Tracking Intracellular Pathways to Abeta Generation

追踪 Abeta 生成的细胞内途径

基本信息

批准号：
9264170
负责人：
Phyllis I Hanson
金额：
$ 22.88万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-15 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9264170
关键词：
Abeta synthesis Address Affect Alzheimer&apos s Disease Amyloid beta-Protein Amyloid beta-Protein Precursor Applications Grants Axon Binding Biological Models Brain Diseases Cell Line Cell membrane Cells Chemistry Cleaved cell Complex Dendrites Development Endocytosis Endosomes Engineering Fluorescence Future Generations Grant Imagery Integral Membrane Protein Knowledge Label Lead Length Location Lysosomes Mediating Methods Modeling Monitor Neurons Nonsense Mutation Outcome Pathogenesis Pathway interactions Pattern Peptides Production Protein Sortings Proteins Recycling Route Senile Plaques Site Sorting - Cell Movement System Therapeutic Time Vesicle Work alpha secretase amyloid precursor protein processing beta secretase extracellular fluorophore genetic variant induced pluripotent stem cell inhibitor/antagonist late endosome neuronal cell body novel protein complex protein transport receptor receptor density secretase small molecule subcellular targeting tool trafficking trans-Golgi Network unnatural amino acids vector

项目摘要

ABSTRACT Amyloid-beta (Aβ)—the principal component of amyloid plaques in Alzheimer’s disease (AD)—is generated by sequential secretase cleavages of the amyloid precursor protein (APP), a type 1 transmembrane protein. Despite three decades of study, the precise subcellular locations of Aβ generation have remained elusive. This is important because not only is Aβ central to AD pathogenesis but reducing Aβ levels has been a major focus in the development of potential AD therapeutics. Determing the precise cellular locations of A generation has been a major bottleneck because current model systems are incapable of tracking A peptide in living cells. We have recently developed a novel APP construct which incorporates an unnatural amino acid in the extracellular Aβ segment of APP, enabling click-chemistry to attach a small molecule fluorophore to A at the plasma membrane. Coincident attachment of distinct fluorescent proteins to intra- and extracellular regions of APP allows the real-time visualization of APP trafficking and Aβ generation in living cells. In this application we will exploit this model system to determine the impact of retromer sorting on APP trafficking and Aβ generation in engineered neural cell lines (Aim 1) and to determine the temporal-spatial dynamics of APP trafficking and Aβ generation within primary neurons (Aim 2). At the completion this grant, we will have a basic understanding of the itineraries of APP that lead to Aβ generation. We will also be able to examine these same pathways in primary neurons (or even iPSCs), and study how these Aβ-generating pathways may be altered by genetic variants or neuronal activity.

抽象的 β 淀粉样蛋白 (Aβ) 是阿尔茨海默病 (AD) 中淀粉样蛋白斑的主要成分，由以下物质产生淀粉样前体蛋白 (APP)（一种 1 型跨膜蛋白）的连续分泌酶裂解。尽管经过三十年的研究，Aβ 生成的精确亚细胞位置仍然难以捉摸。很重要，因为 Aβ 不仅是 AD 发病机制的核心，而且降低 Aβ 水平也一直是人们关注的焦点确定 A 一代的精确细胞位置已成为开发潜在 AD 疗法的重要一步。这是一个主要瓶颈，因为当前的模型系统无法跟踪活细胞中的 A 肽。我们最近开发了一种新型 APP 构建体，它在 APP 的细胞外 Aβ 片段，使点击化学能够将小分子荧光团附着到 A 不同的荧光蛋白同时附着在细胞内和细胞外区域。 APP 允许活细胞中 APP 运输和 Aβ 生成的实时可视化。将利用该模型系统来确定逆转录酶分选对 APP 运输和 Aβ 生成的影响在工程神经细胞系中（目标 1）并确定 APP 贩运和的时空动态初级神经元内 Aβ 的生成（目标 2）完成本次资助后，我们将对 Aβ 产生一个基本的了解。我们还能够研究导致 Aβ 生成的 APP 行程。原代神经元（甚至 iPSC），并研究这些 Aβ 生成途径如何通过遗传改变变异或神经元活动。