创新化学药物硝酮嗪清除α-突触核蛋白和改善线粒体功能治疗帕金森病的机理研究

The pathogenesis of Parkinson’s disease (PD) is complicated. Currently there is no effective treatment for PD. Targeting α-synuclein and mitochondrial dysfunction to protect dopaminergic neurons is the hotspot of PD research. Tetramethylpyrazine nitrone (TBN), a novel derivative of tetramethylpyrazine, significantly protects DA neurons in MPTP/6-OHDA induced rodent models of PD; lowers α-synuclein protein level in human A53T α-synuclein transgenic mice and delays the deterioration of motor behavior. Moreover, TBN can activate PGC-1α/Nrf2 signaling pathway and improve mitochondrial dysfunction. Recent studies revealed that activation of Nrf2 promoted the clearance of α-synuclein via protein degradation systems. We hypothesize that TBN protects dopaminergic neuron through down-regulate α-synuclein and improvement of mitochondrial dysfunction via activating PGC-1α/Nrf2 signaling pathways. How does TBN activate PGC-1α and lower α-synuclein? What're the direct binding targets of TBN? In this proposal, we will use the techniques including molecular pharmacology, chromatin immunoprecipitation (ChIP), animal behavioral tests and molecular genetics to elucidate the mechanisms underlying neuroprotection of TBN. We will also use DARTS (drug affinity-responsive target stability) method to identify the direct binding proteins with TBN. The accomplishment of this project would be expected to uncover the novel molecular mechanisms of TBN for treatment of PD, and provide solid support for developing safe and effective anti-PD drug with intellectual property protection.

帕金森病(PD)发病机制复杂，目前尚无有效治疗手段。靶向α-突触核蛋白(α-Syn)和线粒体保护多巴胺能(DA)神经元是抗PD研究热点。前期我们发现创新药物硝酮嗪(TBN)可保护PD动物模型DA神经元，并降低PD转基因小鼠α-Syn水平，延缓运动行为恶化；TBN可激活PGC-1α/Nrf2通路进而改善线粒体功能。研究报道激活Nrf2能促进蛋白降解系统清除α-Syn。TBN如何激活PGC-1α，上调Nrf2从而清除α-Syn，以及TBN有无直接作用靶点蛋白是本项目需解决的关键科学问题。本项目拟采用细胞和转基因小鼠为研究对象，通过分子药理学、染色质免疫共沉淀、病毒介导的基因长效抑制等手段，研究TBN清除α-Syn和改善线粒体功能的机制；通过药物亲和靶点稳定性(DARTS)技术揭示TBN的直接结合蛋白靶点。本项目有望阐明TBN治疗PD的分子机理，为研发具有自主知识产权抗PD新药提供科学依据。

帕金森病(PD)发病机制复杂，目前尚无有效治疗手段。靶向线粒体和α-突触核蛋白(α-Syn)保护多巴胺能(DA)神经元是抗PD的研究热点。前期我们发现创新药物硝酮嗪(TBN)在MPTP/6-OHDA诱导的PD动物模型能保护DA神经元；降低转基因小鼠α-Syn蛋白水平，延缓运动行为恶化；此外TBN能激活PGC-1α/Nrf2通路改善线粒体功能。最近研究报道激活Nrf2能促进蛋白降解系统清除α-Syn。我们假设TBN通过调控AMPK/SIRT1/MEF2D激活PGC-1α/Nrf2通路促进蛋白降解系统UPS和ALS清除α-Syn，改善线粒体功能，从而保护DA神经元；同时，明确TBN的直接作用靶点蛋白。因此，本项目拟在体外细胞和α-Syn转基因小鼠采用分子药理学等技术，研究TBN清除α-Syn和改善线粒体功能的机制；应用药物亲和靶点稳定性(DARTS)和分子对接技术发现TBN可能直接结合的蛋白靶点。研究结果显示，TBN可抑制MPP+或6-OHDA诱导的SY5Y/A53T细胞及Dox诱导的A53T/PC12细胞中α-Syn蛋白上调；TBN能清除Dox诱导的PC12/A53T细胞模型中的活性氧自由基(ROS)产生；TBN上调Dox诱导PC12/A53T细胞模型中PGC-1α/Nrf2/HO-1信号通路蛋白表达，并促进Nrf2入核；TBN能明显增加6-OHDA大鼠和A53T转基因小鼠黒质致密部(SNpc) PGC-1α、Nrf1/2和HO-1 mRNA表达。研究还发现，TBN主要通过激活chymotrpsin-like活性影响UPS途径清除α-Syn；通过激活Nrf1和Nrf2促进Psmb8蛋白表达；增加MPTP小鼠和A53T小鼠SNpc部位自噬溶酶体数目，提高AMPK和ULK1的磷酸化水平；提高PD模型自噬相关蛋白表达。更重要的是，过表达AMPK和ULK1显著降低α-syn表达；敲低AMPK和ULK1显著抑制TBN对α-syn的降解清除； TBN保证自噬流通路正常；分子对接显示TBN可能作用于脂联素受体1 (AdipoR1)。本项目有望阐明TBN治疗PD的机理，为研发具自主知识产权的抗PD新药提供理论依据。本研究的相关结果在Human Molecular Genetics等SCI期刊共发表研究论文10篇，投稿中研究论文2篇；发表相关综述2篇。