Exploiting EP2 receptor biology to target seizure-related neuroinflammation selectively

项目概要 在人类和动物模型中积累的证据表明，大脑炎症 癫痫持续状态（SE）后发生的疾病可能在长期有害后果中发挥决定性作用， 独立于感染或自身免疫原因。各种因素之间的病理生理学相互作用 炎症分子以及导致其诱导的事件顺序尚未被剖析。 之前的研究指出了环氧合酶 2 (COX-2) 通路在 SE 诱导的炎症中的作用，并且 表明 EP2 受体介导大部分 COX-2 效应。我们最近的工作表明 PGE2 SE 激活附近神经元和骨髓细胞上的 EP2 受体后，神经元释放 EP2 神经元和骨髓细胞的激活可能会引起相反的作用。我们假设 EP2 激活 SE 后神经元上的 EP2 激活具有神经保护作用，而小胶质细胞或入侵单核细胞上的 EP2 激活则产生神经保护作用 参与细胞因子合成和癫痫的后续发展。在这里，我们使用新颖的 HaloTag 技术 用 EP2 拮抗剂和激动剂分别靶向神经元和骨髓细胞来检验这一假设。我们的 具体目标是： 1. 检验以下假设：药物阻断神经元上的 EP2 受体 SE 后骨髓细胞具有相反的作用。 2. 检验阻断髓样EP2受体的假设 细胞干扰癫痫发生的过程。 3. 检验 EP2 介导的假设 神经保护涉及神经元 EP2 受体，利用 cAMP 而不是 β-arrestin 途径，并且 需要 CX3CL1 (fractalkine)。为了实现这些目标，我们采用体外培养模型和体内 SE HaloTag 靶向神经元或小胶质细胞的新型 EP2 拮抗剂和激动剂模型。 进行免疫组织化学、蛋白质印迹、qRT-PCR、细胞活力、脑电图和行为测定。

Project Summary Accumulating evidence in humans and in animal models indicates that inflammation of the brain that develops after status epilepticus (SE) may play a determinant role in long-term detrimental consequences, independent of an infection or auto-immune cause. The pathophysiological interactions among the various inflammatory molecules, and the sequence of events leading to their induction, have not yet been dissected. Previous work pointed to a role for cyclooxygenase-2 (COX-2) pathways in SE-induced inflammation, and showed that the EP2 receptor mediates much of the COX-2 effect. Our recent work suggests that PGE2 released from neurons after SE activates EP2 receptors on nearby neurons and myeloid cells, and that EP2 activation on neurons and myeloid cells might cause opposing effects. We hypothesize that EP2 activation on neurons after SE is neuroprotective, whereas EP2 activation on microglia or invading monocytes results in cytokine synthesis and subsequent development of epilepsy. Here we use a novel HaloTag technology to target neurons and myeloid cells separately with EP2 antagonists and agonists to test this hypothesis. Our specific aims are: 1. To test the hypothesis that pharmacologic block of EP2 receptors on neurons and myeloid cells has opposing effects after SE. 2. To test the hypothesis that blocking EP2 receptors on myeloid cells interferes with the process of epileptogenesis. 3. To test the hypothesis that EP2-mediated neuroprotection involves neuronal EP2 receptors, utilizes a cAMP rather than β-arrestin pathway, and requires CX3CL1 (fractalkine). To address these aims we employ in vitro culture models and in vivo SE models with novel EP2 antagonists and agonists targeted by HaloTag to neurons or microglia. Immunohistochemical, western blot, qRT-PCR, cell viability, EEG and behavioral assays are performed.