天然来源抗帕金森病候选药物特咖宁通过促进线粒体自噬保护多巴胺能神经元作用机制研究

通过自噬清除受损线粒体来保护多巴胺能神经元，是治疗帕金森病（PD）的重要策略。特咖宁（Tc）是本课题组从苦茶中分离的专利化合物，治疗小鼠PD药效显著，已入选国家重大新药创制临床前候选药物，并解决药源问题，但作用机制未明。在体外实验中，我们发现Tc可以激活线粒体Sirt3酶，促进自噬信号分子Cardiolipin（CL）外翻介导线粒体自噬，从而保护多巴胺能神经元。本项目拟建立线粒体损伤的细胞和动物PD模型, 采用线粒体靶向载体实现Tc在线粒体富集，研究其对多巴胺能神经元的保护机制。我们拟建立MALDI-IMS质谱成像等新方法来检测线粒体中CL分子的变化，采用RNAi等技术研究Sirt3对CL翻转酶PSL3/NDPK-D的影响，结合荧光共聚焦和电镜技术全面解析Tc对线粒体自噬的调控分子机制。本项目有望阐明Tc抗PD的作用靶点与机制，为研发安全、有效、具知识产权保护的新型抗PD药物提供科学依据。

Removal of dysfunctional or damaged mitochondria has been considered to be an important strategy to protect dopaminergic neurons and prevent Parkinson's disease.. Theacrine (Tc), a purine alkaloid obtained from Camellia assamica var. kucha, has been selected for preclinical study due to its potent protective effect against MPTP-induced Parkinson's Disease in mouse. The mechanisms through which Tc protects dopaminergic neuron, however, remain elusive. Our preliminary data have shown that Tc could activate mitochondrial deacetylase Sirt3. In addition, Tc treatment facilitated the externalization of CL from the inner mitochondria membrane to the outer leaflet of the outer mitochondrial membrane. Recent discoveries have highlighted that externalized cardiolipin (CL) on outer mitochondria membrane functions as "eat-me" signal for mitophagy. Moreover, Tc enhanced mitophagy in dopaminergic neurons challenged with neuron toxin rotenone. Our central hypothesis is that, by activating Sirt3, Tc enhance mitophagy by modulating phospholipid scramblase 3 (PLS3)/Nucleoside Diphosphate Kinase D (NDPK-D)-dependent CL signaling pathway and therefore protect dopaminergic neurons against mitohocndrial oxidative damage. To test the hypothesis, mitochondrial respiratory chain complex I toxins-induced cell and mouse PD disease models will be utilized. We will employ RNA interference-based loss-of-function study and Signaling Pathway Analysis to explore the involvement of Sirt3, PLS3 and NDPK-D in the neuroprotective effect of Tc. We will establish novel Liquid chromatography-mass spectrometry and MALDI imaging mass-spectrometry to analysis the distribution of CL on inner and outer mitochondrial membrane and assess the mitochondria mass in vivo. Using Confocal Fluorescence Microscopy and Transmission Electronic Microscopy, we will further investigate the degree to which modulation of CL signaling affect the Tc-induced mitophagy. Finally, using a TPP-conjuction, we will selectively target Tc to mitochondria, the site of action, to explore the molecular mechanisms of the neuroprotective effects of Tc. Based on the highly innovative concept that CL is unique signal for mitophagy in Tc-treated dopaminergic neuron, the present project will open new avenues for developing non-dopamine targeting anti-Parkinson's Disease drug.