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

描述（由申请人提供）：淀粉样β肽（ABeta）或特定亚型（ABeta42）的积累增加是所有形式阿尔茨海默病（AD）神经退行性变的主要致病事件。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β的产生。然而，越来越多的证据表明，虽然凋亡诱导的β生成可能发生在中枢神经系统急性损伤后，但由于APP和PS基因的家族性AD突变，其他致病机制可能参与了β的产生。PS基因的克隆导致了一种叫做γ -分泌酶的蛋白酶的初步特征，这种蛋白酶可以在β的c端切割APP。γ -分泌酶是一种蛋白质的异质复合体，其中只有两种成分已被确定，PS和nicastrin。PS1中的FAD突变增加了abeta42:ABeta40的比例，并且可能涉及γ -分泌酶/PS1中许多尚未确定的蛋白质。因此，在即将到来的资助期内，我们建议扩展原始申请的Specific Aim 3，通过探索PS中的γ -分泌酶复合物/PS1和FAD突变如何导致APP成熟和加工的改变并影响β的产生。在我们的初步数据中，我们发现nicastrin和13种未知蛋白与碳酸钠洗涤裂解液中的PS1 C端和n端片段共免疫沉淀，从而代表了潜在的新型膜相关成分和/或γ -分泌酶/PS1复合物的底物。我们还在高尔基体/核内体中发现了一个亚细胞部分，其中包含该复合物及其APP c端底物。我们已经初步确定了复合体中一个未知的蛋白质带。为了进一步研究FAD早老素突变对γ -分泌酶活性的影响，我们建议鉴定和表征γ -分泌酶/PS1复合物的新成分，特别是那些调节β产生和ABeta42/ abetattal比值的成分。我们还将测试编码γ -分泌酶/PS1复合物新组分的基因多态性，以确定与阿尔茨海默病的家族关联。我们计划确定γ -分泌酶/PS1复合物的亚细胞定位并阐明其生理功能。最后，我们进行体外γ -分泌酶测定，并将分离的复合物重组为单层脂质体，以研究其体外活性。这些研究的总体目标是确定PS中100多种FAD突变影响ABeta生成的发病机制，并最终确定阿尔茨海默病中减少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.