Elaine Delvaux

Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation

DNA methylation is a vital component of the epigenetic machinery that orchestrates changes in multiple genes and helps regulate gene expression in all known vertebrates. We evaluated immunoreactivity for two markers of DNA methylation and eight methylation maintenance factors in entorhinal cortex layer II, a region exhibiting substantial Alzheimers disease (AD) pathology in which expression changes have been reported for a wide variety of genes. We show, for the first time, neuronal immunoreactivity for all 10 of the epigenetic markers and factors, with highly significant decrements in AD cases. These decrements were particularly marked in PHF1/PS396 immunoreactive, neurofibrillary tangle-bearing neurons. In addition, two of the DNA methylation maintenance factors, DNMT1 and MBD2, have been reported also to interact with ribosomal RNAs and ribosome synthesis. Consistent with these findings, DNMT1 and MBD2, as well as p66α, exhibited punctate cytoplasmic immunoreactivity that co-localized with the ribosome markers RPL26 and 5.8s rRNA in ND neurons. By contrast, AD neurons generally lacked such staining, and there was a qualitative decrease in RPL26 and 5.8s rRNA immunoreactivity. Collectively, these findings suggest epigenetic dysfunction in AD-vulnerable neurons.

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

Epigenetic dysregulation of gene expression is thought to be critically involved in the pathophysiology of Alzheimers 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.

Epigenetic mechanisms in Alzheimer's disease

Epigenetic modifications help orchestrate sweeping developmental, aging, and disease-causing changes in phenotype by altering transcriptional activity in multiple genes spanning multiple biologic pathways. Although previous epigenetic research has focused primarily on dividing cells, particularly in cancer, recent studies have shown rapid, dynamic, and persistent epigenetic modifications in neurons that have significant neuroendocrine, neurophysiologic, and neurodegenerative consequences. Here, we provide a review of the major mechanisms for epigenetic modification and how they are reportedly altered in aging and Alzheimers disease (AD). Because of their reach across the genome, epigenetic mechanisms may provide a unique integrative framework for the pathologic diversity and complexity of AD.

Aberrant intracellular localization of H3k4me3 demonstrates an early epigenetic phenomenon in Alzheimer's disease

We have previously reported in Alzheimers disease (AD) the mislocalization of epigenetic molecules between the cell nucleus and the cytoplasm. We have extended our finding to include the aberrant localization of histone 3 trimethylation on lysine 4 (H3k4me3), an epigenetic mark associated with actively transcribing genes as well as those poised for transcription. These findings raise the question of where the ectopic localization of H3k4me3 fits within the cascade of cell biological events in the progression of AD. We, therefore, examined the expression and intracellular location of H3k4me3 as a function of Braak stage and also in relation to a series of tau markers that are indicative of disease state. Both lines of evidence showed that ectopic localization of H3k4me3 is early in the course of disease. Because of the known role of H3k4me3 in the expression of synaptic genes, our data suggest an epigenetic role in synaptic deficits early in the course of AD.

Nuclear but not mitochondrial-encoded oxidative phosphorylation genes are altered in aging, mild cognitive impairment, and Alzheimer's disease

We have comprehensively described the expression profiles of mitochondrial DNA and nuclear DNA genes that encode subunits of the respiratory oxidative phosphorylation (OXPHOS) complexes (I–V) in the hippocampus from young controls, age matched, mild cognitively impaired (MCI), and Alzheimers disease (AD) subjects.

Differential processing of amyloid precursor protein in brain and in peripheral blood leukocytes

Because amyloid precursor protein (APP) fragments exist in many tissues throughout the body, including the fluid compartments of blood, they have been the focus of numerous investigations into their potential as a biomarker of Alzheimers disease. Using immunohistochemistry, immunoelectron microscopy, Western blot, and quantitative real-time-polymerase chain reaction (qRT-PCR) analysis we examined whether APP processing in leukocytes is analogous to APP processing in the brain. We show APP immunoreactivity at light and electron microscopic levels in the cytoplasm and nucleus of peripheral blood leukocytes (PBL) yet our Western blot analysis data demonstrated that brain and PBL contain different APP fragments and differentially expressed APP processing enzymes. A Disintegrin and Metalloproteinase domain 10 (ADAM10), nicastrin, and beta-secretase 2 (BACE2) were present in brain but were undetected in PBL. Presenilin 1 and beta-secretase 1 (BACE1) were detected in both tissues but showed different patterns in Western blots. Quantitative PCR results identified Neprilysin as the only processing enzyme we interrogated in which Western and quantitative PCR data coincided. Although our data on differential processing of APP in brain and PBL point to exercising caution when generalizing between blood and brain with regard to mechanisms, they have no implications regarding utility as biomarkers.

Laser-captured microglia in the Alzheimer's and Parkinson's brain reveal unique regional expression profiles and suggest a potential role for hepatitis B in the Alzheimer's brain

Expression array data from dozens of laboratories, including our own, show significant changes in expression of many genes in Alzheimers disease (AD) patients compared with normal controls. These data typically rely on brain homogenates, and information about transcripts specific to microglia and other central nervous system (CNS) cell types, which far outnumber microglia-specific transcripts, is lost. We therefore used single-cell laser capture methods to assess the full range of microglia-specific expression changes that occur in different brain regions (substantia nigra and hippocampus CA1) and disease states (AD, Parkinsons disease, and normal controls). Two novel pathways, neuronal repair and viral processing were identified. Based on KEGG analysis (Kyoto Encyclopedia of Genes and Genomes, a collection of biological pathways), one of the most significant viruses was hepatitis B virus (HBV) (false discovery rate < 0.00000001). Immunohistochemical analysis using HBV-core antibody in HBV-positive control, amnestic mild cognitive impairment, and HBV-positive AD cases show increased HBV immunoreactivity as disease pathology increases. These results are the first, to our knowledge, to show regional differences in human microglia. In addition, these data reveal new functions for microglia and suggest a novel risk factor for AD.

Nuclear but not mitochondrial-encoded OXPHOS genes are altered in aging, mild cognitive impairment, and Alzheimer's disease

We have comprehensively described the expression profiles of mitochondrial DNA and nuclear DNA genes that encode subunits of the respiratory oxidative phosphorylation (OXPHOS) complexes (I–V) in the hippocampus from young controls, age matched, mild cognitively impaired (MCI), and Alzheimers disease (AD) subjects.

The epigenetics of Alzheimer's disease — additional considerations

The critical commentaries following our review of the epigenetics of Alzheimers disease (AD) amplify a number of key points with respect to the role of 1-carbon metabolism, the phenomenon of allele-specific methylation, the potentially critical explanatory link provided by epigenetic mechanisms for genome-wide association and large-scale gene expression array studies, and new therapeutic approaches afforded by epigenetic manipulation in Alzheimers disease.

ANK1 is up-regulated in laser captured microglia in Alzheimer’s brain; the importance of addressing cellular heterogeneity

Recent epigenetic association studies have identified a new gene, ANK1, in the pathogenesis of Alzheimer’s disease (AD). Although strong associations were observed, brain homogenates were used to generate the data, introducing complications because of the range of cell types analyzed. In order to address the issue of cellular heterogeneity in homogenate samples we isolated microglial, astrocytes and neurons by laser capture microdissection from CA1 of hippocampus in the same individuals with a clinical and pathological diagnosis of AD and matched control cases. Using this unique RNAseq data set, we show that in the hippocampus, ANK1 is significantly (p<0.0001) up-regulated 4-fold in AD microglia, but not in neurons or astrocytes from the same individuals. These data provide evidence that microglia are the source of ANK1 differential expression previously identified in homogenate samples in AD.