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

项目总结/摘要 阿尔茨海默病（AD）的神经病理学特征包括细胞外淀粉样蛋白斑块和 细胞内神经纤维缠结。淀粉样蛋白-β肽（Aβ）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）的连续蛋白水解裂解。 最近的研究表明，β-分泌酶通过APP的独立途径内吞， 在早期内体中与APP相互作用，产生β-C-末端片段（β-CTF），其被运输到 典型的内溶酶体途径然后，γ-分泌酶复合物在核内体/多囊泡细胞中切割β-CTF。 Aβ的产生是通过MVB体（MVB）来实现的，其中一些已经被证明是与外泌体一起释放的。工作的 近十年来，已经证明了替代途径在调节Aβ产生中的重要性， APP通过逆转录酶-一种异五聚体蛋白复合物从内溶酶体途径回到高尔基体 其介导跨膜蛋白向高尔基体的逆行转运。SorLA（分选蛋白相关 含有低密度受体A类重复序列的受体），其结合逆转录复合物，也结合 APP;其破坏导致Aβ产生增加。retromer中断如何改变APP处理和 Aβ代尚不清楚。事实上，一份报告指出，Aβ甚至可能在高尔基体中产生。一些 这些先前的研究受到细胞分析的静态性质的限制， 来研究活细胞。最终，一个动态的定量方法来审问APP贩运和从头 Aβ的产生对于理解细胞内运输如何影响AD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前体蛋白Aβ， 跟踪APP和Aβ生成的细胞内分选。我将使用非天然氨基酸（UAA）诱变 点击化学，一种我们实验室首次应用于Aβ/APP的标记技术， APP内的Aβ区域中的分子荧光探针。APP反过来将与荧光蛋白融合， C-末端，以产生双荧光APP构建体; Aβ产生将通过分离C-末端来指示。 两个荧光探针。将调查APP及其片段的贩运和加工途径 在神经细胞系和原代神经元中。APP加工和Aβ生成的真实空间动力学 在神经元的复杂结构中最受赞赏，其中APP在细胞体中合成， 运输下来的树突和轴突的位置和机制，随后的处理步骤 不清楚。这项研究将有助于深入了解AD发病机制的潜在机制。

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.