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: 6741950
• 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/6741950
• 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（由申请方提供）：NMDA亚型谷氨酸受体的过度刺激导致一氧化氮合酶（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凋亡信号通路仍然知之甚少。事件，如蛋白质亚硝基化，线粒体功能障碍和应激激活的p38丝裂原激活蛋白（MAP）激酶的激活已被提出作为NO诱导的神经毒性的下游效应。受影响的神经元被认为是通过细胞凋亡而死亡的，细胞凋亡是一种细胞死亡的形式，涉及细胞死亡蛋白酶的激活，称为半胱天冬酶。然而，半胱天冬酶抑制通常仅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促成细胞死亡。细胞皱缩是所有凋亡性细胞死亡的普遍事件，并且涉及细胞内K+离子的流出。细胞凋亡过程中K+外流的分子机制尚不清楚。本项目的目的将是探讨是否可能激活外向电压门控K+通道和随后的细胞收缩和线粒体损伤通过游离Zn+介导的途径可能构成的早期事件，承诺神经元不可逆的NO诱导的神经毒性。为了实现这些目标，将使用延时去卷积显微镜、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分析原代皮层神经元。在这个项目中将解决的具体问题是：（1）NO是否引起K+外流，电压门控K+通道的增强，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活性和细胞收缩？(3)NO是否引起金属硫蛋白（MT）释放Zn 2+，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NO在广泛的神经退行性疾病中起着重要作用，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，因此从该项目获得的结果可以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的神经元细胞损失提供广泛的意义。