Consistent decrease in global DNA methylation and hydroxymethylation in the hippocampus of Alzheimer's disease patients

Abstract

Epigenetic dysregulation of gene expression is thought to be critically involved in the pathophysiology of Alzheimer's disease (AD). Recent studies indicate that DNA methylation and DNA hydroxymethylation are 2 important epigenetic mechanisms that regulate gene expression in the aging brain. However, very little is known about the levels of markers of DNA methylation and hydroxymethylation in the brains of patients with AD, the cell-type specificity of putative AD-related alterations in these markers, as well as the link between epigenetic alterations and the gross pathology of AD. The present quantitative immunohistochemical study investigated the levels of the 2 most important markers of DNA methylation and hydroxymethylation, that is, 5-methylcytidine (5-mC) and 5-hydroxymethylcytidine (5-hmC), in the hippocampus of AD patients (n = 10) and compared these to non-demented, age-matched controls (n = 10). In addition, the levels of 5-hmC in the hippocampus of a pair of monozygotic twins discordant for AD were assessed. The levels of 5-mC and 5-hmC were furthermore analyzed in a cell-type and hippocampal subregion-specific manner, and were correlated with amyloid plaque load and neurofibrillary tangle load. The results showed robust decreases in the hippocampal levels of 5-mC and 5-hmC in AD patients (19.6% and 20.2%, respectively). Similar results were obtained for the twin with AD when compared to the non-demented co-twin. Moreover, levels of 5-mC as well as the levels of 5-hmC showed a significant negative correlation with amyloid plaque load in the hippocampus (r(p) = -0.539, p = 0.021 for 5-mC and r(p) = -0.558, p = 0.016 for 5-hmC). These human postmortem results thus strengthen the notion that AD is associated with alterations in DNA methylation and hydroxymethylation, and provide a basis for further epigenetic studies identifying the exact genetic loci with aberrant epigenetic signatures.

Fig. 1

Hippocampal 5-methylcytidine (5-mC) immunoreactivity (IR) and Neurotrace counterstain. Representative photomicrograph of 5-mC IR (green) and the corresponding Neurotrace image (red) taken from the CA1 hippocampal subregion. Based on the Neurotrace morphology, a total of 15 neurons and 15 glial cells were delineated within each photomicrograph, and their fluorescence intensity was analyzed using ImageJ software (details in text). Representative neurons are highlighted with a continuous white circle, whereas glial cells are highlighted with a dotted red circle. A total of 15 images per subject for each staining concomitant with 15 corresponding Neutrorace-counterstained images were analyzed. Scale bar = 50 μm.

Fig. 2

Representative photomicrographs of 5-mC immunoreactivity (IR). High magnification representative photomicrographs of the hippocampal DG, CA3, and CA1-2 regions. (A–C) Non-demented control cases (ND) and (D–F) Alzheimer’s disease cases (AD). Note: A loss of 5-mC IR is observed in AD cases when compared to ND controls in all 3 hippocampal subregions. Images were taken with a ×40 objective. Scale bar = 50 μm.

Fig. 3

Representative photomicrographs of 5-hydroxymethylcytosine (5-hmC) immunoreactivity (IR). High magnification representative photomicrographs of the hippocampal DG, CA3, and CA1-2 regions. (A–C) Non-demented control cases (ND) and (D–F) Alzheimer’s disease cases (AD). Note: A decrease of 5-hmC IR is observed in AD cases when compared to ND controls in all 3 hippocampal subregions. Images were taken with a ×40 objective. Scale bar = 50 μm.

Fig. 4

5-mC and 5-hmC hippocampal fluorescence intensities. Mean and standard error of the mean of fluorescence intensity measurements of 5-mC (A) and 5-hmC (B) immunoreactivity (IR). Pooled data from the 3 hippocampal subregions of non-demented control cases (ND; open bars) and Alzheimer’s disease cases (AD; filled bars). The percentage of decrease in each analysis and the significant effects (p < 0.05 in all cases) are indicated with an asterisk in each graph. Abbreviation: AU, arbitrary units.

Fig. 5

Stratified hippocampal 5-mC fluorescence intensities. Mean and standard error of the mean of fluorescence intensity measurements of 5-mC immunoreactivity (IR) (A–C). Pooled data from the non-demented control cases (ND; open bars) and Alzheimer’s disease cases (AD; filled bars) are represented separately for the DG (A), CA3 (B), and CA1-2 (C). The percentage of decrease in each analysis and the significant effects (p < 0.05 in all cases) are indicated with an asterisk in each graph. Abbreviation: AU, arbitrary units.

Fig. 6

Stratified hippocampal 5-hmC fluorescence intensities. Mean and standard error of the mean of fluorescence intensity measurements of 5-hmC immunoreactivity (IR) (A–C). Pooled data from the non-demented control cases (ND; open bars) and Alzheimer’s disease cases (AD; filled bars) are represented separately for the DG (A), CA3 (B), and CA1-2 (C). Percentage of decrease in each analysis and the significant effects (p < 0.05 in all cases) are indicated with an asterisk in each graph. Abbreviation: AU, arbitrary units.

Fig. 7

Hippocampal 5-hmC fluorescence intensities in the monozygotic twin pair discordant for Alzheimer’s disease cases (AD). Mean and standard error of the mean of fluorescence intensity measurements of 5-hmC immunoreactivity (IR) (A and B). Pooled data (A) and stratified data from neurons and glial cells (B) from the non-demented control case (ND; open bars) and Alzheimer’s disease case (twin) (AD; filled bars) are represented for the CA1 hippocampal subregion. Percentages of decrease in each analysis are indicated in each graph. Abbreviation: AU, arbitrary units.

Fig. 8

Correlation analysis between hippocampal 5-mC, 5-hmC immunoreactivity (IR) and amyloid plaque and neurofibrillary tangle loads. Spearman’s correlation coefficients and p values are noted in the bottom of each graph.