Nunomura A, Perry G, Pappolla M A, Wade R, Hirai K, Chiba S, Smith M A
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Neurosci. 1999 Mar 15;19(6):1959-64. doi: 10.1523/JNEUROSCI.19-06-01959.1999.
In this study we used an in situ approach to identify the oxidized nucleosides 8-hydroxydeoxyguanosine (8OHdG) and 8-hydroxyguanosine (8OHG), markers of oxidative damage to DNA and RNA, respectively, in cases of Alzheimer's disease (AD). The goal was to determine whether nuclear and mitochondrial DNA as well as RNA is damaged in AD. Immunoreactivity with monoclonal antibodies 1F7 or 15A3 recognizing both 8OHdG and 8OHG was prominent in the cytoplasm and to a lesser extent in the nucleolus and nuclear envelope in neurons within the hippocampus, subiculum, and entorhinal cortex as well as frontal, temporal, and occipital neocortex in cases of AD, whereas similar structures were immunolabeled only faintly in controls. Relative density measurement showed that there was a significant increase (p < 0.0001) in 8OHdG and 8OHG immunoreactivity with 1F7 in cases of AD (n = 22) as compared with senile (n = 13), presenile (n = 10), or young controls (n = 4). Surprisingly, the oxidized nucleoside was associated predominantly with RNA because immunoreaction was diminished greatly by preincubation in RNase but only slightly by DNase. This is the first evidence of increased RNA oxidation restricted to vulnerable neurons in AD. The subcellular localization of damaged RNA showing cytoplasmic predominance is consistent with the hypothesis that mitochondria may be a major source of reactive oxygen species that cause oxidative damage in AD.
在本研究中,我们采用原位方法,在阿尔茨海默病(AD)病例中鉴定氧化核苷8-羟基脱氧鸟苷(8OHdG)和8-羟基鸟苷(8OHG),它们分别是DNA和RNA氧化损伤的标志物。目的是确定AD中核DNA、线粒体DNA以及RNA是否受损。在AD病例中,海马体、海马下托、内嗅皮质以及额叶、颞叶和枕叶新皮质的神经元细胞质中,与识别8OHdG和8OHG的单克隆抗体1F7或15A3的免疫反应性显著,在核仁和核膜中的反应性较弱,而在对照中,类似结构的免疫标记很微弱。相对密度测量显示,与老年对照组(n = 13)、早老对照组(n = 10)或年轻对照组(n = 4)相比,AD病例(n = 22)中与1F7的8OHdG和8OHG免疫反应性显著增加(p < 0.0001)。令人惊讶的是,氧化核苷主要与RNA相关,因为在RNase中预孵育后免疫反应大大减弱,而在DNase中预孵育后仅略有减弱。这是AD中仅局限于易损神经元的RNA氧化增加的首个证据。受损RNA的亚细胞定位显示以细胞质为主,这与线粒体可能是AD中导致氧化损伤的活性氧主要来源的假设一致。