CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
Free Radic Biol Med. 2012 Nov 15;53(10):1857-67. doi: 10.1016/j.freeradbiomed.2012.09.004. Epub 2012 Sep 14.
Huntington's disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and γ-glutamyl transpeptidase (γ-GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.
亨廷顿病（HD）是一种 CAG 重复紊乱，影响 HD 基因，该基因编码 huntingtin（Htt），其特征是纹状体中明显的细胞死亡。氧化应激先前与 HD 神经退行性变有关，但在表达全长突变 Htt（FL-mHtt）后，主要内源性抗氧化系统谷胱甘肽氧化还原循环的作用研究较少。因此，在这项工作中，我们分析了来自表达突变 Htt 的 HD 基因敲入小鼠的纹状体细胞中的谷胱甘肽系统与野生型细胞相比。突变细胞显示细胞内活性氧（ROS）和半胱天冬酶-3 活性增加，用谷胱甘肽乙酯处理后明显预防。有趣的是，突变细胞显示出还原和氧化形式的谷胱甘肽的细胞内水平增加，并且谷胱甘肽过氧化物酶（GPx）和谷胱甘肽还原酶（GRed）的活性增强。此外，突变细胞中的谷胱甘肽-S-转移酶（GST）和γ-谷氨酰转肽酶（γ-GT）活性也增加。然而，在表达 FL-mHtt 的细胞中，谷氨酸-半胱氨酸连接酶（GCL）和谷胱甘肽合成酶（GS）活性和 GCL 催化亚基水平降低，高度表明谷胱甘肽的从头合成减少。尽管合成减少，但增强的细胞内总谷胱甘肽可能是由于突变细胞中外源性谷胱甘肽减少所致。这与多药耐药蛋白 1（Mrp1）的 mRNA 表达水平和活性降低同时发生，Mrp1 是一种转运蛋白，介导细胞内谷胱甘肽二硫化物和谷胱甘肽缀合物的细胞输出。此外，仅在野生型细胞中抑制 Mrp1 增强了细胞内 GSH。这些数据表明，FL-mHtt 通过降低 Mrp1 的表达来影响谷胱甘肽的输出。数据进一步表明，FL-mHtt 诱导的与 GSH 相关的抗氧化防御机制的增强不足以抵消 HD 纹状体细胞中增加的 ROS 形成和新兴的凋亡特征。
