NO-Induced Neurotoxicity and Apoptotic Cell Shrinkage

NO 诱导的神经毒性和凋亡细胞萎缩

• 批准号: 6637768
• 负责人: Ella R Bossy-Wetzel
• 金额: $ 37.62万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637768
• 关键词: NMDA receptors; animal tissue; apoptosis; biological signal transduction; cell free system; cysteine endopeptidases; cytochrome c; enzyme activity; free radical oxygen; glutamate receptor; immunoprecipitation; membrane potentials; metallothionein; mitochondria; mitogen activated protein kinase; neural degeneration; neurons; neurotoxicology; nitric oxide; nitric oxide synthase; phosphorylation; potassium channel; tissue /cell culture; transfection; voltage /patch clamp; western blottings; zinc

DESCRIPTION (provided by applicant): Excessive stimulation of glutamate receptors of the NMDA sub-type result in the activation of nitric oxide synthase (NOS), the generation of nitric oxide (NO), and neuronal cell death. The apoptotic signaling pathway by which NO exerts its neurotoxic effects remains poorly understood. Events such as protein nitrosylation, mitochondrial dysfunction and activation of stress-activated p38 mitogen activated protein (MAP) kinase have been proposed to act as downstream effectors of NO-induced neurotoxicity. Affected neurons are thought to die by apoptosis, a form of cell death that involves the activation of cell death proteases, known as caspases. However, caspase inhibition often only delays neuronal cell death. Thus cell death determining events, upstream of casr ase activation, are likely to contribute to the commitment to cell death. Cell shrinkage is a universal event of all apoptotic cell death and involves the efflux of intracellular K+ ions. The molecular mechanism that drives K+ efflux during apoptosis is unclear. The purpose of this project will be to explore the possibility whether activation of outward voltage-gated K+ channels and subsequent cell shrinkage and mitochondrial injury via a pathway mediated by free Zn+ may constitute early events that commit neurons irreversibly to NO-induced neurotoxicity. To pursue these goals, primary cerebrocortical neurons will be analyzed using approaches such as time-lapse deconvolution microscopy, whole cell patch-clamp recording, transient transfections, biochemistry, and cell-free systems of apoptosis with isolated mitochondria. Among the specific questions that will be addressed in this project are: (1) Does NO provoke K+ efflux, enhancement of voltage-gated K+ channels, and apoptotic cell shrinkage? (2) Does stress-activated p38 MAP kinase regulate the activity of voltage-gated potassium channels and cell shrinkage? (3) Does NO provoke Zn2+ release from metallothionein (MT) which in turn results in mitochondrial damage, generation of reactive oxygen species, and p38 MAP kinase phosphorylation? Because NO plays an important role in a wide range of neurodegenerative diseases including stroke, Parkinson's disease, Alzheimer's disease, multiple sclerosis, epilepsy, and AIDS dementia, results obtained from this project could provide broad implications for the development of new therapeutic drugs to mitigate or even prevent neuronal cell loss during neurodegeneration.

描述（申请人提供）：过度刺激NMDA亚型谷氨酸受体会导致一氧化氮合酶（NOS）激活、一氧化氮（NO）生成和神经元细胞死亡。 NO 发挥其神经毒性作用的细胞凋亡信号通路仍然知之甚少。蛋白质亚硝基化、线粒体功能障碍和应激激活的 p38 丝裂原激活蛋白 (MAP) 激酶的激活等事件已被认为是 NO 诱导的神经毒性的下游效应器。受影响的神经元被认为是通过细胞凋亡而死亡，这是一种细胞死亡形式，涉及细胞死亡蛋白酶（称为半胱天冬酶）的激活。然而，半胱天冬酶抑制通常只能延迟神经元细胞死亡。因此，casr 酶激活上游的细胞死亡决定事件可能有助于细胞死亡。细胞收缩是所有细胞凋亡的普遍事件，涉及细胞内 K+ 离子的流出。细胞凋亡过程中驱动 K+ 流出的分子机制尚不清楚。该项目的目的是探讨通过游离 Zn+ 介导的途径激活外向电压门控 K+ 通道以及随后的细胞收缩和线粒体损伤是否可能构成早期事件，使神经元不可逆地遭受 NO 诱导的神经毒性。为了实现这些目标，将使用延时解卷积显微镜、全细胞膜片钳记录、瞬时转染、生物化学和分离线粒体的无细胞凋亡系统等方法对原代脑皮质神经元进行分析。该项目将解决的具体问题包括：(1) NO 是否会引起 K+ 外流、电压门控 K+ 通道增强和凋亡细胞收缩？ (2) 应激激活的p38 MAP激酶是否调节电压门控钾通道的活性和细胞收缩？ (3) NO 是否会引起金属硫蛋白 (MT) 释放 Zn2+，进而导致线粒体损伤、活性氧的产生和 p38 MAP 激酶磷酸化？ 由于一氧化氮在多种神经退行性疾病中发挥着重要作用，包括中风、帕金森病、阿尔茨海默病、多发性硬化症、癫痫和艾滋病痴呆，因此该项目获得的结果可能为开发新的治疗药物以减轻甚至预防神经退行性疾病期间神经元细胞损失提供广泛的影响。