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

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 - 补充仪器补助金

• 批准号: 10796611
• 负责人: Maciej J. Stawikowski
• 金额: $ 9.66万
• 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/10796611
• 关键词: Affect; Alzheimer' s Disease; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Astrocytes; Award; Cell membrane; Cell physiology; Cells; Cholesterol; Cholesterol Homeostasis; Endosomes; Environment; Equipment; Fibroblasts; Fluorescent Probes; Functional disorder; Funding; Grant; High Pressure Liquid Chromatography; Homeostasis; Huntington Disease; Impairment; Interdisciplinary Study; Intracellular Transport; Link; Lipids; Lysosomes; Membrane; Metabolism; Methods; Modernization; National Institute of General Medical Sciences; Neurodegenerative Disorders; Neurons; Organelles; Parkinson Disease; Pathogenesis; Play; Property; Proteins; Proteolysis; Regulation; Rejuvenation; Reporter; Reporting; Resources; Stains; Sterols; Synapses; Synaptic Receptors; Synaptic Vesicles; System; Technology; Transgenic Organisms; abeta accumulation; brain cell; cell type; chemical synthesis; computational chemistry; experience; fluorescence imaging; fluorophore; imaging capabilities; inhibitor; instrumentation; live cell imaging; multidisciplinary; neuron loss; novel; parent grant; pharmacologic; postsynaptic; protein distribution; scaffold; secretase; spatiotemporal; student participation; tau Proteins; trafficking; uptake

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.

抽象的 胆固醇（Chol）在神经元功能和存活中起关键作用，细胞chol的破坏 体内平衡与包括帕金森氏病，亨廷顿的主要神经退行性疾病有关 疾病和阿尔茨海默氏病（AD）。胆固醇不仅修饰淀粉样蛋白的贩运和裂解 前体蛋白（APP），但也使β-淀粉样蛋白（AβS）和tau蛋白的聚集恶化。 此外，膜上包裹的胆固醇异常会通过损害 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 越来越多的报告表明，越来越多的报告表明细胞内CHOL 通过膜贩运或新发现的立体转运蛋白的运输提供了更多的本地控制 亚细胞膜胆固醇分布比胆固醇代谢的全球调节。研究 胆固醇的运输，尤其是在活细胞中，需要荧光问题和成像能力，不仅可以 模仿膜胆固醇，但也区分质膜和细胞内室 内体。在这个项目中，我们建议开发一类环境敏感的荧光CHOL问题 （CND）使用1,8-萘酰亚胺荧光团并使用它们来阐明亚细胞分布和运输 Chol及其在AD发病机理中的影响。我们将采取多学科策略 包括计算化学，化学合成和活细胞成像的技术。基于模块化 我们提出的问题的脚手架，我们将生成具有不同属性的新CND。我们将确定 在不同细胞类型中选定的CND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，包括内体，溶酶体和 具有高时空精度的脂质液滴）。为了研究CNDS与内源性CHOL的相似之处，我们会问 如何破坏参与CHOL吸收，细胞内转运的蛋白质的细胞过程和活性， 隔离和分散会影响CND染色。将选定的CND与光谱分开的 突触蔬菜（SVS）和应用的记者，我们将研究不同膜室中的chol 影响主要类型的脑细胞中的SV转换，应用分布和裂解。使用转基因方法，我们 将询问应用程序及其主要的分裂产品如何影响MCHOL贩运和体内平衡。使用 药理学抑制剂，我们将询问应用程序的分泌裂解如何影响mchol。此外，我们会的 询问这种改变与突触功能障碍和神经元丧失的改变相关性如何 在广告中。总而言之，该项目将使膜chol的研究恢复活力，并给参与的学生 跨学科研究经验。