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Altered Biogenesis and MicroRNA Content of Hippocampal Exosomes Following Experimental Status Epilepticus

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Altered Biogenesis and MicroRNA Content of Hippocampal Exosomes Following Experimental Status Epilepticus

Aasia Batool et al. Front Neurosci.

Abstract

Repetitive or prolonged seizures (status epilepticus) can damage neurons within the hippocampus, trigger gliosis, and generate an enduring state of hyperexcitability. Recent studies have suggested that microvesicles including exosomes are released from brain cells following stimulation and tissue injury, conveying contents between cells including microRNAs (miRNAs). Here, we characterized the effects of experimental status epilepticus on the expression of exosome biosynthesis components and analyzed miRNA content in exosome-enriched fractions. Status epilepticus induced by unilateral intra-amygdala kainic acid in mice resulted in acute subfield-specific, bi-directional changes in hippocampal transcripts associated with exosome biosynthesis including up-regulation of endosomal sorting complexes required for transport (ESCRT)-dependent and -independent pathways. Increased expression of exosome components including Alix were detectable in samples obtained 2 weeks after status epilepticus and changes occurred in both the ipsilateral and contralateral hippocampus. RNA sequencing of exosome-enriched fractions prepared using two different techniques detected a rich diversity of conserved miRNAs and showed that status epilepticus selectively alters miRNA contents. We also characterized editing sites of the exosome-enriched miRNAs and found six exosome-enriched miRNAs that were adenosine-to-inosine (ADAR) edited with the majority of the editing events predicted to occur within miRNA seed regions. However, the prevalence of these editing events was not altered by status epilepticus. These studies demonstrate that status epilepticus alters the exosome pathway and its miRNA content, but not editing patterns. Further functional studies will be needed to determine if these changes have pathophysiological significance for epileptogenesis.

Keywords: epileptogenesis; extracellular vesicle; neuroinflammation; non-coding RNA; seizure; temporal lobe epilepsy.

Figures

FIGURE 1
FIGURE 1
Acute changes to exosome biogenesis transcripts after status epilepticus in hippocampal subfields. Graphs show transcript (mRNA) levels 4, 8, and 24 h after status epilepticus (SE) induced by KA compared to control in each subfield of the (A) ipsilateral and (B) contralateral, hippocampus. Levels were normalized to β-actin. Shown are ESCRT-dependent exosome biogenesis transcripts, ESCRT-independent exosome biogenesis transcripts, and potential exosome secretion transcripts. Dotted line indicates control level. Graphs show mean ± SEM. p < 0.05 compared to matching control. n = 4–6 per group; ANOVA with Bonferroni post hoc test.
FIGURE 2
FIGURE 2
Acute regulation of protein levels of exosome biogenesis pathways after status epilepticus in hippocampal subfields. Representative immunoblots and graphs show protein levels of Alix, Tsg101, and Rab27a exosome pathway components 4, 8, and 24 h after SE compared to control (C) in each subfield of the (A) ipsilateral and (B) contralateral, hippocampus. Graphs show mean ± SEM. Protein levels were normalized to tubulin (n = 4–5 per group; ANOVA with Bonferroni post hoc test showed no significant differences).
FIGURE 3
FIGURE 3
Long-term regulation of exosome biogenesis transcripts in hippocampal subfields after status epilepticus. Graphs show mRNA levels of exosome pathway components in samples obtained 2 weeks after SE for each subfield in the (A) ipsilateral and (B) contralateral hippocampus. Transcript levels were normalized to β-actin. Graphs show mean ± SEM. n = 5 per group. p < 0.05, t-test comparing to control.
FIGURE 4
FIGURE 4
Long-term regulation of exosome biogenesis proteins after status epilepticus in hippocampal subfields. Representative western blots and graphs show levels of a set of exosome proteins in samples obtained 2 weeks after SE for each subfield in the (A) ipsilateral and (B) contralateral hippocampus. Protein levels are normalized to tubulin. Graphs show mean ± SEM. n = 5–6 per group. p < 0.05, t-test comparing to control.
FIGURE 5
FIGURE 5
Study design and sequencing of EEF miRNAs. (A) Schematic of experimental design for miRNA sequencing. EEFs were prepared using hippocampi from sets of control (Ctrl) or KA-treated mice using UC- or kit-based methods. EEFs were then RNAse treated to remove non-enclosed miRNAs and processed for RNA sequencing. (B) Top: bar plot showing total number of counts per sample mapping to miRBase_V21. Bottom: Number of confidently called, unique miRNA identified in each sample. (C) Principal component analysis (PCA) plot showing clustering of the 420 and 520 filtered miRNAs from UC and kit samples, respectively. (D) Venn diagram shows the number of detected miRNAs in exosomes post filtration in UC and in kit, highlighting EEF miRNAs common to both methods.
FIGURE 6
FIGURE 6
Abundance, cell origins, and ExoCarta analysis of EEF miRNA content. (A) Amounts of miRNAs identified by small RNA sequencing of EEFs from mouse hippocampus. MiRNAs are ordered on a Matlab-based visual graphic, according to average log2FC across all samples, from highest to lowest abundance. The size of the visual graphic bubble shows the number of reads (CPM) of each miRNA detected in the samples and its color represents the log2FC in KA relative to PBS control levels. Presented are the top 20 miRNA in the UC-derived EEFs, of which 16 are common across all 24 samples (in red). (B) Pie chart depicts the assignment of miRNA based on likely cell type for the common 397 miRNAs detected in both UC and kit EEFs. (C) Venn diagram showing overlap (∼64%; 255 of 397) of the detected miRNA with those listed in the exosome contents database, Exocarta. (D) Top 100 miRNA (most abundant based on average across all 12 samples in UC) expressed in EEFs from both UC and kit. The miRNAs colored red are listed in ExoCarta.
FIGURE 7
FIGURE 7
Differential expression of miRNA in EEFs after status epilepticus. (A) The table shows miRNA which were detected as differentially expressed between control and KA at 24 h and 2-weeks, at unadjusted p-value ≤ 0.05. Highlighted miRNAs are those differentially expressed in EEFs using both UC and kit methods. Red asterisk indicates miRNA which were differentially expressed at an adjusted p-value ≤ 0.05 in UC samples. (B) Conserved miRNA changes after status epilepticus. The graph shows levels of miR-21a-3p, miR-21a-5p, and miR-107-3p (increased at 24 h), and miR-21a-5p and miR-146a-5p (increased at 2-weeks). Of these, significance for miR-21a-3p and miR-21a-5p at 24 h and miR-146a-5p at 2-weeks was only found in the UC method at an adjusted p-value of <0.05. (C) Validation of sequencing results using individual miRNA assays confirming increased miR-21a-5p and -3p. UC, in red; kit, in blue. miRNA levels are normalized to average of 25-3p and 92b-3p. Graphs show mean ± SEM. p < 0.05 comparing to matching control. n = 3 per group using the same samples as were sequenced (ANOVA with Bonferroni post hoc test). (D) Network map of GO terms significantly enriched among the targets of the four miRNAs (miR-21a-3p, miR-21a-5p, miR-107-3p, and miR-146a-5p). The network was generated using ReactomePA R/Bioconductor package. The node color indicates the significance of the enrichment (adjusted p-value) while the node size indicates the number of miRNA targets were found in that GO term category.
FIGURE 8
FIGURE 8
RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site (Li et al., 2018) reported in brain (Alon et al., 2012) and reported in exosomes (Nigita et al., 2018).

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