Cyclooxygenase-2 in Ischemia Neuronal Injury

脑缺血后环氧合酶2(COX2)活化产生的次级前列腺素代谢产物 由于环戊烯酮类前列腺素(CyPGs)可引起神经细胞死亡。在初步研究中，我们 发现脑缺血后脑组织CyPG 15-脱氧-12，14-PGD2浓度升高。我们发现 15-脱氧-12，14-前列腺素D2诱导神经元原代培养细胞死亡不依赖于其对 PPAR？和DP2受体。我们发现CyPG与泛素C末端水解酶-L1(UCH-L1)结合，a 蛋白质泛素化途径中的关键酶在去除未折叠或错误折叠的蛋白质中起重要作用 手机。此外，用CyPGs治疗可抑制UCH-L1并诱导泛素化蛋白的积累 (UB蛋白)。 基于这些数据和其他数据，我们假设脑组织中的CyPG浓度在 缺血和CyPG增加Ub蛋白的积聚，内质网(ER)应激和 通过共价修饰和抑制UCH-L1活性实现缺血后蛋白质聚集。 提出了以下具体目标： 1.确定CyPG与Uch-L1共价结合的位置。测定CyPG加合物的作用 Uch-L1功能的形成、Ub蛋白的积累和内质网应激的产生 神经元培养。 2.鉴定大鼠局灶性脑缺血后脑组织中发现的特异性细胞周期蛋白。确定这些CyPG是否 直接诱导细胞死亡或在体外改变神经元对缺氧缺血性细胞死亡的敏感性。 3.体内研究CyPG对缺血后UCH-L1的抑制作用。确定CyPgs是否与 UCH-L1在体内的表达。确定短暂性局灶性脑缺血后COX2活性产生的CyPgs是否与 Uch-L1，并产生Ub-蛋白的积累、内质网胁迫和蛋白质聚集。 内质网应激、Ub蛋白堆积和蛋白聚集是迟发性神经元死亡的重要原因 死于脑缺血和神经退行性疾病。这些研究旨在阐明一种新的机制，通过 缺血后诱导的内质网应激和Ub蛋白的积聚可能与 中风和神经退行性疾病的发病机制，如帕金森S和阿尔茨海默病S。

Secondary prostaglandin metabolites produced by activation of cyclooxygenase 2 (COX2) after ischemia such as the cyclopentenone prostaglandins (cyPGs) may produce neuronal cell death. In our preliminary studies we find that concentrations of the cyPG 15-deoxy-?12,14-PGD2 are increased in brain after ischemia. We found that 15-deoxy-?12,14-PGD2 induces cell death in neuron-enriched primary cultures independent of its effects on the PPAR? and DP2 receptors. We found that cyPGs bind to ubiquitin C-terminal hydrolase-L1 (UCH-L1), a key enzyme in the protein ubiquitination pathway important in removing unfolded or misfolded proteins from the cell. Furthermore, treatment with cyPGs inhibits UCH-L1 and induces accumulation of ubiquitinated proteins (Ub-proteins) in primary neurons. Based on these and other data we hypothesize that concentrations of cyPGs are increased in brain after ischemia and that cyPGs increase accumulation of Ub-proteins, endoplasmic reticulum (ER) stress and protein aggregation after ischemia by covalent modification and inhibition of UCH-L1 activity. The following specific aims are proposed: 1. Determine the site where cyPGs covalently bind to UCH-L1. Determine the effect of cyPG adduct formation on UCH-L1 function, accumulation of Ub-proteins and production of ER stress in primary neuronal cultures. 2. Identify the specific cyPGs found in rat brain after temporary focal ischemia. Determine if these cyPGs directly induce cell death or modify the susceptibility of neurons to hypoxic ischemic cell death in vitro. 3. Characterize the role of cyPG inhibition of UCH-L1 after ischemia in vivo. Determine if cyPgs bind to UCH-L1 in vivo. Determine if cyPgs produced by COX2 activity after temporary focal ischemia bind to UCH-L1, and produce accumulation of Ub-proteins, ER stress and protein aggregation. ER stress, accumulation of Ub-proteins and protein aggregation are important etiologies of delayed neuronal death in ischemia and neurodegenerative diseases. These studies aim to elucidate a new mechanism by which ER stress and accumulation of Ub-proteins are induced after ischemia that may be relevant to the pathogenesis of stroke and neurodegenerative diseases such as Parkinson¿s and Alzheimer¿s disease.