Jun Kinase Signaling and Apoptosis in Ischemia Stroke

说明： 最近的研究表明，细胞凋亡是缺血性脑损伤的一个重要机制，尤其是在缺血半暗区，神经元受到缺氧的挑战，但获得剩余的血液供应。本项目的最终目标是确定脑缺血时导致Caspase激活和细胞凋亡的信号转导机制。考虑到这一点，建立了一种基于单侧颈总动脉闭塞后缺氧的全局性脑缺血模型。在初步实验中提出了四个主要发现。首先，缺血和缺氧的联合应激触发Caspase-3的激活，导致海马神经元的凋亡。其次，海马区损伤与c-Jun NH2末端激酶(JNK)信号通路的激活有关。第三，神经和心脏特有的JNK3亚型的缺失保护神经元免受缺血凋亡的影响。最后，脑缺血缺氧所致的神经细胞凋亡进入细胞周期的S期，并掺入溴脱氧尿苷。这些结果表明：(1)。神经和心脏特异的JNK3信号通路在缺血性细胞凋亡中起着关键作用。(2)下游机制可能涉及细胞周期检查点异常、转录诱导线粒体释放促死亡因子。 目的1：确定JNK亚型在脑缺血缺氧诱导的细胞凋亡中起重要作用，以及抑制JNK3信号通路是否对脑梗塞有保护作用。我们将测试结构性激活的JNK1亚型在全脑缺血中的作用，以及应激诱导的JNK3在局灶性缺血中的作用。 目的：探讨脑缺血后有丝分裂后神经元重新进入细胞周期的机制，以及这种异常的细胞周期进程是否由JNK信号引起和/或对神经元的凋亡起重要作用。我们将探讨在脑缺血时取消G1/S相变阻滞的细胞机制。此外，还将评估细胞周期进程异常的功能意义和原因。 目的：阐明JNK信号通路促进脑缺血后神经元凋亡的转录和线粒体靶点。该基因芯片技术将用于体外神经元培养系统中，以确定JNK信号的下游促死亡基因。体外Caspase-3裂解实验将被用来测试JNK3的缺失是否减弱了缺氧和神经毒素应激下神经元线粒体细胞色素c的释放。最后，我们将利用双向蛋白质电泳质谱鉴定JNK信号的线粒体靶点。

DESCRIPTION: Recent studies indicate apoptosis is an integral mechanism of ischemic brain damage and is particularly relevant in the ischemic penumbra area where neurons are challenged by hypoxia, but receive residual blood supply. The ultimate goal of this project is to identify the signal transduction mechanisms leading to Caspase activation and apoptosis in cerebral ischemia. With this in mind, a mouse model of global based on unilateral common carotid artery occlusion followed by hypoxia was created. Four major findings are presented in the preliminary experiments. First, the combined stress of ischemia and hypoxia triggers the activation of Caspase-3 causing apoptosis of hippocampal neurons. Second, the hippocampal damage is associated with activation of the c-Jun NH2-terminal kinases (JNK) signaling pathway. Third, deletion of the neural-and heart-specific Jnk3 isoform protects neurons from ischemic apoptosis. Finally, the apoptotic neurons in cerebral ischemia-hypoxia progress into the S-phase of cell cycle and incorporate bromodeoxyuridine (BrdU). These results suggest: (1). The neural-and heart-specific JNK3 signaling pathway plays a pivotal role in ischemic apoptosis. (2) the downstream mechanisms may involve aberrant cell cycle checkpoints, transcriptional induction of mitochondrial release of death-promoting factors. Aim 1: To identify which JNK isoform is important for apoptosis induced by ischemia-hypoxia and whether the inhibition of JNK3 signaling protects against cerebral infarction. We will test the role of the constitutively active JNK1 isoform in global ischemia and the stress-induced JNK3 in focal ischemia. Aim 2: To determine the mechanism by which post-mitotic neurons re-enter the cell cycle in cerebral ischemia and whether the aberrant cell cycle progression is caused by JNK signaling and/or important for neuronal apoptosis. The cellular mechanisms that may abolish the G1/S-phase transition block in cerebral ischemia will be examined. In addition, the functional significance and cause of the aberrant cell cycle progression will be assessed. Aim 3: To elucidate the transcriptional and mitochondrial targets of JNK signaling that promotes neuronal apoptosis in cerebral ischemia. The cDNA microarray technology will be used in an in-vitro neuron culture system to identify the downstream death-promoting genes of JNK signaling. An in-vitro Caspase-3 cleavage assay will be used to test whether the absence of JNK3 attenuates the release of Cytochrome c from mitochondria in neurons under hypoxia and neurotoxin stress. Finally, we will use tw0-dimensional protein electrophoresis of mass spectrometry to identify the mitochondrial targets of JNK signaling.