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.

摘要 胆固醇（Chol）对神经元的功能和存活起着关键作用，细胞Chol的破坏 体内平衡与主要的神经退行性疾病有关，包括帕金森氏病、亨廷顿舞蹈病、 疾病和阿尔茨海默病（AD）。胆固醇不仅改变了淀粉样蛋白的运输和切割 前体蛋白（APP），但也阻止了β-淀粉样肽（Aβs）和Tau蛋白的聚集。 此外，膜包埋胆固醇的异常可以通过损害突触的神经元而引起突触功能障碍。 突触囊泡（SV）的周转和突触后受体的活动。虽然绝大多数研究 Chol的细胞代谢一直是人们关注的焦点，越来越多的研究表明，细胞内Chol 通过膜运输或新发现的固醇转运蛋白的转运提供了更多的局部控制， 亚细胞膜胆固醇分布比胆固醇代谢的整体调节。研究 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， 模拟膜胆固醇，但也区分质膜和细胞内区室， 核内体本研究拟开发一类环境敏感的Chol荧光探针 （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 及其在AD发病机制中的意义。我们将采取多学科战略， 这些技术包括计算化学、化学合成和活细胞成像。基于模块化 我们提出的探针的支架，我们将产生具有不同性质的新的CND。康贝特人将以 选择的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，包括内体、溶酶体和 脂滴），具有高时空精度。为了研究CND与内源性Chol的相似性，我们将问 如何破坏参与Chol摄取，细胞内转运， 螯合和分散将影响CND染色。将选定的CND与光谱可分离的CND组合 报告突触囊泡（SV）和APP，我们将研究如何Chol在不同的膜室 影响主要类型脑细胞中的SV周转、APP分布和裂解。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运输和稳态。使用 对于药理学抑制剂，我们将询问APP的分泌酶裂解如何影响mChol。此外，我们将 我想知道这种mChol调节的改变如何与突触功能障碍和神经元丢失联系在一起， 在AD中。总之，本项目将振兴膜胆固醇的研究，并给予参与的学生 跨学科的研究经验。