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

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

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

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

A Cannabidiol-Regulated MicroRNA Controls Brain Excitability and is a Target for Seizure Control

Epilepsy is a serious neurological disease which is primarily characterised by spontaneous recurrent seizures, affecting approximately 65 million people worldwide. So far there is still no pharmacological cure for the disease and the current anti-seizure drugs (ASDs) only treat the symptoms and do not modify the underlying pathophysiology or prevent the progression of the disease. There remains an urgent need for new therapies for drug-resistant epilepsy, as ~30% of affected people do not respond adequately to current pharmacological treatments. In recent years, cannabidiol (CBD) emerged as a new therapy option for several epileptic encephalopathies, including Dravet syndrome and Lennox-Gastaut syndrome. However, the extent to which CBD is anti-seizure and the mechanism(s) by which CBD mediates its anticonvulsant effects are not elucidated. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and have shown to be involved in the pathophysiology of epilepsy. To date, over 20 miRNAs have been functionally investigated in different animal models of epilepsy and seizures and proven to modulate seizure activity or associated pathologies. The aim of this thesis was to explore whether CBD regulates specific miRNAs and then to use the findings to probe novel actions of miRNAs in the setting of epilepsy. The studies combined in vivo seizure models, small RNA sequencing, in silico microRNA target analysis, ex vivo brain slice electrophysiology and molecular biology.  The first results chapter characterised the anti-seizure properties of CBD in a set of preclinical models of acquired and genetic epilepsies. As expected, dosing mice with CBD protected mice in the pentylenetetrazol (PTZ) model against seizures, both severity and improved survival. CBD also reduced hyperthermia-induced seizure severity in a mouse model of Dravet syndrome (Scn1atm1Kea). However, CBD treatment did not alter status epilepticus (SE) or the occurrence or severity of spontaneous recurrent seizures in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy. In addition to the observed anti-seizure effects, studies here also determined that CBD has a dose-related hypothermic effect in mice. The second results chapter investigated whether cannabinoids, in particular CBD, alter the expression of miRNAs in the mouse hippocampus. Using small RNA sequencing, CBD produced significant changes to the hippocampal expression of miRNAs. This effect was more extensive than for the two closely-related phytocannabinoids cannabidivarin (CBDV) and cannabigerol (CBG). MiRNA-335 was identified as an abundant miRNA downregulated after CBD treatment. MiRNA target interaction analysis identified several epilepsy-related miR-335 targets, including voltage-gated sodium channels (VGSCs). The third results chapter explored how bi-directional modulation of miR-335 affects excitability and the expression of VGSCs. Inhibition of miR-335 using an antimiR (Ant-335) de-repressed various VGSC subtypes in the mouse brain in adult but not young (P21) C57BL/6 mice. Ex vivo brain slice electrophysiology detected changes in the amplitude, and rising phase of action potentials of hippocampal pyramidal cells of Ant-335 treated mice. These changes in the action potential properties were consistent with an effect to increased firing frequency of individual neurons. Furthermore, an AAV9 was designed and deployed to upregulate miR-335 to explore the effects of in vivo overexpression of miR-335 in the brain. In the final results chapter, functional studies investigated how changes to miR-335 levels affect seizures in a set of different mouse models. Inhibition of miR-335 slightly increased the susceptibility to seizures in the intra-amygdala kainic acid model and PTZ model whereas overexpression of miR-335 reduced the severity of PTZ-induced seizures and improved the survival rate. However, modulation of miR-335 produced variable effects on the expression of VGSCs in the different models.  In conclusion, these studies provide evidence that CBD may exert some of its effects via modulation of miRNAs. Moreover, we identify miR-335 as an important regulator of brain excitability through network effects on components that set neuronal excitability thresholds. Findings here point also to complex, context-specific bi-directional effects of this miRNA on targets and brain excitability which are age-dependent. Targeting miR-335 may represent a new therapeutic strategy for the epilepsies, including sodium channelopathies. </p

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 6,451 hours were used to develop and test the Epi-AI approach. EEG recordings were obtained from two mouse models of kainic acid-induced epilepsy (Model 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 Model 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 Model 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 Model 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

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. </p

DataSheet1_Anti-seizure effects of JNJ-54175446 in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy in mice.PDF

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.</p

Data_Sheet_1_Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy.PDF

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.</p

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