Reporter Mice For APP Processing And Transport

用于APP处理和运输的记者小鼠

• 批准号: 8091640
• 负责人: Zoia Muresan
• 金额: $ 19.19万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8091640
• 关键词: Affect; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein Precursor; Antibodies; Axonal Transport; Behavioral; Biochemical; Biological Assay; Biology; C-terminal; Cell Culture Techniques; Cell Fractionation; Cleaved cell; Destinations; Disease; Epitopes; Generations; Goals; Health; Human; Individual; Integral Membrane Protein; Knock-in Mouse; Lead; Length; Location; Methods; Modification; Molecular; Mus; N-terminal; Neurites; Neurodegenerative Disorders; Neurons; Neurosciences; Organelles; Pathology; Pathway interactions; Peptides; Population; Process; Protein Fragment; Protein Precursors; Proteins; Proteolytic Processing; Regulatory Element; Reporter; Research; Role; Seminal; Senile Plaques; Set protein; Site; Sorting - Cell Movement; Testing; Toxic effect; Transgenic Mice; Transport Process; Transport Vesicles; Vesicle; Work; cell motility; design; homologous recombination; image processing; improved; in vivo; mouse model; mutant; novel; overexpression; polypeptide; polypeptide C; promoter; protein expression; protein metabolite; protein transport; secretase; tool; trafficking; treatment strategy

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to characterize and understand the modifications in axonal transport in Alzheimer's disease (AD). Although it was suggested that an abnormal axonal transport could contribute to AD pathology, no systematic study was conducted. Here, we aim to cover this gap. We begin by asking how the amyloid-? precursor protein (APP), a type I transmembrane protein that is at the core of the pathogenic process in AD, is transported to the site(s) of its function within neuronal processes, in normal and in disease-related conditions. This study should have a strong impact on human health. APP, the precursor of the amyloid-? peptide that forms the senile plaques in AD, is proteolytically cleaved, by the action of secretases, into soluble, N-terminal (sAPP) and C-terminal (CTF) polypeptides. We recently showed that, in neurons, proteolytic processing of APP into fragments largely occurs prior to sorting into cargo vesicles, and that the different APP-derived polypeptides are transported independently (by distinct vesicle populations) to different destinations (Muresan et al., 2009. J. Neuroscience, 29: 3565-3578). To begin characterizing APP transport we will start with characterizing the transport of full-length APP and of the APP-derived N- and C-terminal fragments. To achieve this goal, we will generate knock-in mice that express APP, tagged with different reporters at its termini, which will allow separate tracking of the APP's N- and C-terminal fragments. Most importantly, because the dual-tagged APP will be knocked into the endogenous APP locus by homologous recombination (to maintain the endogenous regulatory elements for APP expression), the dual-tagged APP will be expressed at endogenous levels. We propose two Specific Aims. In Specific Aim I, we will generate the knock-in mice that will allow us to analyze the processing, transport, and localization of APP, and investigate the spectrum of proteins that interact with the APP's N- and C-terminal fragments. With in vivo motility assay, in Specific Aim II we will analyze the transport of APP-derived polypeptides in the neurons from the knock-in mice expressing CFP-APP-YFP (with CFP and YFP tagging the APP's N- and C- termini). An additional knock-in mouse expressing FLAG-APP-myc will be useful to determine with biochemical and subcellular fractionation methods the organelles where the APP fragments target, and the different sets of proteins that cotransport or interact with APP polypeptides. Finally, this study will emphasize the relation between the transport, processing, and function(s) of APP, and will increase the understanding of APP biology in the context of AD. PUBLIC HEALTH RELEVANCE: This project is directly relevant to Alzheimer's disease (AD), a neurodegenerative disorder predicted to affect 14 million individuals by 2050. The proposed work will generate novel mouse lines for the in vivo study of the processing and transport of APP, the protein at the core of the pathogenic process in AD. This study will provide unique tools for a better understanding of AD at molecular level, and could potentially lead to the design of novel treatment strategies aimed at improving axonal transport.

描述（由申请人提供）：拟议研究的长期目标是表征和了解阿尔茨海默氏病（AD）中轴突运输的修饰。尽管提出异常的轴突运输可能会导致AD病理学，但没有进行系统的研究。在这里，我们旨在弥补这一差距。我们首先问淀粉样蛋白如何？前体蛋白（APP）是一种在AD中致病过程核心的I型跨膜蛋白，在正常和疾病相关条件下，在神经元过程中运输到其功能的位点。这项研究应该对人类健康产生强大的影响。应用程序，淀粉样蛋白的前体？在AD中形成老年斑块的肽是通过分泌酶的作用将蛋白水解裂解的，可溶于可溶性，N末端（SAPP）和C末端（CTF）多肽。我们最近表明，在神经元中，将应用程序处理成碎片的蛋白水解处理在很大程度上发生在货物囊泡之前，并且不同的App衍生多肽是独立运输（通过不同的囊泡群体）到不同的目的地（Muresan等人，2009年，2009年。为了开始表征应用程序传输，我们将首先表征全长应用程序的传输以及应用程序衍生的N-和C端片段的传输。为了实现这一目标，我们将生成在其终端上用不同记者标记的表达应用的敲击鼠标，这将允许对应用程序的N-和C末端片段进行单独跟踪。最重要的是，由于双重标记的应用程序将通过同源重组（以维持APP表达的内源性调节元件）将其撞到内源性应用程序基因座中，因此双标签的应用将在内源水平上表达。我们提出了两个具体目标。在特定的目标I中，我们将生成敲入小鼠，这将使我们能够分析APP的处理，运输和定位，并研究与APP的N-和C末端片段相互作用的蛋白质谱。使用体内运动测定法，在特定的目标II中，我们将分析表达CFP-APP-YFP的敲入小鼠的神经元中的应用衍生多肽的运输（用CFP和YFP标记App的N-和C-termini）。一种额外的敲入小鼠表达Flag-App-Myc将有助于确定APP片段靶向的细胞器以及共同转移或与App Polypeptides相互作用的不同蛋白质集体组的细胞器。最后，这项研究将强调APP的运输，处理和功能之间的关系，并将在AD的背景下增加对APP生物学的理解。 公共卫生相关性：该项目与阿尔茨海默氏病（AD）直接相关，这是一种预计到2050年会影响1400万个人的神经变性疾病。拟议的工作将为APP的处理和运输，为APP的加工和运输，该蛋白质的蛋白质在AD中的核心。这项研究将提供独特的工具，以更好地了解分子水平的AD，并有可能导致设计旨在改善轴突运输的新型治疗策略。