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

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

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

Chronically dysregulated NOTCH1 interactome in the dentate gyrus after traumatic brain injury

<div><p>Traumatic brain injury (TBI) can result in several dentate gyrus-regulated disabilities. Almost nothing is known about the chronic molecular changes after TBI, and their potential as treatment targets. We hypothesized that chronic transcriptional alterations after TBI are under microRNA (miRNA) control. Expression of miRNAs and their targets in the dentate gyrus was analyzed using microarrays at 3 months after experimental TBI. Of 305 miRNAs present on the miRNA-array, 12 were downregulated (p<0.05). In parallel, 75 of their target genes were upregulated (p<0.05). A bioinformatics analysis of miRNA targets highlighted the dysregulation of the transcription factor NOTCH1 and 39 of its target genes (NOTCH1 interactome). Validation assays confirmed downregulation of miR-139-5p, upregulation of <i>Notch1</i> and its activated protein, and positive enrichment of NOTCH1 target gene expression. These findings demonstrate that miRNA-based transcriptional regulation can be present at chronic time points after TBI, and highlight the NOTCH1 interactome as one of the mechanisms behind the dentate gyrus pathology-related morbidities.</p></div

Expression change and genomic location of differentially regulated microRNAs (miRNAs) in the dentate gyrus at 3 months after Traumatic Brain Injury (TBI).

<p><b>(A)</b> A heat map showing 12 downregulated miRNAs after TBI. Micro RNA names and the heat map color key are shown on the right side of the panel. Each column represents one animal and each row one miRNA. Data was clustered using Euclidean distance measurement. The dendrogram on the top shows the clustering of animals into two groups. Cluster 1 (left) contains 4 TBI rats and cluster 2 (right) contains all 5 controls and 2 TBI animals. Rats in cluster 1 have a clearly downregulated miRNA expression profile (lower expression indicated in blue) as compared to cluster 2. The dendrogram on the left side of the panel clusters the miRNAs into the groups. Color bars between the dendrogram and heat map indicate a cell type in which the particular miRNA is expressed according to previous experiments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172521#pone.0172521.ref036" target="_blank">36</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172521#pone.0172521.ref037" target="_blank">37</a>]. Purple color refers to miRNAs which under normal conditions are neuron-enriched even though they can be present also in other brain cell types (neurons, astrocytes, microglia or oligodendrocytes). (<b>B)</b> A circos plot showing the genomic location (rat genome version RN5) of downregulated miRNAs and mRNAs from the same samples. Of the 12 differentially expressed miRNAs, 7 (58%) belonged to two neuron-enriched clusters at chromosome 6 (Chr6q32). Abbreviations: Max, maximum row z-value; Min, minimum row z-value; TBI, traumatic brain injury.</p

Downregulation of mir-139-5p was associated with an upregulation of the transcription factor <i>Notch1</i>.

<p><b>(A)</b> STRING analysis revealed that NOTCH1 had the most abundant protein-protein interactions with other miRNA-regulated proteins (8/23 interactions). For clarity, proteins without any interactions with other miRNA targets were omitted from the figure. Red color-coding indicates upregulated genes (1.6–1.1-fold as compared to controls, p<0.05). Darker the red color, more upregulated the gene expression. <b>(B).</b> Gene Set Enrichment Analysis (GSEA) showed positive enrichment of 48% (39/82) known (IPA^® database) NOTCH1 target genes in the mRNA microarray (Rank numbers 1-3448/11260, FDR<0.01, enrichment score 0.42), suggesting activation of NOTCH1 protein. <b>(C)</b> Upregulation of <i>Notch1</i> gene expression on the microarray (fold-change 1.16, p<0.01) was replicated by quantitative reverse transcription PCR (RT-qPCR; fold-change 1.53, p<0.01). <b>(D)</b> Downregulated miR-139-5p expression on the microarray (fold-change 0.76, p<0.05). Analysis of miR-139-5p expression with droplet digital PCR (ddPCR) revealed a trend towards downregulation (fold-change 0.83). <b>(E)</b> <i>Notch1</i> and miR-139-5p expression levels were tightly correlated on array analysis (r = -0.80, p<0.01), and importantly, also in <b>(F)</b> PCR replication analysis (r = -0.61, p<0.05). <b>(G)</b> Factor analysis was used to combine the microarray intensity for all 12 downregulated miRNAs to reduce the data, and analysis of the first principal component to <i>Notch1</i> expression led to an even better correlation (r = 0.9, p<0.01) than for miR-139-5p alone. This indicates that one or more other miRNAs regulates the expression of <i>Notch1</i>, either directly or indirectly via their target genes. Statistical significance: *, p<0.05; **, p<0.01; ***, p<0.001. Abbreviations: ddPCR, droplet digital PCR; FAC_1, 1^st principal component from the factor analysis; TBI, traumatic brain injury; r, correlation coefficient; RT-qPCR, quantitative reverse-transcription PCR.</p

Flow-charts for the study design.

<p>Flow-charts showing the randomization of <b>(A)</b> 46 rats into the microRNA, transcriptomics, and immunohistochemical analysis and <b>(B)</b> 11 rats into the bioinformatics and validation experiments. Abbreviations: ddPCR, droplet digital PCR; FPI, fluid-percussion injury; GSEA, Gene Set Enrichment Analysis; IH, immunohistochemistry; IPA, Ingenuity Pathway Analysis; qPCR, quantitative PCR.</p

Neuronal loss after Traumatic Brain Injury (TBI).

<p>Representative photomicrographs of a thionin-stained section from the dentate gyrus of <b>(A)</b> a sham-operated control rat and <b>(B)</b> a rat with lateral fluid-percussion induced (FPI) TBI 3 months earlier. Note that the principal cells located in the granule cell layer and proximal CA3c are well preserved. The loss of hilar neurons, however, was remarkable. Dashed outline shows the dissected tissue used for mRNA and microRNA array analysis. <b>(C)</b> A heat map presenting the gene expression of neuronal marker <i>Hrnbp3</i> and 7 interneuronal markers (<i>Gad1</i>, <i>Gad2</i>, <i>Pvalb</i>, <i>Sst</i>, <i>Cck</i>, <i>Calb2</i>, and <i>Npy</i>) as well as the expression of the 12 downregulated miRNAs. Two rats with TBI clustered together with the controls. Expression of the <i>Hrnbp3</i> gene in these animals was not changed, whereas the gene expression of interneuronal markers was remarkably decreased. Abbreviations: g, granule cell layer; h, hilus; Max, maximum; Min, minimum; mol, molecular layer; TBI, traumatic brain injury.</p