RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer's disease

Abstract

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.

Fig. 1.

Stratum pyramidale of the hippocampal CA1 field immunostained with 1F7 antibody. Oxidized nucleosides, 8OHdG and 8OHG, are abundant in the neuronal cytoplasm for a case of Alzheimer’s disease (age, 76 years; postmortem interval, 4 hr) (A,C, E) although it is virtually undetectable in a control (age, 80 years; postmortem interval, 4 hr) (B, D). Immunolabeling of the nucleoli and nuclear envelopes is moderate and chromatin is faint as compared with the intense reaction in the cytoplasm (C, E). The relative lack of DNA staining as compared with RNA staining can be noted for the nucleolus, whereas the small vacuolar region (arrow), which is rich in DNA, remains unstained, the outer region, which contains rRNA, is stained more strongly (E). Counterstaining with Congo red and viewing under plane polarized light shows birefringent neurofibrillary tangles (outlined with small arrowheads in two neurons) and shows that the immunoreaction is not only absent from neurofibrillary tangles but also reduced in the cytoplasm excluded from the neurofibrillary tangle (F). Images are shown with differential interference contrast optics, except forF. Scale bars: A, B, 100 μm; C, D, 50 μm; E,F, 10 μm.

Fig. 2.

Relative scale of 8OHdG and 8OHG immunoreactivity with 1F7 antibody in prosubiculum neurons of 4 young controls, 10 presenile controls, and 13 senile controls, as defined in Materials and Methods, and in 22 cases of AD. Values shown are the means with SE. The difference among all controls and AD is significant by ANOVA (p < 0.0001), with post hocanalysis showing significant differences between young controls and AD (p = 0.0015), between presenile controls and AD (p = 0.0001), and between senile controls and AD (p = 0.0001).

Fig. 3.

Neuronal immunolabeling with 1F7 antibody in AD (A) is abolished completely by adsorption with purified 8OHG (B). Images are shown with differential interference contrast optics. *indicates landmark blood vessel in adjacent section. Scale bar, 50 μm.

Fig. 4.

Immunoreaction with 1F7 antibody in AD (A) was diminished greatly by the RNase treatment (B) but only slightly by DNase I treatment (C). Arrows indicate the same neurons in adjacent serial sections. Images are shown with differential interference contrast optics. Scale bar, 50 μm.