Oxidative Stress Induced Compensatory Protective Mechanisms in PD

描述（由申请人提供）：我们的提案旨在研究在黑质纹状体多巴胺能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。虽然氧化应激、细胞凋亡在帕金森病（PD）多巴胺能神经元的退行性过程中起重要作用，但caspase-3下游导致多巴胺能神经元退行性变的凋亡信号机制尚不清楚。我们已经发现，PKCd是新的PKC异构体家族的一员，在黑质多巴胺能神经元中高度表达，并且该激酶通过caspase-3蛋白水解裂解永久地将PKCd的催化亚基与调节亚基分离的新机制持续激活。我们证明PKCd蛋白水解激活有助于多巴胺能神经元在氧化损伤过程中的凋亡细胞死亡。在解剖PD细胞培养模型中的氧化信号通路时，我们还意外地发现蛋白激酶D1 （PKD1）被PKCd依赖机制激活，以保护多巴胺能神经元免受早期氧化损伤。因此，我们建议通过细胞培养和PD动物模型以及人类死后PD脑来系统地表征PKCd-PKD1氧化信号在神经退行性过程中的作用。这将通过实现下列具体目标来实现：(i)利用PD细胞培养模型确定氧化应激时多巴胺能神经元中PKD1激活的机制；（ii）在新的转基因‘ mitoppark ’ PD小鼠模型、PKCd敲除（PKCd-/-）小鼠模型和人类死后PD脑中检测线粒体缺陷导致的PKD1和PKCd激活机制。iii)通过诱导型PKD1激酶死亡（PKD1- kd）转基因小鼠模型和组成型活性PKD1过表达模型，证明PKD1对黑质多巴胺能变性的抗凋亡代偿功能。细胞、分子和神经化学的方法将被用来描述这些特定的目标。总之，提出的PKCd-PKD1信号将提供与氧化应激早期代偿性保护反应相关的信号通路的全面信息，以及在长时间氧化损伤的黑质多巴胺能神经元中覆盖保护反应的细胞信号机制。这项工作具有翻译潜力，因为针对所提出的信号通路的神经保护策略可能被证明对PD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. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a major neurodegenerative disorder affecting over a million Americans with an associated annual cost of several billion dollars. Current treatment approaches available for PD are symptomatic and fail to prevent the progression of the neurodegenerative process. The present drugs are limited in their effectiveness to either slow or stop the progressive neurodegenerative processes in PD, largely due to the lack of mechanistic insights into the selective dopaminergic degenerative process. We propose to characterize a previously unexplored oxidative signaling pathway in nigral dopaminergic degeneration, and this signaling pathway could provide a novel target for the development of an effective therapeutic agent.