Investigation of MicroRNA-134 as a Target against Seizures and SUDEP in a Mouse Model of Dravet Syndrome

Dravet syndrome (DS) is a catastrophic form of pediatric epilepsy mainly caused by noninherited mutations in the SCN1A gene. DS patients suffer severe and life-threatening focal and generalized seizures which are often refractory to available anti-seizure medication. Antisense oligonucleotides (ASOs) based approaches may offer treatment opportunities in DS. MicroRNAs are short noncoding RNAs that play a key role in brain structure and function by post-transcriptionally regulating gene expression, including ion channels. Inhibiting miRNA-134 (miR-134) using an antimiR ASO (Ant-134) has been shown to reduce evoked seizures in juvenile and adult mice and reduce epilepsy development in models of focal epilepsy. The present study investigated the levels of miR-134 and whether Ant-134 could protect against hyperthermia-induced seizures, spontaneous seizures and mortality (SUDEP) in F1.Scn1a(1/)tm1kea mice. At P17, animals were intracerebroventricular in-jected with 0.1–1 nmol of Ant-134 and subject to a hyperthermia challenge at postnatal day (P)18. A second cohort of P21 F1.Scn1a(1/)tm1kea mice received Ant-134 and were followed by video and EEG monitoring until P28 to track the incidence of spontaneous seizures and SUDEP. Hippocampal and cortical levels of miR-134 were similar between wild-type (WT) and F1.Scn1a(1/)tm1kea mice. Moreover, Ant-134 had no effect on hyperthermia-induced seizures, spontaneous seizures and SUDEP incidence were unchanged in Ant-134-treated DS mice. These findings suggest that targeting miR-134 does not have therapeutic applications in DS

Spared CA1 pyramidal neuron function and hippocampal performance following antisense knockdown of microRNA-134

OBJECTIVE: Inhibition of microRNA-134 by an oligonucleotide antagomir (ant-134) has been shown to produce powerful antiseizure effects in multiple models of epilepsy. However, to successfully translate the treatment to the clinic, it is important to assess what potential adverse effects it may have on naive brain tissue. METHODS: To investigate this, adult male Sprague-Dawley rats were treated with either ant-134 or a scrambled control sequence. Animals were later assessed for spatial navigation, before ex vivo slices were taken to assess the effects of microRNA-134 knockdown on well-defined measures of intrinsic and synaptic properties. RESULTS: Hippocampal field potential recordings determined that silencing of microRNA-134 by ant-134 injection was associated with a reduction in epileptiform activity following application of 9 mmol/L K+ . Nevertheless, rats performed normally in the novel object location test. Action potential waveforms and miniature excitatory synaptic currents recorded in CA1 pyramidal neurons were unaffected by ant-134. SIGNIFICANCE: These results demonstrate that ant-134 confers a seizure-protective effect without obvious interference with hippocampal neuronal properties or network function. These findings support further development of this novel approach to epilepsy treatment

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

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

Genetic deletion of microRNA-22 blunts the inflammatory transcriptional response to status epilepticus and exacerbates epilepsy in mice

MicroRNAs perform important roles in the post-transcriptional regulation of gene expression. Sequencing as well as functional studies using antisense oligonucleotides indicate important roles for microRNAs during the development of epilepsy through targeting transcripts involved in neuronal structure, gliosis and inflammation. MicroRNA-22 (miR-22) has been reported to protect against the development of epileptogenic brain networks through suppression of neuroinflammatory signalling. Here, we used mice with a genetic deletion of miR-22 to extend these insights. Mice lacking miR-22 displayed normal behaviour and brain structure and developed similar status epilepticus after intraamygdala kainic acid compared to wildtype animals. Continuous EEG monitoring after status epilepticus revealed, however, an accelerated and exacerbated epilepsy phenotype whereby spontaneous seizures began sooner, occurred more frequently and were of longer duration in miR-22-deficient mice. RNA sequencing analysis of the hippocampus during the period of epileptogenesis revealed a specific suppression of inflammatory signalling in the hippocampus of miR-22-deficient mice. Taken together, these findings indicate a role for miR-22 in establishing early inflammatory responses to status epilepticus. Inflammatory signalling may serve anti-epileptogenic functions and cautions the timing of anti-inflammatory interventions for the treatment of status epilepticus

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 recurrent seizures. The anti-epileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. MicroRNAs (miRNAs), a class of small non-coding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a. Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.Significance statementmiRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism-of-action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mTLE. Silencing miR-135a in experimental TLE reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy