Zhu Huaiping, Foretz Marc, Xie Zhonglin, Zhang Miao, Zhu Zhiren, Xing Junjie, Leclerc Jocelyne, Gaudry Murielle, Viollet Benoit, Zou Ming-Hui
Section of Molecular Medicine; Department of Medicine; University of Oklahoma Health Sciences Center; Oklahoma City, OK USA.
Inserm; U1016; Institut Cochin; Paris, France; CNRS; UMR8104; Institut Cochin; Paris, France; Université Paris Descartes; Sorbonne Paris; Paris, France.
Autophagy. 2014 Sep;10(9):1522-34. doi: 10.4161/auto.29197. Epub 2014 Jun 19.
AMP-activated protein kinase α1 knockout (prkaa1(-/-)) mice manifest splenomegaly and anemia. The underlying molecular mechanisms, however, remain to be established. In this study, we tested the hypothesis that defective autophagy-dependent mitochondrial clearance in prkaa1(-/-) mice exacerbates oxidative stress, thereby enhancing erythrocyte destruction. The levels of ULK1 phosphorylation, autophagical flux, mitochondrial contents, and reactive oxygen species (ROS) were examined in human erythroleukemia cell line, K562 cells, as well as prkaa1(-/-) mouse embryonic fibroblasts and erythrocytes. Deletion of Prkaa1 resulted in the inhibition of ULK1 phosphorylation at Ser555, prevented the formation of ULK1 and BECN1- PtdIns3K complexes, and reduced autophagy capacity. The suppression of autophagy was associated with enhanced damaged mitochondrial accumulation and ROS production. Compared with wild-type (WT) mice, prkaa1(-/-) mice exhibited a shortened erythrocyte life span, hemolytic destruction of erythrocytes, splenomegaly, and anemia, all of which were alleviated by the administration of either rapamycin to activate autophagy or Mito-tempol, a mitochondria-targeted antioxidant, to scavenge mitochondrial ROS. Furthermore, transplantation of WT bone marrow into prkaa1(-/-) mice restored mitochondrial removal, reduced intracellular ROS levels, and normalized hematologic parameters and spleen size. Conversely, transplantation of prkaa1 (-/-) bone marrow into WT mice recapitulated the prkaa1(-/-) mouse phenotypes. We conclude that PRKAA1-dependent autophagy-mediated clearance of damaged mitochondria is required for erythrocyte maturation and homeostasis.
AMP激活的蛋白激酶α1基因敲除（prkaa1(-/-)）小鼠表现出脾肿大和贫血。然而，其潜在的分子机制仍有待确定。在本研究中，我们检验了以下假设：prkaa1(-/-)小鼠中自噬依赖性线粒体清除缺陷会加剧氧化应激，从而增强红细胞破坏。我们在人红白血病细胞系K562细胞、prkaa1(-/-)小鼠胚胎成纤维细胞和红细胞中检测了ULK1磷酸化水平、自噬通量、线粒体含量和活性氧（ROS）水平。Prkaa1基因的缺失导致Ser555位点的ULK1磷酸化受到抑制，阻止了ULK1和BECN1-PtdIns3K复合物的形成，并降低了自噬能力。自噬的抑制与受损线粒体积累增加和ROS产生增强有关。与野生型（WT）小鼠相比，prkaa1(-/-)小鼠的红细胞寿命缩短，红细胞发生溶血性破坏，出现脾肿大和贫血，而通过给予雷帕霉素激活自噬或给予线粒体靶向抗氧化剂Mito-tempol清除线粒体ROS，所有这些症状均得到缓解。此外，将WT骨髓移植到prkaa1(-/-)小鼠中可恢复线粒体清除，降低细胞内ROS水平，并使血液学参数和脾脏大小恢复正常。相反，将prkaa1(-/-)骨髓移植到WT小鼠中则重现了prkaa1(-/-)小鼠的表型。我们得出结论，红细胞成熟和内环境稳定需要PRKAA1依赖性自噬介导的受损线粒体清除。
