Investigating intracellular cholesterol distribution and trafficking using novel environment-sensitive cholesterol probes

摘要 胆固醇(Chol)对细胞的功能和生存起着关键作用，细胞胆固醇的破坏 动态平衡与包括帕金森氏症在内的主要神经退行性疾病有关， 亨廷顿病和阿尔茨海默病(AD)。胆固醇不仅改变了人口的贩运和卵裂 淀粉样前体蛋白(APP)，但也恶化β-淀粉样多肽(AβS)和Tau的聚集 蛋白。此外，膜包埋Chol的异常可通过损害突触功能而导致突触功能障碍 突触小泡的周转和突触后受体的活动。虽然绝大多数人 有关CHOL的细胞代谢的研究一直很集中，越来越多的报告表明 通过膜转运或新发现的固醇转运蛋白进行的细胞内Chol运输提供了更多 局部控制亚细胞膜Chol的分布比全局调控Chol代谢。学习 Chol的运输，特别是在活细胞中，需要荧光探针不仅可以模拟Chol，而且还可以 区分质膜和像内小体一样的细胞内隔间。在这个项目中，我们建议 用1，8-萘酰亚胺制备一类环境敏感的荧光胆碱探针 并利用它们来阐明CHOL及其受体的亚细胞分布和转运 在AD发病机制中的意义。我们将采取多学科战略，采用先进技术，包括 计算化学、化学合成和活细胞成像。基于我们的模块化脚手架 建议的探针，我们将产生新的CNDS具有不同的性质理想的不同应用。我们会 确定选定的CNDS在不同细胞类型(3T3成纤维细胞， 星形胶质细胞和神经元)和不同的亚细胞室(例如，不同的细胞器包括 内小体、溶酶体和脂滴)具有很高的时空精度。为了研究CNDS与 内源性Chol，我们将询问Chol如何扰乱细胞过程和参与Chol的蛋白质的活性 摄取、细胞内转运、隔离和分散将影响CNDS染色。组合所选内容 CNDS与突触小泡(SVS)和APP的光谱可分离记者，我们将研究Chol是如何 不同的膜间隔影响主要脑型的SV周转、APP分布和卵裂 细胞。使用转基因方法，我们将研究APP及其主要切割产物如何影响mChol运输 和动态平衡。利用药理学工具，我们将询问APP的分泌酶裂解如何影响mChol。 此外，我们将讨论这种改变的mChol调节如何导致突触功能障碍和 阿尔茨海默病中常见的神经元丢失。总之，该项目将为膜胆固醇的研究注入新的活力 并为参与的学生提供跨学科的研究体验。

Abstract Cholesterol (Chol) plays a key role for cell functionality and survival, the disruption of cellular Chol homeostasis has been linked to major neurodegenerative disorders including Parkinson's disease, Huntington's disease, and Alzheimer's disease (AD). Chol not only modifies the trafficking and cleavage of amyloid precursor protein (APP) but also worsens the aggregation of β-amyloid peptides (Aβs) and Tau protein. Moreover, abnormality of membrane-embedded Chol can cause synaptic dysfunction by impairing the turnover of synaptic vesicles (SVs) and the activities of postsynaptic receptors. While the vast majority studies have been focused on Chol's cellular metabolism, more and more reports have suggested that intracellular Chol transport via membrane trafficking or newly discovered sterol transporters provides more local control of subcellular membrane Chol distribution than the global regulation of Chol metabolism. Studying Chol transport, especially in live cells, requires fluorescent probes that can not only mimic Chol but also distinguish the plasma membrane and intracellular compartments like endosomes. In this project we propose to develop a class of environment-sensitive fluorescent Chol probes (CNDs) using 1,8-naphthalimide fluorophore and employ them to elucidate the subcellular distribution and trafficking of Chol as well as its implication in AD pathogenesis. We will take a multidisciplinary strategy with advanced technologies including computational chemistry, chemical synthesis and live-cell imaging. Based on the modular scaffold of our proposed probes, we will generate new CNDs with distinct properties ideal for different applications. We will determine the uptake, distribution, and dispersion of selected CNDs in different cell types (3T3 fibroblast cells, astrocytes, and neurons) and different subcellular compartments (e.g., different organelles including endosomes, lysosomes and lipid droplets) with high spatiotemporal precision. To study CNDs' resemblance to endogenous Chol, we will ask how disrupting cellular processes and activity of proteins involved in Chol uptake, intracellular transport, sequestration, and dispersion will affect CNDs staining. Combining selected CNDs with spectrally separable reporters for synaptic vesicles (SVs) and APP, we will study how Chol in different membrane compartments affect SV turnover, APP distribution and cleavage in major types of brain cells. Using transgenic methods, we will ask how APP and its major cleavage product affect mChol trafficking and homeostasis. Using pharmacological tools, we will ask how the secretase cleavages of APP affect mChol. Furthermore, we will address how such altered mChol regulation contribute to synaptic dysfunction and neuronal loss commonly found in AD. In summary, this project will rejuvenate the studies of membrane Chol and give the participating students a cross-disciplinary research experience.