Blood-brain barrier function in epilepsy: new targets for therapy

描述（由申请人提供）：对癫痫患者血脑屏障功能障碍的机制缺乏了解。这是一种重要的临床药物，因为它阻止了克服抗癫痫药物（AED）耐药性和减少癫痫发作负担的治疗的发展。我们的长期目标是阐明调节血脑屏障功能的机制，这可能导致治疗癫痫和其他神经系统疾病的新策略。本申请的目的是确定癫痫发作格尔屏障功能障碍的机制，并绘制可能作为修复癫痫血脑屏障靶点的关键信号传导蛋白。实现这些目标预计将增加AED脑摄取并减少癫痫发作负担。我们的中心假设是：1）谷氨酸激活双臂LOX/考克斯通路，从而降低流入转运蛋白的表达和活性，并通过考克斯臂上调流出转运蛋白的表达和活性，2）谷氨酸触发通过LOX臂的屏障渗漏的发展;和3）抑制cPLA 2以阻断LOX/考克斯途径的两个臂逆转屏障功能障碍，从而降低癫痫发作负担。这项研究的基本原理是，确定负责转运蛋白表达和活性变化的机制，以及屏障渗漏将可能提供新的靶点，以改善癫痫治疗和更好地控制癫痫发作。为了实现本申请的目的，我们的假设将通过追求三个具体目标进行测试：1）确定尿素诱导的转运蛋白表达和活性变化的机制; 2）确定尿素诱导的血脑屏障渗漏的机制;和3）开发减少慢性癫痫大鼠癫痫发作的治疗策略。在目标1中，我们将阻断cPLA 2和考克斯-2以逆转顺铂诱导的转运蛋白表达和活性的变化。我们将监测流入和流出转运蛋白的表达和活性，并确定AED脑摄取。在目标2中，我们将抑制cPLA 2和5-LOX以绘制引起糖尿病诱导的屏障渗漏的信号通路。我们将确定紧密连接蛋白和基质金属蛋白酶的表达，并评估屏障渗漏。在目标3中，我们将通过测定流入和流出转运蛋白的表达和活性，评估屏障渗漏，监测癫痫发作频率、持续时间和严重程度，在大鼠慢性癫痫模型中评估cPLA 2抑制对癫痫发作负荷的治疗益处。拟议的研究是创新的，因为它专注于一种新的综合策略，该策略考虑了血脑屏障转运蛋白以及屏障渗漏，并且专门设计用于修复屏障以克服AED抗性并减少癫痫发作。这项拟议的研究意义重大，因为预期的结果将可能提供一种新的策略，以改善耐药性癫痫患者的药物治疗。拟议的研究是转化的，因为cPLA 2抑制剂正在开发中，我们的策略有可能转化为临床。

DESCRIPTION (provided by applicant): There is a lack in understanding the mechanism responsible for blood-brain barrier dysfunction in epilepsy. This is a significant clinical challene since it prevents development of a therapy to overcome antiepileptic drug (AED) resistance and reduce seizure burden. Our long-term goal is to elucidate the mechanisms that regulate blood-brain barrier function, which may lead to new strategies to treat epilepsy and other neuro- logical diseases. The objectives in this application are to identify the mechanism(s) by which seizures trig- ger barrier dysfunction, and map key signaling proteins that can potentially serve as targets to repair the blood-brain barrier in epilepsy. Accomplishing these objectives is expected to increase AED brain uptake and reduce seizure burden. Our central hypothesis is that 1) glutamate activates the two-arm LOX/COX pathway, thereby decreasing expression and activity of influx transporters, and upregulating expression and activity of efflux transporters through the COX arm, that 2) glutamate triggers development of barrier leakage through the LOX arm; and that 3) inhibiting cPLA2 to block both arms of the LOX/COX pathway reverses barrier dysfunction and thereby reduces seizure burden. The rationale for this research is that identifying the mechanism(s) responsible for changes in transporter expression and activity, and barrier leakage will potentially provide new targets to improve epilepsy treatment and better control seizures. To accomplish the objectives of this application, our hypothesis will be tested by pursuing three specific aims: 1) Determine the mechanism of seizure-induced changes of transporter expression and activity; 2) Deter- mine the mechanism of seizure-induced blood-brain barrier leakage; and 3) Develop a therapeutic strategy to reduce seizures in chronic epileptic rats. In Aim 1, we will block cPLA2 and COX-2 to reverse seizure-induced changes in transporter expression and activity. We will monitor influx and efflux transporter expression and activity, and determine AED brain uptake. In Aim 2, we will inhibit cPLA2 and 5-LOX to map the signaling pathway causing seizure-induced barrier leakage. We will determine expression of tight junction proteins and matrix metalloproteases, and assess barrier leakage. In Aim 3, we will evaluate the therapeutic benefit of cPLA2 inhibition on seizure burden in a rat chronic epilepsy model by determining influx and efflux transporter expression and activity, assessing barrier leakage, and monitoring seizure frequency, duration and severity. The proposed research is innovative because it is focused on a new, integrated strategy that considers blood-brain barrier transporters, as well as barrier leakage, and it is designed specifically to repair te barrier to overcome AED resistance and reduce seizures. The proposed research is significant because the expected outcomes will potentially provide a new strategy to improve pharmacotherapy in patients with resistant epilepsy. The proposed research is translational because cPLA2 inhibitors are under development, and our strategy has potential to be translated into the clinic.