Tracking Intracellular Pathways to Abeta Generation

追踪 Abeta 生成的细胞内途径

• 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
• 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' 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

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.

摘要 β-淀粉样蛋白（Aβ）是阿尔茨海默病（AD）中淀粉样蛋白斑块的主要成分， 淀粉样前体蛋白（APP）（一种1型跨膜蛋白）的连续分泌酶裂解。 尽管经过了30年的研究，Aβ生成的精确亚细胞位置仍然难以捉摸。这 重要的是，不仅Aβ是AD发病机制的中心，而且降低Aβ水平一直是主要焦点 开发潜在的AD治疗方法。确定A染色体的精确细胞位置， 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构建体，其将非天然氨基酸掺入到 APP的细胞外Aβ片段，使得点击化学能够将小分子荧光团连接到APP的细胞外A β片段， 质膜不同的荧光蛋白与细胞内和细胞外区域的一致附着 APP允许实时可视化活细胞中的APP运输和Aβ生成。在本申请中，我们 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（目的1），并确定APP运输的时空动力学， 原代神经元内的Aβ生成（目的2）。在完成这项赠款时，我们将有一个基本的了解， 导致Aβ生成的APP的行程。我们也将能够检查这些相同的途径， 原代神经元（甚至是iPSC），并研究这些Aβ生成途径如何被遗传改变 变体或神经元活动。