Oxidatively modified RNA in mild cognitive impairment

Abstract

Studies show increased oxidative damage in the brains of subjects with Alzheimer's disease (AD) and mild cognitive impairment (MCI). To determine if RNA oxidation occurs in MCI, sections of hippocampus/parahippocampal gyrus (HPG) from 5 MCI, 5 late stage AD (LAD) and 5 age-matched normal control (NC) subjects were subjected to immunohistochemistry using antibodies against 8-hydroxyguanine (8-OHG) and 1-N2-propanodeoxyguanosine (NPrG). Confocal microscopy showed 8-OHG and NPrG immunostaining was significantly (p<0.05) elevated in MCI and LAD HPG compared with NC subjects and was predominately associated with neurons identified using the MC-1 antibody that recognizes conformational alterations of tau, which are associated with early neurofibrillary tangle formation. Pretreating sections with RNase or DNase-I showed immunostaining for both adducts was primarily associated with RNA. In addition, levels of both adducts in MCI were comparable to those measured in LAD, suggesting RNA oxidation may be an early event in the pathogenesis of neuron degeneration in AD.

Fig. 1

Confocal micrographs (100X objective) of representative sections of HPG immunostained for 1-N2-propanoguanosine, an exocyclic adduct formed between guanosine and acrolein, anα, β unsaturated aldehydic by-product of lipid peroxidation (1-A1), Tuj-1, a monoclonal antibody against neuron specific tubulin-III (Tuj-1; 1-A2; blue) and GFAP, an astrocyte specific marker (1-A3; red). Figure 1-A4 is a merged image. Note NPrG immunostaining is uniquely associated with β-tubulin-III-1-positive neurons and is minimally associated with GFAP-positive astrocytes. Figure 1-B shows an adjacent section of HPG immunostained for 8-OHG (1-B1; green), Tuj-1 (1-B2; blue) and GFAP (1-B3; red). Figure 1-B4 is a merged image. As was observed for NPrG, immunostaining for 8-OHG was specifically associated with Tuj-1-positive neurons and not with GFAP-positive astrocytes. Scale bar = 20 μm.

Fig. 2

Confocal micrographs of representative sections of MCI HPG immunostained for 1-N2-propanoguanosine (2-A1) and MC-1 (2-A2), an antibody that recognizes conformational changes in tau associated with early neurofibrillary tangle formation. Figure 2-A3 shows a merged image. Note NPrG immunostaining is associated with MC-1 positive neurons although we did observe pronounced NPrG immunostaining in several neurons with minimal MC-1 staining (arrows) suggesting oxidative damage to RNA may precede changes in tau conformation. Confocal images in 2-B show a representative section of MCI HPG pretreated with DNase-I-free RNase prior to immunostaining with MC-1 and NPrG. RNase treatment eliminated NPrG staining (2-B1) but not MC-1 staining (2-B2). Figure 2-C shows preincubation of the NPrG antibody with NPrG diminished immunostaining for the acrolein/guanine adduct (2-C1) but did not inhibit MC-1 staining (2-C2). The scale bar in all images = 50 μm.

Fig. 3

Mean ± SEM fluorescence intensities for NPrG and 8-OHG immunostaining. * p < 0.05.

Fig. 4

Confocal micrographs of representative sections of MCI HPG immunostained for 8-OHG (1) and MC-1 (2). Panel 3 shows a merged image. Figure 4-A1 shows 8-OHG immunostaining is present in neurons immuopositive for MC-1 (4-A2). Confocal images in Figure 4-B show a representative section of MCI HPG pretreated with DNase-I-free RNase prior to immunostaining with MC-1 and 8-OHG. RNase treatment essentially eliminated 8-OHG staining (4-B1) but not MC-1 staining (4-B2). Figure 4-C shows preincubation of the 8-OHG antibody with 8-hydroxyguanosine diminished immunostaining for the acrolein/guanosine adduct (4-C1) but did not inhibit MC-1 staining (4-C2). The scale bar in all images = 50 μm.