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。这是一个重大的临床挑战，因为它阻碍了克服抗癫痫药物（AED）耐药性和减轻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，这可能会导致治疗癫痫和其他神经系统疾病的新策略。本应用程序的目的是确定癫痫发作触发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蛋白。实现这些目标有望增加AED脑吸收和减轻癫痫发作负担。我们的中心假设是：1)谷氨酸激活了两条臂的LOX/COX通路，从而降低了内流转运体的表达和活性，并上调了通过COX臂的外流转运体的表达和活性；2)谷氨酸触发了通过LOX臂的屏障渗漏的发生；3)抑制cPLA2阻断LOX/COX通路的两条臂可逆转屏障功能障碍，从而减轻癫痫发作负担。这项研究的基本原理是确定转运蛋白表达和活性变化的机制，以及屏障泄漏，将有可能为改善癫痫治疗和更好地控制癫痫发作提供新的靶点。为了实现这一应用的目标，我们的假设将通过追求三个具体目标来检验：1)确定癫痫引起的转运蛋白表达和活性变化的机制；2)威慑-挖掘癫痫致血脑屏障渗漏的机制；3)开发减少慢性癫痫大鼠癫痫发作的治疗策略。在Aim 1中，我们将阻断cPLA2和COX-2来逆转癫痫引起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Aim 2中，我们将抑制cPLA2和5-LOX来绘制导致癫痫诱发屏障泄漏的信号通路。我们将测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Aim 3中，我们将通过测定内流和外流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，评估cPLA2抑制对大鼠慢性癫痫模型癫痫发作负担的治疗效果。这项提议的研究是创新的，因为它专注于一种新的、综合的策略，考虑了血脑屏障转运蛋白，以及屏障泄漏，它是专门为修复屏障而设计的，以克服AED抵抗并减少癫痫发作。这项研究具有重要意义，因为预期的结果可能为改善顽固性癫痫患者的药物治疗提供新的策略。由于cPLA2抑制剂正在开发中，我们提出的研究具有转译性，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.