NO-Induced Neurotoxicity and Apoptotic Cell Shrinkage

描述(申请人提供)：过度刺激NMDA亚型的谷氨酸受体会导致一氧化氮合酶(NOS)的激活，一氧化氮(NO)的产生，以及神经细胞的死亡。NO发挥神经毒性作用的细胞凋亡信号通路尚不清楚。蛋白质亚硝化、线粒体功能障碍和应激激活的p38丝裂原激活蛋白(MAP)激酶等事件被认为是NO诱导的神经毒性的下游效应因子。受影响的神经元被认为是通过凋亡而死亡的，这是一种细胞死亡形式，涉及到细胞死亡蛋白酶的激活，即caspase。然而，抑制caspase通常只会延缓神经细胞的死亡。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的承诺。细胞萎缩是所有凋亡性细胞死亡的普遍事件，涉及细胞内K+离子的外流。细胞凋亡过程中K+外流的分子机制尚不清楚。本项目的目的是探讨外向电压门控K+通道的激活以及随后的细胞收缩和线粒体损伤是否可能构成神经元不可逆转地受到NO诱导的神经毒性的早期事件。为了实现这些目标，我们将使用延时去卷积显微镜、全细胞膜片钳记录、瞬时转基因、生物化学以及具有分离线粒体的无细胞凋亡系统等方法来分析原代大脑皮层神经元。本项目将解决的具体问题包括：(1)NO是否引起K+外流、电压门控K+通道的增强和细胞凋亡的萎缩？(2)应激激活的p38蛋白激酶是否调节电压门控钾通道的活性和细胞收缩？(3)NO是否促使锌离子从金属硫蛋白(MT)释放，进而导致线粒体损伤、产生活性氧和p38蛋白激酶磷酸化？ 由于NO在包括中风、帕金森氏病、阿尔茨海默病、多发性硬化症、癫痫和艾滋病痴呆在内的多种神经退行性疾病中发挥着重要作用，该项目的研究结果可能为开发新的治疗药物以减轻甚至防止神经退行性变过程中神经细胞的丢失提供广泛的指导意义。

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.