Ultralong-term single-molecule imaging of amyloid precursor protein (APP) processing in Alzheimer's disease

阿尔茨海默病中淀粉样前体蛋白（APP）加工的超长期单分子成像

• 批准号: 10078232
• 负责人: Chunte Peng
• 金额: $ 11.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078232
• 关键词: Aging; Alzheimer disease prevention; Alzheimer' s Disease; Alzheimer' s disease patient; Amyloid beta-Protein Precursor; Axon; Axonal Transport; Biochemistry; Biological Models; Blinking; Cell membrane; Cells; Chemistry; Cleaved cell; Color; Data; Defect; Deposition; Destinations; Development; Disease Progression; Disease model; Dyes; Enzymes; Future; Gene Proteins; Goals; Healthcare; Hour; Human; Image; Imaging Techniques; Impairment; Individual; Investigation; Ions; Kinesin; Knowledge; Lead; Light; Link; Measures; Mentors; Methods; Mission; Molecular; Motor; Mus; Mutation; Nanotechnology; Neuroglia; Neurons; Optics; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Photobleaching; Planet Earth; Prevention; Production; Property; Proteins; Public Health; Quantum Dots; Research; Resolution; Signal Transduction; Site; System; Techniques; Testing; Therapeutic; Time; Training; Transgenic Mice; Transmembrane Transport; United States; United States National Institutes of Health; Vesicle; Work; amyloid imaging; amyloid peptide; amyloid precursor protein processing; base; beta secretase; career; emission line; fluorophore; human disease; human embryonic stem cell; induced pluripotent stem cell; innovation; insight; luminescence; meter; millisecond; molecular imaging; mouse model; mutant; nanometer; nanoparticle; next generation; novel; overexpression; physical science; prevent; protein transport; receptor; single molecule; skills; spatiotemporal; stem cell technology; trafficking

PROJECT SUMMARY/ABSTRACT Cleavage of amyloid precursor protein (APP) by b-site APP cleaving enzyme-1 (BACE-1) is the rate- limiting step in production of Ab, whose deposition is the pathological hallmark of Alzheimer’s disease (AD). Despite a rapidly growing burden of health care for the aging United States, there is a fundamental gap in understanding how trafficking and mutations of APP influence neuronal function and contribute to AD pathogenesis. Continued existence of this gap represents a critical problem because, until it is filled, AD prevention and treatment based on molecular understanding of the disease progression remains inaccessible. Since neurons can grow axons that are up to a meter long, continuous imaging of APP trafficking and processing in live neurons at the single-molecule level requires extremely photostable fluorophores. Our lab has recently developed a new class of upconversion nanoparticles (UCNPs) that are immensely photostable over months. The overall objective of this project is to use our novel photostable UCNPs to perform single-molecule imaging of the trafficking and processing of APP in live human induced neurons (iNs) – an excellent model system as many human diseases are not fully recapitulated in mouse neurons. The central hypothesis is that mutations of APP lead to impaired axonal transport and render APP more vulnerable for b-cleavage by BACE-1. This hypothesis has been formulated on the basis of previous work on culture mouse neurons and transgenic mouse models. The rationale for the proposed research is that ultralong-term single molecule imaging of WT and mutant APP in human iNs will reveal axonal transport defects caused by AD-associated mutations, providing important insights into their relationship to AD. Guided by strong preliminary data on the novel experimental platform, the hypothesis will be tested by pursuing the three specific aims: 1) Measure the trafficking dynamics of endocytosed APP in human iNs; 2) Determine how axonal transport is impaired by mutations of APP in human iNs; and 3) Visualize the association dynamics of APP and BACE-1 in human iNs. A battery of techniques including single-molecule imaging, nanotechnology, biochemistry and stem cell technology will be used to interrogate APP trafficking. The approach is innovative because it departs from the status quo by utilizing extremely photostable UCNPs to perform long-term single- molecule tracking, novel analysis for non-invasive determination of motor number, and the use of human induced neurons. The proposed research is significant because it is expected to characterize in depth the trafficking and association dynamics of APP and BACE-1 with unprecedented spatiotemporal resolution, as well as uncover the extent to which APP mutations impair axonal transport, thereby shedding light on future prevention and treatment of AD.

项目概要/摘要 b 位点 APP 裂解酶-1 (BACE-1) 对淀粉样前体蛋白 (APP) 的裂解率为： 抗体产生的限制步骤，其沉积是阿尔茨海默病的病理标志 （广告）。尽管老龄化的美国的医疗保健负担迅速增加，但有一个基本的问题 在理解 APP 的贩运和突变如何影响神经元功能并有助于 AD发病机制。这一差距的持续存在是一个关键问题，因为在它被填补之前， 基于对疾病进展的分子理解的 AD 预防和治疗仍然存在 无法访问。由于神经元可以生长长达一米的轴突，因此 APP 可以连续成像 单分子水平上活神经元的运输和处理需要极高的光稳定性 荧光团。我们的实验室最近开发了一种新型上转换纳米粒子（UCNP） 数月内具有极高的耐光性。该项目的总体目标是使用我们的小说 光稳定的 UCNP 可对实时 APP 的运输和加工进行单分子成像 人类诱导神经元 (iNs) – 一个优秀的模型系统，因为许多人类疾病尚未完全了解 在小鼠神经元中重述。核心假设是 APP 突变导致轴突受损 运输并使 APP 更容易受到 BACE-1 的 b 切割。这一假设已被 在之前培养小鼠神经元和转基因小鼠模型的基础上制定。这 拟议研究的基本原理是 WT 和突变 APP 的超长期单分子成像 人类 iNs 将揭示由 AD 相关突变引起的轴突运输缺陷，提供 关于他们与 AD 关系的重要见解。以小说的强有力的初步数据为指导 实验平台上，将通过追求三个具体目标来检验假设：1）测量 内吞 APP 在人类 iN 中的运输动态； 2) 确定轴突运输如何受损 通过人类 iN 中 APP 的突变； 3) 可视化 APP 和 BACE-1 的关联动态 人类 iNs。一系列技术，包括单分子成像、纳米技术、生物化学 干细胞技术将用于审问APP贩运。该方法具有创新性，因为 它摆脱了现状，利用极其耐光的 UCNP 来执行长期单 分子追踪、运动数量非侵入性测定的新颖分析以及人类的使用 诱导神经元。拟议的研究意义重大，因为预计将深入描述 APP 和 BACE-1 的贩运和关联动态具有前所未有的时空 分辨率，以及揭示 APP 突变损害轴突运输的程度，从而 为未来 AD 的预防和治疗提供线索。