ROLE OF APP-RELATED PROTEINS DURING NEURONAL MIGRATION

描述（由申请人提供）：本提案的长期目标是确定正常发育期间与淀粉样前体蛋白（APP）相关的蛋白质的体内功能，并深入了解这些功能的扰动如何导致阿尔茨海默病（AD）的病理学。AD与分泌酶组合引起的APP加工失调有关，这导致产生过量的β-淀粉样蛋白片段（Abeta），其可在神经系统内聚集成淀粉样蛋白斑块。虽然Abeta已被证明具有神经毒性作用，但APP的正常功能也可能被该过程破坏，从而导致AD的病理学。多种体外研究表明，APP作为跨膜受体，能够通过多种细胞内信号通路调节神经元迁移和生长。特别令人信服的是，实验表明，APP 695（被认为是APP的神经元形式）直接结合到异源三聚体G蛋白Go-alpha，并可以调节其活性。然而，由于与哺乳动物神经系统相关的复杂性，以及由于缺乏APP-Go-alpha信号传导的生物学相关测定，这种相互作用的功能分析已经被排除。为了解决这个问题，已经建立了一个模型系统（烟草天蛾的肠神经系统或ENS），其中可以在完整的神经系统内可视化和操纵一组识别的迁移神经元（EP细胞）。EP细胞表达APP的直系同源物（msAPPL，或APP样蛋白），随着神经元的发育，该蛋白会经历受调节的运输和加工。MsAPPL还与果阿在他们的领导过程中互动。初步研究表明，抑制EP细胞中的msAPPL表达会诱导异位、不适当的迁移，这与Go-α介导的信号传导事件的破坏一致。该提案的目标是测试msAPPL作为一种新型Go-α偶联受体的假设：当被内源性配体ENS激活时，它以Go-α依赖性方式调节神经元引导。还将探讨迁移神经元中msAPPL-Go-alpha相互作用的性质以及分泌酶在调节依赖msAPPLE的迁移方面可能发挥的作用。最后，使用ENS作为体内测定系统，将采用表达克隆策略来鉴定msAPPL的候选配体。这些研究将为发育中的神经系统中APP相关信号传导的分子机制提供新的见解，并且它们应该作为未来研究如何破坏APP的正常功能可能有助于AD病理学的基础。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to determine the in vivo functions of proteins related to the amyloid precursor protein (APP) during normal development, and to gain insight into how perturbations of these functions may contribute to the pathology of Alzheimer's Disease (AD). AD is associated with the misregulated processing of APP by a combination of secretases, which results in the generation of excessive beta-amyloid fragments (Abeta) that can aggregate into amyloid plaques within the nervous system. Although Abeta has been shown to have neurotoxic effects, the normal functions of APP may also be disrupted by this process, contributing to the pathology of AD. A variety of studies in vitro have indicated that APP can act as a transmembrane receptor capable of regulating neuronal migration and outgrowth via several candidate intracellular signaling pathways. Particularly compelling are experiments showing that APP695 (considered a neuronal form of APP) binds directly to the heterotrimeric G protein Go-alpha and can regulate its activity. However, a functional analysis of this interaction has been precluded by complexities associated with the mammalian nervous system, and due to the lack of a biologically relevant assay for APP-Go-alpha signaling. To address this issue, a model system (the enteric nervous system or ENS of Manduca sexta) has been established, in which an identified set of migratory neurons (the EP cells) can be visualized and manipulated within the intact nervous system. The EP cells express an orthologue of APP (msAPPL, or APP-Like protein), which undergoes regulated trafficking and processing as the neurons develop. MsAPPL also interacts with Goa in their leading processes. Preliminary studies have shown that inhibiting msAPPL expression in the EP cells induces ectopic, inappropriate migration, consistent with a disruption of Go-alpha- mediated signaling events. The goals of this proposal are to test the hypothesis that msAPPL acts as a novel Go-alpha-coupled receptor: when activated by endogenous ligands the ENS, it regulates neuronal guidance in a Go-alpha-dependent manner. The nature of msAPPL-Go-alpha interactions in the migrating neurons and the role that secretases may play in modulating msAPPL-dependent aspects of migration will also be explored. Lastly, an expression cloning strategy will be employed to identify candidate ligands for msAPPL, using the ENS as an in vivo assay system. These studies will provide new insight into the molecular mechanisms of APP-related signaling in the developing nervous system, and they should serve as a foundation for future research into how disrupting the normal functions of APP may contribute to the pathology of AD.