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并预防疾病， 发病的最早阶段在阿尔茨海默病及其先兆的情况下，轻度认知障碍 尽管许多实验室的研究都支持一个尚未得到证实的假设，即渐进的 淀粉样p-蛋白（A（3））在负责记忆和认知的脑区的积累和寡聚化 引发了这种复杂的综合症。鉴于学术界和学术界花费了大量资源， 制药科学家确定抗淀粉样蛋白疗法，并将其用于人体试验，这是至关重要的， 准确地理解可溶性A| 3开始寡聚化，以及这个过程是否真的诱导了 在MCI和早期AD中观察到的突触功能的微妙妥协。在这个新的RO 1应用程序中， 研究人员进行了富有成效的合作，发现了低n A的自然分泌，|3低聚物 细胞培养，然后证明他们的能力，抑制长时程增强和破坏记忆的生活 动物现在提出在分子水平上严格定义这些最早的A β组装形式， 阐明其对神经元功能的作用机制。根据大量的初步数据， 敏感的生化方法，我们已经开发出分离和研究天然低聚物，我们提出4 相关的具体目标。1.确定天然分泌A的精确分子组成|3 低聚物通过质谱和搜索共价交联，相关的小分子和/或 可能有助于其有效神经元活性的结合蛋白。2.描述的影响， 天然寡聚体对突触形式和功能的影响，包括在器官型海马培养物中，以及 评估它们是否可以在体内诱导AD型tau磷酸化和改变的递质释放，3. 将天然低聚物纯化至均一，内在标记它们并鉴定它们的同源分子 和活脑中的细胞靶点。4.评估3种具体的治疗策略，以减少生产 细胞分泌的寡聚体，从而消除其突触毒性：（3-或γ-分泌酶抑制剂;某些 抗聚集化合物;和伴侣蛋白表达。我们在研究这一问题方面的丰富经验 生理量的人A β寡聚体的无限细胞来源应该使我们能够利用这一点， 一个独特的实验范式，以阐明最早的A（3）的性质和神经元的影响 组装，以及随之而来的治疗意义。与公共卫生的相关性：因为我们的中心 假设是，最早形成的“寡聚体”（双联体、三联体等）淀粉样|3-蛋白质是 微妙的和渐进的记忆障碍，这是早期AD的标志，我们将使用一个独特的 一种实验系统，在该系统中，培养的细胞自然地产生这种早期形式，以破译 这些致病组件的确切性质，确定它们对神经元的损伤机制， 突触的记忆所需，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.