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

描述（由申请人提供）：卒中是美国发病率和死亡率的第三大原因，但目前尚无治疗缺血性脑损伤的有效治疗干预措施。在缺血实验模型中的许多研究表明，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皮质或海马区;然而，纹状体缺血性损伤的细胞机制尚不清楚，纹状体是一个富含多巴胺的脑区，对氧化损伤高度敏感。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，但其在缺血诱导的纹状体细胞死亡信号通路中的确切作用仍有待澄清。同样地，ROS产生、线粒体功能障碍和半胱天冬酶-3活化与缺血诱导的脑损伤有关;然而，导致半胱天冬酶-3活化后的DNA片段化的下游细胞事件目前还没有很好地建立。如初步数据所示，我们已经确定，蛋白激酶C-δ，一个新的PKC亚型家族和氧化应激激酶的成员是高度表达的纹状体神经元，并作为一个关键的底物半胱天冬酶-3。此外，caspase-3对PKC-δ的蛋白水解激活导致激酶的调节和催化亚基的永久解离，从而导致激酶活性持续增加，并增强氧化应激诱导的凋亡损伤。因此，在本建议中，我们将扩展我们的初步研究结果，通过调查以下具体目标：1）系统地表征在原代纹状体神经元培养后的氧-葡萄糖剥夺（OGD）的PKC-δ细胞死亡途径的caspase-3依赖的蛋白水解激活。2)研究多巴胺诱导的氧化损伤是否是OGD纹状体神经元中PKC-δ介导的细胞死亡途径的潜在触发因素。药理学、细胞学和分子学的方法将被用来描述这些特定的目标。拟议的研究不仅将提供深入了解PKC-δ介导的细胞死亡的机制和DA在调节缺血诱导的纹状体细胞崩溃的影响，但也可能导致开发新的治疗脑缺血的干预措施。

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.