R01: Cytosolic phospholipase A2 in amyloid-beta peptide-stimulated cerebral endot

描述（由申请人提供）：本项目的总体目标是研究胞质磷脂酶A2 （cPLA2）在淀粉样蛋白- β肽（Abeta）刺激的脑内皮细胞（CECs）中与膜分子顺序改变和膜系结粘附机制相关的细胞通路中的作用。膜系泊黏附是控制单核细胞跨内皮层迁移的第一个机械步骤；因此，从外周单核细胞分化而来的大脑小胶质细胞的增加加剧了阿尔茨海默病（AD）的进展。在本项目中，我们将验证Ab刺激CECs并导致cPLA2及其上游有丝分裂原活化蛋白激酶（MAPKs）激活的假设，这些激酶通过核因子- kb （NFkB）途径在粘附分子p-选择素的增加中起关键作用，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。这个项目将通过生物化学、生物物理和生物力学的方法来完成。生化方法包括测定NFkB的细胞报告试验，测定MAPKs和cPLA2活化的Western blot分析和定量免疫荧光显微镜（QIM）来定量abeta刺激CECs中的活性氧、粘附分子（p-选择素）和肌动蛋白聚合。对于生物物理方法，LAURDAN的荧光显微镜将用于检查MAPKs和cPLA2在β刺激的CECs中对膜分子秩序的作用。对于生物力学方法，原子力显微镜将用于确定MAPKs、cPLA2和NFkB在β刺激的CECs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来填补该领域的空白

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.