Cyclooxygenase-2: an endogenous neuromodulator in seizures and epileptogenesis

描述（由申请人提供）：花生四烯酸（AA）的代谢物是有效的局部介质，在正常和异常条件下控制广泛的功能。环氧化酶-2 （COX-2）是中枢神经系统（CNS）内AA代谢的关键酶，是许多高度自我处方的非处方抗炎/镇痛药物的治疗靶点。正常情况下在海马CA3区和大脑皮层2/3层的神经元中表达。与这一建议相关的是，惊厥发作活动诱导这些区域以及齿状回（DG）神经元中COX-2表达的上调。这提高了COX-2可能在癫痫中起神经调节作用的可能性，癫痫是一种以自发发作为特征的脑部疾病。事实上，来自大量研究的证据，包括PI实验室的初步和已发表的结果，都支持神经元COX-2活性产物可能具有抗癫痫特性的观点。这一中心假设构成了本提案的拟议研究的基础。Aims 1和Aims 2的研究将测试靶向转基因（TG）神经元COX-2过表达对急性发作阈值和癫痫发生的影响，这是大脑变成癫痫的过程。在这两个目的中，假设COX-2表达的区域特异性上调将减少急性惊厥发作和癫痫发生的发生率。为了验证这一点，Aim 1中的研究将检查海马形成DG或CA3区域神经元中靶向COX-2过表达的后果。Aim 2的补充研究将进一步测试COX-2靶向过表达在海马CA1神经元和皮层兴奋层2/3神经元中的作用。一种允许cre依赖性COX-2上调的新型TG小鼠系将用于这些目的。这些小鼠将与TG小鼠杂交，TG小鼠使用神经元特异性启动子将Cre表达靶定在三个所需脑区的神经元上。在Aim 2中使用的TG Cre系的另一个优点是，它将允许他莫昔芬诱导的Cre表达。因此，对cox -表达的特殊和时间控制将成为可能。这些杂交的双TG小鼠将在急性发作和癫痫发生模型中进行测试。目的3研究将探索从其天然基因控制神经元COX-2表达水平的机制。具体来说，我们假设TIA-1（一种已知可以结合并抑制COX-2 mRNA翻译的mRNA结合蛋白）的缺失将导致神经元中COX-2表达的增强。一种靶向突变小鼠系将用于在纯神经元皮层培养物和癫痫动物模型中验证这一假设。这一提议的三个目标的研究可能会提出新的方法（例如，基因治疗），通过这种方法，癫痫发生或已建立的癫痫可能会被阻止，甚至可能被逆转。

DESCRIPTION (provided by applicant): Metabolites of arachidonic acid (AA) are potent local mediators that control a wide range of functions under both normal and abnormal conditions. Cyclooxygenase-2 (COX-2), a key enzyme in the metabolism of AA within the central nervous system (CNS), is the therapeutic target of many highly self-prescribed over the counter anti-inflammatory/analgesic drugs. It is expressed under normal conditions in neurons of the hippocampal CA3 region and layers 2/3 of the cerebral cortex. Relevant to this proposal, convulsive seizure activity induces upregulation of COX-2 expression in these regions as well as in neurons of the dentate gyrus (DG). This raised the possibility that COX-2 may serve a neuromodulatory function in epilepsy, a brain disorder characterized by spontaneous seizures. Indeed, evidence from numerous studies, including preliminary and published results from the PI's laboratory, supports the notion that products of neuronal COX-2 activity may possess antiepileptic properties. This central hypothesis forms the basis for the proposed studies of this proposal. Studies in Aims 1 and 2 will test the effect of targeted transgenic (TG) overexpression of neuronal COX-2 on acute seizure threshold and epileptogenesis, the process by which the brain becomes epileptic. In both Aims, it is hypothesized that region-specific upregulation of COX-2 expression will reduce incidence of acute convulsive seizures and epileptogenesis. To test this, studies in Aim 1 will examine the consequences of targeted COX-2 overexpression in neurons of the DG or CA3 regions of the hippocampal formation. Complementary studies in Aim 2 will further test the effects of targeted COX-2 overexpression in both the CA1 hippocampal neurons and excitatory layer 2/3 neurons of the cortex. A novel TG mouse line that permits Cre-dependent upregulation of COX-2 will be used in each of these Aims. These mice will be crossed with TG mice that use neuron-specific promoters to target Cre expression to neurons of each of the 3 desired brain areas. An added advantage of the TG Cre line to be used in Aim 2 is that it will permit tamoxifen-induced expression of Cre. Thus both special and temporal control of COX-expression will be possible. Double TG mice from each of these crosses will be tested in models of acute seizures and epileptogenesis. Aim 3 studies will explore the mechanisms that control the level of neuronal COX-2 expression from its native gene. Specifically, it is posited that loss of TIA-1, an mRNA binding protein that is known to bind to and suppress COX-2 mRNA translation, will lead to enhancement of COX-2 expression in neurons. A targeted mutant mouse line will be employed to test this hypothesis in pure cortical cultures of neurons and in animal models of epilepsy. Studies in the three Aims of this proposal may suggest novel ways (e.g., Gene therapy) by which epileptogenesis or established epilepsy may be arrested or perhaps even reversed.