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.

项目摘要/摘要 淀粉样前体蛋白(APP)被b位APP裂解酶-1(BACE-1)裂解的速率是- 限制抗体产生的步骤，抗体的沉积是阿尔茨海默病的病理标志 (Ad)。尽管老龄化的美国医疗保健负担迅速增加，但有一个根本的 对APP的贩运和突变如何影响神经元功能和促进 AD发病机制。这一缺口的持续存在是一个严重的问题，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 无法接近。由于神经元可以长出长达一米长的轴突，因此APP的连续成像 活神经元在单分子水平上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类新的上转换纳米颗粒(UCNPs)， 在几个月内都是非常稳定的。这个项目的总体目标是用我们的小说 光稳定的UCNPs对APP在体内的运输和加工进行单分子成像 人类诱导神经元(INS)-一个很好的模型系统，因为许多人类疾病并不完全 在小鼠神经元中重述。核心假设是APP的突变会导致轴突受损 运输和使APP更容易受到BACE-1的b-裂解。这一假设一直是 在前人工作的基础上制定了小鼠神经元培养和转基因小鼠模型。这个 拟议研究的基本原理是WT和突变APP的超长期单分子成像 INS将揭示AD相关突变引起的轴突运输缺陷，提供 对他们与AD关系的重要见解。以关于这部小说的强劲的初步数据为指导 在实验平台上，将通过追求三个具体目标来验证假设：1)测量 内吞APP在人INS中的转运动力学；2)确定轴突运输如何受损 通过人INS中APP的突变；3)可视化APP与BACE-1在INS中的关联动态 人类入侵。一系列技术，包括单分子成像、纳米技术、生物化学 干细胞技术将被用于审问应用程序的贩运。这种方法是创新的，因为 它改变了现状，利用极具光稳定性的UCNP进行长期的单 分子跟踪，用于非侵入性确定运动数量的新分析，以及使用人类 诱导神经元。这项拟议的研究具有重要意义，因为它有望深入描述 APP和BACE-1的时空转运和关联动态 分辨率，以及揭示APP突变对轴突运输的损害程度，从而 为今后AD的预防和治疗提供参考。