线粒体自噬调控Aβ诱导小胶质细胞炎症反应的机制研究

Although the pathogenesis of Alzheimer’s disease(AD) has not been clarified, Aβ-induced neuroinflammation and mitochondrial damage play an important role in the pathogenesis of AD. Mitophagy is involved in the regulation of inflammation in many diseases, but the role of mitophagy in AD is not clear. Recently, we found that Aβ induced the production of mitochondrial ROS and the NLRP3 inflammasome, resulting in enhanced caspase-1 activity and IL-1β release. Besides, PINK1 expression was significantly reduced and microglia was obviously activated in the 12-month-old APP/PS1 mouse brain compared to wild-type mice. PINK1 overexpression inhibited the upregulation of NLRP3, mitochondrial ROS and Caspase-1 activity as well as reduced the release of IL-1β in Aβ-treated microglia. We hypothesis that the reduction of PINK1 decreases mitophagy and increases ROS production, resulting in the activation of NLRP3 inflammasome and pro-inflammatory cytokines released from microglia. We will use immunofluorescence, western blot, Elisa, Electron microscopy, and other experimental methods to observe the effects of autophagy enhancers, autophagy inhibitors, mitochondrial ROS inducers, ROS scavengers and other drugs on Mitophagy/NLRP3 and inflammation in Aβ-treated microglia. Futhermore, we investigate whether PINK1/Parkin-mediated mitophagy exhibits neuroprotective properties via enhance the clearance of Aβ-induced mitochondrial damage, thereby reducing mitochondrial ROS generation and suppressing NLRP3 activation and inflammation in APP/PS1 trangenic mice.

阿尔茨海默病（AD）发病机制尚未阐明，研究认为Aβ诱导的神经炎症和线粒体损伤在AD发病中起关键作用。线粒体自噬广泛参与多种疾病的小胶质细胞炎症调控，但在AD中作用不明确，申请人前期研究发现Aβ诱导小胶质细胞线粒体膜电位下降，ROS增加，NLRP3炎症小体活化，IL-1β分泌增多，12月龄APP/PS1小鼠大脑PINK1减少、小胶质细胞激活；PINK1过表达抑制Aβ诱导的NLRP3、Caspase-1上调和IL-1β释放。据此推测PINK1下降所致的线粒体自噬受损使小胶质细胞清除Aβ诱导的损伤线粒体能力下降，NLRP3炎症小体活化及炎症因子分泌。我们将从细胞和小鼠水平检测线粒体自噬对Aβ诱导NLRP3活化和小胶质细胞激活的影响，建立PINK1过表达细胞和动物模型，明确PINK1/Parkin介导的线粒体自噬对Aβ诱导的小胶质细胞神经炎症的调控机制，寻找AD治疗新靶点。

阿尔茨海默病（AD）发病机制尚未阐明，研究认为Aβ诱导的神经炎症和线粒体损伤在AD发病中起关键作用。线粒体自噬广泛参与多种疾病的小胶质细胞炎症调控，但在AD中作用不明确，我们首先通过体外实验观察Aβ刺激后小胶质细胞线粒体损伤、NLRP3炎症信号通路和炎症因子分泌的关系，随后观察PINK1基因过表达诱导的线粒体自噬上调对Aβ刺激后小胶质细胞神经炎症的影响。接着进一步在APP/PS1转基因小鼠水平观察PINK1/Parkin介导的线粒体自噬对AD转基因小鼠行为学、线粒体损伤、ROS、NLRP3信号通路、小胶质细胞炎症等的影响，探讨PINK1/Parkin介导的线粒体自噬对Aβ诱导小胶质细胞炎症的调控和神经保护机制。结果发现Aβ诱导原代小胶质细胞线粒体膜电位下降，促进ROS生成，线粒体碎片化增多，线粒体损伤，NLRP3信号通路激活，小胶质细胞激活，炎症因子分泌增加，自噬抑制剂3-MA，线粒体ROS清除剂Edaravone、Mito-Tempo、Methylene Blue发挥抑制神经炎症作用。在APP/PS1转基因小鼠动物模型，PINK1过表达通过增强线粒体自噬，减少线粒体ROS，压抑NLRP3/ASC/Caspase-1信号通路，抑制炎症因子IL-1β和IL-18释放。PINK1过表达对Aβ沉积诱导的小胶质细胞线粒体膜电位下降，ROS升高，线粒体碎片化，线粒体自噬损伤，NLRP3/ASC/Caspase-1信号通路激活，小胶质细胞活化和炎症因子释放均发挥抑制作用。最后观察到APP/PS1成年鼠小胶质细胞，PINK1过表达对Aβ沉积诱导的小胶质细胞线粒体膜电位下降，ROS升高，线粒体自噬损伤，NLRP3/ASC/Caspase-1信号通路激活，小胶质细胞活化和释放炎症因子发挥抑制作用。证实PINK1的表达下降导致PINK1/Parkin介导的线粒体自噬降低，清除Aβ诱导的损伤线粒体能力下降，线粒体ROS产生增多，激活小胶质细胞和NLRP3炎症小体，促进炎症因子IL-1β和IL-18分泌增加。而PINK1过表达则通过增强线粒体自噬，减少ROS产生，发挥抑制Aβ诱导的神经炎症作用。自噬增强剂、线粒体抗氧化剂、NLRP3抑制剂和Caspase-1抑制剂减少Aβ诱导的炎症因子分泌，为AD的治疗提供新靶点。