Pathogenic Mechanisms of Cell-Derived Abeta Oligomers

成功干预人类疾病的一个持久原则是识别-然后预防- 发病机制的最早步骤。在阿尔茨海默病及其先兆的情况下，轻度认知 损害(MCI)，许多实验室的研究支持仍未得到证实的假设，即渐进性 淀粉样蛋白P蛋白(A(3))在大脑记忆和认知区域的积聚和寡聚 会引发这种复杂的综合症。鉴于学术界和企业界正在花费的巨大资源 制药科学家识别抗淀粉样蛋白疗法并将其用于人体试验，这是至关重要的 准确地了解可溶性A|3是如何开始齐聚的，以及这一过程是否实际上诱导了 在MCI和早期AD中可见突触功能的微妙妥协。在这个新的RO1应用程序中， 合作卓有成效地发现低N-A|3寡聚体自然分泌的研究人员 细胞培养，然后展示它们在生活中抑制长时程增强和破坏记忆的能力 动物们现在建议在分子水平上严格定义这些最早的A(3)组装形式和 阐明其对神经功能的作用机制。基于大量的初步数据和 为了分离和研究天然低聚物，我们开发了灵敏的生化方法，我们建议4 相互关联的具体目标。1.测定天然分泌物A|3的精确分子组成 通过质谱学分析齐聚物并搜索共价交联物、相关小分子和/或 结合蛋白，可能有助于其强大的神经元活动。2.描述以下因素的影响 天然低聚物对突触形态和功能的影响，包括在器官型海马区培养中，以及 评估它们是否可以在体内诱导AD型tau磷酸化和改变递质释放，3. 将天然低聚物提纯至均一，对其进行本质标记并鉴定其同源分子 活着的大脑中的细胞靶标。4.评估减少产量的3种具体治疗策略 细胞分泌的寡聚体，从而消除它们的突触毒性：(3-或γ-分泌酶抑制剂；某些 抗聚集化合物和伴侣蛋白的表达。我们在研究这方面的丰富经验 生理数量的人类A的无限细胞来源(3个寡聚体应该使我们能够利用这一点 独特的实验范式来阐明最早的A(3)的性质和神经元效应 集会，伴随着治疗的影响。与公共卫生的相关性：因为我们的核心 假说是最早形成的“寡聚体”(双胞胎、三胞胎等)淀粉样蛋白|3-蛋白质构成了 记忆的细微和渐进性损害是早期AD的标志，我们将使用一种独特的 一种实验系统，在这种实验系统中，培养的细胞自然地产生这种早期形式，以便破译 这些致病集合体的确切性质，确定它们对神经元和 记忆所需的突触，然后用新药阻断这一过程。

An enduring principle for successful intervention in human disease is to identify - andthen prevent - the earliest steps in pathogenesis. In the case of Alzheimer's disease and its harbinger, mild cognitive impairment (MCI), studies from many labs support the still unproven hypothesis that the gradual accumulation and oligomerization of amyloid p-protein (A(3)in brain regions serving memory and cognition initiates this complex syndrome. Given the enormous resources being expended by academic and pharmaceutical scientists to identify anti-amyloid therapies and bring them to human trials, it is crucial to understand precisely how soluble A|3begins to oligomerize and whether this process actually induces the subtle compromise of synaptic function seen in MCI and early AD. In this new RO1 application, investigators who have collaborated productively to discover the natural secretion of low-n A|3oligomers in cell culture and then demonstrate their ability to inhibit long-term potentiation and disrupt memory in living animals now propose to rigorously define at the molecular level these earliest A(3assembly forms and elucidate their mechanisms of action on neuronal function. Based on extensive preliminary data and sensitive biochemical methods we have developed to isolate and study natural oligomers, we propose 4 interrelated Specific Aims. 1. Determine the precise molecular composition of naturally secretedA|3 oligomers by mass spectrometry and search for covalent crosslinks, associated small molecules and/or binding proteins that may contribute to their potent neuronal activity. 2. Characterize the effects of the natural oligomers on synaptic form and function, including in organotypic hippocampal cultures, and assess whether they can induce AD-type tau phosphorylation and altered transmitter release in vivo, 3. Purify the natural oligomers to homogeneity, intrinsically label them and identify their cognate molecular and cellular targets in living brain. 4. Assess 3 specific therapeutic strategies to decrease the production of cell-secreted oligomers and thereby abrogate their synaptotoxicity: (3- or y-secretase inhibitors; certain anti-aggregation compounds; and chaperone expression. Our extensive experience in studying this unlimited cellular source of physiological amounts of human A(3 oligomers should enable us to exploit this unique experimental paradigm to elucidate both the nature and the neuronal effects of the earliest A(3 assemblies, with attendant therapeutic implications. Relevance to Public Health: Because our central hypothesis is that the earliest-forming "oligomers" (doublets, triplets, etc.) of amyloid |3-protein underlie the subtle and progressive impairment of memory that is the hallmark of incipient AD, we will use a unique experimental system in which cultured cells naturally produce such early forms in order to decipher the precise nature of these pathogenic assemblies, identify their mechanism of injury on the neurons and synapses required for memory, and then block this process with novel drugs.