Molecular analysis of altered intracellular dopamine pools in substantia nigra

尽管负责的因素(S)尚未阐明，但一个看似合理的候选因素是多巴胺本身，它很容易氧化产生苯二酚和其他自由基，对培养的神经元的毒性比类似浓度的MPTP更大。有毒的氧自由基多巴胺代谢物可能攻击重要的胞浆蛋白，提供一种信号，使它们容易受到泛素介导的蛋白质分解和降解。泛素阳性的路易小体是帕金森氏病的细胞特征，一些人认为这些包涵体是异常蛋白积聚的结果，主要包括神经丝蛋白以及α-突触核蛋白和CDK5。因此，多巴胺氧化代谢的异常不仅可能导致黑质神经元的选择性变性，还可能导致路易小体的形成，并在溶酶体/内小体隔室内黑色素的形成中发挥已证实的作用。在这项提案中，我们概述了旨在测试囊泡和胞质多巴胺池中的异常如何在出生后来源的培养中启动黑质神经元退化的实验。我们将使用四种工具来改变黑质细胞内的多巴胺。1)黑质培养来源于VMAT2基因敲除小鼠，这些小鼠不能在突触小泡中隔离多巴胺。2)甲基苯丙胺，它将多巴胺从突触小泡重新分配到胞浆中，增加了胞浆池和可供释放的递质，最终导致氧自由基介导的突触变性。3)L-多巴，它迅速转化为胞质多巴胺，导致囊泡多巴胺库和量子大小(每个突触小泡胞吐释放的分子数量)增加。我们还将使用来自α-突触核蛋白基因敲除小鼠的神经元培养来确定该蛋白是否参与了神经黑色素和泛素包涵体的生物合成。值得注意的是，这些方法可以用来诱导细胞内神经黑色素和细胞内泛素化包涵体的形成，为研究这些特征提供了第一个体外系统。这些研究承诺提供与帕金森氏病相关的神经退行性变和细胞生物学的信息，为研究活的、可延展的系统中的干预提供机会。

While the pathogenesis of idiopathic Parkinson's Disease remains enigmatic, there is considerable evidence associating the cognition with heightened oxidative stress. Although the agent(s) responsible has not been elucidated, a plausible candidate is dopamine itself, which readily oxidizes to produce quinones and other free radicals, and is more toxic to cultured neurons than similar concentrations of MPTP. Toxic oxyradical dopamine metabolites may attack essential cytosolic proteins, providing a signal that renders them subject to ubiquitin-mediated proteolysis and degradation. Ubiquitin-positive Lewy bodies are the characteristic cellular hallmarks of Parkinson's Disease and it is thought by some that these inclusions result from an accumulation of abnormal proteins, prominently including neurofilament proteins as well as alpha-synuclein and cdk5. It is therefore possible that aberrant dopamine oxidative metabolism not only contributes to the selective degeneration of substantia nigra neurons but also to formation of Lewy bodies, as well as playing a well-established role in neuromelanin formation in lysosomal/endosomal compartments. In this proposal, we outline experiments designed to test how aberrations in vesicular and cytosolic dopamine pools initiate degeneration of substantia nigra neurons in postnatally-derived cultures. We will use four tools to alter substantia nigra intracellular dopamine. 1) Substantia nigra cultures derived from VMAT2 knockout mice, which are unable to sequester dopamine in synaptic vesicles. 2) Methamphetamine, which redistributes dopamine from synaptic vesicles to the cytosol, increasing the cytosolic pool and the transmitter available for release and eventually results in oxyradical-mediated synaptic degeneration. 3) L-DOPA, which is rapidly converted to cytosolic dopamine, resulting in an elevated vesicular dopamine pool and quantal size (the number of molecules released per synaptic vesicle exocytosis). We will also use neuronal cultures derived from alpha-synuclein knockout mice to determine if this protein is involved in biosynthesis of neuromelanin and ubiquitin inclusions. Remarkably, these approaches can be used to induce formation of intracellular neuromelanin and intracellular ubiquitinated inclusions, providing the first in vitro system for study of these features. These studies promise to provide information on neurodegeneration and the cell biology related to Parkinson's Disease in a preparation that provides the opportunity to study interventions in a living, malleable system.