NEURONAL DEGENERATION: MECHANISMS AND PREVENTION

描述(申请人提供)：神经元退化和死亡是许多神经系统疾病的特征，有相当多的证据表明氧化应激在中风/缺血中起着重要作用。该项目的长期目标是阐明控制自由基引发的神经元凋亡的分子机制，并利用这些分子数据设计治疗干预措施。我们使用培养的原代神经元中SOD1的下调作为氧化应激的模型，在该模型中可以检测导致神经元凋亡的途径。SOD1的下调通过过氧亚硝酸盐介导的途径导致死亡。这一途径需要caspase-1的激活，进而导致IL-1β的产生，IL-1β从细胞中释放出来，以自分泌的方式作用于IL-1受体，促进细胞死亡。我们也有证据表明，caspase-8和-7的激活是死亡继续进行所必需的，但出乎意料的不是caspase-3。这提示了一种新的caspase级联反应，其中caspase-1是顶端caspase，导致自分泌受体激活，然后激活caspase-8，最后激活执行者caspase-7。未能激活caspase-3在神经元死亡的级联反应中以前从未见过，并支持设计特定的药物干预这一途径的可行性。我们现在提出的假设是，存在NO和IL-1β的反馈环，IL-1β是自由基死亡途径的重要组成部分，并且存在由caspase-1激活启动的新的caspase级联反应。验证这些假说的具体目的如下：1.确定SOD1下调途径和缺血中关键分子的诱导/激活和定位的时间进程。2.确定SOD1下调后和缺血后nNOS的诱导。3.确定SOD1下调后和缺血后IL-1β的诱导是如何调节的。4.研究SOD1下调后caspase-8和-7的激活情况。5.检测SOD1下调后caspase-3及其他caspase的激活/调节情况。

DESCRIPTION (provided by applicant): Neuronal degeneration and death are the hallmarks of many neurological diseases and there is considerable evidence that oxidative stress plays an important role in stroke/ischemia. The long term goals of this project are to elucidate the molecular mechanisms governing free radical-initiated apoptotic neuronal death and to utilize this molecular data for the design of therapeutic interventions. We have used the down-regulation of SOD1 in cultured primary neurons as a model of oxidative stress in which the pathways leading to neuronal apoptosis can be examined. Down-regulation of SOD1 leads to a death via a peroxynitrite-mediated pathway. This pathway requires activation of caspase-1 which in turn leads to the generation of IL-1beta which is released from the cell and acts in an autocrine manner on the IL-1 receptor to potentiate cell death. We also have evidence that activation of caspases-8 and -7, but unexpectedly not caspase-3, are required for death to proceed. This suggests a novel caspase cascade in which caspase-1 is the apical caspase leading to autocrine receptor activation followed by activation of caspase-8 and finally to activation of the executioner caspase, caspase-7. The failure to activate caspase-3 has not previously been seen in neuronal death cascades and supports the feasibility of designing specific agents to intervene in this pathway. We now propose the hypotheses that there is a feedback loop of NO and IL-1beta which is an essential component of the free radical death pathway and that there is a novel caspase cascade initiated by caspase-1 activation. These hypotheses will be examined with the following specific aims: 1. To determine the time course of induction/activation and localization of key molecules in the SOD1 down-regulation pathway and in ischemia. 2. To determine how the induction of nNOS is regulated after SOD1 down-regulation and after ischemia. 3. To determine how the induction of IL-1beta is regulated after SOD1 down-regulation and after ischemia. 4. To determine how caspases-8 and -7 are activated after SOD1 down-regulation. 5. To determine if there is activation/regulation of caspase-3 and other caspases after SOD1 down-regulation.