Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Cζ
Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Cζ
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DOI：
10.1046/j.1471-4159.2003.01846.x
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发表时间：
2003-07-01
影响因子：
4.7
通讯作者：
Koistinaho, J
中科院分区：
文献类型：
--
作者：
Koponen, S;Kurkinen, K;Koistinaho, J
刺激N-甲基-d-天冬氨酸(NMDA)受体的兴奋性毒性导致包括中风在内的脑损伤中的神经元死亡。一些证据表明，蛋白激酶C(PKC)亚型在NMDA兴奋性毒性中起作用。我们使用了经典PKCs的特异性多肽抑制剂(α、β和伽马)、新的PKCs Delta和epsilon以及非典型的PKcheeta，以确定哪些亚种参与了NMDA诱导的细胞死亡。从出生15天的小鼠胚胎制备神经细胞培养，并将其移植到星形胶质细胞单层上。体外培养2周后，将神经细胞暴露于100um N-甲基-D-天冬氨酸中5min，24 h后通过测定乳酸脱氢酶释放和双苯甲亚胺染色检测细胞存活率。当针对经典PKCs(α、β和伽马)或新型PKC(Delta或epsilon)的抑制剂没有作用时，PKcheeta抑制剂完全阻止NMDA诱导的神经元坏死性死亡。共聚焦显微镜证实，NMDA诱导了PKcheeta易位，该易位可被PKcheeta抑制剂阻断。这些多肽不影响NMDA引起的细胞内游离钙离子的变化。原位杂交实验证明，大鼠局灶性脑缺血后，大脑皮层中可表达PKcheeta基因。综上所述，这些结果表明，在NMDA诱导的皮层神经元死亡过程中，PKcheeta的激活是一个下游信号。
Excitotoxicity through stimulation of N -methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 mum NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.