NO-Induced Neurotoxicity and Apoptotic Cell Shrinkage

描述（由申请人提供）：NMDA亚型谷氨酸受体的过度刺激导致一氧化氮合酶（NOS）的激活，一氧化氮（NO）的产生和神经元细胞死亡。一氧化氮发挥其神经毒性作用的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丝裂原活化蛋白（MAP）激酶的激活等事件被认为是一氧化氮诱导的神经毒性的下游效应物。受影响的神经元被认为死于细胞凋亡，这是一种细胞死亡的形式，涉及细胞死亡蛋白酶的激活，称为半胱天蛋白酶。然而，半胱天冬酶的抑制往往只是延缓神经元细胞的死亡。因此，细胞死亡决定事件，上游的casr酶激活，可能有助于承诺细胞死亡。细胞收缩是所有凋亡细胞死亡的普遍事件，涉及细胞内K+离子的外排。细胞凋亡过程中驱动K+外排的分子机制尚不清楚。本项目的目的是探索通过游离Zn+介导的途径激活外向电压门控K+通道以及随后的细胞萎缩和线粒体损伤是否可能构成使神经元不可逆地发生no诱导的神经毒性的早期事件。为了实现这些目标，将使用延时反褶积显微镜、全细胞膜片钳记录、瞬时转染、生物化学和分离线粒体的细胞凋亡无细胞系统等方法分析初级脑皮质神经元。该项目将解决的具体问题包括：(1)NO是否会引起K+外排、电压门控K+通道的增强和凋亡细胞的收缩？(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是否会刺激金属硫蛋白（MT）释放Zn2+，从而导致线粒体损伤、活性氧的产生和p38 MAP激酶磷酸化？

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.