Pathogenic Mechanisms of Cell-Derived Abeta Oligomers

描述(由申请人提供)：成功干预人类疾病的一个持久原则是识别--然后预防--发病的最早步骤。在阿尔茨海默病及其先兆--轻度认知障碍(MCI)的案例中，许多实验室的研究支持仍未得到证实的假设，即淀粉样蛋白(AB)在负责记忆和认知的大脑区域逐渐积累和寡聚化引发了这种复杂的综合征。鉴于学术和制药科学家花费了大量资源来确定抗淀粉样蛋白疗法并将其应用于人体试验，准确地了解可溶性AB是如何开始寡聚的，以及这一过程是否真的导致了MCI和早期AD所见的突触功能的微妙妥协，是至关重要的。在这一新的RO1应用中，研究人员合作发现了细胞培养中低n AB寡聚体的自然分泌，然后证明了他们在活动物中抑制长期增强和干扰记忆的能力，现在建议在分子水平上严格定义这些最早的AB组装形式，并阐明它们对神经元功能的作用机制。基于大量的初步数据和我们开发的分离和研究天然低聚物的灵敏生化方法，我们提出了4个相互关联的特定目标。1.用质谱仪测定天然分泌的AB寡聚体的精确分子组成，寻找可能与其强大的神经元活性有关的共价交联物、相关小分子和/或结合蛋白。2.鉴定天然寡聚体对突触形态和功能的影响，包括在器官型海马培养中的作用，并评估它们是否能够在活体内诱导AD型tau磷酸化和改变递质释放；3.纯化天然寡聚体，对其进行内在标记，并鉴定其在活脑中的同源分子和细胞靶点。4.评估3种特定的治疗策略，以减少细胞分泌的寡聚体的产生，从而消除它们的突触毒性：(B-或Y-分泌酶抑制剂；某些抗聚集化合物；以及伴侣的表达。我们在研究人类AB寡聚体生理数量的无限细胞来源方面的丰富经验，应该使我们能够利用这一独特的实验范式来阐明最早的AB组装的性质和神经元效应，以及伴随而来的治疗意义。与公共卫生相关：因为我们的中心假设是最早形成的“寡聚体”(双胞胎、三胞胎等)由于淀粉样蛋白B蛋白是早期AD的标志-记忆的微妙和进行性损害的基础，我们将使用一种独特的实验系统，在该系统中培养的细胞自然产生这种早期形式，以破译这些致病组件的确切性质，识别它们对记忆所需的神经元和突触的损伤机制，然后用新型药物阻断这一过程。

DESCRIPTION (provided by applicant): An enduring principle for successful intervention in human disease is to identify - and then 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 (AB) 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 AB begins 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 AB oligomers 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 AB assembly 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 secreted AB 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: (B- 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 AB oligomers should enable us to exploit this unique experimental paradigm to elucidate both the nature and the neuronal effects of the earliest AB assemblies, with attendant therapeutic implications. Relevance to Public Health: Because our central hypothesis is that the earliest-forming "oligomers" (doublets, triplets, etc.) of amyloid B-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.