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

描述(申请人提供)：本项目的总体目标是研究胞浆磷脂酶A2(CPLA2)在与淀粉样β蛋白(Abeta)刺激的脑内皮细胞(CECs)膜分子顺序和膜系留黏附机制改变相关的细胞通路中的作用。膜系留黏附是控制单核细胞跨内皮层迁移的第一个机械性步骤；因此，大脑中从外周单核细胞分化而来的小胶质细胞增加加剧了阿尔茨海默病(AD)的进展。在本项目中，我们将验证以下假设：抗体刺激CECs并导致cPLA2及其上游丝裂原激活蛋白激酶(MAPKs)的激活，而cPLA2及其上游丝裂原激活蛋白激酶(MAPKs)通过核因子-kB(NFkB)途径在黏附分子p-选择素的增加中发挥关键作用，从而促进肌动蛋白聚合，改变细胞膜的分子顺序，以及膜系绳黏附机制。这个项目将通过生化、生物物理和生物力学的方法来完成。生化方法包括细胞报告素法检测NFkB，Western印迹分析表征MAPKs和cPLA2的激活，定量免疫荧光显微镜(QIM)定量检测Abeta刺激的CECs中的活性氧物种、黏附分子(p-选择素)和肌动蛋白聚合。对于生物物理的方法，Laurdan的荧光显微镜将被用来研究MAPKs和cPLA2在Abeta刺激的CECs中对膜分子秩序的作用。对于生物力学方法，将使用原子力显微镜来确定MAPKs、cPLA2和NFkB在Abeta刺激的CECs细胞膜黏附改变中的作用。该项目的成果将通过提供参与机制来填补这一领域的空白 Abeta诱导的cPLA2相关通路与细胞膜系绳黏附机制的关系。由于膜系绳黏附是单核细胞迁移的决定性机械步骤，单核细胞可以进一步分化为小胶质细胞，从而加剧AD脑内的氧化和神经炎症状况，该项目有望有助于我们了解AD脑内的氧化和炎症。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。

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.