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

摘要 胆固醇(Chol)对神经元的功能和存活起着关键作用，细胞胆固醇的破坏 动态平衡与包括帕金森氏症、亨廷顿氏症在内的主要神经退行性疾病有关 疾病和阿尔茨海默病(AD)。胆固醇不仅改变淀粉样蛋白的运输和裂解 前体蛋白(APP)，但也恶化β-淀粉样多肽(Aβ，S)和牛磺酸蛋白的聚集。 此外，膜包埋胆固醇的异常可通过损害 突触小泡的周转和突触后受体的活动。虽然绝大多数人都在学习 CHOL的细胞代谢一直是人们关注的焦点，越来越多的报道表明细胞内CHOL 通过膜转运或新发现的类固醇转运体提供更多的局部控制 亚细胞膜胆固醇的分布比全球胆固醇代谢的调节要强。学习 胆固醇的运输，特别是在活细胞中，需要荧光探针和成像能力，不仅可以 模拟膜胆固醇，但也区分质膜和细胞内的隔室 内噬菌体。在这个项目中，我们计划开发一类环境敏感的荧光胆碱探针 (CNDS)使用1，8-萘酰亚胺荧光团，并用它们来阐明亚细胞的分布和运输 Chol的表达及其在AD发病中的意义。我们将采取多学科战略，以先进的 包括计算化学、化学合成和活细胞成像在内的技术。基于模块化的 在我们提议的探针的支架上，我们将生成具有不同性质的新的CNDS。我们将确定 所选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的相似性，我们将问 如何扰乱细胞过程和参与Chol摄取、细胞内转运、 隔离性、分散性会影响CNDS染色。将选定的CNDS与光谱可分离的CNDS相结合 对于突触小泡(SVS)和APP，我们将研究Chol如何在不同的膜室中 影响主要类型脑细胞的SV周转、APP分布和分裂。使用转基因方法，我们 将询问APP及其主要切割产品如何影响mChol交易和动态平衡。vbl.使用 药物抑制剂，我们将询问APP的分泌酶裂解是如何影响mChol的。此外，我们还将 询问这种改变的mChol调节与突触功能障碍和神经元丢失之间的关系 在公元后。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 跨学科的研究经验。

Abstract Cholesterol (Chol) plays a key role for neuronal 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). Cholesterol 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 cholesterol 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 cholesterol distribution than the global regulation of cholesterol metabolism. Studying cholesterol transport, especially in live cells, requires fluorescent probes and imaging capability that can not only mimic membrane cholesterol 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 use 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 different properties. 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 inhibitors, we will ask how the secretase cleavages of APP affect mChol. Furthermore, we will ask how such altered mChol regulation associates with 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 an interdisciplinary research experience.