Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy.

The diagnosis of epilepsy is complex and challenging and would benefit from the availability of molecular biomarkers, ideally measurable in a biofluid such as blood. Experimental and human epilepsy are associated with altered brain and blood levels of various microRNAs (miRNAs). Evidence is lacking, however, as to whether any of the circulating pool of miRNAs originates from the brain. To explore the link between circulating miRNAs and the pathophysiology of epilepsy, we first sequenced argonaute 2 (Ago2)-bound miRNAs in plasma samples collected from mice subject to status epilepticus induced by intraamygdala microinjection of kainic acid. This identified time-dependent changes in plasma levels of miRNAs with known neuronal and microglial-cell origins. To explore whether the circulating miRNAs had originated from the brain, we generated mice expressing FLAG-Ago2 in neurons or microglia using tamoxifen-inducible Thy1 or Cx3cr1 promoters, respectively. FLAG immunoprecipitates from the plasma of these mice after seizures contained miRNAs, including let-7i-5p and miR-19b-3p. Taken together, these studies confirm that a portion of the circulating pool of miRNAs in experimental epilepsy originates from the brain, increasing support for miRNAs as mechanistic biomarkers of epilepsy.

Antagomir-mediated suppression of microRNA-134 reduces kainic acid-induced seizures in immature mice.

MicroRNAs are short non-coding RNAs that negatively regulate protein levels and perform important roles in establishing and maintaining neuronal network function. Previous studies in adult rodents have detected upregulation of microRNA-134 after prolonged seizures (status epilepticus) and demonstrated that silencing microRNA-134 using antisense oligonucleotides, termed antagomirs, has potent and long-lasting seizure-suppressive effects. Here we investigated whether targeting microRNA-134 can reduce or delay acute seizures in the immature brain. Status epilepticus was induced in 21 day-old (P21) male mice by systemic injection of 5 mg/kg kainic acid. This triggered prolonged electrographic seizures and select bilateral neuronal death within the CA3 subfield of the hippocampus. Expression of microRNA-134 and functional loading to Argonaute-2 was not significantly changed in the hippocampus after seizures in the model. Nevertheless, when levels of microRNA-134 were reduced by prior intracerebroventricular injection of an antagomir, kainic acid-induced seizures were delayed and less severe and mice displayed reduced neuronal death in the hippocampus. These studies demonstrate targeting microRNA-134 may have therapeutic applications for the treatment of seizures in children.

Genome-wide microRNA profiling of plasma from three different animal models identifies biomarkers of temporal lobe epilepsy.

Epilepsy diagnosis is complex, requires a team of specialists and relies on in-depth patient and family history, MRI-imaging and EEG monitoring. There is therefore an unmet clinical need for a non-invasive, molecular-based, biomarker to either predict the development of epilepsy or diagnose a patient with epilepsy who may not have had a witnessed seizure. Recent studies have demonstrated a role for microRNAs in the pathogenesis of epilepsy. MicroRNAs are short non-coding RNA molecules which negatively regulate gene expression, exerting profound influence on target pathways and cellular processes. The presence of microRNAs in biofluids, ease of detection, resistance to degradation and functional role in epilepsy render them excellent candidate biomarkers. Here we performed the first multi-model, genome-wide profiling of plasma microRNAs during epileptogenesis and in chronic temporal lobe epilepsy animals. From video-EEG monitored rats and mice we serially sampled blood samples and identified a set of dysregulated microRNAs comprising increased miR-93-5p, miR-142-5p, miR-182-5p, miR-199a-3p and decreased miR-574-3p during one or both phases. Validation studies found miR-93-5p, miR-199a-3p and miR-574-3p were also dysregulated in plasma from patients with intractable temporal lobe epilepsy. Treatment of mice with common anti-epileptic drugs did not alter the expression levels of any of the five miRNAs identified, however administration of an anti-epileptogenic microRNA treatment prevented dysregulation of several of these miRNAs. The miRNAs were detected within the Argonuate2-RISC complex from both neurons and microglia indicating these miRNA biomarker candidates can likely be traced back to specific brain cell types. The current studies identify additional circulating microRNA biomarkers of experimental and human epilepsy which may support diagnosis of temporal lobe epilepsy via a quick, cost-effective rapid molecular-based test.

Epigenomically Bistable Regions across Neuron-Specific Genes Govern Neuron Eligibility to a Coding Ensemble in the Hippocampus.

Sensory inputs activate sparse neuronal ensembles in the dentate gyrus of the hippocampus, but how eligibility of individual neurons to recruitment is determined remains elusive. We identify thousands of largely bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during mouse dentate gyrus development. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromises novel context-induced neuronal activation. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhances intrinsic excitability. Single-nucleus profiling reveals enrichment of specific epialleles related to a subset of primarily exonic, bistable regions in activated neurons. Genes displaying both differential methylation and expression in activated neurons define a network of proteins regulating neuronal excitability and structural plasticity. We propose a model in which bistable regions create neuron heterogeneity and constellations of exonic methylation, which may contribute to cell-specific gene expression, excitability, and eligibility to a coding ensemble.

MicroRNAs as biomarkers and treatment targets in status epilepticus.

Microribonucleic acids (miRNAs) are short noncoding ribonucleic acids (RNAs) that have been proposed as potential biomarkers for epilepsy, acute seizures, and status epilepticus. Various properties support their potential in this regard, including relative stability and amenability to rapid quantitation in biofluids. Several miRNAs are enriched in the brain and within specific cell types. Dysregulation of miRNAs has been reported in brain regions damaged by status epilepticus and in resected brain tissue from patients with drug-resistant epilepsy. Silencing miRNAs using antisense-like oligonucleotides termed antagomirs has been reported to suppress evoked and spontaneous seizures in animal models, indicating therapeutic applications. The prospect of miRNAs as mechanistic biomarkers is supported by recent studies showing blood levels of brain-enriched miRNAs increase after status epilepticus in rodents, and clinical studies have identified miRNAs upregulated in human cerebrospinal fluid after status epilepticus. It remains unproven, however, whether there are miRNAs that uniquely identify acute seizures, chronic epilepsy, or the process of epileptogenesis. Finally, efforts have turned to the challenge of proving that some of the circulating miRNAs actually originate from the brain. New models that feature a biochemically-labeled protein involved in miRNA function and restricted to specific brain cell types offer opportunities to resolve this issue. This review summarizes recent progress on miRNAs as diagnostic biomarkers of status epilepticus and considers some of the unanswered questions and future directions. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures.

Dual-center, dual-platform microRNA profiling identifies potential plasma biomarkers of adult temporal lobe epilepsy.

BACKGROUND: There are no blood-based molecular biomarkers of temporal lobe epilepsy (TLE) to support clinical diagnosis. MicroRNAs are short noncoding RNAs with strong biomarker potential due to their cell-specific expression, mechanistic links to brain excitability, and stable detection in biofluids. Altered levels of circulating microRNAs have been reported in human epilepsy, but most studies collected samples from one clinical site, used a single profiling platform or conducted minimal validation. METHOD: Using a case-control design, we collected plasma samples from video-electroencephalogram-monitored adult TLE patients at epilepsy specialist centers in two countries, performed genome-wide PCR-based and RNA sequencing during the discovery phase and validated findings in a large (>250) cohort of samples that included patients with psychogenic non-epileptic seizures (PNES). FINDINGS: After profiling and validation, we identified miR-27a-3p, miR-328-3p and miR-654-3p with biomarker potential. Plasma levels of these microRNAs were also changed in a mouse model of TLE but were not different to healthy controls in PNES patients. We determined copy number of the three microRNAs in plasma and demonstrate their rapid detection using an electrochemical RNA microfluidic disk as a prototype point-of-care device. Analysis of the microRNAs within the exosome-enriched fraction provided high diagnostic accuracy while Argonaute-bound miR-328-3p selectively increased in patient samples after seizures. In situ hybridization localized miR-27a-3p and miR-328-3p within neurons in human brain and bioinformatics predicted targets linked to growth factor signaling and apoptosis. INTERPRETATION: This study demonstrates the biomarker potential of circulating microRNAs for epilepsy diagnosis and mechanistic links to underlying pathomechanisms.

Correction to: Understanding Plain English Summaries: A Comparison of two Approaches to Improve the Quality of Plain English Summaries in Research.

[This corrects the article DOI: 10.1186/s40900-017-0064-0.].

Understanding Plain English summaries. A comparison of two approaches to improve the quality of Plain English summaries in research reports.

PLAIN ENGLISH SUMMARY: There is a need for the authors of research reports to be able to communicate their work clearly and effectively to readers who are not familiar with the research area. The National Institute for Health Research (NIHR), along with a number of other funding bodies and journals, require researchers to write short lay summaries, often termed plain English summaries (PESs), to make research accessible to the general public. Because many researchers write using technical, specialised language, particularly in scientific reports, writing PESs can be challenging. In this study we looked at how to improve the quality of PESs. We took PESs which had been submitted to the NIHR Journals Library and asked authors to rewrite them using new guidance. We also asked an independent medical writer to edit the summaries. We measured the quality of these three versions (original summary, rewritten summary and edited summary) in two ways. First, we asked a group of people who were not specialists in the subject area to read and rate how easy the summaries were to understand. Secondly, we used a well-known measure called the Flesch reading ease score to assess how easy the PESs were to read. We found that there was no difference in how easy people found the summaries to understand across the three versions. However, the PESs that were rewritten by the authors and that were edited by the independent medical writer were both easier to read than the originals. This shows that PESs can be improved and for organisations who feel that employing an independent writer to edit summaries, providing clear, practical guidance to authors may be a cost-effective alternative. BACKGROUND: Plain English summaries (PES) or lay summaries are often included as part of research reports and journal articles. These summaries are vital to ensure that research findings are accessible and available to non-specialist audiences, for example patients and members of the public. Writing a PES requires the adoption of a different style than is generally used in a traditional scientific report, and researchers can find this challenging. This study explored two possible ways to improve the quality of PESs in the NIHR Journals Library: 1) Providing enhanced guidance to authors and asking them to rewrite the PES and 2) Employing an independent medical writer to edit the PES. METHODS: We compared the three versions of the PES (original, author rewritten and independent writer edited) to assess 1) how easy they were to understand and 2) how easy they were to read. In order to establish how easy PESs were to understand, a group of 60 public reviewers read a set of summaries and rated them on a four point scale from "Did not understand" to "Understood all". The Flesch reading ease score was used to measure how easy the summaries were to read. RESULTS: Results indicated no significant difference across the three versions of the PES in terms of ease of understanding. However, both the author rewritten and independent writer edited versions were significantly easier to read than the original. There was no significant difference in ease of reading between these two versions. CONCLUSION: These findings suggest that employing independent medical writers to edit PESs and providing clear, practical guidance to authors are two ways in which the readability of PESs could be improved. Results have implications for journal editors and publishers seeking to enhance accessibility and availability of research findings.