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

描述(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方法的发展。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疾病的新策略。本应用的目的是确定癫痫发作触发屏障功能障碍的机制(S)，并绘制可能作为癫痫血脑屏障修复靶点的关键信号蛋白。实现这些目标有望增加AED的大脑摄取量，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/COX途径，从而降低内流转运体的表达和活性，上调通过COX臂的外流转运体的表达和活性；2)谷氨酸触发通过LOX臂的屏障渗漏；以及3)抑制cPLA2以阻断LOX/COX通路的两个臂，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(S)和屏障渗漏将潜在地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，我们的假设将通过三个具体目标进行验证：1)确定癫痫发作诱导转运体表达和活性变化的机制；2)确定癫痫发作导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。在目标1中，我们将阻断cPLA2和COX-2来逆转癫痫发作诱导的转运蛋白表达和活性的变化。我们将监测内流和外流转运体的表达和活性，并确定AED的脑摄取。在目标2中，我们将抑制cPLA2和5-LOX来映射导致癫痫诱导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。在目标3中，我们将通过检测内流和外流转运体的表达和活性，评估屏障渗漏，以及监测癫痫发作的频率、持续时间和严重程度，来评估cPLA2抑制大鼠慢性癫痫模型癫痫发作负担的疗效。这项拟议的研究具有创新性，因为它侧重于一种新的综合战略，该战略考虑了血脑屏障转运体以及屏障渗漏，并专门设计用于修复TE屏障，以克服AED抵抗和减少癫痫发作。这项拟议的研究意义重大，因为预期结果可能会为改善难治性癫痫患者的药物治疗提供一种新的策略。拟议的研究是翻译的，因为cPLA2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.