Dynamic miRNA changes during the process of epileptogenesis in an infantile and adult-onset model

Abstract

Temporal lobe epilepsy (TLE) is the most common epilepsy type. TLE onset in infancy aggravates features like severity, drug responsiveness, or development of comorbidities. These aggravations may arise from altered micro RNA (miRNA) expression specific to the early onset of the disease. Although the miRNA involvement in TLE is widely studied, the relationship between the onset-age and miRNA expression has not been addressed. Here, we investigated the miRNA profile of infantile and adult-onset TLE in rats combining sequencing and PCR. Since miRNA expression changes with the disease progression, we scrutinized miRNA dynamics across three stages: acute, latent, and chronic. We report that infantile-onset TLE leads to changes in the expression of fewer miRNAs across these stages. Interestingly, the miRNA profile in the acute stage of infantile-onset TLE overlaps in dysregulation of miR-132-5p, -205, and -211-3p with the chronic stage of the disease starting in adulthood. The analysis of putative targets linked the majority of dysregulated miRNAs with pathways involved in epilepsy. Our profiling uncovered miRNA expression characteristic for infantile and adulthood-onset epileptogenesis, suggesting the distinct biology underlying TLE in the onset age-dependent matter. Our results indicate the necessity of addressing the onset age as an important parameter in future epilepsy research.

Figure 1

Timelines and Multidimensional Scaling (MDS) of complete miRNA expression. Timelines depict the age of animals (as a number of postnatal days P) at the status epilepticus (SE) induction and tissue collection separately for individual onset ages (infancy and adulthood). Tissue collection timepoints (24 h, 7 days, and 3 months) correspond with epileptogenesis stages: acute, latent, and chronic. The MDS plot is a clustering method to compute the relative variability of expression among samples and visualize the level of their similarity. The MDS plots were constructed using miRNA expression data (normalized read count from sequencing) and used “Biological coefficient of variation” (BCV) to compute distances. Points represent samples of the adult-onset (blue) and infantile-onset (violet) onset of epilepsy and their controls (adult—green, infantile—red). Individual plots depict clustering in a specific stage of epileptogenesis.

Figure 2

Expression changes of all miRNAs detected in adult and infantile-onset of epileptogenesis. The volcano plots display changes in miRNA expression after SE induced in adulthood (a) and infancy (b). Scattered points represent individual miRNAs identified by DESeq2. The x-axis specifies the log2 fold-changes (log2(FC)), and the y-axis specifies the negative logarithm to the base 10 of the p-values. Green vertical and horizontal dashed lines reflect the filtering criteria (log2(FC) =  ± 0.48 and p-value = 0.05). log2(FC) >  + 0.48 indicates miRNA levels increased by > 1.4 times in SE-treated rats, whereas log2(FC) <  − 0.48 indicates miRNA levels reduced by > 1.4 times in SE-treated rats. miRNAs significantly dysregulated in the acute stage of epilepsy are depicted by red circle points. miRNAs dysregulated in the latent stage are represented as blue square points, and in the chronic stage, they are represented as green triangle points. Non-significant miRNAs are depicted in black. Ten miRNAs with the highest fold-change difference between SE and control animals in adult and infantile-onset group respectively are labeled.

Figure 3

The visualization of expression and clustering analyses of dysregulated miRNAs. Heatmap shows expression profiles for PCR-validated differentially expressed miRNAs in infantile and adult-onset epileptogenesis. Rows represent individual miRNAs, while columns correspond to samples (labelled R + randomly assigned sample number) from control (Ctrl; gold) and post-status epilepticus (SE; purple) rats. Each heatmap is split into blocks representing the periods between status epilepticus (SE) induction and sample collection: 24 h (dark gray), 7 days (gray), and 3 months (light gray). The visualized expression was normalized to a row-wise Z-score (subtracting the row mean from each cell, and then dividing the value by the standard deviation of the row). The Z-score color range was further scaled to − 3 to 3 for better visualization. The initial expression values were log10(expression + 0.1) transformed prior to Z-score normalization. The miRNA clustering dendrogram is shown on the left and revealed miRNAs with similar expression patterns across collection periods.

Figure 4

miRNAs with altered expression in adult and infantile-onset epilepsy-summary diagram. The diagram summarizes the occurrence of dysregulated miRNAs after status epilepticus (SE) induced in adulthood (left; adult rat) and infancy (right; rat pup). The inner parts specify the stage of epileptogenesis with a short description of seizure occurrence. The middle parts contain the lists of dysregulated miRNAs in the respective stages. Upregulated miRNAs are displayed in red, while downregulated miRNA after SE are shown in blue. The outer parts list the predicted pathways affected by dysregulated miRNAs in the given stage.