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α-突触核蛋白调节多巴胺转运蛋白的机制和功能后果。本申请中描述的工作集中在α-突触核蛋白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。所提出的研究将潜在地定义多巴胺转运蛋白调节的分子机制，从而当α-突触核蛋白过表达时，神经元损伤上游的多巴胺能传递。我们假设α-突触核蛋白与多巴胺转运蛋白的C-末端结构域相互作用以改变转运蛋白的离子渗透性，从而增加多巴胺流出并减少底物摄取，而不影响多巴胺转运蛋白表面水平。该项目将通过以下具体目标来解决这一假设：1）确定α-突触核蛋白是否调节多巴胺转运蛋白的生物物理特性，从而调节其功能，例如多巴胺转运蛋白介导的全细胞电流、底物的正向和反向转运，以及这些功能是否由转运蛋白表面水平的改变介导; 2）确定α-突触核蛋白对多巴胺转运蛋白功能的调节是否是通过与多巴胺转运蛋白的C-末端结构域上的钙钙调蛋白激酶II α（CaMK II 1）的共享结合结构域与多巴胺转运蛋白的物理相互作用;和3）确定α-突触核蛋白过表达对人多能细胞中多巴胺转运蛋白功能的影响，所述人多能细胞分化为源自帕金森病患者和正常受试者的成纤维细胞的中脑多巴胺能神经元。我们将同时使用全细胞膜片钳电生理与安培定量释放多巴胺通过多巴胺转运蛋白，和实时测量底物摄取研究α-突触核蛋白调节多巴胺转运蛋白与高的时间和空间分辨率的小鼠中脑多巴胺能神经元的原代培养物。使用荧光共振能量转移，生物化学和免疫沉淀策略，我们将确定a-突触核蛋白过度表达的多巴胺转运蛋白与CaMKII 1的关联的调节作用，除了在这些条件下的转运蛋白的磷酸化状态。此外，这些创新的方法将被部署，以确定从正常的人类受试者和患有特发性帕金森病和帕金森病与α-突触核蛋白三倍的个体获得的人中脑多巴胺能神经元中的多巴胺转运蛋白功能的α-突触核蛋白调节的后果。由于α-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。 公共卫生相关性：我们研究的长期目标是了解α-突触核蛋白调节多巴胺转运蛋白（DAT）的机制和功能后果。DAT对于维持多巴胺能传递的时间和空间维度是必不可少的，而α-突触核蛋白通过与DAT的物理相互作用参与多巴胺能传递。我们的假设得到了永生化DA神经元的初步数据的支持，表明α-突触核蛋白与DAT的C-末端结构域的相互作用通过改变DAT的离子渗透性以增加DAT-1来调节多巴胺能传递。介导的DA流出并减少DA摄取，而不影响DAT表面分布，这可能导致与神经退行性疾病有关的多巴胺能传递的改变。

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 (CaMKII1) 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 pluripotent 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 real-time measurement of substrate uptake to study a-synuclein regulation of the dopamine transporter with high temporal and spatial resolution 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 CaMKII1, 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. PUBLIC HEALTH RELEVANCE: The long-term goal of our studies is to understand the mechanism and functional consequences of regulation of the dopamine transporter (DAT) by a-synuclein. DAT is essential for maintaining the temporal and spatial dimension of dopaminergic transmission, while a-synuclein is involved in dopaminergic transmission through a physical interaction with DAT. Our hypothesis is supported by preliminary data in immortalized DA neurons indicating a-synuclein interaction with the C-terminus domain of DAT regulates dopaminergic transmission by altering the ionic permeability of DAT to increase DAT-mediated DA efflux and decrease DA uptake without an effect on DAT surface distribution that potentially leads to alterations in dopaminergic transmission implicated in neurodegenerative diseases.