Etiology matters - genomic DNA methylation patterns in three rat models of acquired epilepsy

This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation, and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns

Alterations in miRNA Levels in the Dentate Gyrus in Epileptic Rats

<div><p>The aim of this study was to characterize changes in miRNA expression in the epileptic dentate gyrus. Status epilepticus evoked by amygdala stimulation was used to induce epilepsy in rats. The dentate gyri were isolated at 7 d, 14 d, 30 d and 90 d after stimulation (n=5). Sham-operated time-matched controls were prepared for each time point (n=5). The miRNA expression was evaluated using Exiqon microarrays. Additionally, mRNA from the same animals was profiled using Affymetrix microarrays. We detected miRNA expression signatures that differentiate between control and epileptic animals. Significant changes in miRNA expression between stimulated and sham operated animals were observed at 7 and 30 d following stimulation. Moreover, we found that there are ensembles of miRNAs that change expression levels over time. Analysis of the mRNA expression from the same animals revealed that the expression of several mRNAs that are potential targets for miRNA with altered expression level is regulated in the expected direction. The functional characterization of miRNAs and their potential mRNA targets indicate that miRNA can participate in several molecular events that occur in epileptic tissue, including immune response and neuronal plasticity. This is the first report on changes in the expression of miRNA and the potential functional impact of these changes in the dentate gyrus of epileptic animals. Complex changes in the expression of miRNAs suggest an important role for miRNA in the molecular mechanisms of epilepsy.</p> </div

Correlation matrix of expression levels between miRNAs with expression that differs significantly between sham and stimulated animals

<p>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076051#pone-0076051-t001" target="_blank">Table 1</a>.) The color and size of the circles in the matrix code for level of correlation; red represents positive correlation and blue represents negative correlation. Numerical values of correlations are presented in the lower left part of the matrix. Values that do not reach statistical significance are crossed. </p

Clustering analysis of the miRNA with altered expression levels in epileptic animals.

<p>(<b>A</b>) Clusters represent groups of miRNAs displaying similar alterations in expression over time after stimulation. Colors of lines within the clusters indicate the membership values of the expression profile to current cluster. Red and violet are high membership values and blue and green are low membership values. Members of each cluster are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076051#pone.0076051.s001" target="_blank">Table S1</a>. (<b>B</b>) Functional analysis of miRNA belonging to individual clusters. Functions associated with top networks according to Ingenuity Pathways are listed. (<b>C</b>) Functions belonging to “Neurological Disorder” and “Nervous System Development and Function” categories as defined by Ingenuity Pathways in individual miRNA clusters. Functions related to brain cancer and tumors are not included. Lists of functions with respective miRNAs are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076051#pone.0076051.s002" target="_blank">Table S2</a>.</p

Expression levels of selected miRNAs in individual sham-operated and stimulated animals.

<p>The bottom and the top of the box indicate the first and the third quartile, the band within the box indicates the median and the ends of the whiskers represent the lowest and the highest datum still within 1.5 IQR (interquartile range) of the lower and upper quartile, respectively. Each dot represents one animal. Blue are sham operated animals, and red are stimulated animals.</p

miRNA expression profiles in the dentate gyrus of epileptic and sham-operated control animals at different times after SE.

<p>(<b>A</b>) Principle Component Analysis (PCA) of microarray data derived from epileptic (red) and sham-operated control animals (blue) at 7 d (square), 14 d (circle), 30 d (triangle), and 90 d (cross) after status epilepticus. Each mark represents an individual animal. Note that epileptic animals are separate from the controls. (<b>B</b>) A heatmap of the 66 miRNAs with altered expression levels in epileptic animals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076051#pone-0076051-t001" target="_blank">Table 1</a>). Each column represents an individual animal and each row represents an individual miRNA. Colors on the heatmap represent the Z-score: higher – red, lower – green. Red color in the bar over the heatmap panel represents epileptic animals, and blue represents sham-operated control animals.</p

miR-124-3p is a chronic regulator of gene expression after brain injury

Traumatic brain injury (TBI) initiates molecular and cellular pathologies that underlie post-injury morbidities, including hippocampus-related memory decline and epileptogenesis. Non-coding small RNAs are master regulators of gene expression with the potential to affect multiple molecular pathways. To evaluate whether hippocampal gene expression networks are chronically regulated by microRNAs after TBI, we sampled the dentate gyrus of rats with severe TBI induced by lateral fluid-percussion injury 3 months earlier. Ingenuity pathway analysis revealed 30 upregulated miR-124-3p targets, suggesting that miR-124-3p is downregulated post-TBI (z-score = − 5.146, p < 0.05). Droplet digital polymerase chain reaction (ddPCR) and in situ hybridization confirmed the chronic downregulation of miR-124-3p (p < 0.05). Quantitative PCR analysis of two targets, Plp2 and Stat3, indicated that their upregulation correlated with the miR-124-3p downregulation (r = − 0.647, p < 0.05; r = − 0.629, p < 0.05, respectively). Immunohistochemical staining of STAT3 confirmed the increased protein expression. STRING analysis showed that 9 of the 30 miR-124-3p targets belonged to a STAT3 network. Reactome analysis and data mining connected the targets especially to inflammation and signal transduction. L1000CDS2 software revealed drugs (e.g., importazole, trichostatin A, and IKK-16) that could reverse the observed molecular changes. The translational value of our data was emphasized by in situ hybridization showing chronic post-traumatic downregulation of miR-124-3p in the dentate gyrus of TBI patients. Analysis of another brain injury model, status epilepticus, highlighted the fact that chronic downregulation of miR-124 is a common phenomenon after brain injury. Together, our findings indicate that miR-124-3p is a chronic modulator of molecular networks relevant to post-injury hippocampal pathologies in experimental models and in humans