Muñoz Patricia, Cardenas Sergio, Huenchuguala Sandro, Briceño Andrea, Couve Eduardo, Paris Irmgard, Segura-Aguilar Juan
*Department of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile; Faculty of Engineering, Central University of Chile; Department of Biology, University of Valparaiso, Chile; and Department Basic Sciences, University of Santo Tomas, Viña del Mar, Chile.
*Department of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile; Faculty of Engineering, Central University of Chile; Department of Biology, University of Valparaiso, Chile; and Department Basic Sciences, University of Santo Tomas, Viña del Mar, Chile *Department of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile; Faculty of Engineering, Central University of Chile; Department of Biology, University of Valparaiso, Chile; and Department Basic Sciences, University of Santo Tomas, Viña del Mar, Chile.
Toxicol Sci. 2015 May;145(1):37-47. doi: 10.1093/toxsci/kfv016. Epub 2015 Jan 28.
It was reported that aminochrome induces the formation of alpha synuclein (SNCA) oligomers during dopamine oxidation. We found that DT-diaphorase (NQO1) prevents the formation of SNCA oligomers in the presence of aminochrome determined by Western blot, transmission electron microscopy, circular dichroism, and thioflavin T fluorescence, suggesting a protective role of NQO1 by preventing the formation of SNCA oligomers in dopaminergic neurons. In order to test NQO1 protective role in SNCA neurotoxicity in cellular model, we overexpressed SNCA in both RCSN-3 cells (wild-type) and RCSN-3Nq7 cells, which have constitutive expression of a siRNA against NQO1. The expression of SNCA in RCSN-3SNCA and RCSN-3Nq7SNCA cells increased 4.2- and 4.4-fold, respectively. The overexpression of SNCA in RCSN-3Nq7SNCA cells induces a significant increase in cell death of 2.8- and 3.2-fold when they were incubated with 50 and 70 µM aminochrome, respectively. The cell death was found to be of apoptotic character determined by annexin/propidium iodide technique with flow cytometry and DNA laddering. A Western blot demonstrated that SNCA in RCSN-3SNCA is only found in monomer form both in the presence of 20 µM aminochrome or cell culture medium contrasting with RCSN-3Nq7SNCA cells where the majority SNCA is found as oligomer. The antioligomer compound scyllo-inositol induced a significant decrease in aminochrome-induced cell death in RCSN-3Nq7SNCA cells in comparison to cells incubated in the absence of scyllo-inositol. Our results suggest that NQO1 seems to play an important role in the prevention of aminochrome-induced SNCA oligomer formation and SNCA oligomers neurotoxicity in dopaminergic neurons.
据报道，在多巴胺氧化过程中，氨基色素会诱导α-突触核蛋白（SNCA）寡聚体的形成。我们发现，通过蛋白质免疫印迹法、透射电子显微镜、圆二色光谱和硫黄素T荧光检测发现，在有氨基色素存在的情况下，DT-黄递酶（NQO1）可阻止SNCA寡聚体的形成，这表明NQO1通过阻止多巴胺能神经元中SNCA寡聚体的形成发挥保护作用。为了在细胞模型中测试NQO1对SNCA神经毒性的保护作用，我们在RCSN-3细胞（野生型）和RCSN-3Nq7细胞中过表达SNCA，RCSN-3Nq7细胞组成型表达针对NQO1的小干扰RNA。RCSN-3SNCA和RCSN-3Nq7SNCA细胞中SNCA的表达分别增加了4.2倍和4.4倍。当RCSN-3Nq7SNCA细胞分别与50和70μM氨基色素孵育时，SNCA的过表达导致细胞死亡显著增加，分别为2.8倍和3.2倍。通过膜联蛋白/碘化丙啶技术结合流式细胞术和DNA梯状条带分析发现，细胞死亡具有凋亡特征。蛋白质免疫印迹法表明，在存在20μM氨基色素或细胞培养基的情况下，RCSN-3SNCA中的SNCA仅以单体形式存在，这与RCSN-3Nq7SNCA细胞形成对比，在RCSN-3Nq7SNCA细胞中，大多数SNCA以寡聚体形式存在。与未添加异肌醇孵育的细胞相比，抗寡聚体化合物异肌醇可显著降低氨基色素诱导的RCSN-3Nq7SNCA细胞死亡。我们的结果表明，NQO1似乎在预防氨基色素诱导的多巴胺能神经元中SNCA寡聚体形成和SNCA寡聚体神经毒性方面发挥重要作用。
