Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.
The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia.
Katarina
K
Arsikin
Tamara
T
Kravic-Stevovic
Maja
M
Jovanovic
Biljana
B
Ristic
Gordana
G
Tovilovic
Nevena
N
Zogovic
Vladimir
V
Bumbasirevic
Vladimir
V
Trajkovic
Ljubica
L
Harhaji-Trajkovic
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000111:Acetylcysteine / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism*; D016627:Oxidopamine / Q000494:pharmacology*; D010766:Phosphorylation; D034741:RNA, Small Interfering; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D016627:Oxidopamine; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D055372:AMP-Activated Protein Kinases; D020123:Sirolimus; D000111:Acetylcysteine