/PS1. Thus, in the coming funding period, we propose to expand upon Specific Aim 3 of the original application, by exploring how the gamma-secretase complex/PS1 and FAD mutations in PS lead to alterations in the maturation and processing of APP and affect ABeta production. In our preliminary data, we show that nicastrin and thirteen unknown proteins coimmunoprecipitate with PS1 C- and N-terminal fragments from a sodium carbonate-washed lysate, thereby representing potentially novel membrane-associated components and/or substrates of the gamma-secretase/PS1 complex. We have also identified a subcellular fraction in the Golgi/endosomes harboring the complex, together with its APP C-terminal substrates. We have tentatively already identified one of the unknown protein bands in the complex. To follow up on these findings and extend our studies of the effect of FAD presenilin mutations on gamma-secretase activity, we propose to identify and characterize novel components of the gamma-secretase/PS1 complex, especially those that modulate ABeta production and the ABeta42/ABetatotal ratio. We will also test polymorphisms in genes that encode novel components of the gamma-secretase/PS1 complex, for family-based association with Alzheimer's disease. We plan to determine the subcellular localization and elucidate the physiological functions of the gamma-secretase/PS1 complex. Finally, we are performing in vitro gamma-secretase assays and reconstituting the isolated complex into unilamellar liposomes to study its activity in vitro. The overall goal of these studies is to define the pathogenetic mechanism by which more than 100 FAD mutations in PS affect ABeta generation, and to ultimately identify potential targets for reducing ABeta generation in Alzheimer's disease.