Alpha-synuclein Regulates Dopamine Transporter Functions

描述（由申请人提供）：本提案的总体目标是了解a-synuclein调节多巴胺转运体的机制和功能后果。本应用程序中描述的工作集中在a-synuclein过表达是否影响多巴胺转运蛋白活性，导致多巴胺能传递改变的问题上。这些研究将有可能确定多巴胺转运蛋白调控的分子机制，从而确定当a-synuclein过表达时多巴胺能在神经元损伤上游的传递。我们假设a-synuclein与多巴胺转运体的c端结构域相互作用，改变转运体的离子渗透性，从而增加多巴胺外排，减少底物摄取，而不影响多巴胺转运体表面水平。该项目将通过以下具体目标来解决这一假设：1)确定a-synuclein是否调节多巴胺转运体的生物物理特性，从而确定其功能，如多巴胺转运体介导的全细胞电流，底物的正向和反向运输，以及这些功能是否由转运体表面水平的改变介导；2)确定a-synuclein对多巴胺转运蛋白功能的调控是否通过多巴胺转运蛋白c端域与钙调蛋白激酶II α (CaMKIIa)共享结合域与多巴胺转运蛋白的物理相互作用；3)确定a-synuclein过表达对帕金森病患者和正常人成纤维细胞分化为中脑多巴胺能神经元的人多能细胞多巴胺转运蛋白功能的影响。在小鼠中脑多巴胺能神经元原代培养中，我们将同时使用全细胞膜片钳电生理技术，通过多巴胺转运体释放多巴胺的电流定量和底物摄取的测量来研究a-突触核蛋白对多巴胺转运体的调节。利用荧光共振能量转移、生化和免疫沉淀策略，我们将确定a-synuclein过表达对多巴胺转运体与CaMKIIa关联的调节作用，以及转运体在这些条件下的磷酸化状态。此外，这些创新的方法将被用于确定a-突触核蛋白调节人类中脑多巴胺能神经元多巴胺转运蛋白功能的后果，这些神经元来自正常人、特发性帕金森病患者和a-突触核蛋白三分型帕金森病患者。由于a-突触核蛋白和多巴胺转运蛋白都与神经退行性疾病如帕金森病有关，我们的研究结果将为我们对这些疾病状态的机制知识提供重要的见解，并可用于开发疾病建模和靶向药物发现的新策略。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the mechanism and functional consequence of regulation of the dopamine transporter by a-synuclein. The work described in this application is focused on the problem of whether a-synuclein over-expression affects the dopamine transporter activity, leading to alterations in dopaminergic transmission. The proposed studies will potentially define the molecular mechanisms of dopamine transporter regulation and thus dopaminergic transmission upstream of neuronal injury when a-synuclein is over-expressed. We hypothesize that a-synuclein interacts with the C-terminus domain of the dopamine transporter to alter the ionic permeability of the transporter, thus increasing dopamine efflux and decreasing substrate uptake without an effect on dopamine transporter surface levels. The project will address this hypothesis with the following specific aims: 1) to determine whether a-synuclein modulates the biophysical properties of the dopamine transporter, and therefore its functions such as dopamine transporter-mediated whole cell currents, forward and reverse transport of the substrate, and whether these functions are mediated by alterations in surface levels of the transporter; 2) to determine whether a-synuclein regulation of dopamine transporter function is through a physical interaction with the dopamine transporter via a shared binding domain with calcium calmodulin kinase II alpha (CaMKIIa) on the C-terminus domain of the dopamine transporter; and 3) to determine the impact of a-synuclein over-expression on dopamine transporter function in human pleuripotent cells differentiated to midbrain dopaminergic neurons derived from fibroblasts obtained from Parkinson's disease patients and normal subjects. We will use simultaneous whole cell patch clamp electrophysiology with amperometric quantification of released dopamine via the dopamine transporter, and measurement of substrate uptake to study a-synuclein regulation of the dopamine transporter in primary cultures of mouse midbrain dopaminergic neurons. Using Fluorescence Resonance Energy Transfer, biochemical, and immunoprecipitation strategies, we will determine the regulatory role of a-synuclein over-expression on association of the dopamine transporter with CaMKIIa, in addition to the phosphorylation state of the transporter under these conditions. Furthermore, these innovative approaches will be deployed to determine the consequences of a-synuclein regulation of dopamine transporter function in human midbrain dopaminergic neurons obtained from normal human subjects and individuals with idiopathic Parkinson's disease and Parkinson's disease with a-synuclein triplication. As both a-synuclein and the dopamine transporter have been implicated in neurodegenerative diseases such as Parkinson's disease, results from our studies will provide important insight into our mechanistic knowledge of these disease states and could be used to develop novel strategies in disease modeling and targeted drug discovery.