Neurotoxicity Mechanisms of Reactive Intermediates

描述（由申请人提供）：本申请测试了a-突触核蛋白的生物化学不同寡聚物部分通过抑制伴侣介导的自噬（CMA）功能诱导神经元变性的假设。a-突触核蛋白（a- syn）已被确定为散发性和家族性帕金森病（PD）及相关神经退行性疾病患者大脑中蛋白质包涵体的主要成分之一。多巴胺代谢失调导致胞质多巴胺氧化增加，也被认为是PD选择性神经元功能障碍和死亡的致病机制之一。我们已经提供了氧化多巴胺与a-syn相互作用的证据，并提出这种相互作用统一了导致PD和以a-syn包涵体为特征的疾病的两种潜在神经毒性机制。尽管在疾病的发病机制中具有深远的意义，但a-syn与多巴胺在体内的相互作用尚未得到评估。因此，我们将通过注射递送不受多巴胺反馈抑制的n端R37E， R38E突变（TH- rree）的人酪氨酸羟化酶（TH） cDNA的慢病毒载体，提高具有小鼠PrP启动子驱动的致病性A53T突变的人a-syn小鼠黑质中的多巴胺水平。在这些小鼠中，我们将：1)比较和对比多巴胺对a-syn低聚物的区域形成和生化特性的影响。实验将确定可溶性a-syn低聚物的区域分布、生化和生物物理性质。实验还将测试低聚物对神经元功能障碍、囊泡关联和a-syn聚集的体外影响。2)评估a-syn与CMA的区域关联，并确定增加多巴胺水平对体内的影响。实验将验证一种新的假设，即与氧化多巴胺诱导的a-syn低聚物相互作用导致的CMA功能受损，通过s -亚硝基化甘油醛磷酸脱氢酶的积累导致神经元功能障碍。这一假设将a-syn低聚物阻断CMA的作用与先前建立的一氧化氮诱导的多巴胺能神经元死亡的作用结合起来。3)研究增加多巴胺水平对A53T -syn表达小鼠表型的影响。表型的发生将在对多巴胺神经元活力、包涵体形成和星形胶质形成进行全面的免疫组织化学和生化分析后确定。

DESCRIPTION (provided by applicant): This application tests the hypothesis that biochemically distinct oligomers of a-synuclein induce neuron degeneration in part by inhibiting the function of chaperone-mediated autophagy (CMA). a-Synuclein (a- syn) has been identified as one of the major components of the protein inclusions in the brains of subjects with sporadic and familial forms of Parkinson's disease (PD) and related neurodegenerative disorders. Deregulation in dopamine metabolism resulting in increased cytosolic dopamine that undergoes oxidation has been also considered as contributing pathogenic mechanism for the selective neuron dysfunction and death in PD. We have provided evidence that oxidized dopamine interacts with a-syn and propose that this interaction unifies two potential neurotoxic mechanisms responsible for PD and disorders characterized by a-syn inclusions. Despite the profound implications in the pathogenesis of disease the interaction of a-syn with dopamine in vivo has not been evaluated. Therefore we will elevate the levels of dopamine in the substantia nigra of the mice expressing human a-syn with the pathogenic A53T mutation driven by the mouse PrP promoter by injecting lentiviral vectors that deliver cDNA of human tyrosine hydroxylase (TH) with N-terminus R37E, R38E mutation (TH-RREE), which is not feed-back inhibited by dopamine. In these mice we will then: 1) Compare and contrast the effects of dopamine on regional formation and biochemical properties of a-syn oligomers. Experiments will define the regional distribution, biochemical and biophysical properties of the soluble a-syn oligomers. Experiments will also test the ex vivo effects of oligomers on neuron dysfunction, vesicular association and a-syn aggregation. 2) Evaluate the regional association of a-syn with CMA and determine the in vivo effects of increasing dopamine levels. Experiments will test the novel hypothesis that compromised function of CMA resulting from the interaction with oxidized dopamine-induced a-syn oligomers leads to neuron dysfunction by the accumulation of S-nitrosylated glyceraldehyde phosphate dehydrogenase. This hypothesis unites the effect of a-syn oligomers in blocking CMA with the previously established effects of nitric oxide-induced dopaminergic neuron death. 3) Investigate the effect of increasing the levels of dopamine on the phenotype of A53T a-syn expressing mice. Onset of phenotype will be determined followed by a comprehensive immunohistochemical and biochemical analysis of the brains for dopamine neuron viability, inclusion formation, and astrogliosis.