PILOT STUDY--ASTROCYTE GENE EXPRESSION AND METHYL MERCURY NEUROTOXICITY

甲基汞中毒是有据可查的化学物质。 人类历史上的灾难。大量使用甲基汞的理由 对中枢神经系统(CNS)的毒性受到强烈支持 通过体内和体外研究。只是最近才有 星形胶质细胞被发现优先积聚甲基汞和它们的 甲基汞致神经细胞反应的病因学研究 毒性。暴露于甲基汞可导致星形胶质细胞肿胀 伴随谷氨酸摄取的抑制，调节的变化 体积减少，兴奋性氨基酸释放增加 (EaaS)，如谷氨酸。这一理论的主要工作假设 协作性提案假设， 甲基汞与细胞外液浓度升高有关 谷氨酸，反过来，导致激活神经元的N-甲基-D- 天冬氨酸受体。为增加的 胞外谷氨酸的浓度包括酶的改变 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体的水平。我们最初的 该方法将应用最先进的分子生物学 谷氨酰胺合成酶和谷氨酰胺酶的分离方法 研究它们在急性、慢性和原位甲基汞中的活性 暴露的星形胶质细胞。谷氨酰胺合成酶、谷氨酰胺酶水平 谷氨酸转运体将由Western 印迹技术。这两种基因在对照组中的表达 甲基汞处理的细胞将与可获得性相关 各自的信使RNA，通过RNA过量来量化 溶液杂交法。我们还将研究是否可能 谷氨酰胺合成酶编码基因的突变， 谷氨酰胺酶与谷氨酸转运体的PCR-SSCP研究 (聚合酶链式单链构象多态)和 甲基汞暴露组和对照组星形胶质细胞的双脱氧DNA测序。 这项合作研究的总体长期目标是 继续阐明甲基汞的神经毒性机制和 星形胶质细胞失控的潜在有害影响 谷氨酸动态平衡在其病因中的作用。

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.