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

使用新型环境敏感胆固醇探针研究细胞内胆固醇分布和运输

• 批准号: 10522716
• 负责人: Maciej J. Stawikowski
• 金额: $ 43.56万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522716
• 关键词: Address; Affect; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Astrocytes; Biological; Biophysics; Cardiovascular Diseases; Cell Survival; Cell membrane; Cell physiology; Cells; Cellular Structures; Cellular biology; Chemicals; Cholesterol; Cholesterol Homeostasis; Clinical; Collaborations; Dyes; Endosomes; Environment; Exhibits; Fibroblasts; Fluorescence Microscopy; Fluorescent Probes; Functional disorder; Head; Homeostasis; Huntington Disease; Image Analysis; Impairment; Interdisciplinary Study; Intracellular Transport; Label; Link; Lipids; Liposomes; Lysosomes; Measures; Membrane; Membrane Proteins; Membrane Transport Proteins; Metabolism; Methods; Micelles; Modeling; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Parkinson Disease; Pathogenesis; Pathologic; Pathway interactions; Pharmacology; Physiological; Play; Property; Proteins; Proteolysis; Publishing; Regulation; Reporter; Reporting; Research; Research Personnel; Resolution; Role; Stains; Sterols; Students; Synapses; Synaptic Vesicles; Technology; Testing; Transgenic Organisms; Vesicle; Work; abeta accumulation; amyloid peptide; base; biophysical properties; brain cell; cell type; cellular imaging; chemical synthesis; cholesterol trafficking; cholesterol transporters; computational chemistry; design; experience; fluorescence imaging; fluorophore; imaging capabilities; inhibitor; insight; live cell imaging; molecular dynamics; multidisciplinary; neuron loss; novel; postsynaptic; protein distribution; receptor; scaffold; secretase; spatiotemporal; tau Proteins; tool; trafficking; uptake

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.

抽象的 胆固醇（Chol）在细胞功能和存活中起关键作用，细胞chol的破坏 稳态与包括帕金森氏病的主要神经退行性疾病有关， 亨廷顿氏病和阿尔茨海默氏病（AD）。 Chol不仅修改了贩运和裂解 淀粉样蛋白前体蛋白（APP），但也会使β-淀粉样肽（AβS）和TAU的聚集恶化 蛋白质。此外，膜包裹的chol的异常会通过损害而引起突触功能障碍 突触蔬菜（SVS）的营业额和突触后受体的活性。而绝大多数 研究集中在Chol的细胞代谢上，越来越多的报告表明 细胞内CHOL传输通过膜运输或新发现的立体转运蛋白提供更多 亚细胞膜CHOL分布的局部控制，而不是全球对CHOL代谢的调节。研究 CHOL运输，尤其是在活细胞中，需要荧光问题，不仅可以模仿Chol，还需要 区分质膜和细胞内室（如内体）。在这个项目中，我们提出了 使用1,8-萘甲酰亚胺开发一类环境敏感的荧光CHOL问题（CND） 荧光团并雇用它们来阐明Chol的亚细胞分布和运输及其 在AD发病机理中的含义。我们将采用高级技术的多学科策略 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 提出的问题，我们将生成具有不同应用程序的不同属性的新CND。我们将 确定所选CND在不同细胞类型中的摄取，分布和分散（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 内体，溶酶体和脂质液滴），具有高时空精度。研究CND与 内源性chol，我们将询问如何破坏参与Chol的蛋白质的细胞过程和活性 摄取，细胞内转运，隔离和色散会影响CND染色。合并选定的 带有频谱分开的记者突触蔬菜（SVS）和应用程序的CND，我们将研究CHOL的方式 不同的膜室影响SV的周转，应用程序分布和裂解主要类型的大脑 细胞。使用转基因方法，我们将询问应用程序及其主要裂解产品如何影响MCHOL贩运 和稳态。使用药品工具，我们将询问应用程序的分裂如何影响mchol。 此外，我们将解决这种改变的MCHOL调节如何导致突触功能障碍和 AD中通常发现的神经元损失。总而言之，该项目将使膜chol的研究恢复活力 并为参与的学生提供跨学科的研究经验。