PILOT STUDY--ASTROCYTE GENE EXPRESSION AND METHYL MERCURY NEUROTOXICITY

试点研究--星形胶质细胞基因表达和甲基汞神经毒性

• 批准号: 6123348
• 负责人: KIM H TAN
• 金额: $ 17.24万
• 依托单位: WINSTON-SALEM STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 1999-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6123348
• 关键词: NMDA receptors; astrocytes; cytotoxicity; environmental toxicology; enzyme activity; gene expression; gene mutation; glutaminase; glutamine; laboratory rat; methylmercury; neurotoxicology; nucleic acid sequence; polymerase chain reaction; single strand conformation polymorphism; solution hybridization; western blottings

Methylmercury (MeHg) poisonings are well documented chemical disasters in human history. The case for significant MeHg toxicity to the central nervous system (CNS) is strongly supported by both in vivo and in vitro studies. Only recently have astrocytes been found to preferentially accumulate MeHg and their responses considered in the etiology of MeHg-induced neuronal toxicity. Exposure to MeHg results in astrocytic swelling with concomitant inhibition of glutamate uptake, changes in regulatory volume decrease, and increased release of excitatory amino acids (EAAs), such as glutamate. The primary working hypothesis of this collaborative proposal postulates that the neurotoxic effect of MeHg is associated with elevated concentrations of extracellular glutamate, in turn, leading to activation of neuronal N-methyl-D- aspartate receptors. Mechanisms invoked for the increased concentration of extracellular glutamate include altered enzymatic activities of glutamine synthetase and glutaminase, as well as the levels of these enzymes and glutamate transporters. Our initial approach will e apply state-of-the-art molecular biology approaches to isolate glutamine synthetase and glutaminase, and study their activities in acutely-, chronically-, and in situ-MeHg exposed astrocytes. Levels of glutamine synthetase, glutaminase and the glutamate transporters will be determined by Western blotting technique. Expression of these genes both in controls and MeHg-treated cells will be correlated to the availability of the respective messenger RNA, quantified by means of RNA-excess solution hybridization method. We will also study possible mutations of the genes coding for glutamine synthetase, glutaminase and the glutamate transporters by means of PCR-SSCP (PCR-directed single strand conformational polymorphism) and dideoxy DNA sequencing in controls and MeHg exposed astrocytes. The overall long term objective of this collaborative research is to continue to elucidate the mechanisms of MeHg neurotoxicity and the potential deleterious effects of astrocytic failure to control glutamate homeostasis in its etiology.

甲基汞 (MeHg) 中毒是有据可查的化学物质 人类历史上的灾难。 大量甲基汞的情况 强烈支持对中枢神经系统（CNS）的毒性 通过体内和体外研究。 直到最近才有 研究发现星形胶质细胞优先积累甲基汞及其 甲基汞诱发神经元病因学中考虑的反应 毒性。 接触甲基汞会导致星形胶质细胞肿胀 谷氨酸吸收的同时抑制，调节的变化 体积减少，兴奋性氨基酸释放增加 (EAA)，例如谷氨酸。 本研究的主要工作假设 合作提案假设神经毒性作用 MeHg 与细胞外浓度升高有关 谷氨酸反过来导致神经元 N-甲基-D- 的激活 天冬氨酸受体。 为增加而调用的机制 细胞外谷氨酸浓度包括改变的酶 谷氨酰胺合成酶和谷氨酰胺酶的活性，以及 这些酶和谷氨酸转运蛋白的水平。 我们最初的 该方法将应用最先进的分子生物学 分离谷氨酰胺合成酶和谷氨酰胺酶的方法，以及 研究它们在急性、慢性和原位甲基汞中的活动 暴露的星形胶质细胞。 谷氨酰胺合成酶、谷氨酰胺酶水平 谷氨酸转运蛋白将由西方决定 印迹技术。 这些基因在对照中的表达 和经甲基汞处理的细胞将与以下物质的可用性相关： 各自的信使 RNA，通过 RNA 过量进行定量 溶液杂交法。 我们也会研究可能的 编码谷氨酰胺合成酶的基因突变， 通过 PCR-SSCP 检测谷氨酰胺酶和谷氨酸转运蛋白 （PCR 指导的单链构象多态性）和 对对照和暴露于甲基汞的星形胶质细胞进行双脱氧 DNA 测序。 这项合作研究的总体长期目标是 继续阐明甲基汞神经毒性的机制 星形胶质细胞失控的潜在有害影响 谷氨酸稳态的病因学。