Oxidative Stress Induced Compensatory Protective Mechanisms in PD

描述（由申请人提供）：我们的建议旨在研究在黑质纹状体多巴胺能系统中氧化损伤的早期阶段由PKCd-PKD 1介导的新型补偿性细胞存活信号通路。虽然氧化应激、细胞凋亡在帕金森病（PD）多巴胺能神经元的变性过程中是重要的，但对导致多巴胺能神经元变性的caspase-3下游的细胞凋亡信号传导机制知之甚少。我们已经确定，PKCd，一个新的PKC亚型家族的成员，是高表达的黑质多巴胺能神经元，和激酶持续激活通过一种新的机制，caspase-3蛋白水解切割永久解离PKCd的调节亚基的催化亚基。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细胞凋亡。在剖析PD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（PKD 1）通过PKCd依赖性机制被激活，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PD动物模型以及人死后PD脑系统地表征PKCd-PKD 1氧化信号在黑质变性过程中的作用。这将通过实现以下具体目标来实现：（ii）在新的转基因“MitoPark”PD小鼠模型、PKCd敲除（PKCd-/-）小鼠模型和人死后PD脑中检查由线粒体缺陷引起的PKD 1和PKCd活化机制，iii）使用诱导型PKD 1激酶死亡（PKD 1-KD）转基因小鼠模型和组成型活性PKD 1过表达模型证明PKD 1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绘这些具体的目标。总之，建议PKCd-PKD 1信号将提供全面的信息与补偿性保护反应相关的信号通路在氧化应激的早期阶段，以及细胞信号机制，在黑质多巴胺能神经元的长期氧化损伤过程中覆盖的保护反应。拟议的工作具有转化潜力，因为针对拟议信号通路的神经保护策略可能被证明对PD有效。 公共卫生相关性：帕金森氏病（PD）是一种主要的神经退行性疾病，影响超过一百万美国人，相关的年度费用为数十亿美元。目前可用于PD的治疗方法是对症的，并且不能防止神经退行性过程的进展。目前的药物在减缓或停止PD中进行性神经退行性过程的有效性方面受到限制，这主要是由于缺乏对选择性多巴胺能退行性过程的机制见解。我们提出了一个以前未探索的黑质多巴胺能变性的氧化信号通路的特点，这个信号通路可以提供一个新的目标，开发一个有效的治疗剂。

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.