AMPK与MFN2相互作用及其生物学功能

The production of energy needs a healthy mitochondrial pool and the disordered energy state inside the cell also triggers mitochondrial deformation and dysfunction. AMPK, an energy sensor, is activated in response to energy deprivation, and mitochondrial fission then occurs. Mitophagy is also blocked in AMPK-null cells. However, the relationship between cellular energy and mitochondrial dynamics remains largely unknown. We have found AMPK interacts with MFN2 and the latter is phosphorylated and mitophagy then occurs upon AMPK activation or ETC inhibition. Conditional deletion of MFN2 in mouse skeletal muscle shows abnormal metabolism, swollen mitochondria and accumulation of autolysosomes. We therefore propose that MFN2 is a novel mitochondrial substrate of AMPK, which coordinately regulate mitochondrial fission and mitophagy, thereby affecting the function of skeletal muscle. Based on above findings, we will investigate the interaction of AMPK and MFN2 and their roles in mitochondrial dynamics, metabolism and the physiological function of skeletal muscle by using CRISPR/CAS9, immunoprecipitation, in vitro kinase assay, electron microscopy techniques, etc. Our study will provide novel insight for better understanding the molecular link between cellular energetics and mitochondrial dynamics.

健康的线粒体是细胞能量产生的基础，细胞能量失衡亦会导致线粒体形态和功能改变。细胞能量失衡时AMPK活化，线粒体断裂；而AMPK敲除则抑制线粒体自噬，但能量代谢与线粒体动态之间的关系仍不清楚。申请人最近发现AMPK与MFN2相互作用；药物诱导AMPK活化或线粒体呼吸链障碍时，MFN2被磷酸化、线粒体发生自噬；骨骼肌缺乏MFN2后小鼠代谢异常、骨骼肌细胞线粒体肿胀、自噬体累积。由此推测： MFN2是AMPK在线粒体上的新底物，AMPK和MFN2协同作用调节线粒体分裂、促进线粒体自噬，影响骨骼肌功能。本项目拟运用CRISPR/CAS9基因敲除、免疫共沉淀、体外磷酸化、透射电镜等技术，在分子、细胞和动物模型三个层面研究AMPK和MFN2的相互作用，探讨它们在调节线粒体动态变化、代谢、骨骼肌功能等生物学过程中的作用机制。本项目的完成将为理解细胞能量变化和线粒体形态功能变化之间的联系提供新线索。

能量损失可以激活细胞内的能量传感器AMPK（AMP活化蛋白激酶），进而诱导巨自噬/自噬。MAM（线粒体相关内质网膜结构）在线粒体分裂和自噬中起关键作用，而MFN2（线粒体融合蛋2）是MAM的束缚蛋白，然而AMPK与MFN2的相互作用在调节能量响应的自噬中的分子机制目前仍不清楚。在本项目研究中，我们发现能量压力不仅会触发线粒体分裂和自噬，而且更重要的是会增加MAM的数量，而这个过程需要AMPK参与。在能量压力下，当线粒体分裂发生时，大量的AMPK从细胞质转移到MAM和线粒体上，并且AMPK能直接与MFN2相互作用。与野生型MEF（小鼠胚胎成纤维细胞）相比，MFN2缺失（mfn2-/-）的MEF细胞对能量压力产生自噬的应激反应显着减弱，而在MFN2缺失细胞中重新表达MFN2可以挽救这些细胞的自噬缺陷。在MFN2缺陷型细胞中，MAM的丰度也大大降低。功能实验表明，MFN2缺失细胞的耗氧率和糖酵解功能明显减弱，而MFN1缺失的细胞不明显，并且MFN2对于能量应激下的细胞存活也很重要。动物实验显示，骨骼肌特异性敲除MFN2小鼠的氧化应激、耗氧量、糖耐受和胰岛素指标发生变化，并且其骨骼肌细胞中发生线粒体肿胀和自噬累积。总而言之，我们的研究建立了能量传感器AMPK和MAM系链MFN2之间的分子联系，并揭示了AMPK和MFN2在能量应激诱导的自噬和MAM动力学中的重要作用。