Jun Kinase Signaling and Apoptosis in Ischemia Stroke

缺血性中风中的 Jun 激酶信号转导和细胞凋亡

• 批准号: 6699667
• 负责人: Chia-Yi Kuan
• 金额: $ 31.85万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6699667
• 关键词: JUN kinase; apoptosis; biological signal transduction; brain circulation; cell cycle; cerebral ischemia /hypoxia; cerebrovascular occlusions; cyclin dependent kinase; cysteine endopeptidases; disease /disorder model; enzyme activity; gel electrophoresis; gene deletion mutation; genetic transcription; hippocampus; kinase inhibitor; laboratory mouse; mass spectrometry; microarray technology; mitochondria; neurons; neuroprotectants; neurotoxins; phosphorylation; protein isoforms; tissue /cell culture

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.

产品说明： 最近的研究表明，细胞凋亡是缺血性脑损伤的一个不可分割的机制，特别是在缺血半暗带区，神经元受到缺氧的挑战，但接受残余的血液供应。本课题的最终目的是研究缺血性脑损伤中Caspase激活和凋亡的信号转导机制。考虑到这一点，创建了基于单侧颈总动脉闭塞随后缺氧的整体小鼠模型。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，引起海马神经元的凋亡。其次，海马损伤与c-Jun NH 2末端激酶（JNK）信号通路的激活有关。第三，神经和心脏特异性Jnk 3同种型的缺失保护神经元免于缺血性凋亡。最后，脑缺血缺氧时凋亡的神经元进入细胞周期的S期，并掺入溴脱氧尿苷（BrdU）。这些结果表明：（1）.神经和心脏特异性JNK 3信号通路在缺血性细胞凋亡中起着关键作用。(2)下游机制可能涉及异常的细胞周期检查点、转录诱导线粒体释放促死亡因子。 目标1：明确哪种JNK亚型在缺血缺氧诱导的细胞凋亡中起重要作用，以及抑制JNK 3信号通路是否对脑梗死有保护作用。我们将测试组成型活性JNK 1亚型在全脑缺血中的作用和应激诱导的JNK 3在局灶性缺血中的作用。 目标二：明确脑缺血时有丝分裂后神经元重新进入细胞周期的机制，以及细胞周期进程的异常是否由JNK信号转导引起和/或对神经元凋亡有重要作用。将检查可能消除脑缺血中的G1/S-相转换阻滞的细胞机制。此外，还将评估异常细胞周期进展的功能意义和原因。 目的3：阐明JNK信号通路促进脑缺血神经元凋亡的转录和线粒体靶点。cDNA微阵列技术将用于体外神经元培养系统，以确定JNK信号转导的下游促死亡基因。将使用体外胱天蛋白酶-3切割测定来测试JNK 3的不存在是否减弱细胞色素c从缺氧和神经毒素应激下的神经元中的线粒体的释放。最后，我们将使用二维蛋白质电泳质谱来鉴定JNK信号转导的线粒体靶点。