Oxidative Stress, PKC-delta Activation and Striatal Ischemic Cell Death

描述(申请人提供)：中风是美国发病率和死亡率的第三大原因，但目前还没有有效的治疗措施来治疗由缺血引起的脑损伤。大量的缺血实验模型研究表明，氧化应激和细胞凋亡是缺血诱导细胞死亡的关键介质。这些研究大多集中在大脑皮层或海马区；然而，纹状体缺血性损伤背后的细胞机制尚不清楚，纹状体是一个富含多巴胺的大脑区域，非常容易受到氧化损伤。虽然多巴胺被认为是氧化应激诱导的纹状体神经元损伤的关键介质，但其在纹状体缺血诱导的细胞死亡信号通路中的确切作用仍不清楚。同样，ROS的产生、线粒体功能障碍和caspase-3的激活与脑缺血损伤有关；然而，caspase-3激活后导致DNA片段化的下游细胞事件目前还没有得到很好的证实。根据初步的数据，我们已经确定蛋白激酶C-Delta是新的PKC亚型家族的成员，也是一种氧化应激激酶，在纹状体神经元中高表达，并作为caspase-3的关键底物。此外，caspase-3对PKC-Delta的蛋白水解性激活导致调节亚基和催化亚基的永久性解离，从而导致激酶活性的持续增强，并加强了氧化应激诱导的细胞凋亡损伤。因此，在目前的提案中，我们将通过以下具体目标来扩展我们的初步发现：1)系统地表征原代培养的纹状体神经元缺氧缺糖(OGD)后依赖caspase-3的蛋白水解酶激活PKC-Delta细胞死亡途径。2)探讨多巴胺诱导的氧化损伤是否是OGD所致纹状体神经元PKC-Delta介导的细胞死亡途径的潜在触发因素。药理学、细胞和分子方法将被用来描述这些特定的目的。这些研究不仅将对PKC-Delta介导的细胞死亡的机制以及DA在调节缺血诱导的纹状体细胞崩溃中的影响提供深入的认识，而且还可能导致开发治疗脑缺血的新的治疗干预措施。

DESCRIPTION (provided by applicant): Stroke is the third leading cause of morbidity and mortality in the US, yet effective therapeutic interventions for the treatment of ischemia-induced brain damage are not currently available. Numerous studies in experimental models of ischemia have implicated oxidative stress and apoptosis as key mediators of ischemia-induced cell death. The majority of these studies have focused on either cortical or hippocampal regions; however, the cellular mechanisms underlying ischemic damage to the striatum, a dopamine enriched brain region that is highly susceptible to oxidative damage, is unclear. Although dopamine is considered to be a key mediator of oxidative stress-induced neuronal injury to the striatum, its exact role in ischemia-induced cell death signaling pathways in the striatum remain to be clarified. Likewise, ROS generation, mitochondrial dysfunction and caspase-3 activation have been implicated in ischemia- induced brain damage; however, the downstream cellular events that lead to DNA fragmentation subsequent to caspase-3 activation are currently not well established. As outlined in the preliminary data, we have identified that protein kinase C-delta, a member of the novel PKC isoform family and an oxidative stress kinase is highly expressed in the striatal neurons and serves as a key substrate for caspase-3. Furthermore, the proteolytic activation of PKC-delta by caspase-3 results in the permanent dissociation of regulatory and catalytic subunits of the kinase, thereby resulting in persistently increased kinase activity, and potentiation of oxidative stress-induced apoptotic damage. Therefore, in the present proposal we will extend our preliminary findings, by investigating the following specific aims: 1) To systematically characterize the caspase-3 dependent proteolytic activation of PKC-delta cell death pathways in primary striatal neuronal cultures following oxygen-glucose deprivation (OGD). 2) To investigate whether dopamine-induced oxidative insult is a potential trigger for PKC-delta mediated cell death pathways in striatal neurons subjected to OGD. Pharmacological, cellular and molecular approaches will be utlilized to delineate these specific aims. The proposed studies will not only provide insights into the mechanism of PKC-delta mediated cell death and the influence of DA in modulating ischemia-induced striatal cellular collapse but may also lead to the development of novel thereapeutic interventions for the treatment of cerebral ischemia.