Cytosolic Phospholipase A2 in Amyloid-beta Peptide-stimulated Cerebral Endothelial cells

淀粉样β肽刺激的脑内皮细胞中的胞质磷脂酶A2

• 批准号: 9268545
• 负责人: JAMES C LEE
• 金额: $ 30.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9268545
• 关键词: Actins; Adhesions; Alzheimer' s Disease; Amyloid beta-Protein; Astrocytes; Atomic Force Microscopy; Biochemical; Biological Assay; Biomechanics; Biophysics; Brain; Brain Diseases; Cell Adhesion; Cell Adhesion Molecules; Cell Membrane Alteration; Cell Nucleus; Cell membrane; Cell surface; Cells; Cellular Membrane; Cerebrovascular system; Cerebrum; Characteristics; Cognitive deficits; Coupling; Cytosolic Phospholipase A2; Data; Development; Disease; Disease Progression; Endothelial Cells; Endothelium; F-Actin; Fluorescence Microscopy; Goals; Human; Immunofluorescence Microscopy; In Vitro; Inflammation; Inflammatory; Knockout Mice; Label; Laser Scanning Confocal Microscopy; MAPK14 gene; MAPK8 gene; Measurement; Measures; Mechanics; Mediating; Membrane; Microglia; Mitogen-Activated Protein Kinases; Molecular; NADPH Oxidase; Nuclear; Nuclear Translocation; P-Selectin; Pathway interactions; Peripheral; Phospholipids; Play; Probability; Process; Production; Reactive Oxygen Species; Reporter; Reporting; Resolution; Role; Selectins; Techniques; Testing; Therapeutic; Western Blotting; abeta deposition; biophysical analysis; biophysical techniques; brain endothelial cell; dihydroethidium; inhibitor/antagonist; innovation; insight; mitogen-activated protein kinase p38; monocyte; mouse model; nanometer; neuroinflammation; novel; oxidation; peptide B; polymerization; public health relevance; response

DESCRIPTION (provided by applicant): The overall goal of this project is to investigate the role of cytosolic phospholipase A2 (cPLA2) in the cellular pathways associated with alterations of membrane molecular order and membrane tethering adhesion mechanics in amyloid-beta peptide (Abeta)-stimulated cerebral endothelial cells (CECs). Membrane tethering adhesion is the first mechanical step governing the transmigration of monocytes across the endothelial layer; thus, increased microglial cells in brains differentiated from peripheral monocytes exacerbate the progression of Alzheimer's disease (AD). In this project, we will test the hypothesis that Ab stimulates CECs and results in activation of cPLA2 and its upstream mitogen-activated protein kinases (MAPKs) which play a critical role in the increase in adhesion molecules, p-selectin, through the nuclear factor-kB (NFkB) pathway, and subsequently enhanced actin polymerization, and alteration of the molecular order of cell membranes, and membrane tether adhesion mechanics.. This project will be accomplished by biochemical, biophysical, and biomechanical approaches. Biochemical approaches include cell reporter assay to measure NFkB, Western blot analysis to characterize activation of MAPKs and cPLA2 and quantitative immunofluorescence microscopy (QIM) to quantify reactive oxygen species, adhesion molecules (p-selectin), and actin polymerization in Abeta-stimulated CECs. For the biophysical approach, fluorescence microscopy of LAURDAN will be applied to examine the role of MAPKs and cPLA2 on membrane molecular order in Abeta-stimulated CECs. For the biomechanical approach, atomic force microscopy will be used to determine the role of MAPKs, cPLA2, and NFkB in alterations of cell membrane adhesion in Abeta-stimulated CECs. Results derived from this project will fill the gap in the field by providing the mechanism for involvement of cPLA2 activation and the relationship between Abeta-induced cPLA2-related pathway and membrane tether adhesion mechanics in CECs. Since membrane tether adhesion is a determining mechanical step for transmigration of monocytes, and monocytes can further differentiate into microglial cells which exacerbate oxidation and neuro-inflammation conditions in AD brains, this project is expected to contribute to our understanding of the oxidation and inflammation in AD brains. Ultimately, information derived from this project will provide new insights into therapeutic strategies for AD treatment and progression of the disease.

描述（由申请人提供）：本项目的总体目标是研究胞浆磷脂酶A2（cPLA 2）在淀粉样β肽（Abeta）刺激的脑内皮细胞（CEC）中与膜分子顺序和膜束缚粘附机制改变相关的细胞通路中的作用。膜栓系粘附是控制单核细胞穿过内皮层的迁移的第一个机械步骤;因此，从外周单核细胞分化的脑中增加的小胶质细胞加剧了阿尔茨海默病（AD）的进展。在这个项目中，我们将测试这样的假设，即Ab刺激CEC并导致cPLA 2及其上游丝裂原活化蛋白激酶（MAPK）的活化，其在粘附分子P-选择素的增加中起关键作用，通过核因子-kB（NFkB）途径，随后增强肌动蛋白聚合，改变细胞膜的分子顺序，以及膜系链粘附机制。 该项目将通过生物化学，生物物理和生物力学的方法来完成。生物化学方法包括测量NF κ B的细胞报告基因测定、表征MAPK和cPLA 2活化的蛋白质印迹分析以及定量免疫荧光显微术（QIM），以定量活性氧物质、粘附分子（p-选择素）和A β刺激的CEC中的肌动蛋白聚合。对于生物物理方法，将应用LAURDAN的荧光显微镜检查MAPK和cPLA 2对Abeta刺激的CEC中膜分子秩序的作用。对于生物力学方法，原子力显微镜将用于确定MAPK、cPLA 2和NF κ B在Abeta刺激的CEC中细胞膜粘附改变中的作用。 该项目的成果将通过提供参与机制填补该领域的差距 cPLA 2激活的机制以及Abeta诱导的cPLA 2相关通路与CEC中膜系链粘附力学之间的关系。由于膜系链粘附是单核细胞迁移的决定性机械步骤，并且单核细胞可以进一步分化为小胶质细胞，从而加剧AD脑中的氧化和神经炎症状况，因此该项目有望有助于我们了解AD脑中的氧化和炎症。最终，来自该项目的信息将为AD治疗和疾病进展的治疗策略提供新的见解。