Alpha-synuclein Regulates Dopamine Transporter Functions

描述(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体的机制和功能结果。本申请中描述的工作集中在a-突触核蛋白过度表达是否影响多巴胺转运体活性，从而导致多巴胺能传递的改变的问题。这项拟议的研究可能会确定当α-突触核蛋白过度表达时，多巴胺转运体调节的分子机制，从而在神经元损伤的上游进行多巴胺能传递。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，改变转运蛋白的离子通透性，从而增加多巴胺的外流，减少底物摄取，而不影响多巴胺转运蛋白的表面水平。该项目将以以下具体目标解决这一假说：1)确定a-突触核蛋白是否调节多巴胺转运体的生物物理性质，从而调节其功能，如多巴胺转运体介导的全细胞电流、底物的正向和反向转运，以及这些功能是否由转运体表面水平的变化所介导；2)确定a-突触核蛋白对多巴胺转运体功能的调控是否通过与多巴胺转运体通过与多巴胺转运体C-末端区域上的钙钙调素蛋白激酶IIα(CaMKII1)的共同结合结构域进行物理相互作用来实现；3)研究α-突触核蛋白过度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多巴胺转运体功能的影响。我们将同时使用全细胞膜片钳电生理学和安培定量多巴胺转运体释放的方法，以及底物摄取的实时测量，以高时间和空间分辨率研究α-突触核蛋白对原代培养的小鼠中脑多巴胺能神经元转运体的调节。利用荧光共振能量转移、生化和免疫沉淀技术，我们将确定在这些条件下，除了转运蛋白的磷酸化状态外，α-突触核蛋白的过度表达对多巴胺转运体与CaMKII1的关联的调节作用。此外，这些创新的方法将被用于确定a-突触核蛋白对人中脑多巴胺能神经元中多巴胺转运体功能的调节的后果，这些多巴胺能神经元来自正常人和特发性帕金森病患者以及具有a-突触核蛋白三倍体的帕金森病患者。由于α-突触核蛋白和多巴胺转运体都与帕金森氏病等神经退行性疾病有关，我们的研究结果将为我们提供对这些疾病状态的机制知识的重要见解，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体(DAT)的机制和功能后果。DAT是维持多巴胺能传递的时间和空间维度所必需的，而α-突触核蛋白通过与DAT的物理作用参与多巴胺能传递。我们的假说得到了永生化DA神经元的初步数据的支持，表明α-突触核蛋白与DAT的C末端结构域相互作用，通过改变DAT的离子通透性来调节多巴胺能传递，从而增加DAT介导的DA流出，减少DA摄取，而不影响DAT的表面分布，从而潜在地导致神经退行性疾病所涉及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.