Cytosolic Phospholipase A2 Alpha in Stroke Injury

描述（由申请人提供）：本项目的长期目标是确定酶胞质磷脂酶A2 α（cPLA 2a）在卒中后损伤演变中的作用。cPLA 2a催化花生四烯酸从膜的细胞内释放。脑缺血再灌注激活磷脂酶A2，导致花生四烯酸及其类花生酸代谢物水平升高。我们发现cPLA 2a缺陷小鼠在局灶性脑缺血/再灌注后神经元损伤明显减少。我们现在已经确定了KIDS-cPLA 2a，这是cPLA 2a的一种新形式，它在齿状回中特异性诱导并保护神经元。我们假设（a）cPLA 2 a活性通过放大炎症而增强中风损伤;（B）cPLA 2 a和KIDS-cPLA 2 a的水平在脑缺血/再灌注后增加;（c）cPLA 2 a和KIDS-cPLA 2 a之间的平衡影响缺血/再灌注后海马中的细胞命运.目的1将测试在短暂性大脑中动脉闭塞后，与对照组相比，cPLA 2a敲除小鼠或用特异性cPLA 2a抑制剂治疗的小鼠是否具有减小的梗死面积、改变的脑灌注和基因诱导。结果还将定义cPLA 2a抑制的治疗时间窗。目的2将相关的cPLA 2在类花生酸合成的脑诱导，和炎症标记物缺血后使用敏感的分子，免疫组织化学和生物化学测定在我们的实验室开发。灵敏的质谱法将鉴定缺血/再灌注损伤的cPLA 2a依赖性脂质介质。目的3将测试cPLA 2a和KIDS-cPLA 2a是否在短暂性全脑缺血后在海马中被诱导，并测量小鼠的分子和行为结果。将使用我们实验室创建的慢病毒载体在培养和器官型切片培养中对海马神经元进行进一步检查。由于治疗效果有限，中风损伤是一个主要的公共卫生问题。这些研究的结果将表明，针对cPLA 2a酶的药物是否可以用于减少中风后的损伤，以及新蛋白KIDS-cPLA 2a是否有潜力作为中风的新脑特异性治疗。

DESCRIPTION (provided by applicant): The long-term objective of this project is to determine the roles of the enzyme cytosolic phospholipase A2 alpha (cPLA2a) in the evolution of injury following stroke. cPLA2a catalyzes the intracellular release of arachidonic acid from membranes. Cerebral ischemia-reperfusion activates phospholipase A2 resulting in increased levels of arachidonic acid and its eicosanoid metabolites. We showed that cPLA2a-deficient mice suffer significantly less neuronal injury after focal cerebral ischemia/reperfusion. We have now identified KIDS- cPLA2a, a novel form of cPLA2a, which is specifically induced in the dentate gyrus and protects neurons. We hypothesize (a) that cPLA2a activity enhances stroke injury by amplifying inflammation; (b) that levels of both cPLA2a and KIDS- cPLA2a are increased following cerebral ischemia/reperfusion, (c) that the balance between cPLA2a and KIDS- cPLA2a influences cell fate in the hippocampus following ischemia/reperfusion. Aim 1 will test if cPLA2a knockouts or mice treated with specific cPLA2a inhibitors have decreased infarct size, altered cerebral perfusion, and gene induction, as compared to controls after transient middle cerebral artery occlusion. Results will also define a therapeutic time window for cPLA2a inhibition. Aim 2 will correlate the cerebral induction of cPLA2at eicosanoid synthesis, and markers of inflammation following ischemia using sensitive molecular, immunohistochemical and biochemical assays developed in our labs. Sensitive mass spectrometry will identify cPLA2a-dependent lipid mediators of ischemia/reperfusion injury. Aim 3 will test if cPLA2a and KIDS- cPLA2a are induced in the hippocampus following transient global ischemia and measure molecular and behavioral outcomes in mice. Further examination in hippocampal neurons in culture and in organotypic slice culture will be performed using lentivirus vectors created in our lab. Stroke injury is a major public health problem because of the limited effectiveness of treatments. The results of these studies will show if drugs aimed against the enzyme cPLA2a can be used to decrease injury following stroke and if the new protein, KIDS- cPLA2a has potential as a new brain-specific therapy for stroke.