Altered serum microRNA expression profiles in neonatal seizures

Poster presented at the inaugural INFANT research day - June 16, 2015, Cork University Maternity Hospital, Cork Ireland<div><br></div><div><br></div><div><div>Seizures are more common in the neonatal period than at any other time of life and are linked to poor neurological outcomes including cerebral palsy, lowered IQ and later-life epilepsy. Many neonatal seizures are sub-clinical and difficult to diagnose. Accurate detection requires continuous EEG monitoring, with equipment and expertise not available outside specialist centres. A blood based biomarker of neonatal seizures is urgently needed.</div><div><br></div><div>MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. They are present in biofluids such as blood under normal conditions, and injury to the brain, including seizures, results in unique profiles. We wished to examine the microRNA profile of infants with EEG confirmed neonatal seizures</div><div><br></div><div>MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. They are present in biofluids such as blood under normal conditions, and injury to the brain, including seizures, results in unique profiles. High-throughput RT-qPCR  (OpenArray), was used to profile microRNA in umbilical cord and post natal serum samples at 24h, 48h and 72h, from babies with hypoxic ischaemic encephalopathy, the most common cause of seizures in full-term babies, with and without seizures, and normal controls. </div><div><br></div><div>MicroRNA profiles were significantly altered in infants with HIE and neonatal seizures, compared to HIE without seizures and normal controls. Three microRNAs were differentially expressed at all time-points. These microRNAs include known brain-expressed microRNAs implicated in epilepsy in mature and immature human and rodent brains. </div><div><br></div><div>MicroRNA alterations are present and detectable in umbilical cord blood prior to onset of neonatal seizures and persist throughout the first 72 hours after birth.</div></div><div><br></div

RNA sequencing of synaptic and cytoplasmic Upf1-bound transcripts supports contribution of nonsense-mediated decay to epileptogenesis.

The nonsense mediated decay (NMD) pathway is a critical surveillance mechanism for identifying aberrant mRNA transcripts. It is unknown, however, whether the NMD system is affected by seizures in vivo and whether changes confer beneficial or maladaptive responses that influence long-term outcomes such the network alterations that produce spontaneous recurrent seizures. Here we explored the responses of the NMD pathway to prolonged seizures (status epilepticus) and investigated the effects of NMD inhibition on epilepsy in mice. Status epilepticus led to increased protein levels of Up-frameshift suppressor 1 homolog (Upf1) within the mouse hippocampus. Upf1 protein levels were also higher in resected hippocampus from patients with intractable temporal lobe epilepsy. Immunoprecipitation of Upf1-bound RNA from the cytoplasmic and synaptosomal compartments followed by RNA sequencing identified unique populations of NMD-associated transcripts and altered levels after status epilepticus, including known substrates such as Arc as well as novel targets including Inhba and Npas4. Finally, long-term video-EEG recordings determined that pharmacologic interference in the NMD pathway after status epilepticus reduced the later occurrence of spontaneous seizures in mice. These findings suggest compartment-specific recruitment and differential loading of transcripts by NMD pathway components may contribute to the process of epileptogenesis.</p

Computational discovery of plasma microRNA profiles as biomarkers of temporal lobe epilepsy

<div>Presented at ISMB/ECCB 2015 - July 10 – 14, 2015, Dublin, Ireland</div><div><br></div><div><br></div>Epilepsy is a common neurological disorder affecting approximately 1% of the population and is characterised by recurrent unprovoked seizures. The lack of a clinically accepted biomarker for epilepsy diagnosis as well as the incomplete and vague history often provided by the patient is responsible for up to 30% misdiagnosis. MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. MicroRNAs are important contributors to brain function and emerging animal and human data suggest microRNAs control multiple pathways in epilepsy. MicroRNAs are also detectable in various body fluids and their stability as well as link to disease mechanism makes them potentially ideal molecular biomarkers of epilepsy. We determined plasma levels of over 800 microRNAs collected from 20 healthy volunteers and 20 epilepsy patients using highthroughput real-time quantitative reverse transcription PCR. Computational analysis included normalisation, clustering, differential expression analysis, target prediction and pathway analysis. A number of significantly differentially expressed microRNAs were identified between control and epilepsy samples including known brain-expressed microRNAs implicated in epilepsy. Furthermore, we applied feature selection with machine learning algorithms, including support vector machines and bidirectional recurrent neural networks, to build a microRNAs-based predictor of epilepsy, validated on an independent test set. This analyse showed that these classifiers may be useful in supporting the existence of a set of microRNAs implicated in disease pathogenesis that may be biomarkers of human epilepsy

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

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

Systemic delivery of antagomirs during blood-brain barrier disruption is disease-modifying in experimental epilepsy

Oligonucleotide therapies offer precision treatments for a variety of neurological diseases, including epilepsy, but their deployment is hampered by the blood-brain barrier (BBB). Previous studies showed that intracerebroventricular injection of an antisense oligonucleotide (antagomir) targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in animal models of epilepsy. In this study, we used assays of serum protein and tracer extravasation to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically injected Ant-134 into the brain parenchyma. Intraperitoneal and intravenous injection of Ant-134 reached the hippocampus and blocked seizure-induced upregulation of miR-134. A single intraperitoneal injection of Ant-134 at 2 h after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at 3 months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. In vivo knockdown of LIM kinase-1 (Limk-1) increased seizure frequency in Ant-134-treated mice, implicating de-repression of Limk-1 in the antagomir mechanism. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces long-lasting seizure-suppressive effects after systemic injection in mice when timed with BBB disruption and may be a clinically viable approach for this and other disease-modifying microRNA therapie

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