PRESENILIN BIOLOGY & THE MECHANISM OF ALZHEIMERS DISEASE

正常生物学和病理生物学的相互作用 关于阿尔茨海默氏症的研究特别好地说明了 两种直接与遗传有关的蛋白质的研究 阿尔茨海默病的发病机制：早老素和β-淀粉样蛋白前体 蛋白质(APP)。在本计划中，四个独立的项目拨款 多年来每个实验室都为 阿尔茨海默病发病机制的阐明正合力广泛应用 分子和细胞生物学、神经病理学和 用动物模型解决有关早老素的关键悬而未决的问题 以及它们在AD发病机制中的作用。我们计划的中心愿景是 利用这四个成熟实验室的组合专业知识和 他们拥有大量的技术和试剂来进行检查 详细介绍了早老素的生物学特性，以及它们与其他 功能重要的神经元蛋白(包括APP、Notch和 连环蛋白及其在最常见和最具侵袭性形式中的致病作用 以基因为基础的AD。主要调查人员，他们已经 过去曾多次合作，一直在一起会面 几个月来定期讨论共同的科学问题 兴趣、共享未发布的数据、交换试剂、交叉验证 发现和设计新的协作实验，最引人注目的 它们已被纳入本计划。在我们众多的 具体目标(组织为4个项目)，我们将：1)描述 我们转基因小鼠中PS1的详细细胞和亚细胞解剖 用原位杂交和共聚焦技术表达wt和突变体PS1 使用新开发的试剂进行显微镜检查；2)评估AD样病理 这些小鼠和我们的小鼠与PDAPP V717F杂交产生的新小鼠 转基因小鼠，使用现代定量体视学；3)检查 PS的复杂内蛋白分解，包括一种令人兴奋的新的细胞凋亡 我们最近确定的途径，以及PS如何改变这一点 突变，但在细胞和转基因小鼠中；4)使用PS突变作为 一种确定难以捉摸的伽马分泌酶处理机制的途径 APP，鉴于突变体PS对Abeta42的高度选择性 生产和我们最近演示的APP直接交互 在内质网和高尔基体中同时有PS1和PS2；以及5)表征细胞 连环蛋白家族中一个新成员的生物学研究 PS在体内的相互作用，并评估其在细胞信号转导和 它是否参与了PS-APP复合体。这些只是其中的一小部分 关于结构和功能的悬而未决的问题 我们将接洽早产儿。我们的实验将得到3个项目的支持 核心，包括一个用于培育和维护转基因小鼠的核心，以及一个 这将表征和分配非常大的DNA阵列 构建、稳定的细胞系、探针和抗体，并将进行 敏感的阿贝塔ELISAs。我们提议的实验是由假设驱动的 而且，在每一种情况下，都是基于强劲的初步数据。我们相信我们的 结合经验和我们敬业的前辈和后辈 科学家将使我们能够成功地执行高度集成的 基础与应用分子神经生物学专业将有直接 对了解阿尔茨海默病的发病机制和治疗有重要意义。

The interplay of normal and pathological biology that characterizes research on Alzheimer's disease is particularly well illustrated by studies of two proteins that have been directly implicated in the genetic mechanisms of AD: the presenilins (PS) and the beta-amyloid precursor protein (APP). In this Program Project grant, four independent laboratories that have each contributed productively over many years to the elucidation of the mechanisms of AD are joining forces to apply a wide range of techniques in a molecular and cell biology, neuropathology and animal modeling to address key unresolved questions about the presenilins and their role in AD pathogenesis. The central vision of our Program is to use the combined expertise of these four well-established laboratories and their extensive array of techniques and reagents they possess to examine in detail the biology of the presenilins, their interactions with other functionally important neuronal proteins (including APP, Notch and the catenins and their pathogenic role in the most common and aggressive form of genetically based AD. The principal investigators, who have collaborated on numerous occasions in the past, have been meeting together regularly for many months to discuss scientific questions of mutual interest, share unpublished data, exchange reagents, cross-validate findings and design new collaborative experiments, the most compelling of which have been incorporated into this Program. Among our numerous Specific Aims (organized into 4 projects), we will: 1) characterize in detail cellular and subcellular anatomy of PS1 in our transgenic mice expressing wt versus mutant PS1, using in situ hybridization and confocal microscopy with newly developed reagents; 2) assess AD-like pathology in these mice and new mice resulting from crossing our mice with PDAPP V717F transgenic mice, using modern quantitative stereology; 3) examine the complex endoproteolysis of PS, including an exciting novel apoptotic pathway we have recently identified, and how this is changed by PS mutations, but in cells and in transgenic mice; 4) use the PS mutations as a route to defining the elusive mechanism of gamma-secretase processing of APP, in view of the highly selective effect of mutant PS on Abeta42 production and our recent demonstration of a direct interaction of APP with both PS1 and PS2 in the ER and Golgi; and 5) characterize the cell biology of a novel member of the catenin family we recently cloned as a PS-interaction in vivo and assess how it functions in cell signaling and whether it participates in the PS-APP complexes. These are but a few of the unanswered questions about the structure and function of the presenilins we will approach. Our experiments will be supported by 3 Cores, including one for breeding and maintaining transgenic mice, and one that will characterize and distribute a very large array of DNA constructs, stable cell lines, probes and antibodies and will conduct sensitive Abeta ELISAs. Our proposed experiments are hypothesis-driven and, in each case, based on strong preliminary data. We believe our combined experiences and our committed group of senior and junior scientists will enable us to successfully execute a highly integrated program of basic and applied molecular neurobiology that will have direct implications for understanding the mechanism and treatment of AD.