Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by progressive neuropathology and cognitive decline. We describe a cross-tissue analysis of methylomic variation in AD using samples from three independent human post-mortem brain cohorts. We identify a differentially methylated region in the ankyrin 1 (ANK1) gene that is associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as significantly hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex) but not in the cerebellum, a region largely protected from neurodegeneration in AD, nor whole blood obtained pre-mortem, from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents the first epigenome-wide association study (EWAS) of AD employing a sequential replication design across multiple tissues, and highlights the power of this approach for identifying methylomic variation associated with complex disease.

We present robust evidence for elevated DNA methylation associated with Alzheimer's disease neuropathology spanning the HOXA gene cluster on chromosome 7. These data add to the growing evidence highlighting a role for epigenetic variation in Alzheimer's disease, implicating the HOX gene family as a target for future investigation.

Epigenetic processes play a key role in the central nervous system and altered levels of 5-methylcytosine have been associated with a number of neurological phenotypes, including Alzheimer’s disease (AD). Recently three additional cytosine modifications have been identified (5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine), which are thought to be intermediate steps in the demethylation of 5-methylcytosine to unmodified cytosine. Little is known about the frequency of these modifications in the human brain during health or disease. In this study we used immunofluorescence to confirm the presence of each modification in human brain and investigate their cross-tissue abundance in Alzheimer’s disease patients and elderly control samples. We identify a significant AD-associated decrease in global 5-hydroxymethylcytosine in entorhinal cortex and cerebellum, and differences in 5-formylcytosine levels between brain regions. Our study further implicates a role for epigenetic alterations in AD.

Although mutations within the TREM2 gene have been robustly associated with Alzheimer’s disease, it is not known whether alterations in the regulation of this gene are also involved in pathogenesis. Here we present data demonstrating increased DNA methylation in the superior temporal gyrus in AD brain at a CpG site located 289bp upstream of the transcription start site of the TREM2 gene in three independent study cohorts using two different technologies (Illumina Infinium 450K methylation beadchip and pyrosequencing). A meta-analysis across all three cohorts reveals consistent Alzheimer’s disease associated hypermethylation (p=3.47E-08). This study highlights that extending genetic studies of TREM2 in Alzheimer’s disease to investigate epigenetic changes may nominate additional mechanisms by which disruption to this gene increases risk.

Prolonged social isolation has negative effects on brain and behavior in humans and other social organisms, but neural mechanisms leading to these effects are not understood. Here we tested the hypothesis that even brief periods of social isolation can alter gene expression and DNA methylation in higher cognitive centers of the brain, focusing on the auditory/associative forebrain of the highly social zebra finch. Using RNA sequencing, we first identified genes that individually increase or decrease expression after isolation and observed general repression of gene sets annotated for neurotrophin pathways and axonal guidance functions. We then pursued 4 genes of large effect size: EGR1 and BDNF (decreased by isolation) and FKBP5 and UTS2B (increased). By in situ hybridization, each gene responded in different cell subsets, arguing against a single cellular mechanism. To test whether effects were specific to the social component of the isolation experience, we compared gene expression in birds isolated either alone or with a single familiar partner. Partner inclusion ameliorated the effect of solo isolation on EGR1 and BDNF, but not on FKBP5 and UTS2B nor on circulating corticosterone. By bisulfite sequencing analysis of auditory forebrain DNA, isolation caused changes in methylation of a subset of differentially expressed genes, including BDNF. Thus, social isolation has rapid consequences on gene activity in a higher integrative center of the brain, triggering epigenetic mechanisms that may influence processing of ongoing experience.