Tracking APP and De Novo Aβ Generation in Live Cells Using Fluorogenic Click Chemistry

使用荧光点击化学跟踪活细胞中的 APP 和 De Novo Aβ 生成

• 批准号: 9318187
• 负责人: Leah Czerniewski
• 金额: $ 3.16万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9318187
• 关键词: Alzheimer' s Disease; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Appearance; Architecture; Axon; Back; Baculoviridae; Baculoviruses; Binding; C-terminal; Carrier Proteins; Cell Line; Cell surface; Cells; Chemistry; Cleaved cell; Complex; Data; Dementia; Dendrites; Deposition; Early Endosome; Endocytosis; Endosomes; Fluorescence; Fluorescent Probes; Generations; Golgi Apparatus; Grant; Label; Location; Lysosomes; Mediating; Multivesicular Body; Mutagenesis; Nature; Neurofibrillary Tangles; Neurons; Pathogenesis; Pathway interactions; Production; Protein Precursors; Protein Sortings; Proteins; Reporting; Research; Route; Senile Plaques; Site; Sorting - Cell Movement; System; Techniques; Work; amyloid precursor protein processing; base; beta secretase; exosome; extracellular; gamma secretase; hyperphosphorylated tau; insight; live cell imaging; microdevice; neuronal cell body; novel strategies; polarized cell; protein complex; protein transport; receptor; receptor density; retrograde transport; small molecule; trafficking; unnatural amino acids

PROJECT SUMMARY/ABSTRACT Defining neuropathological features of Alzheimer’s disease (AD) include extracellular amyloid plaques and intracellular neurofibrillary tangles. The amyloid-β peptide (Aβ), the principal component of amyloid plaques, is derived from the serial proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. Recent studies have demonstrated that β-secretase, endocytosed via an independent pathway from APP, interacts with APP in early endosomes to produce β-C-terminal fragment (β-CTF), which is trafficked down the canonical endolysosome pathway. The γ-secretase complex then cleaves β-CTF in endosomes/multi-vesicular bodies (MVBs) to generate Aβ, some of which has been shown to be released with exosomes. Work over the last decade has demonstrated the importance of an alternate pathway in modulating Aβ production by diverting APP away from the endo-lysosome pathway back to Golgi via retromers—a heteropentameric protein complex which mediates retrograde transport of transmembrane proteins to the Golgi. SorLA (sorting protein-related receptor containing low-density receptor class A repeats), which binds to the retromer complex, also binds APP; and its disruption results in increased Aβ production. How retromer disruption alters APP processing and Aβ generation is unclear. Indeed, one report indicates that Aβ might even be generated in the Golgi. Some of these previous studies have been limited by the static nature of the cellular analysis—few probes are available to study in living cells. Ultimately, a dynamic quantitative approach to interrogate APP trafficking and de novo Aβ generation will be important for understanding how intracellular trafficking influences AD pathogenesis. In this grant, I propose to use a novel strategy to label Aβ within its precursor protein in living cells to track the intracellular sorting of APP and Aβ generation. I will use unnatural amino acid (UAA) mutagenesis and click chemistry, a labeling technique first applied to Aβ/APP by our lab, to site-specifically insert a small molecule fluorescent probe in the Aβ region within APP. APP, in turn, will be fused to a fluorescent protein in the C-terminus to create a bifluorescent APP construct; Aβ production will be indicated by separation of the two fluorescent probes. The trafficking and processing pathways of APP and its fragments will be investigated in both neural cell lines and primary neurons. The true spatial dynamics of APP processing and Aβ generation are most appreciated in the complex architecture of neurons, where APP is synthesized in the cell body and transported down dendrites and axons where the location and mechanisms of subsequent processing steps are unclear. This research will give insight into the underlying mechanisms of AD pathogenesis.

项目摘要/摘要 阿尔茨海默病(AD)的定义神经病理特征包括细胞外淀粉样斑块和 细胞内神经原纤维缠绕。淀粉样蛋白-β肽(A-β)是淀粉样斑块的主要成分， 来源于β-和γ-分泌酶对淀粉样前体蛋白(APP)的一系列蛋白质分解。 最近的研究表明，β分泌酶通过一条独立于APP的途径内吞， 与早期内体中的APP相互作用产生β-C-末端片段(β-CTF)，该片段沿 典型的内溶酶体途径。γ-分泌酶复合体随后在内体/多囊泡中裂解β-ctf 体内(MVB)产生Aβ，其中一些已被证明与外切体一起释放。在工作中 在过去的十年中，已经证明了另一条途径在通过分流来调节Aβ产生方面的重要性 APP通过一种异五聚体蛋白复合体--逆转录--远离内切溶酶体途径返回高尔基体 它介导跨膜蛋白逆行运输到高尔基体。SOLA(与蛋白质相关的分类 包含低密度受体A类重复的受体)，其与逆转录复合体结合，也结合 APP；它的中断导致Aβ产量增加。回溯颠覆如何改变应用程序处理和 β的一代人还不清楚。事实上，一份报告表明，高尔基山脉甚至可能产生β。其中一些 这些先前的研究受到细胞分析的静态性质的限制--几乎没有可用的探针 在活细胞中研究。最终，一种动态量化的方法来审问应用程序贩运和从头开始 一代β对于理解细胞内转运如何影响AD的发病机制将是重要的。 在这项资助中，我建议使用一种新的策略来标记活细胞中的β前体蛋白，以 跟踪APP和Aβ代的细胞内分选。我将使用非天然氨基酸(UAA)诱变 点击化学，这是我们实验室首次应用于β/APP的一种标记技术，在网站上插入一个小的 APP内Aβ区的分子荧光探针。反过来，APP将与一种荧光蛋白融合在一起 C-末端创建双荧光APP构建；β的生产将通过分离 两个荧光探头。将调查APP及其碎片的贩运和加工途径 在神经细胞系和原代神经元中。APP处理的真实空间动态和β一代 在神经元的复杂结构中，APP是在细胞体中合成的，并且 向下运输树突和轴突，其中后续处理步骤的位置和机制 都不清楚。本研究将有助于揭示AD发病的潜在机制。