Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia.
Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia.
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DOI：
10.1038/cddis.2014.152
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发表时间：
2014-04-17
影响因子：
9
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缺血诱导的细胞死亡是中风后残疾或死亡的主要原因。确定缺血诱导的关键内在保护机制对于开发有效的卒中治疗至关重要。在此，我们报道了14-3-3γ是大脑皮层神经元中选择性缺血诱导的存活因子，其通过下调细胞核中Bax依赖的14-3-3γ/β-catenin Ser 37直接相互作用来减少细胞死亡。通过定量PCR、western blot和荧光免疫染色测定，14- 3-3γ在原代大脑皮质神经元中在氧-葡萄糖剥夺（OGD）后上调，而其他14-3-3亚型则没有。通过在体缺血性中风模型证实了OGD对皮质神经元14-3-3γ的选择性诱导。单独敲低14-3-3γ或抑制14-3-3/受体相互作用足以诱导正常培养的神经元中的细胞死亡并加剧OGD诱导的神经元死亡。在培养的神经元中，14-3-3γ的异位过表达显著减少OGD诱导的细胞死亡。免疫共沉淀和荧光共振能量转移证实内源性14-3-3γ直接结合更多的β-catenin Ser 37，而不结合p-Bad、p-Ask-1、p-p53和Bax。在OGD过程中，β-catenin Ser 37显著增加，而β-catenin Ser 45无明显变化，这与Bax升高有关。OGD促进14-3-3γ进入细胞核，与细胞核p-β-catenin Ser 37的增加有关。在皮质神经元中，14-3-3γ的过表达显著降低Bax表达，而14-3-3γ的敲低增加Bax表达。在Ser 37（S37 A）处消除β-连环蛋白磷酸化显著减少了OGD后神经元中的Bax和细胞死亡。最后，14-3-3γ过表达完全抑制了β-连环蛋白增强的Bax和OGD后神经元中的细胞死亡。基于这些数据，我们提出14-3-3γ/p-β-catenin Ser 37/Bax轴决定了缺血期间神经元的存活或死亡，为缺血性卒中以及其他相关神经系统疾病提供了新的治疗靶点。
Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen–glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia, providing novel therapeutic targets for ischemic stroke as well as other related neurological diseases.