Neurotoxicity Mechanisms of Reactive Intermediates

描述（由申请人提供）：该应用测试了以下假设：生物化学上不同的α-突触核蛋白低聚物部分通过抑制伴侣介导的自噬（CMA）功能来诱导神经元变性。α-突触核蛋白（α- syn）已被鉴定为患有散发性和家族性形式的帕金森病（PD）和相关神经变性病症的受试者的脑中蛋白质内含物的主要组分之一。多巴胺代谢的失调导致细胞溶质多巴胺增加，经历氧化也被认为是PD中选择性神经元功能障碍和死亡的致病机制。我们已经提供了氧化多巴胺与α-syn相互作用的证据，并提出这种相互作用统一了两个潜在的神经毒性机制，负责PD和疾病的特征在于α-syn夹杂物。 尽管在疾病的发病机制中具有深刻的意义，但尚未评估α-syn与多巴胺在体内的相互作用。因此，我们将通过注射慢病毒载体来提高表达具有由小鼠PrP启动子驱动的致病性A53 T突变的人a-syn的小鼠的黑质中的多巴胺水平，所述慢病毒载体递送具有N-末端R37 E、R38 E突变（TH-RREE）的人酪氨酸羟化酶（TH）的cDNA，其不被多巴胺反馈抑制。在这些小鼠中，我们将：1）比较和对比多巴胺对a-syn寡聚体的区域形成和生化性质的影响。 实验将确定可溶性α-syn寡聚体的区域分布、生物化学和生物物理性质。实验还将测试寡聚体对神经元功能障碍、囊泡缔合和α-syn聚集的离体作用。2)评估a-syn与CMA的区域关联，并确定增加多巴胺水平的体内效应。实验将检验新的假设，即由与氧化多巴胺诱导的α-syn寡聚体的相互作用引起的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α-syn寡聚体在阻断CMA中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表达A53 T a-syn的小鼠的表型的影响。将确定表型的发作，然后对大脑进行多巴胺神经元活力、包涵体形成和星形胶质细胞增生的全面免疫组织化学和生化分析。

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.