基于神经细胞氧化应激关联mTOR通路抑制的帕金森病发生及雷帕霉素调控分子机理研究

Oxidative stress-induced neuronal cell apoptosis is associated with neurodegenerative diseases such as Parkinson's disease (PD), etc. The previous work from our Lab found that hydrogen peroxide imitation of oxidative stress elicited neuronal apoptosis via induction of reactive oxygen species (ROS) and inhibition of mammalian target of rapamycin (mTOR) pathway; 6-hydroxydopamine (6-OHDA) inhibition of mTOR pathway was related to apoptosis of neuronal cells. The aim of this study, by experimental models for Parkinson's disease, is to synthetically study the relation of neuronal NADPH oxidase 2 (NOX2) and its regulatory proteins including p22phox, p40phox, p47phox, p67phox, and Rac1 to ROS production as well as their actions in mTOR signaling suppression, and to dissect manifestations of neuronal ROS and apoptosis post upregulation or downregulation of key signal molecules such as raptor and rictor as well as their downstream effector molecules in TORC1 and TORC2, respectively, to deeply understand moleculer mechanism (s) of neuronal oxidative stress contributing to mTOR signaling suppression during the process of PD development. Then, the study on signal transduction pathways involved in rapamycin prevention of neuronal apoptosis in PD development by intervention of ROS production are conducted to reveal molecular mechanism (s) for its regulative actions, as well as its significance and applied value, thereby giving new idea and theory for prevention of oxidative stress-induced neurodegenerative diseases.

氧化应激诱导神经细胞凋亡与神经变性疾病如帕金森病（PD）等发生有关系。本课题组前期工作发现过氧化氢模拟氧化应激通过诱导ROS和抑制mTOR通路致神经细胞凋亡；6-羟多巴胺抑制mTOR通路与神经细胞凋亡有关。本项目拟通过PD实验模型，综合研究PD发生中神经细胞NOX2和它的调节蛋白p22phox、p40phox、p47phox、p67phox、Rac1表达与ROS产生关系以及它们在mTOR通路抑制中作用；解析mTOR复合物TORC1和TORC2中关键信号分子Raptor和Rictor以及其介导的下游效应分子在下调或上调后，神经细胞ROS和凋亡表现，深入阐明神经细胞氧化应激涉及mTOR通路抑制的PD发生分子机理。然后，研究雷帕霉素干预ROS产生抗PD发生中神经细胞凋亡涉及的信号转导通路，揭示其发挥调控作用分子机制及其理论意义和应用价值，为防止氧化应激诱导的神经变性相关疾病提供新思路和理论依据。

氧化应激诱导神经细胞凋亡与神经变性疾病如帕金森病（PD）等发生有关系。本项目通过PD诱导剂（6-羟多巴胺、MPTP/MPP+、鱼藤酮）建立的体内和体外PD模型，并以镉染毒实验模型作为阳性对照，综合研究了神经细胞NOX2和它的调节蛋白p22phox、p40phox、p47phox、p67phox、Rac1表达与ROS/H2O2产生关系及其在mTOR通路抑制/激活中作用；解析了mTOR复合物mTORC1和mTORC2中Raptor和Rictor以及其介导的下游效应分子在上调或下调后，神经细胞ROS/H2O2和凋亡表现；开展了镉或PD模型中钙信号转导和ROS/H2O2产生关系及其在激活或抑制mTOR通路致神经细胞凋亡中作用研究。其中每个环节包含了探讨NAC、雷帕霉素、白藜芦醇、雷公藤红素通过调控神经细胞氧化应激-MAPK/mTOR信号转导网络抗凋亡分子机理。结果显示：PD发生中的AMPK激活和Akt失活导致mTOR介导S6K1和4E-BP1/eIF4E通路抑制关联神经细胞死亡，这与神经细胞NOX2和它的调节蛋白p22phox、p40phox、p47phox、p67phox、Rac1表达上调引起的H2O2过量产生有关系；鱼藤酮诱导线粒体H2O2和[Ca2+]i升高相互作用激活CaMKII导致mTOR通路抑制和神经细胞凋亡。研究证明，NAC和雷帕霉素体内应用通过抑制ROS介导mTOR通路激活抗镉诱导小鼠大脑神经元凋亡；雷帕霉素通过干预mTORC1/2、mTOR-PTEN/PP2A-Erk1/2和ROS-PP2-MAPK信号转导网络阻止镉诱导神经细胞凋亡。我们注意到，白藜芦醇通过PP2A和PP5阻止镉激活Erk1/2和JNK通路防止神经细胞凋亡。我们发现，雷公藤红素靶向PTEN-Akt/mTOR、ROS-AMPK/mTOR和Ca2+/CaMKII-Akt/mTOR通路防止镉诱导神经细胞死亡；雷公藤红素通过抑制NOX2、p22phox、p67phox、p40phox、p47phox、Rac1表达和ROS产生，干预PP5-JNK通路改善镉诱导神经细胞凋亡。这些证据强烈地呈现了调控PP5/PP2A-MAPK和/或AMPK/Akt-mTOR信号转导网络、或操纵胞内ROS/H2O2和/或Ca2+水平、应用NAC、雷帕霉素、白藜芦醇、雷公藤红素可以为防止氧化应激诱导的神经变性疾病开发利用。