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与淀粉样蛋白前体蛋白（APP）相关的蛋白质在正常发育过程中的体内功能，并深入了解这些功能的扰动如何有助于阿尔茨海默病（AD）的病理。AD与分泌酶组合对APP加工的错误调控有关，导致过量的β -淀粉样蛋白片段（Abeta）的产生，这些片段可以在神经系统内聚集成淀粉样斑块。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这一过程破坏，从而导致AD的病理。各种体外研究表明，APP可以作为一种跨膜受体，通过几种候选的细胞内信号通路调节神经元的迁移和生长。特别令人信服的是，实验表明APP695（被认为是APP的一种神经元形式）直接与异源三聚体G蛋白go - α结合，并能调节其活性。然而，由于哺乳动物神经系统的复杂性，以及缺乏与app - go - α信号相关的生物学分析，这种相互作用的功能分析一直被排除。为了解决这个问题，我们建立了一个模型系统（Manduca sexta的肠神经系统或ENS），在这个模型系统中，一组已识别的迁移神经元（EP细胞）可以在完整的神经系统中被可视化和操作。EP细胞表达APP的同源物（msAPPL，或APP样蛋白），该蛋白在神经元发育过程中受到调控的转运和加工。MsAPPL还在其领导流程中与Goa进行交互。初步研究表明，抑制EP细胞中msAPPL的表达可诱导异位、不适当的迁移，这与go - α介导的信号事件的破坏一致。本提案的目的是验证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依赖的方式调节神经元引导。我们还将探讨迁移神经元中msappll - go - α相互作用的性质，以及分泌酶在调节迁移过程中msappll依赖性方面可能发挥的作用。最后，利用ENS作为体内检测系统，采用表达克隆策略鉴定msAPPL的候选配体。这些研究将为了解发育中的神经系统中APP相关信号的分子机制提供新的见解，并为进一步研究APP正常功能的破坏如何导致AD的病理奠定基础。

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.