PATHOGENIC MECHANISMS OF NEURONAL CELL DEATH IN PD

这是四个项目小组中的第二个项目，该小组正在研究与帕金森病（PD）相关的线粒体功能和线粒体缺陷的多个方面，帕金森病是一种主要的神经退行性疾病，至少折磨着100万美国人。项目2主要研究PD患者神经元进行性神经元细胞死亡的发病机制。该研究将使用混合模型神经元和培养的原代神经元进行。混合模型神经元是由克隆的人类神经细胞系缺乏线粒体DNA （mtDNA）和患者来源的线粒体构建的。该项目将检验一个单一的中心假设：线粒体参与氧化应激、Ca2+稳态和调节信号与帕金森病衍生cybrids （PD cybrids）易感性增加有关。线粒体在钙信号网络的维持和效能中起着重要的作用，尤其是在神经元中。这反映在线粒体和网络中包括内质网在内的其他细胞元件之间的特定形态和功能关系上。钙信号网络在协调胞外分泌、细胞代谢、基因表达和细胞器间通讯等方面发挥着重要作用。线粒体还可以防御细胞毒性或兴奋性毒性事件，并被认为是凋亡信号的关键来源，包括细胞色素c的释放。实验将通过以下几个目的来研究帕金森病患者细胞系中细胞死亡易感性的改变与钙信号网络中线粒体功能的改变之间的关系：1)杂交体ETC复合物I活性缺陷、Ca2+稳态和细胞死亡易感性之间存在什么定量关系？神经保护是否包括恢复正常的Ca2+稳态？2) ETC复合体I活性的长期抑制与急性抑制的影响是否不同？关于Ca2+稳态？细胞死亡?生成氧自由基？线粒体形态和细胞器间相互作用？3) PD细胞器中线粒体与其他细胞器的形态和功能关系是如何受到影响的？4)慢性鱼藤酮治疗后PD细胞系和原代神经元的线粒体运动是如何改变的？5) PD细胞系线粒体缺陷如何影响溶酶体活性、蛋白质泛素化、降解和类似路易体的包涵体的形成？获得的结果将揭示这一重要疾病在精心控制的模型系统中可能发病机制的新方面，并可能揭示治疗靶点。

This is the second Project in a four Project group which is examining multiple aspects of mitochondrial functioning and mitochondrial defects associated with Parkinson's Disease (PD), a major neurodegenerative disease afflicting at least 1 million Americans. Project 2 is focused upon the pathogenetic mechanisms leading to the progressive neuronal cell death of the neurons affected in PD. The study will be conducted using cybrid model neurons and primary neurons in culture. Cybrid model neurons are constructed from a clonal human neural cell line lacking mitochondrial DNA (mtDNA) and patient-derived mitochondria. The project will examine a single central hypotheses: Mitochondrial involvement in oxidative stress, Ca2+ homeostasis and regulatory signaling is linked to increased vulnerability in Parkinson's disease- derived cybrids (PD cybrids). Mitochondria play an important role in the maintenance play an important role in the maintenance and efficacy of the calcium signaling network, especially in neurons. This is reflected in specific morphological and functional relationships between mitochondria and other cellular elements in the network, including the endoplasmic reticulum. The calcium signaling network plays a critical role coordinating exocytotic secretion, cellular metabolism, gene expression and inter-organelle communication. Mitochondria also function as a defense against cytotoxic of excitotoxic events and are implicated as a critical source of apoptotic signals including release of cytochrome c. Experiments will investigate the relationship between altered vulnerability to cell death in the cybrids derived from patients with Parkinson's disease and altered mitochondrial functioning in the calcium signaling network by addressing several aims: 1) What quantitative relationship exists between defects in ETC complex I activity, Ca2+ homeostasis and vulnerability to cell death in cybrids? Does neuroprotection involve restoration in normal Ca2+ homeostasis? 2) Does the impact of long-term inhibition of ETC complex I activity differ from that of acute inhibition? With respect to Ca2+ homeostasis? Cell death? Generation of oxyradicals? Mitochondrial morphology and inter- organelle interactions? 3) How are morphological and functional relationships of mitochondrial of mitochondria to other organelles affected in the PD cybrids? 4) How is mitochondrial movement altered in PD cybrids and in primary neurons after chronic rotenone treatment? 5) How do the mitochondrial defects in PD cybrids affect lysosomal activity, protein ubiquitination, degradation and the formation of inclusions that resemble Lewy bodies? The results gained will reveal novel aspects of the probable pathogenesis of this important disease in a carefully controlled model systems and may reveal therapeutic targets.