Understanding Mechanisms of a-synuclein pathology

项目摘要-项目2：了解α-突触核蛋白的病理机制 α-突触核蛋白的遗传和生化异常直接参与家族性和家族性高血压的发病机制。 散发性帕金森氏病(PD)。α-突触核蛋白诱导的潜在机制 人们对神经退行性变知之甚少。α-突触核蛋白的家族突变以及氧化和 亚硝化应激在α-突触核蛋白病理中的作用部分是通过增强的寡聚化、纤颤和 聚合。在上一个供资周期中，我们与项目1合作表明，非 受体酪氨酸激酶c-Abl可能参与了帕金森病的发病过程。从这些研究中得出了 令人兴奋的初步发现，c-Abl使α-突触核蛋白在酪氨酸39处磷酸化。然而，潜在的角色 酪氨酸39、α-突触核蛋白和c-Abl的激活在帕金森病发病机制中的作用尚不清楚。我们会研究 α-突触核蛋白酪氨酸39位磷酸化和c-Abl激活在α-1诱导的DA神经元死亡中的作用 突触核蛋白及其在体外和体内α-突触核蛋白聚集中的作用。有了蛋白质组学核心D， 临床核心B和神经病理核心C，我们将调查磷酸化水平是否 酪氨酸39位的α-突触核蛋白可作为α-突触核蛋白诱导的进展和/或病理标记物 人类帕金森病的神经退变和α-突触核蛋白病理。在这些研究中，我们将评估 人A53Tα-突触核蛋白中α-突触核蛋白酪氨酸39磷酸化及c-Abl的激活状态 转基因模型、腺相关病毒-WT或A53Tα-突触核蛋白DA神经元缺失模型和人 应用磷酸酪氨酸39α-突触核蛋白抗体和磁共振波谱检测帕金森病患者的尸检组织 (多反应监测)质谱学。错误折叠的α-突触核蛋白的细胞间传递可能 散发性帕金森病患者多巴胺能神经元的退行性改变及其机制 内源性α-突触核蛋白在纤维中的募集和破坏尚不清楚。因为我们的初步数据 提示c-Abl对α-突触核蛋白的酪氨酸39磷酸化促进了α-突触核蛋白的纤颤。 将研究WT对缺磷的Y39F和模拟磷酸的α-突触核蛋白Y39E的能力，以及 α-突触核蛋白PFF模型中细胞间传递的c-Abl缺陷和DA神经元变性 散发性帕金森病。最后，我们将探讨α-突触核蛋白pff在退行性DA中引起的蛋白质组学变化。 结合SILAM(稳定同位素标记)的高级尖峰质谱学方法的神经元 在哺乳动物中)。这些研究将为α-突触核蛋白的发病机制提供新的机制见解 诱导神经变性，并可能导致新的治疗靶点和生物标志物的开发 帕金森病的治疗。

PROJECT SUMMARY - PROJECT 2: UNDERSTANDING MECHANISMS OF α -SYNUCLEIN PATHOLOGY Genetic and biochemical abnormalities of α-synuclein are directly implicated in the pathogenesis of familial and sporadic forms of Parkinson's disease (PD). The underlying mechanisms of α-synuclein-induced neurodegeneration are poorly understood. Familial mutations in α-synuclein as well as oxidative and nitrosative stress contribute to α-synuclein pathology, in part, via enhanced oligomerization, fibrillation and aggregation. During the last funding cycle, we showed in collaboration with Project 1 that activation of the non- receptor tyrosine kinase, c-Abl may contributes to the pathogenesis of PD. From these studies emerged the exciting preliminary findings that c-Abl phosphorylates α-synuclein at tyrosine 39. However, the potential roles of tyrosine 39 α-synuclein and c-Abl activation in pathogenesis of PD has not been explored. We will study the roles of phosphorylation of α-synuclein at tyrosine 39 and c-Abl activation in the death of DA neurons due to α- synuclein, as well as, their roles in aggregation of α-synuclein in vitro and in vivo. With the Proteomics Core D, the Clinical Core B and the Neuropathology Core C, we will investigate whether the levels of phosphorylation of α-synuclein at tyrosine 39 can serve as a progression and/or pathologic maker of α-synuclein-induced neurodegeneration and of α-synuclein pathology in human PD. For these studies, we will assess the levels of tyrosine 39 phosphorylation of α-synuclein and the activation state of c-Abl in human A53T α-synuclein transgenic model, the adeno-associated virus-WT or A53T α-synuclein model with DA neuron loss, and human post-mortem tissues from PD patients via a phosphospecific tyrosine 39 α-synuclein antibody and MRM (Multiple Reaction Monitoring) mass spectrometry. Cell-to-cell transmission of misfolded α-synuclein may contribute to the degeneration of DA neurons in sporadic PD and the mechanisms accounting for the recruitment and the corruption of endogenous α-synuclein into fibrils are not known. Since our preliminary data suggests that tyrosine 39 phosphorylation of α-synuclein by c-Abl promotes the fibrillation of α-synuclein, we will study the ability of WT versus phospho-deficient Y39F and phospho-mimetic α-synuclein Y39E, as well as c-Abl deficiency in cell-to-cell transmission and degeneration of DA neurons in the α-synuclein PFF model of sporadic PD. Finally, we will explore proteomic changes induced by α-synuclein PFFs in degenerating DA neurons via advanced spike-in mass spectrometry approaches combined with SILAM (Stable Isotope Labeling in Mammals). These studies will provide new mechanistic insights into the pathogenesis of α-synuclein induced neurodegeneration and may lead to the development of novel therapeutic targets and biomarkers for the treatment of PD.