Mechanisms of neuronal death during epileptogenesis

癫痫发生过程中神经元死亡的机制

• 批准号: 9116953
• 负责人: Kevin J. Staley
• 金额: $ 42.76万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9116953
• 关键词: Accounting; Address; Aerobic; Affect; Anticonvulsants; Astrocytes; Bax protein; Biological Assay; Biological Markers; Biotinylation; Calcium; Caspase; Cell Death; Cell membrane; Chronic; Collaborations; Communities; Coxibs; Culture Media; Custom; Data; Double-Blind Method; Electroencephalography; Elements; Epilepsy; Epileptogenesis; Fluorescence Microscopy; Functional disorder; Galactose; Generations; Glucose; Glycolysis; Grant; Health; Hippocampus (Brain); Immunohistochemistry; In Situ; In Vitro; Injury; Investigation; Ion Pumps; Ion Transport; Lactate Dehydrogenase; Lead; Light; Measures; Membrane; Membrane Potentials; Microscope; Microscopy; Mitochondria; Modeling; Mus; NADH; Nature; Neurons; PTGS2 gene; Pharmaceutical Preparations; Pharmacology; Post-Traumatic Epilepsy; Preparation; Production; Propidium Diiodide; Proteins; Publishing; Reporter; Research; Resolution; Seizures; Slice; Small Interfering RNA; Sodium; Speed; Staining method; Stains; Status Epilepticus; Testing; Therapeutic; Time; base; celecoxib; cell type; fluorophore; in vivo; innovation; kainate; killings; mitochondrial membrane; neuron loss; prevent; ratiometric; screening; tool; two-photon

 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.

 描述（由申请人提供）：在上一个资助周期中，我们表征、验证并发表了一种独特的体外癫痫发生研究工具-海马器官型切片培养物，其在体外1周后发生自发性癫痫发作。我们使用这种制剂对500多种药物浓度组合进行盲筛选，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都快几个数量级。然后，我们在红藻氨酸盐慢性癫痫模型中使用双盲体内EEG测试来证实先导化合物塞来昔布的抗惊厥作用。使这些速度成为可能的关键创新是使用废培养基中的乳酸盐和乳酸脱氢酶（LDH）水平分别作为癫痫负荷和细胞死亡的测定。在寻找乳酸增加的原因时，我们发现了持续的神经元膜渗漏，在细胞死亡的组织化学证据之前几天增加了细胞质钠和钙（↑ Nai & Cai）。新的数据表明，COX 2诱导导致Bax（一种典型的线粒体透化蛋白）易位到细胞质膜，在那里它形成允许Na+和Ca 2+进入的孔。我们建议测试以下病理生理学：创伤性或发作性损伤诱导Ca 2+依赖性Bax易位到胞质膜，在那里它创建一个渐进的Ca 2+和Na+泄漏，应该杀死神经元。然而，神经元由于其独特的高离子转运能力而存活一段时间。离子转运消耗大量ATP，并且乳酸产生是ATP产生的结果。渗漏的进展最终导致膜去极化，这可能有助于癫痫发作和细胞死亡。我们将通过关联癫痫发作、乳酸和Aim 1中ATP的产生来验证这些想法。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、图像发生和细胞死亡的后果。我们建议在器官型切片模型中使用Na+、Ca 2+、ATP、NADH、乳酸盐、半胱天冬酶和膜电位的比率荧光报告基因的细胞类型特异性表达，以及多光子和定制的低光、宽视场显微镜，以以前不可行的时间和空间分辨率来解决这些问题。