PRESENILIN BIOLOGY & THE MECHANISM OF ALZHEIMERS DISEASE

正常和病理生物学的相互作用， 关于阿尔茨海默病的研究， 两种蛋白质的研究直接涉及遗传 AD的机制：早老素（PS）和β-淀粉样蛋白前体 蛋白（APP）。在这个项目中，四个独立的项目 多年来，每个实验室都做出了富有成效的贡献， AD机制的阐明正在合力广泛应用于 分子和细胞生物学、神经病理学和 动物模型，以解决有关早老蛋白的关键未解决的问题 及其在AD发病机制中的作用。我们计划的核心愿景是 利用这四个成熟实验室的综合专业知识， 他们拥有的大量技术和试剂 详细介绍了早老素的生物学，它们与其他 功能上重要的神经元蛋白（包括APP、Notch和 连环蛋白及其在最常见和侵袭性形式中的致病作用 基于基因的AD主要调查人员， 过去曾多次合作， 几个月来定期讨论科学问题， 兴趣、共享未发表数据、交换试剂、交叉验证 研究结果和设计新的合作实验，最引人注目的 已纳入本方案。在我们众多的 具体目标（分为4个项目），我们将：1）描述 我们的转基因小鼠中PS1的详细细胞和亚细胞解剖 表达野生型与突变型PS1，使用原位杂交和共聚焦显微镜， 用新开发的试剂进行显微镜检查; 2）评估AD样病理学， 这些小鼠和新的小鼠来自于我们的小鼠与PDAPP V717 F杂交 转基因小鼠，使用现代定量体视学; 3）检查 复杂的PS内蛋白水解，包括一个令人兴奋的新的凋亡 我们最近发现的途径，以及PS如何改变这一途径 突变，但在细胞和转基因小鼠中; 4）使用PS突变作为 一条确定γ-分泌酶加工的难以捉摸的机制的途径， APP，鉴于突变体PS对A β 42的高度选择性作用， 我们最近制作的一个演示APP直接互动 在ER和高尔基体中具有PS1和PS2;以及5）表征细胞 我们最近克隆了一个连环蛋白家族的新成员， 体内PS相互作用，并评估其如何在细胞信号传导中发挥作用， 它是否参与了PS-APP复合物。这些只是 关于结构和功能的未回答的问题 早老素类药物。我们的实验将得到3 核心，包括一个用于繁殖和维持转基因小鼠， 它将描述和分配一个非常大的DNA阵列， 构建体、稳定的细胞系、探针和抗体， 灵敏的Abeta ELISA。我们提出的实验是假设驱动的 而且，在每一种情况下，都是基于强有力的初步数据。我们相信我们的 结合经验和我们的承诺组的高级和初级 科学家们将使我们能够成功地执行一个高度集成的 基础和应用分子神经生物学计划，将有直接的 对了解AD的机制和治疗的意义。

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past, have been meeting together regularly for many months to discuss scientific questions of mutual interest, share unpublished data, exchange reagents, cross-validate findings and design new collaborative experiments, the most compelling of which have been incorporated into this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.