Pathogenic Mechanisms of Cell-Derived Abeta Oligomers

描述（由申请人提供）：成功干预人类疾病的持久原则是识别-然后预防-发病机制的最早阶段。在阿尔茨海默氏病及其先兆轻度认知障碍（MCI）的情况下，许多实验室的研究支持尚未证实的假设，即淀粉样蛋白P-蛋白（AB）在大脑区域的逐渐积累和寡聚化服务于记忆和认知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用于人体试验，因此准确了解可溶性AB如何开始寡聚化以及该过程是否真的诱导了MCI和早期AD中所见的突触功能的微妙妥协至关重要。在这一新的RO 1应用中，研究人员进行了富有成效的合作，发现了细胞培养中低n AB寡聚体的自然分泌，然后证明了它们抑制长时程增强和破坏活体动物记忆的能力，现在他们提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。基于广泛的初步数据和敏感的生化方法，我们已经开发出分离和研究天然寡聚体，我们提出了4个相互关联的具体目标。1.通过质谱测定天然分泌的AB寡聚体的精确分子组成，并寻找可能有助于其有效神经元活性的共价交联、相关小分子和/或结合蛋白。2.表征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，并评估它们是否可以诱导AD型tau磷酸化和改变体内递质释放。将天然寡聚物纯化至均一，内在标记它们并在活脑中鉴定其同源分子和细胞靶标。4.评估减少细胞分泌的寡聚体的产生从而消除其突触毒性的3种特定治疗策略：（B-或γ-分泌酶抑制剂;某些抗聚集化合物;和伴侣蛋白表达。我们在研究这种无限的细胞来源的生理量的人类AB低聚物的丰富经验，应使我们能够利用这种独特的实验范式，以阐明最早的AB组件的性质和神经元的影响，伴随着治疗的影响。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）由于淀粉样蛋白B蛋白是记忆的微妙和进行性损害的基础，这是早期AD的标志，我们将使用一个独特的实验系统，其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，确定它们对记忆所需的神经元和突触的损伤机制，然后用新药阻断这一过程。

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.