Proteomic analysis of microglial contribution to mouse strain-dependent dopaminergic neurotoxicity
Proteomic analysis of microglial contribution to mouse strain-dependent dopaminergic neurotoxicity
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作者：
Mclaughlin, P;Zhou, Y;Zhang, J
虽然帕金森病（PD）的发病机制仍不清楚，但在PD动物模型和PD患者中，小胶质细胞活化似乎与增强的神经变性相关。在实验中，C57 BL/6和SWR/J小鼠表现出由帕金森病毒物1-甲基-4-苯基.2，3，6-四氢吡啶（MPTP）诱导的多巴胺能（DA能）神经变性程度的显著差异。本研究的目的是确定这两种小鼠品系之间小胶质细胞活化的差异是否可以提供对毒物诱导的神经元死亡中观察到的变异性的了解，随后使用高通量蛋白质组学方法，将稳定同位素标记与细胞培养物中的氨基酸（SILAC）与液相色谱和串联质谱相结合，比较C57 BL/6和SWR/J小鼠在用经典的小胶质细胞激活剂脂多糖（LIPS）刺激后的小胶质细胞蛋白质组。我们发现，在原代神经元-小胶质细胞共培养中，LPS诱导的DA能神经毒性比从C57 BL/6小鼠脑中分离的小胶质细胞的毒性大2岁。在蛋白质组学分析后，我们发现，在鉴定和定量的1000多种蛋白质中，400种在这两种小鼠之间的相对丰度中显示出显著差异。在C57 BL/6小鼠中具有相对较高水平的几种蛋白质先前已经涉及LPS介导的小胶质细胞活化，包括参与考克斯-2途径和前列腺素E-2（PGE（2））产生的那些。为了验证我们的蛋白质组学结果，我们用半定量Western印迹证实了C57 BL/6与SWR/J小胶质细胞中iNOS表达水平的增加。我们的蛋白质组学发现数据的进一步分析可能会揭示许多新的蛋白质参与炎症介导的神经毒性在PD。(c)2006威利-利斯公司
Although the pathogenesis of Parkinson's disease (PD) remains unknown, it appears that microglial activation is associated with enhanced neurodegeneration in animal models of PD as well as in PD patients. Experimentally, C57BL/6 and SWR/J mice demonstrate striking differences in the extent of dopaminergic (DAergic) neurodegeneration induced by a parkinsonian toxicant 1-methyl-4-phenyl.2,3,6-tetrahydropyridine (MPTP). The purpose of this study was to determine whether differences in microglial activation between these two strains of mice could provide insight into the variability seen in toxicant induced neuronal death, and subsequently to use a high-throughput proteomic method, combining stable isotope labeling with amino acids in cell culture (SILAC) with liquid chromatography and tandem mass spectrometry, to compare the microglial proteomes of C57BL/6 and SWR/J mice after stimulation with a classical microglial activator, lipopolysaccharide (LIPS). We found that DAergic neurotoxicity induced by LPS in a primary neuron-microglia coculture was two old greater with microglia isolated from the brains of C57BL/6 mice compared with that of SWR/J mice. Upon proteomic analysis we found that, out of over 1000 proteins dentified and quantified, 400 displayed a significant difference in their relative abundance between these two murine;trains. Several proteins, which had relatively higher levels n C57BL/6 mice, have previously been implicated in LPS-mediated microglial activation, including those involved in;he COX-2 pathway and in prostaglandin E-2 (PGE(2)) production. To validate our proteomic results we confirmed the increased expression level of iNOS in C57BL/6 vs. SWR/J microglia with semiquantitative Western blot. Further analysis of our proteomic discovery data will likely reveal numerous novel proteins involved in inflammation-mediated neurotoxicity in PD. (c) 2006 Wiley-Liss, Inc.