STRUCTURE/FUNCTION OF ALZHEIMERS AMYLOID-B AGGREGATES

描述：(摘自申请者摘要)阿尔茨海默病(AD)是一种 毁灭性的进行性神经退行性疾病的特征是 认知能力下降，在尸检中，其独特的大脑病理， 神经原纤维缠结和神经炎斑块。主要蛋白质组分 神经炎斑块中的一种多肽，从机械上讲，被称为淀粉样β蛋白(AB) 通过其前体蛋白(APP)的突变与阿尔茨海默病有关。这些 APP突变可导致AB显著过量，并是致病因素 对于早发性家族性AD(FAD)。这里提出的研究涉及到 与C.Finch博士(南加州大学)和W.Klein博士(西北大学)密切合作 U)。本研究的目的是确定AB的结构 聚集，并精确地确定负责的分子相互作用 用于AB引起的AD相关神经元反应。另一个目标是 表征AB与另外两个斑块相互作用的结构方面 阿尔茨海默病患者脑内载脂蛋白E和载脂蛋白J的升高 组织。与AD相关神经元相关的分子相互作用的定义 反应对于识别减慢或阻断药物靶点是必不可少的 疾病病理学的进展。一个主要的障碍 了解AB的活动是实验室面临的最大困难 重现神经毒性或细胞信号传递实验。这些 困难源于AB的本质，AB可以组装成各种类型 不同的形式，其中只有一部分能引起生物反应。 因此，另一个重要目标是确定具体的 特定AB肽类似物结构的实验条件 需要制备一贯有效的AB形式(S)。 本研究的具体目的是：1)确定AB的结构 使用扫描探头，在系统变化的条件下形成纤维 显微镜。评估AB类似物的纤维结构变化，包括 AB初级序列中的特定功能变化。2)定义 功能化AB聚集体的生物相关表面拓扑结构 AB肽类似物与化学或生物化学修饰的原子力 显微镜探头尖端。3)与AB(氧化作用)的神经元效应相关 应激、tau磷酸化、细胞分离)具有纤维结构和 地形。4)确定实验参数和/或制备AB肽 能够形成具有以下特性的小的、可溶的AB聚集体的类似物 具有特定的、可重复的神经毒性特性。5)分析互动 在AB和apoJ或apoE之间的扫描探针显微镜和使用AB类似物 以确定这些相互作用的结构要求。评估 这些络合物的生物学特性。6)研究AB与 通过扫描探针显微镜、整体电子显微镜和 共聚焦荧光成像。 这些研究中使用的关键方法是扫描探针显微镜，以及 新型AB类似物的设计与合成。聚集动力学将是 用吸收/比浊法测量。AB的生物活性 将被测量的聚集体包括使用氧化还原的氧化应激 底物四甲基偶氮唑蓝，诱导分化过程中tau的磷酸化 用新开发的化学发光三明治检测神经母细胞瘤细胞 免疫分析和神经母细胞瘤与纤维连接蛋白或层粘连蛋白的脱粘。 扫描探针显微镜、整体电子显微镜和共聚焦显微镜 荧光显微镜将用于研究直接接触和 AB聚集体与神经元表面分子的相互作用。

DESCRIPTION: (from applicant's abstract) Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disease characterized by cognitive decline, and at autopsy, its distinct brain pathologies, neurofibrillary tangles and neuritic plaques. The major protein component of neuritic plaques is a peptide known as amyloid beta (AB), mechanistically linked to AD by virtue of mutations in its precursor protein (APP). These APP mutations can give rise to significant excesses of AB and are causative for early onset familial AD (FAD). The research proposed here involves close collaborations with Dr. C. Finch (USC) and Dr. W. Klein (Northwestern U.). The objective of this research is to define the structure of AB aggregates and precisely determine the molecular interactions responsible for AD-relevant neuronal responses elicited by AB. A further objective is to characterize structural aspects of AB interactions with two other plaque components, apolipoprotein E (apoE) and apoJ, which are elevated in AD brain tissue. Definition of molecular interactions linked to AD-relevant neuronal responses is essential for identification of drug targets to slow or block the progression of the disease pathology. A major impediment to understanding AB's activity is the great difficulty laboratories have in reproducing neurotoxicity or cellular signaling experiments. These difficulties stem from the very nature of AB, which assembles into a variety of distinct forms, only some of which can elicit biological responses. Therefore, another important objective is to ascertain the specific experimental conditions or particular AB peptide analogue structures required to prepare consistently active form(s) of AB. The specific aims of this research are: 1) Determine the structure of AB fibrils formed under systematically varied conditions, using scanning probe microscopy. Assess structural changes in fibrils of AB analogues containing specific functional changes in AB primary sequence. 2) Define the biologically relevant surface topology of AB aggregates using functionalized AB peptide analogs and chemically or biochemically modified atomic force microscope probe tips. 3) Correlate the neuronal effects of AB (oxidative stress, tau phosphorylation, cell detachment) with fibril structure and topography. 4) Establish experimental parameters and/or prepare AB peptide analogs that enable formation of small, soluble AB aggregates possessing specific, reproducible neurotoxic properties. 5) Analyze the interactions between AB and apoJ or apoE by scanning probe microscopy and use AB analogs to determine the structural requirements for these interactions. Assess biological properties of these complexes. 6) Study AB interactions with neurons by scanning probe microscopy, whole mount electron microscopy and confocal fluorescence imaging. The key methods used in these studies are scanning probe microscopy, and design and synthesis of novel AB analogues. Aggregation kinetics will be measured by absorption/turbidimetry. The biological activities of AB aggregates that will be measured include oxidative stress using the redox substrate MTT, induction of tau phosphorylation in differentiated neuroblastoma cells using a newly developed chemiluminescent sandwich immunoassay, and neuroblastoma disadhesion from fibronectin or laminin. Scanning probe microscopy, whole mount electron microscopy and confocal fluorescence microscopy will be used to study the direct contact and interactions of AB aggregates and neuronal surface molecules.