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

描述（由申请人提供）：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缺血性脑损伤的干预措施。大量缺血实验模型的研究表明，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层或海马区域；然而，纹状体缺血损伤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，对氧化损伤非常敏感。尽管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有待阐明。同样，ROS的产生、线粒体功能障碍和caspase-3激活与缺血引起的脑损伤有关；然而，导致caspase-3激活后DNA断裂的下游细胞事件目前还没有很好地确定。正如在初步数据中概述的那样，我们已经确定蛋白激酶c - δ，一种新的PKC异构体家族成员和氧化应激激酶在纹状体神经元中高度表达，并作为caspase-3的关键底物。此外，caspase-3对PKC-delta的蛋白水解激活导致激酶的调节和催化亚基永久解离，从而导致激酶活性持续升高，并增强氧化应激诱导的凋亡损伤。因此，在目前的提议中，我们将通过研究以下具体目标来扩展我们的初步发现：1)系统地表征氧-葡萄糖剥夺（OGD）后初级纹状体神经元培养中pkc - δ细胞死亡途径的caspase-3依赖性蛋白水解激活。2)探讨多巴胺诱导的氧化损伤是否可能触发OGD下纹状体神经元中pkc - δ介导的细胞死亡通路。药理学、细胞和分子方法将被用来描述这些特定的目标。这些研究不仅将深入了解pkc - δ介导的细胞死亡机制以及DA在调节缺血诱导的纹状体细胞塌陷中的作用，还可能导致脑缺血治疗的新型治疗干预措施的发展。

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.