Sanders Laurie H, McCoy Jennifer, Hu Xiaoping, Mastroberardino Pier G, Dickinson Bryan C, Chang Christopher J, Chu Charleen T, Van Houten Bennett, Greenamyre J T
Pittsburgh Institute for Neurodegenerative Diseases and Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Department of Genetics, Erasmus MC, Rotterdam, The Netherlands.
Neurobiol Dis. 2014 Oct;70:214-23. doi: 10.1016/j.nbd.2014.06.014. Epub 2014 Jun 27.
DNA damage can cause (and result from) oxidative stress and mitochondrial impairment, both of which are implicated in the pathogenesis of Parkinson's disease (PD). We therefore examined the role of mitochondrial DNA (mtDNA) damage in human postmortem brain tissue and in in vivo and in vitro models of PD, using a newly adapted histochemical assay for abasic sites and a quantitative polymerase chain reaction (QPCR)-based assay. We identified the molecular identity of mtDNA damage to be apurinic/apyrimidinic (abasic) sites in substantia nigra dopamine neurons, but not in cortical neurons from postmortem PD specimens. To model the systemic mitochondrial impairment of PD, rats were exposed to the pesticide rotenone. After rotenone treatment that does not cause neurodegeneration, abasic sites were visualized in nigral neurons, but not in cortex. Using a QPCR-based assay, a single rotenone dose induced mtDNA damage in midbrain neurons, but not in cortical neurons; similar results were obtained in vitro in cultured neurons. Importantly, these results indicate that mtDNA damage is detectable prior to any signs of degeneration - and is produced selectively in midbrain neurons under conditions of mitochondrial impairment. The selective vulnerability of midbrain neurons to mtDNA damage was not due to differential effects of rotenone on complex I since rotenone suppressed respiration equally in midbrain and cortical neurons. However, in response to complex I inhibition, midbrain neurons produced more mitochondrial H2O2 than cortical neurons. We report selective mtDNA damage as a molecular marker of vulnerable nigral neurons in PD and suggest that this may result from intrinsic differences in how these neurons respond to complex I defects. Further, the persistence of abasic sites suggests an ineffective base excision repair response in PD.
DNA损伤可导致氧化应激和线粒体损伤（同时也是氧化应激和线粒体损伤的结果），这两者都与帕金森病（PD）的发病机制有关。因此，我们使用一种新改良的针对无碱基位点的组织化学检测方法和一种基于定量聚合酶链反应（QPCR）的检测方法，研究了线粒体DNA（mtDNA）损伤在人类尸检脑组织以及PD的体内和体外模型中的作用。我们确定mtDNA损伤的分子特征为黑质多巴胺能神经元中的无嘌呤/无嘧啶（无碱基）位点，而在PD尸检标本的皮质神经元中则未发现。为了模拟PD的全身性线粒体损伤，将大鼠暴露于农药鱼藤酮。在未引起神经退行性变的鱼藤酮处理后，在黑质神经元中可观察到无碱基位点，而在皮质中则未观察到。使用基于QPCR的检测方法，单次鱼藤酮剂量可诱导中脑神经元中的mtDNA损伤，但在皮质神经元中未诱导；在体外培养的神经元中也获得了类似结果。重要的是，这些结果表明，在任何退变迹象出现之前即可检测到mtDNA损伤，并且在出现线粒体损伤的情况下，mtDNA损伤在中脑神经元中选择性产生。中脑神经元对mtDNA损伤的选择性易损性并非由于鱼藤酮对复合体I的不同作用，因为鱼藤酮对中脑和皮质神经元的呼吸抑制作用相同。然而，在复合体I受到抑制时，中脑神经元产生的线粒体过氧化氢比皮质神经元更多。我们报告选择性mtDNA损伤是PD中易损黑质神经元的分子标志物，并表明这可能是由于这些神经元对复合体I缺陷的反应存在内在差异所致。此外，无碱基位点的持续存在表明PD中碱基切除修复反应无效。
