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胞浆磷脂酶A2α(CPLA2a)在中风后损伤演变中的作用。CPLA2a催化细胞内膜花生四烯酸的释放。脑缺血再灌注激活磷脂酶A2，导致花生四烯酸及其二十烷类代谢物水平升高。我们发现，cPLA2a基因缺陷的小鼠在局灶性脑缺血/再灌注后遭受的神经元损伤明显较少。我们现在已经确定了KIDS-cPLA2a，这是cPLA2a的一种新形式，它特异性地在齿状回中诱导，并保护神经元。我们假设(A)cPLA2a活性通过放大炎症而增强中风损伤；(B)脑缺血/再灌流后cPLA2a和KIds-cPLA2a的水平都增加，(C)cPLA2a和kIds-cPLA2a之间的平衡影响缺血/再灌流后海马区细胞的命运。目的1将测试cPLA2a基因敲除或用特定cPLA2a抑制剂治疗的小鼠在短暂性大脑中动脉闭塞后是否与对照组相比缩小梗塞范围、改变脑血流灌注和基因诱导。结果还将确定抑制cPLA2a的治疗时间窗口。目的2利用我们实验室开发的敏感分子、免疫组织化学和生化分析，将脑内诱导cPLA2合成二十烷类化合物与脑缺血后炎症标志物之间的关系联系起来。灵敏质谱仪将鉴定cPLA2a依赖的缺血/再灌注损伤的脂质介质。Aim 3将测试短暂性全脑缺血后是否在海马区诱导cPLA2a和KILDs-cPLA2a，并测量小鼠的分子和行为结果。在培养的海马神经元和器官切片培养中的进一步检查将使用我们实验室创建的慢病毒载体进行。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cPLA2a酶的药物是否可以用来减少中风后的伤害，以及这种新的蛋白质-cPLA2a是否有潜力成为治疗中风的新的大脑特异性疗法。

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.