Wang Qilong, Wu Shengnan, Zhu Huaiping, Ding Ye, Dai Xiaoyan, Ouyang Changhan, Han Young-Min, Xie Zhonglin, Zou Ming-Hui
a Center for Molecular and Translational Medicine, Georgia State University , Atlanta , GA USA.
Autophagy. 2017 Feb;13(2):404-422. doi: 10.1080/15548627.2016.1263776. Epub 2017 Jan 13.
PRKAA (protein kinase, AMP-activated, α catalytic subunit) regulates mitochondrial biogenesis, function, and turnover. However, the molecular mechanisms by which PRKAA regulates mitochondrial dynamics remain poorly characterized. Here, we report that PRKAA regulated mitochondrial fission via the autophagy-dependent degradation of DNM1L (dynamin 1-like). Deletion of Prkaa1/AMPKα1 or Prkaa2/AMPKα2 resulted in defective autophagy, DNM1L accumulation, and aberrant mitochondrial fragmentation in the mouse aortic endothelium. Furthermore, autophagy inhibition by chloroquine treatment or ATG7 small interfering RNA (siRNA) transfection, upregulated DNM1L expression and triggered DNM1L-mediated mitochondrial fragmentation. In contrast, autophagy activation by overexpression of ATG7 or chronic administration of rapamycin, the MTOR inhibitor, promoted DNM1L degradation and attenuated mitochondrial fragmentation in Prkaa2-deficient (prkaa2) mice, suggesting that defective autophagy contributes to enhanced DNM1L expression and mitochondrial fragmentation. Additionally, the autophagic receptor protein SQSTM1/p62, which bound to DNM1L and led to its translocation into the autophagosome, was involved in DNM1L degradation by the autophagy-lysosome pathway. Gene silencing of SQSTM1 markedly reduced the association between SQSTM1 and DNM1L, impaired the degradation of DNM1L, and enhanced mitochondrial fragmentation in PRKAA-deficient endothelial cells. Finally, the genetic (DNM1L siRNA) or pharmacological (mdivi-1) inhibition of DNMA1L ablated mitochondrial fragmentation in the mouse aortic endothelium and prevented the acetylcholine-induced relaxation of isolated mouse aortas. This suggests that aberrant DNM1L is responsible for enhanced mitochondrial fragmentation and endothelial dysfunction in prkaa knockout mice. Overall, our results show that PRKAA deletion promoted mitochondrial fragmentation in vascular endothelial cells by inhibiting the autophagy-dependent degradation of DNM1L.
PRKAA（蛋白激酶，AMP激活的α催化亚基）调节线粒体的生物发生、功能及周转。然而，PRKAA调节线粒体动力学的分子机制仍不清楚。在此，我们报道PRKAA通过自噬依赖性降解DNM1L（动力蛋白1样蛋白）来调节线粒体分裂。敲除Prkaa1/AMPKα1或Prkaa2/AMPKα2会导致小鼠主动脉内皮细胞自噬缺陷、DNM1L积累及线粒体异常碎片化。此外，用氯喹处理或转染ATG7小干扰RNA（siRNA）抑制自噬，会上调DNM1L表达并引发DNM1L介导的线粒体碎片化。相反，过表达ATG7或长期给予雷帕霉素（MTOR抑制剂）激活自噬，可促进Prkaa2缺陷（prkaa2）小鼠中DNM1L的降解并减轻线粒体碎片化，这表明自噬缺陷导致DNM1L表达增强和线粒体碎片化。此外，自噬受体蛋白SQSTM1/p62与DNM1L结合并导致其转运至自噬体，参与了自噬-溶酶体途径对DNM1L的降解。沉默SQSTM1基因显著降低了SQSTM1与DNM1L之间的结合，损害了DNM1L的降解，并增强了PRKAA缺陷内皮细胞中的线粒体碎片化。最后，对DNMA1L进行基因（DNM1L siRNA）或药理学（mdivi-1）抑制，消除了小鼠主动脉内皮细胞中的线粒体碎片化，并阻止了乙酰胆碱诱导的离体小鼠主动脉舒张。这表明异常的DNM1L是导致prkaa基因敲除小鼠线粒体碎片化增强和内皮功能障碍的原因。总体而言，我们的结果表明，PRKAA缺失通过抑制自噬依赖性降解DNM1L促进血管内皮细胞中的线粒体碎片化。
