title: Akt and mTOR mediate programmed necrosis in neurons.
abstract: Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3β, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.
authors.affiliation: 1] Department of Pediatric Critical Care Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [3] Department of Anatomy, Histology and Embryology, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Q
authors.initials: Q
authors.lastname: Liu
authors.affiliation: 1] Department of Pediatric Critical Care Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Qiu
authors.affiliation: 1] Department of Pediatric Critical Care Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [3] Department of Rheumatology, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: M
authors.initials: M
authors.lastname: Liang
authors.affiliation: 1] Department of Pediatric Critical Care Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Golinski
authors.affiliation: 1] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: K
authors.initials: K
authors.lastname: van Leyen
authors.affiliation: 1] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Jung
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: You
authors.affiliation: 1] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: E H
authors.initials: EH
authors.lastname: Lo
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Degterev
authors.affiliation: 1] Department of Pediatric Critical Care Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA [2] Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Whalen
mesh_terms: D000818:Animals; D061945:Caspase Inhibitors / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006624:Hippocampus / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D009336:Necrosis; D009474:Neurons / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology; D018384:Oxidative Stress; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D038601:Ribosomal Protein S6 / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D061945:Caspase Inhibitors; D007093:Imidazoles; D007211:Indoles; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D038601:Ribosomal Protein S6; D014409:Tumor Necrosis Factor-alpha; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; C507699:necrostatin-1; C546843:mTOR protein, mouse; C514862:Akt1 protein, mouse; C494922:Akt2 protein, mouse; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; C485615:Ripk3 protein, mouse; D058570:TOR Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3