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.

Figure 1

TNFα/zVAD induces necrosis in HT22 cells. (a, b) TNFα and zVAD dose–response curves. Cell death was assessed by propidium iodide (PI) and Hoechst staining. (c) Representative images of HT22 cells treated with DMSO or TNFα (1 ng/ml)/zVAD (50 μM) for 4 h. (d) HMGB1 translocation was detected in cells (blue: Hoechst dye; green: HMGB1 antibody), and in culture media by western blot. (e) Transmission electron microscopy of HT22 cells treated with TNFα/zVAD for 4 or 24 h shows necrotic morphology including swollen mitochondria, cytoplasmic clearing, membrane damage, and chromatinolysis. M, mitochondrion; N, nucleus. (All data are presented as mean±S.E.M. from 3–5 independent experiments. *P<0.05; **P<0.01; ***P<0.001 versus DMSO group.) Scale bars: c, 100 μm; d, 10 μm; e, 1 μm

Figure 2

Cell death induced by TNFα/zVAD in HT22 cells is necroptosis. (a) Dose dependent inhibition of TNFα/zVAD-induced cell death by necrostatin-1. Data are mean±S.E.M. from three independent experiments. *P<0.01; **P<0.001 versus TNFα (1 ng/ml)/zVAD (50 uM) (TZ) alone. (b) The specific RIPK1 kinase inhibitor, 7-Cl-O-necrostatin-1 (Nec-1) but not inactive analog (7-Cl-O-Nec-1; Nec-1i) prevented cell death induced by TNFα and zVAD. p=ns TZ versus TZ Nec-1i. (c) Knockdown of RIPK3 by specific siRNA suppressed TNFα/zVAD-induced cell death. *P<0.001 versus negative siRNA treated with TNFα/zVAD. (d) Western blot analysis of RIPK3 knockdown. (e) Representative immunoprecipitation experiment (n=3) showing RIPK1–RIPK3 complex assembly in TNFα/zVAD-treated HT22 cells. Complex assembly was inhibited by Nec-1. T, TNFa (1 ng/ml); TZN, TNFα (1 ng/ml)/zVAD (50 μM)/Nec-1(30 μM). Data are mean and S.E.M. of three independent experiments

Figure 3

Cell death induced by TNFα/zVAD is associated with oxidative stress. (a) Representative × 200 photomicrographs showing MitoSox Red and Hoechst fluorescence in HT22 cells 4 h after treatment with dimethylsulfoxide vehicle (DMSO), TNFα/zVAD (TZ), or TNFα/zVAD treatment in the presence or absence of Akt/mTOR inhibitors (TZAR, TZ+Akt inhibitor VIII (10 uM)+rapamycin (100 nM)) or necrostatin-1 (30 uM; TZN). (b) MitoSox-positive cells were quantitated by fluorescence microscopy. ANOVA P<0.0001. *P<0.05 versus TZ for all groups. Data are from four independent experiments. (c) Protective effects of betahydroxyanisole (BHA), LOXBlock-1 (LOX-1) inhibitor, baicalein, and rotenone on TNFα/zVAD-induced cell death. Data are from three independent experiments. *P<0.05; **P<0.01 versus TNFα/zVAD (TZ) group. TZN, TNFα/zVAD/Nec-1 (30 μM)

Figure 4

Activation of Akt/mTOR after TNFα/zVAD treatment. Representative immunoblots (a, b) and densitometry results (c, d) of Akt/mTOR activation in HT22 cells after TNFα/zVAD or vehicle dimethylsulfoxide treatment. (a) Time course of phosphorylation events downstream of Akt/mTOR (n=3 independent experiments). (c) Densitometry data from a. (b) Synergistic effect of TNFα and ZVAD is required to activate Akt/mTOR signaling in HT22 cells. (d) Densitometry data from b. (e) Representative western blot analysis of Akt phosphorylation 2 h after treatment with TNF/cyclohexamide to induce apoptotic cell death. No change in pAkt-473 or total Akt was observed using apoptotic stimuli. *P<0.05; **P<0.01; ***P<0.001 versus DMSO group. Data are from n=3 independent experiments. Abbreviations: phospho-Ser-473-Akt (p-Akt-473), phospho-Ser308-Akt (p-Akt-308), phospho-Ser9-GSK-3β (p-GSK-3β), phospho-Ser2448-mTOR (p-mTOR) and phospho-Ser235/236 S6 (p-S6)

Figure 5

Dual inhibition of Akt and mTOR inhibits necroptosis induced by TNFα/zVAD. (a) Akt and mTOR inhibitors reduced cell death induced by TNFα/zVAD as detected by propidium iodide (PI) and Hoechst staining. (b, c) Representative immunoblots and densitometry for phosphorylated Akt, GSK-3β, FoxO1, mTOR, and S6 in HT22 cells after TNFα/zVAD in the presence or absence of Akt inhibitor VIII (10 uM) and rapamycin (100 nM) (data are three independent experiments, *P<0.01; **P<0.001 compared with TZ group). Abbreviations: TNFα (1 ng/ml)/zVAD (50 μM)/Akt inhibitor VIII (10 μM) (TZA), TNFα (1 ng/ml)/zVAD (50 μM)/rapamycin (100 nM) (TZR), TNFα (1 ng/ml)/zVAD (50 μM)/Akt inhibitor VIII (10 μM)/ rapamycin (100 nM) (TZAR)

Figure 6

Knockdown of Akt and mTOR inhibits necroptosis induced by TNFα/zVAD. (a) HT22 cells expressed Akt1 and Akt2 but not Akt3 determined by immunoblot. Mouse lung fibroblasts were used as Akt3 positive control. (b, c) siRNA-mediated knockdown of mTOR, Akt1, and Akt2 in HT22 cells was confirmed by immunoblot using reagents identifying total Akt and mTOR. (d) Cell death was determined in control cells and in cells with knockdown of Akt1/2, mTOR, or triple knockdown of mTOR and Akt1/Akt2 together. Data are representative of three independent experiments, *P<0.05 versus control (CTL) siRNA

Figure 7

RIPK1–RIPK3 complex assembly occurs upstream of Akt/mTOR activation and reactive oxygen species (ROS) generation after TNFα/zVAD treatment. Interaction between RIPK1–RIPK3 (a) and RIPK1–Akt (b) was detected by immunoprecipitation (IP) and western blot analysis. (c) RIPK1–p-Akt-473 interaction (arrow) was also detected at 2 h after TNF/ZVAD (TZ) treatment. Detection of p-Akt-473 was abolished by pretreatment with necrostatin-1 (TZN, 30 uM). Control experiments showed the presence of RIPK1 in immunoprecipitants from all of the groups except those immunoprecipitated with irrelevant IgG. DMSO, dimethylsulfoxide control. Data are representative of three independent experiments