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

项目总结/摘要 淀粉样前体蛋白（APP）被B位点APP裂解酶-1（BACE-1）裂解的速率为 抗体产生的限制步骤，其沉积是阿尔茨海默病的病理标志 （AD）。尽管美国老龄化的医疗保健负担迅速增加，但有一个基本的 在理解APP的运输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继续存在是一个关键问题，因为在填补这一差距之前， 基于对疾病进展的分子理解的AD预防和治疗仍然是 难以接近由于神经元可以长出长达一米的轴突， 活神经元在单分子水平上的运输和加工需要极其光稳定的 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耐光的。这个项目的总体目标是利用我们的小说 光稳定的UCNPs进行单分子成像的运输和加工的APP在生活中 人类诱导神经元（iN）-一个很好的模型系统，因为许多人类疾病并不完全 在小鼠神经元中重现。核心假设是APP突变导致轴突受损， 运输并使APP更容易被BACE-1裂解。这一假设一直被 在前人对培养小鼠神经元和转基因小鼠模型研究的基础上，的 拟议研究的基本原理是WT和突变APP的超长期单分子成像 在人类iN中的研究将揭示由AD相关突变引起的轴突运输缺陷， 他们与AD的关系的重要见解。根据小说的初步数据 实验平台，假设将通过追求三个具体目标进行测试：1）测量 内吞APP在人iNs中的运输动力学; 2）确定轴突运输如何受损 通过APP在人iN中的突变;和3）可视化APP和BACE-1在人iN中的结合动力学， 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讯APP贩运。这种方法是创新的，因为 它从现状出发，利用极耐光的UCNPs进行长期的单- 分子追踪、用于非侵入性确定运动数量的新分析以及人类 诱导神经元拟议的研究是有意义的，因为它有望深入表征 APP和BACE-1的运输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的预防和治疗提供了线索。

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.