Molecular analysis of altered intracellular dopamine pools in substantia nigra

虽然特发性帕金森病的发病机制仍然是谜，但有相当多的证据表明认知与氧化应激升高有关。虽然尚未阐明起作用的因素，但一个可能的候选因素是多巴胺本身，它很容易氧化产生醌和其他自由基，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必需的细胞质蛋白，提供一个信号，使它们受到泛素介导的蛋白水解和降解。泛素阳性的路易小体是帕金森病的特征性细胞标志，一些人认为这些包涵体是由异常蛋白的积累引起的，主要包括神经丝蛋白、α -突触核蛋白和cdk5。因此，异常的多巴胺氧化代谢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/内体区室的神经黑色素形成中发挥着既定的作用。在这一建议中，我们概述了旨在测试在后天培养中，囊泡和胞质多巴胺池的畸变如何引发黑质神经元的变性的实验。我们将使用四种工具来改变黑质细胞内多巴胺。1)来自VMAT2基因敲除小鼠的黑质培养物，这些小鼠无法在突触囊泡中隔离多巴胺。2)甲基苯丙胺，将多巴胺从突触囊泡重新分配到胞质中，增加胞质池和可释放的递质，最终导致氧化自由基介导的突触变性。3) L-DOPA，迅速转化为胞质多巴胺，导致囊泡多巴胺池和量子大小（每个突触囊泡胞吐释放的分子数量）升高。我们还将使用来自α -突触核蛋白敲除小鼠的神经元培养物来确定该蛋白是否参与神经黑色素和泛素包涵体的生物合成。值得注意的是，这些方法可用于诱导细胞内神经黑色素和细胞内泛素化包裹体的形成，为研究这些特征提供了第一个体外系统。这些研究有望提供与帕金森氏病相关的神经变性和细胞生物学信息，为研究活的、可塑的系统中的干预措施提供机会。

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.