Molecular mechanism of Presenilin-1 (PS1) linked Alzheimer's Disease pathology

描述(申请人提供)：本申请的长期目标是了解家族性阿尔茨海默病(FAD)中早老素(PS)相关的Ap40和Ap42产生的分子基础。由于多通道跨膜蛋白结晶的复杂性和难度，人们对PS/y-分泌酶复合体的结构知之甚少。我们假设PS/y-分泌酶以不同的构象存在，导致APP与催化部位的不同排列，从而产生不同的AB物种。我们的观察，在完整细胞中使用了一种新的基于FRET的技术以及生化方法；提供了检查完整细胞中PS1/y-分泌酶构象以及确定PS1和y-分泌酶底物(包括APP和Notch)的接近程度的方法。我们的观察支持一个具有以下重要的可测试特征的模型：成熟的PS1与其他Y-分泌酶复合体成员结合，并采用N-末端和C-末端(NT和CT)紧密相连的构象；APP在有利于AB40和AB42的条件下特定地呈现到Y-分泌酶活性部位的变化。我们将从几个临床重要的角度来测试这个模型。FAD相关的PS1突变导致Ap42/40比值升高，但机制尚不清楚。我们的初步数据表明，FAD PS1突变使PS1 NT和CT更加紧密，这代表着与PS1/y-分泌酶活性部位与APP对齐改变有关的病理性构象变化，有利于Ap42的切割。我们将测试几个模型来定义导致这种观察到的构象变化(AIML)的精确分子机制。一些非类固醇抗炎药已被证明选择性地降低AB42，这是一种与FAD突变相反的表型。我们将测试这一假设，即选择性改变AB42/40比率的药物可以通过与FAD突变相反的方式变构调节PS1/y-分泌酶和PS1/APP相互作用的构象来做到这一点。我们将探索Ap42降低γ-分泌酶的变构调节剂是否可以“修复”致病的FAD突变体PS1构象(AIM2)。最后，我们建议检验这样一种假设，即激活γ-分泌酶和/或其底物的生理刺激选择性地改变神经元中PS1/y-分泌酶与底物的相互作用。我们将利用新的FRET技术的高空间分辨率，荧光寿命成像显微镜(FLiM)，它是唯一适合于监测完整和/或活细胞中蛋白质和复合体的相对构象变化的技术，以显示这些变化发生在完整细胞的哪些亚细胞室(Aim3)，并最终目标是将这些研究扩展到小鼠大脑。所获得的结果将为了解PS1/y-分泌酶复合体的构象变化及其与细胞内底物的相互作用提供重要的新见解，从而有助于设计变构调节剂作为一种治疗方法。更广泛地说，监测PS1构象和y-分泌酶-底物接近程度的分析将有助于开发可能被证明对了解疾病相关蛋白质构象变化有用的方法。

DESCRIPTION (provided by applicant): The long term goal of this application is to understand the molecular basis of presenilin (PS)-linked production of Ap40 and Ap42 in familial Alzheimer's disease (FAD). Very little is known about the structure of the PS/y-secretase complex due to the complexity and difficulty of crystallizing multipass transmembrane proteins. We hypothesize that PS/y-secretase exists in different conformations, leading to different alignments of APP with the catalytic site, resulting in the production of different AB species. Our observations, using a novel FRET based technique in intact cells as well as biochemical approaches; provide methods to examine PS1/y-secretase conformation in intact cells as well as to determine proximity of PS1 and y-secretase substrates, including APP and Notch. Our observations support a model with the following important, testable features: mature PS1 associates with other y-secretase complex members and adopts a conformation with N- and C-termini (NT and CT) close together; and the specific presentation of APP to the y-secretase active site changes under conditions favoring AB40 vs. AB42. We will test this model from several clinically important perspectives. FAD-linked PS1 mutations lead to elevations in Ap42/4o ratio, but the mechanism is unknown. Our preliminary data suggest that FAD PS1 mutations bring PS1 NT and CT even closer together, and that this represents a pathogenic change in conformation associated with an alteration in the alignment of the active site of PS1/y-secretase with APP, favoring cleavage at Ap42. We will test several models to define the precise molecular mechanism that leads to this observed change in conformation (Aiml). Some nonsteroidal anti-inflammatory drugs have been shown to selectively lower AB42, a phenotype opposite to FAD mutations. We will test the hypothesis that pharmacological agents that selectively alter the AB42/40 ratio can do so by allosterically modulating the conformation of PS1/y-secretase and PS1/APP interactions in the opposite way to FAD mutations. We will explore whether Ap42 lowering allosteric modulators of the y-secretase can "fix" the pathogenic FAD mutant PS1 conformation (Aim2). Finally, we propose to test the hypothesis that physiologic stimuli that activate y-secretase and/or its substrate selectively change PS1 /y-secretase interactions with substrates in neurons. We will take advantage of the high spatial resolution of the new FRET technique, Fluorescence Lifetime Imaging Microscopy (FLIM), which is uniquely suited for monitoring relative conformational changes of proteins and complexes in intact and/or live cells, to show in what subcellular compartments in intact cells these changes occur (Aim3), and ultimately aim to extend these studies to the mouse brain. The results obtained will provide important new insights into conformational changes in the PS1/y-secretase complexes and its interaction with substrates within a cellular context, and thus will help in the design of allosteric modulators of y-secretase function as a therapeutic approach. More generally, the assay of monitoring PS1 conformation and y-secretase -substrate proximity will help to help develop methodologies that may prove useful in understanding disease-related protein conformation changes.