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. 2013 Aug 1;4(8):e754.
doi: 10.1038/cddis.2013.280.

GADD34 induces cell death through inactivation of Akt following traumatic brain injury

J M Farook  1 , J Shields, A Tawfik, S Markand, T Sen, S B Smith, D Brann, K M Dhandapani, N Sen
Affiliations

GADD34 induces cell death through inactivation of Akt following traumatic brain injury

J M Farook et al. Cell Death Dis. .

Erratum in

  • Cell Death Dis. 2013;4:e813

Abstract

Neuronal cell death contributes significantly to the pathology of traumatic brain injury (TBI) irrespective of the mode or severity of the injury. Activation of a pro-survival protein, Akt, is known to be regulated by an E3 ligase TRAF6 through a process of ubiquitination-coupled phosphorylation at its T308 residue. Here we show that upregulation of a pro-apototic protein, GADD34, attenuates TRAF6-mediated Akt activation in a controlled cortical impact model of TBI in mice. TBI induces the expression of GADD34 by stimulating binding of a stress inducible transcription factor, ATF4, to the GADD34 promoter. GADD34 then binds with TRAF6 and prevents its interaction with Akt. This event leads to retention of Akt in the cytosol and prevents phosphorylation at the T308 position. Finally, in vivo depletion of GADD34 using a lentiviral knockdown approach leads to a rescue of Akt activation and markedly attenuates TBI-induced cell death.

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Figures

Figure 1
Figure 1
Inactivation of Akt is associated with cell death following TBI. (a) TUNEL staining was done to identify cell death at 12 and 24 h after TBI. Quantitative analysis shows that TUNEL staining was increased more than twofold after 24 h post TBI in the pericontusional cortex. (b and c) Phosphorylation of Akt (T308) was determined by western blot and immunofluorescent microscopy. Changes in phosphorylation status of Akt (P-Akt T308) was measured quantitatively. (d) Phosphorylation of downstream proteins of Akt, such as GSK3B, Foxo3a and BAD was determined by western blot analysis 12 and 24 h post TBI. (e) Membrane and cytosolic fraction of Akt was determined in the cortex at 12 and 24 h post Sham or TBI in mice. Level of both cytosolic and membrane Akt was determined at 12 and 24 h post TBI quantitatively. *P<0.01, n=3, one-way ANOVA, mean±S.E.M.
Figure 2
Figure 2
Effect of TRAF6 on Akt activation and cell death (a) Binding between TRAF6 and Akt was determined by co-immunoprecipitation assay at 12 and 24 h post TBI. (b) Ubiquitination assay of Akt was done using cell extracts with or without NMDA treatment. (c) Cell death was measured in primary neurons after overexpression of TRAF6 in the presence or absence of Akt inside cells. *P<0.01, n=3, one-way ANOVA, mean±S.E.M.
Figure 3
Figure 3
GADD34 interacts with TRAF6 following TBI (a and b) Interaction of GADD34 and TRAF6 was determined by both western blot analysis (a) and confocal microscopy (b) at 12 and 24 h post TBI. (c) Protein levels of GADD34 was monitored by immunofluorescent staining through confocal microscopy. (d) mRNA level of GADD34 was monitored by RT-PCR analysis at 12 and 24 h post TBI
Figure 4
Figure 4
Both ER and oxidative stress regulates GADD34 levels following TBI. (a) Changes in the level of phosphorylation of PERK (P-PERK) and eIF2alpha (P-eIF2alpha) were determined through western blot analysis. (b) Changes in oxidative stress associated with TBI was measured by staining coronal sections of cortex with H2DCFDA dye. The intensity was quantified. (c) Western blot analysis to measure protein level of ATF4 following sham and TBI. (d) ATF4 binding to GADD34 promoter was determined by chromatin immunoprecipitation analysis. (e) Effect of GSH and salubrinol on the level of ATF4 and GADD34 that was induced by NMDA treatment in primary neuron. *P<0.01, n=3, one-way ANOVA, mean±S.E.M.
Figure 5
Figure 5
GADD34 causes inactivation of Akt at post TBI. (a) Overexpression of Flag-GADD34 causes a decrease in TRAF6-Akt interaction in primary cortical neurons. (b) Western blot analysis of phopho-Akt (T308) in primary neurons with or without overexpression of Flag-GADD34. (c) Western blot analysis of Akt in membrane or cytosolic fractions with or without overexpression of Flag-GADD34. (d) Western blot analysis to detect interaction between TRAF6 and Akt after depletion of GADD34 in primary neurons. (e) Detection of phosphorylation status of Akt (T308) after doing RNAi knockdown of GADD34 in primary neurons. (f) Depletion of GADD34 causes a decrease in cell death in primary neurons after treatment with NMDA. *P<0.01, n=3, one-way ANOVA, mean±S.E.M.
Figure 6
Figure 6
Depletion of GADD34 provides neuroprotection against TBI (a) Confocal microscopic analysis to determine expression of lentiviral RNAi particles in cortex. (b) Western blot analysis to detect levels of GADD34 in sham and TBI samples. (c) Western blot analysis to measure interaction between TRAF6 and Akt at 12 and 24 h post TBI in mice overexpressing either control or GADD34 RNAi particles in brain. (d) Analysis of phosphorylation of Akt (T308) at 12 and 24 h post TBI in mice overexpressing either control or GADD34 RNAi particles in brain. (e) Depletion of GADD34 decreases the number of TUNEL-positive neurons in cortex at 12 and 24 h after TBI. *P<0.01, n=3, one-way ANOVA, mean±S.E.M.
Figure 7
Figure 7
Schematic representation of how inactivation of Akt leads to cell death following TBI. (a) Under Sham condition, an E3 ligase TRAF6 binds with Akt and facilitates its ubiquitination and phosphorylation to provide neuroprotection. However, under TBI condition (b), ATF4 levels are augmented, which leads to an increase in the level of GADD34 in cells. GADD34 competitively interacts with TRAF6 and prevents interaction between TRAF6 and Akt, which leads to cell death.
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References

    1. Nortje J, Menon DK. Traumatic brain injury: physiology, mechanisms, and outcome. Curr Opin Neurol. 2004;17:711–718. - PubMed
    1. Saatman KE, Duhaime AC, Bullock R, Maas AI, Valadka A, Manley GT. Classification of traumatic brain injury for targeted therapies. J Neurotrauma. 2008;25:719–738. - PMC - PubMed
    1. Maas AI, Stocchetti N, Bullock R. Moderate and severe traumatic brain injury in adults. Lancet Neurol. 2008;7:728–741. - PubMed
    1. Raghupathi R, Graham DI, McIntosh TK. Apoptosis after traumatic brain injury. J Neurotrauma. 2000;17:927–938. - PubMed
    1. Biegon A, Fry PA, Paden CM, Alexandrovich A, Tsenter J, Shohami E. Dynamic changes in N-methyl-D-aspartate receptors after closed head injury in mice: Implications for treatment of neurological and cognitive deficits. Proc Natl Acad Sci USA. 2004;101:5117–5122. - PMC - PubMed

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