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

摘要 胆固醇（Chol）对细胞功能和存活起着关键作用，细胞Chol 体内平衡与主要的神经退行性疾病有关， 亨廷顿氏病和阿尔茨海默氏病（AD）。Chol不仅改变了 淀粉样前体蛋白（APP），但也阻止β-淀粉样肽（Aβs）和Tau的聚集 蛋白此外，膜包埋Chol的异常可通过损害神经元的功能而引起突触功能障碍。 突触囊泡（SV）的更新和突触后受体的活性。虽然绝大多数 研究一直集中在Chol的细胞代谢上，越来越多的报道表明， 通过膜运输或新发现的固醇转运蛋白的细胞内Chol转运提供了更多 局部控制亚细胞膜Chol分布比全局调节Chol代谢。研究 Chol的转运，尤其是在活细胞中，需要不仅能模拟Chol， 区分质膜和细胞内区室，如内体。在这个项目中，我们建议 以1，8-萘酰亚胺为探针，制备了一类环境敏感的Chol荧光探针 荧光团，并利用它们来阐明Chol的亚细胞分布和运输，以及其 在AD发病机制中意义。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 提出的探针，我们将产生新的CND具有不同的属性，适合不同的应用。我们将 确定所选CND在不同细胞类型（3 T3成纤维细胞， 星形胶质细胞和神经元）和不同的亚细胞区室（例如，不同的细胞器， 核内体、溶酶体和脂滴）。为了研究CND与 内源性胆固醇，我们将问如何破坏细胞过程和参与胆固醇的蛋白质的活性 摄取、细胞内转运、螯合和分散将影响CND染色。合并选定的 CND与突触囊泡（SV）和APP的光谱可分离报告基因，我们将研究Chol在 不同的膜隔室影响SV周转，APP分布和裂解在主要类型的脑 细胞使用转基因方法，我们将询问APP及其主要裂解产物如何影响mChol运输 和体内平衡。使用药理学工具，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， AD中常见的神经元缺失。总之，本项目将使膜胆固醇的研究重新焕发活力， 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.