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（由适用提供）：在上一个赠款周期中，我们表征，验证和发布了一种独特的体外工具，用于研究癫痫生成 - 海马有机切片培养物，在体外1周后会产生自发性癫痫发作。我们使用这种准备工作来盲目筛选超过500种药物浓缩的组合，以在慢性，创伤后癫痫的活性下，其速度比任何其他用于慢性癫痫的治疗性测试策略要快。然后，我们在慢性癫痫的Kainate模型中使用了双盲EEG测试，以确认铅化合物Celecoxib的抗惊厥作用。使这些速度成为可能的关键创新是在消费培养基中使用乳酸和乳酸脱氢酶（LDH）水平分别用作癫痫发作和细胞死亡的主张。在寻找乳酸增加的原因时，我们发现了持续的神经元泄漏，在组织化学证据的细胞死亡证据之前，会增加细胞质钠和钙（↑NAI＆CAI）。新数据表明COX2诱导导致Bax（一种规范的线粒体通透性蛋白）转移到细胞质膜，在那里它形成了吸收Na+和Ca2+的孔。我们建议测试以下病理生理学：创伤性或发作性损伤会诱导Ca2+依赖性的Bax易位到胞质膜，在此产生渐进的Ca2+和Na+ Na+泄漏，应杀死神经元。但是，由于神经元的独特高离子运输能力，神经元生存了一段时间。离子运输会消耗大量ATP，而乳酸产生是ATP产生的结果。泄漏的进展最终导致膜沉积，这可能导致截瘫和细胞死亡。我们将通过在AIM 1中与癫痫发作，乳酸和ATP产生相关联测试这些想法。在AIM 2中，我们将确定膜泄漏的性质。在AIM 3中，我们将评估膜泄漏对ATP产生，膜电位，诊所和细胞死亡的后果。我们建议在有机切片模型中使用Na+，Ca2+，ATP，NADH，NADH，NADH，NADH，NADH，NADH，NADH，乳酸和膜电位的细胞类型的特异性表达，以及以前不在临时和空间的问题上解决这些问题，以解决这些问题，以解决这些问题，以解决这些问题。