Oxidative Stress Induced Compensatory Protective Mechanisms in PD

描述(申请人提供)：我们的建议旨在研究在黑质纹状体多巴胺系统氧化损伤的早期阶段，由PKCd-PKD1介导的一种新的代偿性细胞生存信号通路。尽管氧化应激、细胞凋亡在帕金森病(PD)多巴胺能神经元的退变过程中起重要作用，但caspase-3下游导致多巴胺能神经元退变的凋亡信号机制却知之甚少。我们已经证实，新的PKC亚型家族成员PKCd在黑质多巴胺能神经元中高表达，并且通过caspase-3蛋白水解性裂解的新机制持久地激活该激酶，使其催化亚基与PKCd调节亚基永久分离。我们证明了在氧化损伤过程中，PKCd蛋白水解性激活导致了多巴胺能神经元的细胞死亡。在剖析帕金森病细胞培养模型中的氧化信号通路的同时，我们还意外地发现，蛋白激酶D1(PKD1)是由一种依赖PKCd的机制激活的，以保护多巴胺能神经元免受氧化损伤的早期阶段。因此，我们建议使用细胞培养和帕金森病动物模型以及人死后帕金森病脑组织来系统地研究黑质退化过程中PKCd-PKD1氧化信号的特征。为实现这一目标，将通过以下具体目标来实现：(I)利用PD的细胞培养模型来确定氧化应激期间多巴胺能神经元中PKD1的激活机制；(Ii)在新的转基因‘MitoPark’PD小鼠模型、PKCd基因敲除(PKCd-/-)小鼠模型和人死后PD大脑中检测由线粒体缺陷引起的PKD1和PKCd的激活机制；iii)利用可诱导的PKD1K-Dead(PKD1-KD)转基因小鼠模型和持续激活的PKD1过表达模型来证明PKD1对黑质多巴胺能变性具有抗细胞凋亡的代偿功能。将使用细胞、分子和神经化学方法来描述这些特定的目标。总之，所提出的PKCd-PKD1信号将提供与氧化应激早期阶段的代偿保护反应相关的信号通路的全面信息，以及在黑质多巴胺能神经元中长期氧化损伤时覆盖保护反应的细胞信号机制。这项拟议的工作具有翻译潜力，因为针对拟议的信号通路的神经保护策略可能被证明对帕金森病有效。

DESCRIPTION (provided by applicant): Our proposal aims to study a novel compensatory cell survival signaling pathway mediated by PKCd- PKD1 during the early stages of oxidative damage in the nigrostriatal dopaminergic system. Although oxidative stress, apoptosis are known to be important in the degenerative process in dopaminergic neurons in Parkinson's disease (PD), the apoptotic signaling mechanisms downstream of caspase-3 that contribute to the degeneration of dopaminergic neurons are poorly understood. We have identified that PKCd, a member of the novel PKC isoform family, is highly expressed in nigral dopaminergic neurons, and the kinase is persistently activated via a novel mechanism by which caspase-3 proteolytically cleaves to permanently dissociate the catalytic subunit from the regulatory subunit of PKCd. We demonstrated that PKCd proteolytic activation contributes to apoptotic cell death of dopaminergic neurons during oxidative damage. While dissecting the oxidative signaling pathway in cell culture models of PD, we also unexpectedly identified that protein kinase D1 (PKD1) is activated by a PKCd dependent mechanism to protect dopaminergic neurons from the early stages of oxidative insult. Therefore, we propose to systematically characterize the PKCd-PKD1 oxidative signaling in nigral degenerative processes using cell culture and animal models of PD as well as human postmortem PD brains. This will be accomplished by pursuing the following specific aims: (i) To determine the mechanism of PKD1 activation in dopaminergic neurons during oxidative stress using cell culture models of PD, (ii) To examine PKD1 and PKCd activation mechanisms resulting from mitochondrial defects in a new transgenic 'MitoPark' PD mouse model, in a PKCd knockout (PKCd-/-) mouse model and in human postmortem PD brains, iii) o demonstrate the anti-apoptotic compensatory function of PKD1 against nigral dopaminergic degeneration using an inducible PKD1 kinase-dead (PKD1-KD) transgenic mouse model and constitutively active PKD1over-expressing model. Cellular, molecular and neurochemical approaches will be used to delineate these specific aims. Together, the proposed PKCd-PKD1 signaling will provide comprehensive information about signaling pathways associated with compensatory protective responses at the early stages of oxidative stress, as well as the cell signaling mechanisms that override the protective responses during prolonged oxidative insult in nigral dopaminergic neurons. The proposed work has translational potential, as neuroprotective strategies targeting the proposed signaling pathway may prove to be effective against PD.