Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury

Long-term molecular changes in the brain resulting from blast exposure may be mediated by epigenetic changes, such as deoxyribonucleic acid (DNA) methylation, that regulate gene expression. Aberrant regulation of gene expression is associated with behavioral abnormalities, where DNA methylation bridges environmental signals to sustained changes in gene expression. We assessed DNA methylation changes in the brains of rats exposed to three 74.5 kPa blast overpressure events, conditions that have been associated with long-term anxiogenic manifestations weeks or months following the initial exposures. Rat frontal cortex eight months post-exposure was used for cell sorting of whole brain tissue into neurons and glia. We interrogated DNA methylation profiles in these cells using Expanded Reduced Representation Bisulfite Sequencing. We obtained data for millions of cytosines, showing distinct methylation profiles for neurons and glia and an increase in global methylation in neuronal versus glial cells (p<10(-7)). We detected DNA methylation perturbations in blast overpressure-exposed animals, compared with sham blast controls, within 458 and 379 genes in neurons and glia, respectively. Differentially methylated neuronal genes showed enrichment in cell death and survival and nervous system development and function, including genes involved in transforming growth factor β and nitric oxide signaling. Functional validation via gene expression analysis of 30 differentially methylated neuronal and glial genes showed a 1.2 fold change in gene expression of the serotonin N-acetyltransferase gene (Aanat) in blast animals (p<0.05). These data provide the first genome-based evidence for changes in DNA methylation induced in response to multiple blast overpressure exposures. In particular, increased methylation and decreased gene expression were observed in the Aanat gene, which is involved in converting serotonin to the circadian hormone melatonin and is implicated in sleep disturbance and depression associated with traumatic brain injury.