α-突触核蛋白调控uptake 2转运体： 多巴胺受体激动剂抗帕金森降效机制研究

The overexpression of α-synuclein (α-Syn) and its aggregation is one of the most important causes for Parkinson’s disease (PD). In clinic, concerns have been increasingly attracted due to reduced therapeutic effect and tolerance to dopamine (DA) agonists in the treatment of early stage of PD. However, the underlying mechanism remains unknown. Regulation of uptake 1 transporters mediated by α-Syn plays a critical role in the process of neurodegenerative diseases. The high similarity of brain distribution and function between uptake 1 and uptake 2 transporters (OCT2/3 and PMAT) has been reported. In this study, pramipexole and ropinirole will be chosen as model drugs. Initially, effect of α-Syn on uptake 2 transporters mediated cellular uptake of pramipexole and ropinirole will be investigated using co-transfected cells, mouse synaptosomes and LUHMES cells. Furthermore, α-Syn null and normal PD mouse models will be constructed to elucidate the molecular pharmacokinetic mechanism for decreased therapeutic effect of pramipexole and ropinirole mediated by regulation of uptake 2 transporter function. Finally, the molecular biological mechanism of α-Syn mediated OCT2/3 and PMAT activity alteration will be explored at both cell and whole animal levels. The research results will elucidate the mechanism for declined therapeutic efficacy of DA agonists, which provides novel ideas and strategies to solve the drug tolerance during clinical PD treatment.

α-突触核蛋白（α-Syn）的过表达及其毒性聚集是帕金森疾病（PD）发生的重要诱因之一。用于早期PD治疗的多巴胺受体激动剂因降效及耐受而在临床备受关注，但其发生机制尚不明确。已有文献报道，α-Syn可调控uptake 1转运体功能并在神经退行性疾病进程中占有重要地位。鉴于uptake 2（OCT2/3、PMAT）与uptake 1转运体脑内分布与功能高度相似，本项目以普拉克索和罗匹尼罗为研究对象，分别采用共转染细胞、小鼠突触小体、人源LUHMES细胞模型研究α-Syn对uptake 2转运体介导药物细胞摄取的影响；整体动物模型阐释α-Syn调控 uptake 2转运体功能从而导致药物降效的分子药动学机制；从细胞与整体动物水平探讨α-Syn影响uptake 2转运体功能的分子生物学机理。研究结果将揭示多巴胺受体激动剂抗PD降效机制，为解决临床抗PD治疗耐受问题提供新的思路与策略。

本项目建立了普拉克索、罗匹尼罗与多巴胺LC-MS/MS生物分析方法，依据生物样品分析方法验证的指导原则，对建立的LC-MS/MS分析方法进行方法验证，包括专属性、标准曲线、定量下限、精密度、准确度、基质效应、残留效应、回收率及稳定性。采用小鼠、大鼠等动物模型，利用药代动力学血药浓度、脑切片与脑部微透析研究方法阐明普拉克索、罗匹尼罗入脑的药动学特点；利用转染细胞、BV2细胞、PD小鼠突触小体模型研究多巴胺和普拉克索/罗匹尼罗基于OCT2/3的相互作用以及α-Syn调控uptake 2转运体的分子生物学机制；利用蛋白质组学与分子对接技术预测、筛选α-Syn、OCT2、OCT3等互作蛋白以及活性位点。本项目以多巴胺受体激动剂普拉克索、罗匹尼罗为研究对象，分别采用转染细胞、离体突触小体与整体动物模型揭示多巴胺受体激动剂抗PD降效的分子药动学机制，为解决临床抗PD治疗耐受问题提供新的思路与策略。