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AMPK contributes to autophagosome maturation and lysosomal fusion.

ABSTRACT: AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK ?1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK ?1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK ?1 knockout cells. The transient expression of AMPK ?1 can rescue autophagosome maturation. These results indicate that AMPK ?1 is required for efficient autophagosome maturation and lysosomal fusion.

SUBMITTER: Jang M

PROVIDER: S-EPMC6107659 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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AMPK contributes to autophagosome maturation and lysosomal fusion.

Jang Minsu M Park Rackhyun R Kim Hyunju H Namkoong Sim S Jo Daum D Huh Yang Hoon YH Jang Ik-Soon IS Lee Jin I JI Park Junsoo J

Scientific reports 20180823 1

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-indep ...[more]

PMID: 30140075

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Project description:Crizotinib, a small-molecule tyrosine kinase inhibitor targeting ALK, MET and ROS1, is the first-line drug for ALK-positive metastatic non-small cell lung cancer and is associated with severe, sometimes fatal, cases of cardiac failure, which increases the risk of mortality. However, the underlying mechanism remains unclear, which causes the lack of therapeutic strategy. We established in vitro and in vivo models for crizotinib-induced cardiotoxicity and found that crizotinib caused left ventricular dysfunction, myocardial injury and pathological remodeling in mice and induced cardiomyocyte apoptosis and mitochondrial injury. In addition, we found that crizotinib prevented the degradation of MET protein by interrupting autophagosome-lysosome fusion and silence of MET or re-activating macroautophagy/autophagy flux rescued the cardiomyocytes death and mitochondrial injury caused by crizotinib, suggesting that impaired autophagy activity is the key reason for crizotinib-induced cardiotoxicity. We further confirmed that recovering the phosphorylation of PRKAA/AMPK (Ser485/491) by metformin re-activated autophagy flux in cardiomyocytes and metformin rescued crizotinib-induced cardiomyocyte injury and cardiac complications. In summary, we revealed a novel mechanism for crizotinib-induced cardiotoxicity, wherein the crizotinib-impaired autophagy process causes cardiomyocyte death and cardiac injury by inhibiting the degradation of MET protein, demonstrated a new function of impeded autophagosome-lysosome fusion in drugs-induced cardiotoxicity, pointed out the essential role of the phosphorylation of PRKAA (Ser485/491) in autophagosome-lysosome fusion and confirmed metformin as a potential therapeutic strategy for crizotinib-induced cardiotoxicity.Abbreviations and Acronyms: AAV: adeno-associated virus; ACAC/ACC: acetyl-Co A carboxylase; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; CHX: cycloheximide; CKMB: creatine kinase myocardial band; CQ: chloroquine; c-PARP: cleaved poly (ADP-ribose) polymerase; DAPI: 4'6-diamidino-2-phenylindole; EF: ejection fraction; FOXO: forkhead box O; FS: fractional shortening; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HF: heart failure; HW: TL: ratio of heart weight to tibia length; IR: ischemia-reperfusion; KEGG: Kyoto encyclopedia of genes and genomes; LAMP2: lysosomal-associated membrane protein 2; LDH: lactate dehydrogenase; MCMs: mouse cardiomyocytes; MMP: mitochondrial membrane potential; mtDNA: mitochondrial DNA; MYH6: myosin, heavy peptide 6, cardiac muscle, alpha; MYH7: myosin, heavy peptide 7, cardiac muscle, beta; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; PI: propidium iodide; PI3K: phosphoinositide 3-kinase; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; qPCR: quantitative real-time PCR; SD: standard deviation; SRB: sulforhodamine B; TKI: tyrosine kinase inhibitor; WGA: wheat germ agglutinin.

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AMPK contributes to autophagosome maturation and lysosomal fusion.

Publications

AMPK contributes to autophagosome maturation and lysosomal fusion.

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