STRESS ACTIVATED PROTEIN KINASE IN NEURONAL APOPTOSIS

谷氨酸的兴奋性毒性是包括急性缺血和慢性神经变性在内的许多神经系统疾病的可能机制。我们实验室最近的工作表明，缺乏神经亚型应激激活蛋白激酶SAPK/JNK(JNK3)的小鼠对红藻氨酸诱导的癫痫发作、AP-1转录活性和海马神经元凋亡具有显著的抵抗作用。这些结果有力地表明SAPK/JNK信号通路在谷氨酸兴奋性毒性的发病机制中起着关键作用。基于这一假设，本方案包括两套密切相关的体外和体内实验。具体目的I：JNK介导的谷氨酸神经毒性机制(体外研究)。需要解决的关键问题包括：(1)JNK通过基因调控和从头合成介导的神经毒性；(2)通过JNK信号调节细胞内钙振荡；(3)JNK信号与氧化应激之间的相互作用；(4)JNK信号激活途径的特异性。这些潜在的机制将在分离的神经元的原代培养中使用形态学、生理学和生化方法的组合进行测试。特定目的II：阻断JNK信号通路的神经保护(体内研究)抑制JNK信号通路的潜在临床应用可能包括：(1)防止缺血凋亡；(2)减轻肌萎缩侧索硬化症的慢性谷氨酸兴奋毒性；(3)防止神经变性；(4)提高移植治疗中移植神经元的存活率。已经为每个具体目标获得了可行性的初步证据。潜在的应用将在神经病理学的实验模型中进行测试，使用缺乏神经特异性JNK3亚型的野生型和突变小鼠。总之，本提案的总体目标既是为了了解JNK介导的谷氨酸神经毒性的机制，也是为了测试针对JNK信号通路进行治疗干预的临床潜力。

Glutamate excitotoxicity is the presumed mechanism underlying many neurological disorders including acute ischemia and chronic neurodegeneration. Recent work from our laboratories demonstrated that mice lacking the neural isoform of the stress activated protein kinsase SAPK/JNK (JNK3) has remarkable resistance to kainic acid-induced seizures, AP-1 transcriptional activity and apoptosis of hippocampal neurons. These results strongly suggest that the SAPK/JNK signaling pathway is a critical component in the pathogenesis of glutamate excitotoxicity. Based on this hypothesis, the present proposal comprises two sets of closely related in vitro and in vivo experiments. Specific Aim I: Mechanism of JNK -mediated glutamate neurotoxicity ( in vitro studies). Keys issues to be addressed include: (1) the JNK - mediated neurotoxicity through gene regulation and de novo biosynthesis; (2) the regulation of intracellular calcium oscillation through JNK signaling; (3) the interplay between JNK - signaling And oxidative stress, (4) the specificity of activation route of JNK signaling. These potential mechanisms will be tested in primary culture of dissociated neurons using a combination of morphological, physiological and biochemical approaches. Specific Aim II: Neuroprotection of the blockade JNK signaling pathway (in vivo studies) Potential clinical applications of inhibition of the JNK signaling may include: (1) the prevention of ischemic apoptosis; (2) attenuation of chronic glutamate excitotoxicity in amytrophic lateral sclerosis; (3) the prevention of neurodegneration (4) enhanced survival of grafted neurons in transplantation therapy. Preliminary evidence of feasibility has been obtained for each Specific Aim. The potential applications will be tested in experimental models of neuropathology using wildtype and mutant mice lacking the neural-specific JNK3 isoform. In summary, the overall goal of the present proposal is both to understand the mechanism underlying JNK- mediated glutamate neurotoxicity and to test the clinical potential of targeting the JNK signaling pathway for therapeutic intervention.