NEURONAL DEGENERATION: MECHANISMS AND PREVENTION

描述（由申请人提供）：神经元变性和死亡是许多神经系统疾病的标志，有相当多的证据表明氧化应激在卒中/缺血中起重要作用。该项目的长期目标是阐明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。我们已经使用了在培养的原代神经元中的SOD 1的下调作为氧化应激的模型，其中可以检查导致神经元凋亡的途径。SOD 1的下调通过过氧亚硝酸盐介导的途径导致死亡。该途径需要激活半胱天冬酶-1，半胱天冬酶-1又导致产生IL-1 β，IL-1 β从细胞中释放并以自分泌方式作用于IL-1受体以增强细胞死亡。我们也有证据表明，caspase-8和-7的激活，但出乎意料的不是caspase-3，是死亡进行所必需的。这表明了一种新的半胱天冬酶级联反应，其中半胱天冬酶-1是顶端半胱天冬酶，导致自分泌受体活化，随后活化半胱天冬酶-8，最后活化刽子手半胱天冬酶，半胱天冬酶-7。以前在神经元死亡级联反应中没有发现激活半胱天冬酶-3的失败，这支持了设计特异性药物干预这一途径的可行性。我们现在提出的假设，有一个反馈回路的NO和IL-1 β，这是一个重要组成部分的自由基死亡途径，并有一个新的caspase级联启动caspase-1激活。这些假设将与以下具体目标进行审查：1。确定SOD 1下调途径和缺血中关键分子的诱导/激活和定位的时间过程。2.确定在SOD 1下调和缺血后如何调节nNOS的诱导。3.确定SOD 1下调和缺血后IL-1 β的诱导是如何调节的。4.确定在SOD 1下调后caspase-8和-7如何被激活。5.确定SOD 1下调后是否存在caspase-3和其他caspase的激活/调节。

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.