Altered biogenesis and microRNA content of hippocampal exosomes following experimental status epilepticus.

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

Antagonizing increased miR-135a levels at the chronic stage of experimental TLE reduces spontaneous recurrent seizures

Mesial Temporal Lobe Epilepsy (mTLE) is a chronic neurological disease characterized by 54 recurrent seizures. The anti-epileptic drugs currently available to treat mTLE are ineffective 55 in one-third of patients and lack disease-modifying effects. MicroRNAs (miRNAs), a class of 56 small non-coding RNAs which control gene expression at the post-transcriptional level, play 57 a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of 58 miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is 59 uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. 60 Furthermore, the functional role and downstream targets of most epilepsy-associated 61 miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated 62 within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs 63 after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage 64 of experimental mTLE in male mice. Further, by using an unbiased approach combining 65 immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of 66 miR-135a, including Mef2a. Mef2 proteins are key regulators of excitatory synapse density. 67 Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and 68 experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. 69 Together, our data show that miR-135a is target for reducing seizure activity in chronic 70 epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and 71 thereby affect synaptic function and plasticity</p

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-β signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-β signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.</p