Impact of JQ1 treatment on seizures, hippocampal gene expression, and gliosis in a mouse model of temporal lobe epilepsy.

Epilepsy is a neurological disease characterised by recurrent seizures with complex aetiology. Temporal lobe epilepsy, the most common form in adults, can be acquired following brain insults including trauma, stroke, infection or sustained status epilepticus. The mechanisms that give rise to the formation and maintenance of hyperexcitable networks following acquired insults remain unknown, yet an extensive body of literature points towards persistent gene and epigenomic dysregulation as a potential mediator of this dysfunction. While much is known about the function of specific classes of epigenetic regulators (writers and erasers) in epilepsy, much less is known about the enzymes, which read the epigenome and modulate gene expression accordingly. Here, we explore the potential role for the epigenetic reader bromodomain and extra-terminal domain (BET) proteins in epilepsy. Using the intra-amygdala kainic acid model of temporal lobe epilepsy, we initially identified widespread dysregulation of important epigenetic regulators including EZH2 and REST as well as altered BRD4 expression in chronically epileptic mice. BRD4 activity was also notably affected by epilepsy-provoking insults as seen by elevated binding to and transcriptional regulation of the immediate early gene Fos. Despite influencing early aspects of epileptogenesis, blocking BET protein activity with JQ1 had no overt effects on epilepsy development in mice but did alter glial reactivity and influence gene expression patterns, promoting various neurotransmitter signalling mechanisms and inflammatory pathways in the hippocampus. Together, these results confirm that epigenetic reader activity is affected by epilepsy-provoking brain insults and that BET activity may exert cell-specific actions on inflammation in epilepsy.

WONOEP appraisal: Modeling early onset epilepsies.

Epilepsy has a peak incidence during the neonatal to early childhood period. These early onset epilepsies may be severe conditions frequently associated with comorbidities such as developmental deficits and intellectual disability and, in a significant percentage of patients, may be medication-resistant. The use of adult rodent models in the exploration of mechanisms and treatments for early life epilepsies is challenging, as it ignores significant age-specific developmental differences. More recently, models developed in immature animals, such as rodent pups, or in three-dimensional organoids may more closely model aspects of the immature brain and could result in more translatable findings. Although models are not perfect, they may offer a more controlled screening platform in studies of mechanisms and treatments, which cannot be done in pediatric patient cohorts. On the other hand, more simplified models with higher throughput capacities are required to deal with the large number of epilepsy candidate genes and the need for new treatment options. Therefore, a combination of different modeling approaches will be beneficial in addressing the unmet needs of pediatric epilepsy patients. In this review, we summarize the discussions on this topic that occurred during the XVI Workshop on Neurobiology of Epilepsy, organized in 2022 by the Neurobiology Commission of the International League Against Epilepsy. We provide an overview of selected models of early onset epilepsies, discussing their advantages and disadvantages. Heterologous expression models provide initial functional insights, and zebrafish, rodent models, and brain organoids present increasingly complex platforms for modeling and validating epilepsy-related phenomena. Together, these models offer valuable insights into early onset epilepsies and accelerate hypothesis generation and therapy discovery.

Integrative network analysis of miRNA-mRNA expression profiles during epileptogenesis in rats reveals therapeutic targets after emergence of first spontaneous seizure.

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) - short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.

WONOEP 2022: Neurotechnology for the diagnosis of epilepsy.

Epilepsy's myriad causes and clinical presentations ensure that accurate diagnoses and targeted treatments remain a challenge. Advanced neurotechnologies are needed to better characterize individual patients across multiple modalities and analytical techniques. At the XVIth Workshop on Neurobiology of Epilepsy: Early Onset Epilepsies: Neurobiology and Novel Therapeutic Strategies (WONOEP 2022), the session on "advanced tools" highlighted a range of approaches, from molecular phenotyping of genetic epilepsy models and resected tissue samples to imaging-guided localization of epileptogenic tissue for surgical resection of focal malformations. These tools integrate cutting edge research, clinical data acquisition, and advanced computational methods to leverage the rich information contained within increasingly large datasets. A number of common challenges and opportunities emerged, including the need for multidisciplinary collaboration, multimodal integration, potential ethical challenges, and the multistage path to clinical translation. Despite these challenges, advanced epilepsy neurotechnologies offer the potential to improve our understanding of the underlying causes of epilepsy and our capacity to provide patient-specific treatment.

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.

Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.

Immunoproteasome deficiency results in age-dependent development of epilepsy.

The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating the degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly not expressed in the healthy brain. In this study, we show an age-dependent increase in polyubiquitination in the brains of wild-type mice, accompanied by an induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout mice), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged triple-knockout mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, the aged triple-knockout mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during ageing.

Differential microRNA editing may drive target pathway switching in human temporal lobe epilepsy.

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

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.

CPEB4-CLOCK crosstalk during temporal lobe epilepsy.

OBJECTIVE: Posttranscriptional mechanisms are increasingly recognized as important contributors to the formation of hyperexcitable networks in epilepsy. Messenger RNA (mRNA) polyadenylation is a key regulatory mechanism governing protein expression by enhancing mRNA stability and translation. Previous studies have shown large-scale changes in mRNA polyadenylation in the hippocampus of mice during epilepsy development. The cytoplasmic polyadenylation element-binding protein CPEB4 was found to drive epilepsy-induced poly(A) tail changes, and mice lacking CPEB4 develop a more severe seizure and epilepsy phenotype. The mechanisms controlling CPEB4 function and the downstream pathways that influence the recurrence of spontaneous seizures in epilepsy remain poorly understood. METHODS: Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma. RESULTS: Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day. SIGNIFICANCE: Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.

A novel protocol to derive cervical motor neurons from induced pluripotent stem cells for amyotrophic lateral sclerosis.

Sporadic amyotrophic lateral sclerosis (sALS) is the majority of ALS, and the lack of appropriate disease models has hindered its research. Induced pluripotent stem cell (iPSC) technology now permits derivation of iPSCs from somatic cells of sALS patients to investigate disease phenotypes and mechanisms. Most existing differentiation protocols are time-consuming or low efficient in generating motor neurons (MNs). Here we report a rapid and simple protocol to differentiate MNs in monolayer culture using small molecules, which led to nearly pure neural stem cells in 6 days, robust OLIG2+ pMNs (73%-91%) in 12 days, enriched CHAT+ cervical spinal MNs (sMNs) (88%-97%) in 18 days, and functionally mature sMNs in 28 days. This simple and reproducible protocol permitted the identification of hyperexcitability phenotypes in our sALS iPSC-derived sMNs, and its application in neurodegenerative diseases should facilitate in vitro disease modeling, drug screening, and the development of cell therapy.

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.

Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived astrocytes.

Activation of the ATP-gated P2X7 receptor (P2X7R), implicated in numerous diseases of the brain, can trigger diverse responses such as the release of pro-inflammatory cytokines, modulation of neurotransmission, cell proliferation or cell death. However, despite the known species-specific differences in its pharmacological properties, to date, most functional studies on P2X7R responses have been analyzed in cells from rodents or immortalised cell lines. To assess the endogenous and functional expression of P2X7Rs in human astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into GFAP and S100 ß-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining. P2X7R protein expression was also confirmed by Western blot. Importantly, stimulation with the potent non-selective P2X7R agonist 2',3'-O-(benzoyl-4-benzoyl)-adenosine 5'- triphosphate (BzATP) or endogenous agonist ATP induced robust calcium rises in hiPSC-derived astrocytes which were blocked by the selective P2X7R antagonists AFC-5128 or JNJ-47965567. Our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived astrocytes and support their in vitro utility in investigating the role of the P2X7R and drug screening in disorders of the central nervous system (CNS).

Taking action on climate change: Testimonials and position statement from the International League Against Epilepsy Climate Change Commission.

The release of the 2021 Intergovernmental Panel on Climate Change (IPCC) report makes clear that human activities have resulted in significant alterations in global climate. There is no doubt that climate change is upon us; chronic global warming has been punctuated by more frequent extreme weather events. Humanity will have to mitigate climate change and adapt to these changing conditions or face dire consequences. One under-appreciated aspect of this global crisis is its impact on healthcare, particularly people with epilepsy and temperature-sensitive seizures. As members of the inaugural International League Against Epilepsy (ILAE) Climate Change Commission, we recount the personal motivations that have led each team member to decide to take action, in the hope that our journeys as ordinary clinicians and scientists will help persuade others that they too can act to foster change within their spheres of influence.

The P2X7 receptor contributes to seizures and inflammation-driven long-lasting brain hyperexcitability following hypoxia in neonatal mice.

BACKGROUND AND PURPOSE: Neonatal seizures represent a clinical emergency. However, current anti-seizure medications fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation, and evidence suggests that P2X7R contributes to seizures and epilepsy in adults. However, no genetic proof has yet been provided to determine what contribution P2X7R makes to neonatal seizures, its effects on inflammatory signalling during neonatal seizures, and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. EXPERIMENTAL APPROACH: Neonatal seizures were induced by global hypoxia in 7-day-old mouse pups (P7). The role of P2X7Rs during seizures was analysed in P2X7R-overexpressing and knockout mice. Treatment of wild-type mice after hypoxia with the P2X7R antagonist JNJ-47965567 was used to determine the effects of the P2X7R on long-lasting brain hyperexcitability. Cell type-specific P2X7R expression was analysed in P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal downstream signalling. KEY RESULTS: P2X7R deletion reduced seizure severity, whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7R deficiency led to an anti-inflammatory phenotype in microglia, and treatment of mice with a P2X7R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knockout mice after neonatal hypoxia, including a down-regulation of genes implicated in inflammation and glutamatergic signalling. CONCLUSION AND IMPLICATIONS: Treatments based on targeting the P2X7R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.

Detection of MicroRNAs in Brain Slices Using In Situ Hybridization.

MicroRNAs are key posttranscriptional regulators of protein levels in cells. The brain is particularly enriched in microRNAs, and important roles have been demonstrated for these noncoding RNAs in various neurological disorders. To this end, visualization of microRNAs in specific cell types and subcellular compartments within tissue sections provides researchers with essential insights that support understanding of the cell and molecular mechanisms of microRNAs in brain diseases. In this chapter we describe an in situ hybridization protocol for the detection of microRNAs in mouse brain sections, which provides cellular resolution of the expression of microRNAs in the brain.

Shaping the future of European epilepsy research: Final meeting report from EPICLUSTER.

Collaboration is essential to the conduct of basic, applied and clinical research and its translation into the technologies and treatments urgently needed to improve the lives of people living with brain diseases and the health professionals who care for them. EPICLUSTER was formed in 2019 by the European Brain Research Area (EBRA) to support the coordination of epilepsy research in Europe. A key objective was to provide a platform to discuss shared research priorities by bringing together scientists and clinicians with multiple stakeholders including patient organisations and industry and the networks and infrastructures that provide healthcare and support research. Additional objectives were to facilitate access and sharing of data and biosamples, working together to ensure epilepsy is a priority for research funding, and embedding a culture of public and patient involvement (PPI) among epilepsy researchers. In this meeting report, we summarise the shared research priorities discussed by the leadership of EPICLUSTER at the recent final meeting. We also briefly review the discussion on patient and industry priorities, guidance on starting PPI for epilepsy researchers, and the sustainability of funding and infrastructures needed to ensure a comprehensive stakeholder-embedded community for epilepsy research.

Increased uptake of the P2X7 receptor radiotracer 18 F-JNJ-64413739 in the brain and peripheral organs according to the severity of status epilepticus in male mice.

OBJECTIVE: The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested 18 F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis. METHODS: Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of 18 F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. 18 F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy. RESULTS: P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control. SIGNIFICANCE: Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.

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.