Tracking Intracellular Pathways to Abeta Generation

摘要 淀粉样β蛋白(Aβ)是阿尔茨海默病(AD)中淀粉样斑块的主要成分，由 淀粉样前体蛋白(APP)是一种1型跨膜蛋白，其顺序分泌裂解。 尽管进行了30年的研究，但Aβ一代的确切亚细胞位置仍然难以捉摸。这 这一点很重要，因为Aβ不仅是AD发病机制的核心，而且降低Aβ水平一直是主要关注的焦点 在开发潜在的阿尔茨海默病治疗药物方面。确定一代人的精确细胞位置 这是一个主要的瓶颈，因为目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的APP结构，它在 细胞外APP的β片段，使点击化学能够将小分子荧光团连接到上 质膜。不同的荧光蛋白与胞内区和胞外区的结合 APP允许实时可视化APP交易和活细胞中的β一代。在此应用程序中，我们 将利用此模型系统来确定回溯排序对应用程序交易和β生成的影响 在工程化神经细胞系(目标1)中，并确定APP贩运和 原代神经元内的β代(目标2)。在完成这笔赠款后，我们将有一个基本的了解 通向β一代的APP路线。我们还将能够研究这些相同的路径 原代神经元(甚至是IPSCs)，并研究这些Aβ产生途径如何通过基因改变 变异或神经元活动。

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.