MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

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.

RET overexpression leads to increased brain metastatic competency in luminal breast cancer.

BACKGROUND: Breast cancer brain metastasis is a rising occurrence, necessitating a better understanding of the mechanisms involved for effective management. Breast cancer brain metastases diverge notably from the primary tumor, with gains in kinase and concomitant losses of steroid signaling observed. In this study, we explored the role of the kinase receptor RET in promoting breast cancer brain metastases and provide a rationale for targeting this receptor. METHODS: RET expression was characterized in a cohort of patients with primary and brain metastatic tumors. RET functionality was assessed using pharmacological inhibition and gene silencing in patient-derived brain metastatic tumor explants and in vivo models, organoid models, and brain organotypic cultures. RNA sequencing was used to uncover novel brain metastatic relevant RET mechanisms of action. RESULTS: A statistically significant enrichment of RET in brain metastases was observed in estrogen receptor-positive breast cancer, where it played a role in promoting cancer cell adhesion, survival, and outgrowth in the brain. In vivo, RET overexpression enhanced brain metastatic competency in patient-derived models. At a mechanistic level, RET overexpression was found to enhance the activation of gene programs involved in cell adhesion, requiring EGFR cooperation to deliver a pro-brain metastatic phenotype. CONCLUSION: Our results illustrate, for the first time, the role of RET in regulating colonization and outgrowth of breast cancer brain metastasis and provide data to support the use of RET inhibitors in the management strategy for patients with breast cancer brain metastases.

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.

Anti-seizure effects of JNJ-54175446 in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy in mice.

There remains a need for new drug targets for treatment-resistant temporal lobe epilepsy. The ATP-gated P2X7 receptor coordinates neuroinflammatory responses to tissue injury. Previous studies in mice reported that the P2X7 receptor antagonist JNJ-47965567 suppressed spontaneous seizures in the intraamygdala kainic acid model of epilepsy and reduced attendant gliosis in the hippocampus. The drug-resistance profile of this model is not fully characterised, however, and newer P2X7 receptor antagonists with superior pharmacokinetic profiles have recently entered clinical trials. Using telemetry-based continuous EEG recordings in mice, we demonstrate that spontaneous recurrent seizures in the intraamygdala kainic acid model are refractory to the common anti-seizure medicine levetiracetam. In contrast, once-daily dosing of JNJ-54175446 (30 mg/kg, intraperitoneal) resulted in a significant reduction in spontaneous recurrent seizures which lasted several days after the end of drug administration. Using a combination of immunohistochemistry and ex vivo radiotracer assay, we find that JNJ-54175446-treated mice at the end of recordings display a reduction in astrogliosis and altered microglia process morphology within the ipsilateral CA3 subfield of the hippocampus, but no difference in P2X7 receptor surface expression. The present study extends the characterisation of the drug-resistance profile of the intraamygdala kainic acid model in mice and provides further evidence that targeting the P2X7 receptor may have therapeutic applications in the treatment of temporal lobe epilepsy.

Distinct dynamics of neuronal activity during concurrent motor planning and execution.

The smooth conduct of movements requires simultaneous motor planning and execution according to internal goals. So far it remains unknown how such movement plans are modified without interfering with ongoing movements. Previous studies have isolated planning and execution-related neuronal activity by separating behavioral planning and movement periods in time by sensory cues. Here, we separate continuous self-paced motor planning from motor execution statistically, by experimentally minimizing the repetitiveness of the movements. This approach shows that, in the rat sensorimotor cortex, neuronal motor planning processes evolve with slower dynamics than movement-related responses. Fast-evolving neuronal activity precees skilled forelimb movements and is nested within slower dynamics. We capture this effect via high-pass filtering and confirm the results with optogenetic stimulations. The various dynamics combined with adaptation-based high-pass filtering provide a simple principle for separating concurrent motor planning and execution.

BICS01 Mediates Reversible Anti-seizure Effects in Brain Slice Models of Epilepsy.

Drug-resistant epilepsy remains a significant clinical and societal burden, with one third of people with epilepsy continuing to experience seizures despite the availability of around 30 anti-seizure drugs (ASDs). Further, ASDs often have substantial adverse effects, including impacts on learning and memory. Therefore, it is important to develop new ASDs, which may be more potent or better tolerated. Here, we report the preliminary preclinical evaluation of BICS01, a synthetic product based on a natural compound, as a potential ASD. To model seizure-like activity in vitro, we prepared hippocampal slices from adult male Sprague Dawley rats, and elicited epileptiform bursting using high extracellular potassium. BICS01 (200 µM) rapidly and reversibly reduced the frequency of epileptiform bursting but did not change broad measures of network excitability or affect short-term synaptic facilitation. BICS01 was well tolerated following systemic injection at up to 1,000 mg/kg. However, we did not observe any protective effect of systemic BICS01 injection against acute seizures evoked by pentylenetetrazol. These results indicate that BICS01 is able to acutely reduce epileptiform activity in hippocampal networks. Further preclinical development studies to enhance pharmacokinetics and accumulation in the brain, as well as studies to understand the mechanism of action, are now required.

Detection of spontaneous seizures in EEGs in multiple experimental mouse models of epilepsy.

Objective.Electroencephalography (EEG) is a key tool for non-invasive recording of brain activity and the diagnosis of epilepsy. EEG monitoring is also widely employed in rodent models to track epilepsy development and evaluate experimental therapies and interventions. Whereas automated seizure detection algorithms have been developed for clinical EEG, preclinical versions face challenges of inter-model differences and lack of EEG standardization, leaving researchers relying on time-consuming visual annotation of signals.Approach.In this study, a machine learning-based seizure detection approach, 'Epi-AI', which can semi-automate EEG analysis in multiple mouse models of epilepsy was developed. Twenty-six mice with a total EEG recording duration of 6451 h were used to develop and test the Epi-AI approach. EEG recordings were obtained from two mouse models of kainic acid-induced epilepsy (Models I and III), a genetic model of Dravet syndrome (Model II) and a pilocarpine mouse model of epilepsy (Model IV). The Epi-AI algorithm was compared against two threshold-based approaches for seizure detection, a local Teager-Kaiser energy operator (TKEO) approach and a global Teager-Kaiser energy operator-discrete wavelet transform (TKEO-DWT) combination approach.Main results.Epi-AI demonstrated a superior sensitivity, 91.4%-98.8%, and specificity, 93.1%-98.8%, in Models I-III, to both of the threshold-based approaches which performed well on individual mouse models but did not generalise well across models. The performance of the TKEO approach in Models I-III ranged from 66.9%-91.3% sensitivity and 60.8%-97.5% specificity to detect spontaneous seizures when compared with expert annotations. The sensitivity and specificity of the TKEO-DWT approach were marginally better than the TKEO approach in Models I-III at 73.2%-80.1% and 75.8%-98.1%, respectively. When tested on EEG from Model IV which was not used in developing the Epi-AI approach, Epi-AI was able to identify seizures with 76.3% sensitivity and 98.1% specificity.Significance.Epi-AI has the potential to provide fast, objective and reproducible semi-automated analysis of multiple types of seizure in long-duration EEG recordings in rodents.