Mechanisms of neuronal death during epileptogenesis

 描述(由申请人提供)：在上一个资助周期中，我们鉴定、验证并发布了一种用于癫痫发生研究的独特的体外工具--在体外1周后发生自发性癫痫的海马体器型切片培养。我们使用这种制剂对500多种药物浓度组合进行盲目筛选，以确定其在慢性创伤后癫痫中的活性，其速度比任何其他慢性癫痫治疗测试策略快几个数量级。然后，我们在海人酸慢性癫痫模型中进行了体内双盲脑电测试，以证实先导化合物塞来昔布的抗惊厥作用。使这种速度成为可能的一项关键创新是使用废液中的乳酸和乳酸脱氢酶(LDH)水平分别作为癫痫负担和细胞死亡的测试。在寻找乳酸增加的原因时，我们在细胞死亡的组织化学证据前几天发现了持续的神经细胞膜泄漏，增加了细胞质中的钠和钙(↑NaI&CaI)。新的数据表明，COX2诱导导致典型的线粒体通透性蛋白Bax移位到细胞膜上，在那里它形成了容纳Na+和Ca~(2+)的孔。我们建议测试以下病理生理学：创伤或发作性损伤诱导钙依赖的Bax易位到细胞膜，在那里它产生进行性的钙和钠离子泄漏，应该杀死神经元。然而，神经元由于其独特的高离子传输能力而存活一段时间。离子转运消耗大量的三磷酸腺苷，而乳酸的产生是三磷酸腺苷生成的结果。泄漏的进展最终导致膜去极化，这可能有助于细胞分裂和细胞死亡。我们将通过在目标1中将癫痫发作、乳酸和三磷酸腺苷产生联系起来来测试这些想法。在目标2中，我们将确定膜泄漏的性质。在目标3中，我们将评估膜泄漏对ATP产生、膜电位、细胞分裂和细胞死亡的影响。我们建议使用细胞型切片模型中Na+、Ca2+、ATP、NADH、乳酸、半胱氨酸天冬氨酸酶和膜电位的比率、荧光报告的细胞类型特异性表达，以及多光子和定制的微光、广场显微镜来解决这些问题，这在以前是不可行的。

 DESCRIPTION (provided by applicant): In the last grant cycle, we characterized, validated and published a unique in vitro tool for the study of epileptogenesis - hippocampal organotypic slice cultures that develop spontaneous seizures after 1 week in vitro. We used this preparation to blindly screen over 500 drug-concentration combinations for activity in chronic, post-traumatic epilepsy at speeds that are orders of magnitude faster than any other therapeutic testing strategy for chronic epilepsy. We then used double-blind in vivo EEG testing in the kainate model of chronic epilepsy to confirm the anticonvulsant effect of a lead compound, celecoxib. A key innovation that made these speeds possible was the use of lactate and lactate dehydrogenase (LDH) levels in spent media as assays for seizure burden and cell death, respectively. In searching for the cause of the increase in lactate, we found a persistent neuronal membrane leak that increases cytoplasmic sodium and calcium (↑ Nai & Cai) days before histochemical evidence of cell death. New data indicate that COX2 induction leads to translocation of Bax, a canonical mitochondrial permeabilizing protein, to the cytoplasmic membrane, where it forms pores that admit Na+ and Ca2+. We propose to test the following pathophysiology: traumatic or ictal injury induces Ca2+-dependent Bax translocation to the cytosolic membrane, where it creates a progressive Ca2+ and Na+ leak that should kill the neuron. However, neurons survive for some time due to their uniquely high ion transport capacity. The ion transport consumes a lot of ATP, and lactate production is a consequence of ATP generation. Progression of the leak eventually leads to membrane depolarization, which may contribute to ictogenesis, and cell death. We will test these ideas by correlating seizures, lactate, and ATP production in Aim 1. In Aim 2 we will establish the nature of the membrane leak. In Aim 3 we will evaluate the consequences of the membrane leak on ATP production, membrane potential, ictogenesis, and cell death. We propose to use cell-type specific expression of ratiometric, fluorescent reporters of Na+, Ca2+, ATP, NADH, lactate, caspase and membrane potential in the organotypic slice model, together with multiphoton and custom- built low-light, wide-field microscopes to address these questions at temporal and spatial resolutions that have not previously been feasible.