A molecular technique for detecting the liberation of intracellular zinc in cultured neurons
A molecular technique for detecting the liberation of intracellular zinc in cultured neurons
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DOI：
10.1016/j.jneumeth.2004.02.018
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发表时间：
2004-08-30
影响因子：
3
通讯作者：
Aizenman, E
中科院分区：
文献类型：
--
作者：
Hara, H;Aizenman, E
我们之前报道过氧化刺激会从金属蛋白中释放 Zn2+，这种现象可以引发神经元细胞死亡。许多细胞类型中过量的细胞内 Zn2+ 通过金属反应元件 (MRE) 结合转录因子 1 (MTF-1) 的激活和核转位，触发编码金属结合蛋白（例如金属硫蛋白）的基因表达。 Cd2+ 强烈诱导非神经元细胞中 MTF-1 的核转位，但它是通过将 Zn2+ 从细胞内的金属结合位点取代并增加该离子的细胞内浓度来实现的。在这里，我们描述了使用 MRE 驱动的荧光素酶报告基因表达作为检测细胞内锌浓度增加的敏感分子测定。短暂暴露于 Zn2+ 或 Cd2+ 后，原代皮质神经元中会诱导 MRE 反式激活。当神经元同时暴露于 Cd2+ 和 NMDA 时，观察到 MRE 反式激活增强，因为这种金属可以渗透穿过受体通道。无论神经元是否与含有 MTF-1 的质粒共转染，都观察到荧光素酶表达，表明存在内源性 MTF-1 样蛋白。事实上，RT-PCR 显示 MTF-1 I mRNA 存在于神经元中。相反，MTF-1缺陷的dko7细胞仅在与MTF-1共转染时观察到MIZE反式激活。我们的结果表明，Cd2+ 可以通过从细胞内结合位点释放 Zn2+ 来有效诱导神经元中 MRE 的反式激活。 (C) 2004 Elsevier B.V. 保留所有权利。
We have previously reported that oxidative stimuli liberate Zn2+ from metalloproteins, a phenomenon that can trigger neuronal cell death. Excessive intracellular Zn2+ in many cell types triggers the expression of genes that encode metal binding proteins, such as metallothionein, via the activation and nuclear translocation of metal response element (MRE)-binding transcription factor-1 (MTF-1). Cd2+ strongly induces nuclear translocation of MTF-1 in non-neuronal cells, but it does so by displacing Zn2+ from its metal binding sites within the cell and increasing the intracellular concentration of this ion. Here, we describe the use of MRE-driven expression of a luciferase reporter gene as a sensitive molecular assay for detecting increases in intracellular zinc concentrations. MRE transactivation was induced in primary cortical neurons upon brief exposure to Zn2+ or Cd2+. Enhanced MRE transactivation was observed upon co-exposure of neurons to Cd2+ together with NMDA, as this metal can permeate through the receptor channel. Luciferase expression was observed regardless of whether or not neurons had been co-transfected with an MTF-1-containing plasmid, suggesting the presence of an endogenous MTF-1-like protein. Indeed, RT-PCR revealed that MTF-1 I mRNA is present in neurons. In contrast, MTF-1 deficient dko7 cells were only observed to have MIZE transactivation when co-transfected with MTF-1. Our results indicate that Cd2+ can effectively induce transactivation of MRE in neurons by liberating Zn2+ from its intracellular binding sites. (C) 2004 Elsevier B.V. All rights reserved.