Search

Objective: Exposure to chemical warfare nerve agents (CWNAs), such as soman (GD), can induce status epilepticus (SE) that becomes refractory to benzodiazepines when treatment is delayed, leading to increased risk of epileptogenesis, severe neuropathology, and long-term behavioral and cognitive deficits. Rodent models, widely used to evaluate novel medical countermeasures (MCMs) against CWNA exposure, normally express plasma carboxylesterase, an enzyme involved in the metabolism of certain organophosphorus compounds. To better predict the efficacy of novel MCMs against CWNA exposure in human casualties, it is crucial to use appropriate animal models that mirror the human condition. We present a comprehensive characterization of the seizurogenic, epileptogenic, and neuropathologic effects of GD exposure with delayed anticonvulsant treatment in the plasma carboxylesterase knockout (ES1-/-) mouse., Methods: Electroencephalography (EEG) electrode-implanted ES1-/- and wild-type (C57BL/6) mice were exposed to various seizure-inducing doses of GD, treated with atropine sulfate and the oxime HI-6 at 1 minute after exposure, and administered midazolam at 15-30 minutes following the onset of seizure activity. The latency of acute seizure onset and spontaneous recurrent seizures (SRS) was assessed, as were changes in EEG power spectra. At 2 weeks after GD exposure, neurodegeneration and neuroinflammation were assessed., Results: GD-exposed ES1-/- mice displayed a dose-dependent response in seizure severity. Only ES1-/- mice exposed to the highest tested dose of GD developed SE, subchronic alterations in EEG power spectra, and SRS. Degree of neuronal cell loss and neuroinflammation were dose-dependent; no significant neuropathology was observed in C57BL/6 mice or ES1-/- mice exposed to lower GD doses., Significance: The US Food and Drug Administration (FDA) animal rule requires the use of relevant animal models for the advancement of MCMs against CWNAs. We present evidence that argues for the use of the ES1-/- mouse model to screen anticonvulsant, antiepileptic, and/or neuroprotective drugs against GD-induced toxicity, as well as to identify mechanisms of GD-induced epileptogenesis., (© 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

Synaptic vesicle protein 2A (SV2A) has become an attractive target of investigation because of its role in the pathophysiology of epilepsy; SV2A is expressed ubiquitously throughout the brain in all nerve terminals independently of their neurotransmitter content and plays an important but poorly defined role in neurotransmission. Previous studies have shown that modifications in the SV2A protein expression could be a direct consequence of disease severity. Furthermore, these SV2A modifications may depend on specific changes in the nerve tissue following the induction of epilepsy and might be present in both excitatory and inhibitory terminals. Thus, we evaluated SV2A protein expression throughout the hippocampi of lithium-pilocarpine rats after status epilepticus (SE) and during early and late epilepsy. In addition, we determined the γ-aminobutyric acid (GABA)ergic or glutamatergic nature associated with SV2A modifications. Wistar rats were treated with lithium-pilocarpine to induce SE and subsequently were shown to present spontaneous recurrent seizures (SRS). Later, we conducted an exhaustive semi-quantitative analysis of SV2A optical density (OD) throughout the hippocampus by immunohistochemistry. Levels of the SV2A protein were substantially increased in layers formed by principal neurons after SE, mainly because of GABAergic activity. No changes were observed in the early stage of epilepsy. In the late stage of epilepsy, there were minor changes in SV2A OD compared with the robust modifications of SE; however, SV2A protein expression generally showed an increment reaching significant differences in two dendritic layers and hilus, without clear modifications of GABAergic or glutamatergic systems. Our results suggest that the SV2A variations may depend on several factors, such as neuronal activity, and might appear in both excitatory and inhibitory systems depending on the epilepsy stage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Epigenetics refers broadly to processes that influence medium- to long-term expression of genes through changes to the readability and accessibility of the genome to transcription. The mediators include DNA methylation, posttranslational modification of histone proteins, and noncoding RNAs. The present review focuses on recent findings showing epigenetic processes influence susceptibility to and severity of status epilepticus, while status epilepticus in turn drives changes to epigenetic marks that affect the gene expression landscape underlying epileptogenesis. Experimental status epilepticus triggers select, spatiotemporal hypo- and hypermethylation changes throughout the genome. Experimental manipulations that alter DNA methylation after status epilepticus are associated with amelioration of cognitive and hyperexcitability phenotypes. Prolonged seizures also modify chromatin compaction via acetylation and other changes to histones and their expression. Finally, short noncoding RNAs called microRNAs target networks of genes through posttranscriptional and other mechanisms and their modulation can alter status epilepticus as well as serve as potential molecular biomarkers. Long noncoding RNAs have also been targeted to influence expression of genes affecting electrophysiological functions of neurons. In summary, epigenetic processes can modulate status epilepticus, and status epilepticus imposes changes on the epigenome, which together provide novel insight into pathomechanisms, therapy, and biomarkers for status epilepticus., (Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.)

Argininemia is a rare hereditary disease due to a deficiency of hepatic arginase, which is the last enzyme of the urea cycle and hydrolyzes arginine to ornithine and urea. Herein we report a patient with arginase I (ARG1) deficiency who presented with recurrent nonconvulsive status epilepticus and liver failure. A novel homozygous frameshift mutation c.703_707delGGACTinsAGACTGGACC (p.G235Rfs*20) was detected.

Objectives: We describe a 13-year-old girl with a past medical history of epilepsy, intellectual impairment, dysphagia with gastric tube dependence, and autism spectrum disorder who presented with focal status epilepticus., Methods: Video-electroencephalography revealed left occipital pseudoperiodic epileptiform discharges and frequent seizures originating from the left hemisphere. The seizure was refractory to antiepileptic medications and pharmacologic coma. Subsequently, left occipital lobectomy was done. Extensive evaluation including whole exome sequencing, histopathologic examination of brain and muscle samples, mitochondrial DNA content analysis of tissue sample was completed to detect the etiology., Results: Skeletal muscle mitochondrial DNA content (qPCR) analysis showed approximately 37% of the mean value of age and tissue matched control group consistent with a mitochondrial depletion syndrome. Microscopic examination of the brain showed cortical abnormalities that largely consisted of infarct-like pathology in a laminar manner, abnormalities of neuronal distribution, and white matter changes. Compound heterozygous mutations of the CARS2 gene were identified by whole exome sequencing; V52G variant [p.Val52Gly (GTG>GGG):c.155 T>G in exon 1] was inherited from the mother and T188M variant[p.Thr188Met (ACG>ATG): c.563 C>T in exon 5] was inherited from the father., Conclusion: This is the first detailed clinicopathologic description of the Alpers-Huttenlocher syndrome phenotype from CARS mutations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Epileptogenesis, induced by status epilepticus (SE), is a chronic process, and intervention in this progress may prevent chronic epilepsy. It has been proposed that DNA methylation might be related with epileptogenesis. RASgrf1 has a differentially methylated region at the promoter which can silence gene expression. We have previously observed the down-regulation of RASgrf1 in epilepsy patients and proved that hypermethylation of RASgrf1 reaches maximal level at the latent period in mice after kainate-induced SE (KA mice), with corresponding alteration of RASgrf1 expression. In the present study, N-phthalyl-L-tryptophan (RG108), a DNA methyltransferase inhibitor, was applied in KA mice at latent phase and the behavior, electroencephalogram and pathological changes were observed in chronic phase. Methylation and expression of RASgrf1 were determined by polymerase chain reaction (PCR), western blotting, and bisulfite sequencing PCR. The results showed that the incidence of spontaneous recurrent seizures (SRS) was significantly lower in the RG108 group than the normal saline (NS) group. Subgroup analysis showed significant hypermethylation and lower expression of RASgrf1 in the RG108-SRS subgroup and the NS-SRS subgroup but not in the RG108-NSRS (no SRS) subgroup and the NS-NSRS subgroup compared with the control group. No significant difference was found between the RG108-SRS and NS-SRS subgroups. Meanwhile, hippocampal neuronal loss was observed in RG108-SRS and NS-SRS subgroups. We thus demonstrated that RG108 could modify the progression of epileptogenesis after KA induced SE and prevent chronic epilepsy. Meanwhile, hypermethylation of RASgrf1 after KA induced SE could be reversed with corresponding changes of RASgrf1 expression. Additionally, we speculated that RASgrf1 might be a potential epigenetic mediator in epileptogenesis and chronic epilepsy.

Inflammation is implicated in epileptogenesis. Activated microglia and macrophages (MG/MΦ) are found in the brains of patients with epilepsy-related diseases and animal models of epilepsy. It is not yet known how the MG/MΦ activation phenotype affects pathological changes in the brain after a single seizure. In this study, we had 2 main purposes: first, to characterize post-status epilepticus (SE) inflammation by tracking MG/MΦ polarization, and, second, to explore the role of an innate immune receptor adaptor protein, namely, myeloid differentiation primary response gene 88 (MyD88), in the induction of SE in a mouse model. A lithium-pilocarpine model of seizure conditions was generated in C57BL/6 mice. The intensity and distribution of MG/MΦ polarization were tracked by fluorescent immunohistochemistry and Western blotting for the polarization markers inducible nitrogen oxygenized synthase, arginase-1, CD163, and mannose receptor. We observed steadily increasing M1 MG/MΦ along with MyD88 signal upregulation after SE in the hippocampi of mice, whereas the M2 marker arginase-1 was localized mainly in astrocytes rather than in MG/MΦ. Inhibition or gene knockout of MyD88 reduced M1 MG/MΦ and gliosis although increasing M2 MG/MΦ in the hippocampi of SE mice. MyD88 inhibition also augmented glutamate transporter 1 expression and reduced N-methyl-D-aspartate receptor NR1 subunit expression in the hippocampus to protect pyramidal neurons from apoptosis. These data suggest that MG/MΦ polarization after SE impacts the pathological outcome of the hippocampus via MyD88 signaling and point to MyD88 as a potential neuroprotective target for epilepsy therapy.

Seizure-triggered maladaptive neural plasticity and neuroinflammation occur during the latent period as a key underlying event in epilepsy chronicization. Previously, we showed that α-tocopherol (α-T) reduces hippocampal neuroglial activation and neurodegeneration in the rat model of kainic acid (KA)-induced status epilepticus (SE). These findings allowed us to postulate an antiepileptogenic potential for α-T in hippocampal excitotoxicity, in line with clinical evidence showing that α-T improves seizure control in drug-resistant patients. To explore neurobiological correlates of the α-T antiepileptogenic role, rats were injected with such vitamin during the latent period starting right after KA-induced SE, and the effects on circuitry excitability, neuroinflammation, neuronal death, and microRNA (miRNA) expression were investigated in the hippocampus. Results show that in α-T-treated epileptic rats, (1) the number of population spikes elicited by pyramidal neurons, as well as the latency to the onset of epileptiform-like network activity recover to control levels; (2) neuronal death is almost prevented; (3) down-regulation of claudin, a blood-brain barrier protein, is fully reversed; (4) neuroinflammation processes are quenched (as indicated by the decrease of TNF-α, IL-1β, GFAP, IBA-1, and increase of IL-6); (5) miR-146a, miR-124, and miR-126 expression is coherently modulated in hippocampus and serum by α-T. These findings support the potential of a timely intervention with α-T in clinical management of SE to reduce epileptogenesis, thus preventing chronic epilepsy development. In addition, we suggest that the analysis of miRNA levels in serum could provide clinicians with a tool to evaluate disease evolution and the efficacy of α-T therapy in SE.

Temporal lobe epilepsy (TLE) is the chronic and pharmacoresistant form of epilepsy observed in humans. The current literature is insufficient in explicating the comprehensive mechanisms underlying its pathogenesis and advancement. Consequently, the development of a suitable animal model mimicking the clinical characteristics is required. Further, the relevance of status epilepticus (SE) to animal models is dubious. SE occurs rarely in people; most epilepsy patients never experience it. The present review summarizes the established animal models of SE and TLE, along with a brief discussion of the animal models that have the distinctiveness and carries the possibility to be developed as effective models for TLE. The review not only covers the basic requirements, mechanisms, and methods of induction of each model but also focuses upon their major limitations and possible modifications for their future use. A detailed discussion on chemical, electrical, and hypoxic/ischemic models as well as a brief explanation on the genetic models, most of which are characterized by development of SE followed by neurodegeneration, is presented.

Glycogen synthase kinase-3 (GSK-3) is ubiquitously expressed throughout the brain and involved in vital molecular pathways such as cell survival and synaptic reorganization and has emerged as a potential drug target for brain diseases. A causal role for GSK-3, in particular the brain-enriched GSK-3β isoform, has been demonstrated in neurodegenerative diseases such as Alzheimer's and Huntington's, and in psychiatric diseases. Recent studies have also linked GSK-3 dysregulation to neuropathological outcomes in epilepsy. To date, however, there has been no genetic evidence for the involvement of GSK-3 in seizure-induced pathology. Status epilepticus (prolonged, damaging seizure) was induced via a microinjection of kainic acid into the amygdala of mice. Studies were conducted using two transgenic mouse lines: a neuron-specific GSK-3β overexpression and a neuron-specific dominant-negative GSK-3β (GSK-3β-DN) expression in order to determine the effects of increased or decreased GSK-3β activity, respectively, on seizures and attendant pathological changes in the hippocampus. GSK-3 inhibitors were also employed to support the genetic approach. Status epilepticus resulted in a spatiotemporal regulation of GSK-3 expression and activity in the hippocampus, with decreased GSK-3 activity evident in non-damaged hippocampal areas. Consistent with this, overexpression of GSK-3β exacerbated status epilepticus-induced neurodegeneration in mice. Surprisingly, decreasing GSK-3 activity, either via overexpression of GSK-3β-DN or through the use of specific GSK-3 inhibitors, also exacerbated hippocampal damage and increased seizure severity during status epilepticus. In conclusion, our results demonstrate that the brain has limited tolerance for modulation of GSK-3 activity in the setting of epileptic brain injury. These findings caution against targeting GSK-3 as a treatment strategy for epilepsy or other neurologic disorders where neuronal hyperexcitability is an underlying pathomechanism.

Background: The dynamin 1-like gene ( DNM1L) encodes a GTPase that mediates mitochondrial and peroxisomal fission and fusion. We report a new clinical presentation associated with a DNM1L pathogenic variant and review the literature., Results: A 13-year-old boy with mild developmental delays and paroxysmal dystonia presented acutely with multifocal myoclonic super-refractory status epilepticus. Despite sustained and aggressive treatment, seizures persisted and care was ultimately withdrawn in the context of extensive global cortical atrophy. Rapid trio-whole exome sequencing revealed a de novo heterozygous c.1207C>T (p.R403C) pathogenic variant in DNM1L. Immunofluorescence staining of fibroblast mitochondria revealed abnormally elongated and tubular morphology., Conclusions: This case highlights the diagnostic importance of rapid whole exome sequencing within a critical care setting and reveals the expanding phenotypic spectrum associated with DNM1L variants. This now includes progressive paroxysmal dystonia and adolescent-onset super-refractory myoclonic status epilepticus contributing to strikingly rapid and progressive cortical atrophy and death.

This study aimed to identify key genes (microRNA and messenger RNA (mRNA)) and associated signaling-regulated pathways in a drug-induced epilepsy model in mice by microarray profiling. The related microarray dataset of seizures was obtained from the NCBI Gene Expression Omnibus database (GEO), and differentially expressed genes (DEGs) between two control samples or multi-treated samples and samples were analyzed using the statistical software R. To identify the expected function of DEGs, Gene Set Enrichment Analysis (GSEA) was utilized to conduct Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The interaction relationship between microRNAs (miRNAs) and mRNAs in normal and epilepsy mouse models was identified using Cytoscape software. TargetScan7.1 was applied to determine the binding sites of DEGs. The dual-luciferase assay was used to verify the target relationship between miRNA and mRNA. Four miRNAs were identified as differentially expressed genes in both 24-h and 28-day status epilepticus (SE)-treated samples. Ppp2ca expression in the mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the pilocarpine-induced SE mouse model. The expression of Ppp2ca was also downregulated in the kinase-induced SE model group compared with that in the untreated group and MAP kinase (MEK) inhibitor-treated group of mice. KEGG pathway analysis indicated that the MAPK signaling pathway was upregulated in the kinase-induced SE model group compared with that in both the untreated group and the MEK inhibitor-treated group of mice. miR-203 had a targeted relationship with Ppp2ca in both humans and mice. The miR-203-3p target Ppp2ca aggravates the seizures of the SE model in mice.

Status epilepticus (SE) is an abnormally prolonged seizure that results from either a failure of mechanisms that terminate seizures or from initiating mechanisms that inherently lead to prolonged seizures. Here we report that mice experiencing a 3 hours of SE caused by pilocarpine exhibit a rapid increase in expression of type 2 iodothyronine deiodinase gene (Dio2) and a decrease in the expression of type 3 iodothyronine deiodinase gene in hippocampus, amygdala and prefrontal cortex. Type 3 iodothyronine deiodinase in hippocampal sections was seen concentrated in the neuronal nuclei, typical of ischemic injury of the brain. An unbiased analysis of the hippocampal transcriptome of mice undergoing 3 hours of SE revealed a number of genes, including those involved with response to oxidative stress, cellular homeostasis, cell signaling, and mitochondrial structure. In contrast, in mice with targeted disruption of Dio2 in astrocytes (Astro D2KO mouse), the highly induced genes in the hippocampus were related to inflammation, apoptosis, and cell death. We propose that Dio2 induction caused by SE accelerates production of T3 in different areas of the central nervous system and modifies the hippocampal gene expression profile, affecting the balance between adaptive and maladaptive mechanisms.

Background: Electrical status epilepticus during slow-wave sleep (ESESS) which is also known as continuous spike-wave of slow sleep (CSWSS) is type of electroencephalographic (EEG) pattern which is seen in ESESS/CSWSS/epilepsy aphasia spectrum. This EEG pattern can occur alone or with other syndromes. Its etiology is not clear, however, brain malformations, immune disorders, and genetic etiologies are suspected to contribute. We aimed to perform a systematic review of all genetic etiologies which have been reported to associate with ESESS/CSWSS/epilepsy-aphasia spectrum. We further aimed to identify the common underlying pathway which can explain it. To our knowledge, there is no available systematic review of genetic etiologies of ESESS/CSWSS/epilepsy-aphasia spectrum. MEDLINE, EMBASE, PubMed and Cochrane review database were searched, using terms specific to electrical status epilepticus during sleep or continuous spike-wave discharges during slow sleep or epilepsy-aphasia spectrum and of studies of genetic etiologies. These included monogenic mutations and copy number variations (CNVs). For each suspected dosage-sensitive gene, further studies were performed through OMIM and PubMed database., Results: Twenty-six studies out of the 136 identified studies satisfied our inclusion criteria. I51 cases were identified among those 26 studies. 16 studies reported 11 monogenic mutations: SCN2A (N = 6), NHE6/SLC9A6 (N = 1), DRPLA/ ATN1 (N = 1), Neuroserpin/SRPX2 (N = 1), OPA3 (N = 1), KCNQ2 (N = 2), KCNA2 (N = 5), GRIN2A (N = 34), CNKSR2 (N = 2), SLC6A1 (N = 2) and KCNB1 (N = 5). 10 studies reported 89 CNVs including 9 recurrent ones: Xp22.12 deletion encompassing CNKSR2 (N = 6), 16p13 deletion encompassing GRIN2A (N = 4), 15q11.2-13.1 duplication (N = 15), 3q29 duplication (N = 11), 11p13 duplication (N = 2), 10q21.3 deletion (N = 2), 3q25 deletion (N = 2), 8p23.3 deletion (N = 2) and 9p24.2 (N = 2). 68 of the reported genetic etiologies including monogenic mutations and CNVs were detected in patients with ESESS/CSWSS/epilepsy aphasia spectrum solely. The most common underlying pathway was channelopathy (N = 56)., Conclusions: Our review suggests that genetic etiologies have a role to play in the occurrence of ESESS/CSWSS/epilepsy-aphasia spectrum. The common underlying pathway is channelopathy. Therefore we propose more genetic studies to be done for more discoveries which can pave a way for proper drug identification. We also suggest development of common cut-off value for spike-wave index to ensure common language among clinicians and researchers.

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.

Aberrant cell cycle re-entry promotes neuronal death in various neurological diseases. Thus, cyclin-dependent kinases (CDKs) seem to be one of potential therapeutic targets to prevent neuronal loss. In the present study, we investigated the involvements of CDK4, CDK5 and p27 Kip1 (an endogenous CDK inhibitor) in status epilepticus (SE)-induced neuronal death. Following SE, CDK4 expression was increased in CA1 neurons, while CDK5 was decreased. Most of TUNEL-positive neurons showed CDK4 expression, but less CDK5 expression. Flavopiridol (a CDK4 inhibitor) attenuated TUNEL signal and CDK4 expression in CA1 neurons following SE. CDK5 inhibitors did not affect these phenomena. Both flavopiridol and leptomycin B (an inhibitor of chromosome region maintenance 1) mitigated SE-induced neuronal death by inhibiting nucleocytoplasmic p27 Kip1 translocation. These findings suggest that SE may lead to nucleocytoplasmic p27 Kip1 export that initiates CDK4, not CDK5, induction, which an abortive and fatal cell cycle re-entry progress in CA1 neurons., (Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.)

Temporal lobe epilepsy (TLE) is one of the most common types of intractable epilepsy, characterized by hippocampal neuron damage and hippocampal sclerosis. Long noncoding RNAs (lncRNAs) have been increasingly recognized as posttranscriptional regulators. However, their expression levels and functions in TLE remain largely unknown. In the present study, TLE rat model is used to explore the expression profiles of lncRNAs in the hippocampus of epileptic rats using microarray analysis. Our results demonstrate that H19 is the most pronouncedly differentiated lncRNA, significantly upregulated in the latent period of TLE. Moreover, the in vivo studies using gain- and loss-of-function approaches reveal that the overexpression of H19 aggravates SE-induced neuron apoptosis in the hippocampus, while inhibition of H19 protects the rats from SE-induced cellular injury. Finally, we show that H19 might function as a competing endogenous RNA to sponge microRNA let-7b in the regulation of cellular apoptosis. Overall, our study reveals a novel lncRNA H19-mediated mechanism in seizure-induced neural damage and provides a new target in developing lncRNA-based strategies to reduce seizure-induced brain injury.

Insights into the dynamic changes in molecular processes occurring in the brain during epileptogenesis can substantially improve our understanding of their pathogenetic relevance. In this context, neuroinflammation is a potential mechanism of epileptogenesis which has recently been investigated in animal models by MRI or PET molecular imaging. Here, we developed an alternative and complementary molecular imaging strategy by designing a serotype 8 recombinant adeno-associated virus (AAV8) harboring promoter fragments of the GFAP or IL-1β promoter and a luciferase reporter gene. Mice were injected intrahippocampally with rAAV8 and treated with intracortical kainic acid to induce status epilepticus (SE) and hence epileptogenesis. In vivo bioluminescence imaging combined with immunohistochemistry revealed a significant activation of the GFAP promoter 24 h and 3 days after kainate-induced SE. For IL-1β, we identified the promoter region required for studying cell-specific induction of the promoter in longitudinal studies. We conclude that the GFAP promoter fragment represents a useful tool for monitoring the in vivo activation of astrocytes with an inflammatory phenotype during epileptogenesis, or under other pathophysiological conditions.

Recently, we have reported that heat shock protein B1 (HSPB1) and purinergic receptor P2X7 (P2RX7) are involved in astroglial autophagy (clasmatodendrosis), following status epilepticus (SE). However, the underlying mechanisms of astroglial autophagy have not been completely established. In the present study, we found that the lacking of P2rx7 led to prolonged astroglial HSPB1 induction due to impaired mitogen-activated protein kinase 1/2 (MAPK1/2)-mediated specificity protein 1 (SP1) phosphorylation, following kainic acid-induced SE. Subsequently, the upregulated HSPB1 itself evoked ER stress and exerted protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1, AMPK1)/unc-51 such as autophagy activating kinase 1 (ULK1)- and AKT serine/threonine kinase 1 (AKT1)/glycogen synthase kinase 3 beta (GSK3B)/SH3-domain GRB2-like B1 (SH3GLB1)-mediated autophagic pathways, independent of mechanistic target of rapamycin (MTOR) activity in astrocytes. These findings provide a novel purinergic suppression mechanism to link chaperone expression to autophagy in astrocytes. Therefore, we suggest that P2RX7 may play an important role in the regulation of autophagy by the fine-tuning of HSPB1 expression.

Reorganization of the neurovascular unit has been suggested in the epileptic brain, although the dynamics and functional significance remain unclear. Here, we tracked the in vivo dynamics of perivascular mural cells as a function of electroencephalogram (EEG) activity following status epilepticus. We segmented the cortical vascular bed to provide a size- and type-specific analysis of mural cell plasticity topologically. We find that mural cells are added and removed from veins, arterioles, and capillaries after seizure induction. Loss of mural cells is proportional to seizure severity and vascular pathology (e.g., rigidity, perfusion, and permeability). Treatment with platelet-derived growth factor subunits BB (PDGF-BB) reduced mural cell loss, vascular pathology, and epileptiform EEG activity. We propose that perivascular mural cells play a pivotal role in seizures and are potential targets for reducing pathophysiology., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Ring chromosome 20 (R20) syndrome is a chromosomal disorder characterized mainly by drug-resistant frontal lobe seizures, recurrent nonconvulsive status epilepticus (NCSE), and typical EEG features. The aim of this study was to investigate if this triad is common and specific to all patients with R20., Methods: In this cross-sectional study (from 2000 to 2011), we selected patients who fulfilled at least two out of three criteria: drug-resistant frontal lobe seizures, recurrent NCSE, and characteristic electroencephalography (EEG) features. In all patients, diagnosis was based on karyotype analysis of at least 100 metaphases., Results: We identified 36 patients who met at least two of the selected criteria: six patients (16.7%) with R20 and 30 (83.3%) without R20 (non-R20). All patients with R20 met all three criteria. Eleven (36.7%) patients without R20, however, also displayed the full triad. In 19 patients without R20 (63.3%), one of the three clinical features was missing: frontal lobe seizures were not resistant to antiepileptic drugs (AED) in four (13.3%), recurrent NCSE was missing in six (20%), and nine (30%) patients did not have typical EEG features. Based on this data, specificity was 63.3%, positive predictive value was 35.3%, and sensitivity and negative predictive values were 100%. Additionally, a review of all publications describing the R20 phenotype revealed that 81.98% of patients with R20 display the full electroclinical triad., Conclusions: In our study, all patients with R20 displayed the three electroclinical characteristics. This is in line with previous reports (presenting high sensitivity and negative predictive value). However, these features can also be observed in other epilepsies and are not specific to R20. Our findings suggest that in the presence of the full triad of symptoms, karyotype analysis focused on chromosome 20 should be conducted., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Soman (GD) exposure results in status epilepticus (SE) that leads to neurodegeneration, neuroinflammation, and behavioral consequences including learning and memory deficits. The neuroinflammatory response is characterized by the upregulation of the pro-inflammatory cytokine, interleukin-1 (IL-1), which mediates the expression of other neurotoxic cytokines induced after GD exposure. However, the specific role of IL-1 signaling has not been defined in terms of the consequences of GD-induced SE. Therefore, the purpose of this study was to regulate IL-1 signaling and study the behavioral deficits and neurodegeneration that occur after convulsion onset. Wild type (WT), IL-1 receptor (IL-1R1) knockout (KO), and IL-1 receptor antagonist (IL-1Ra) KO mice were exposed to a convulsive dose of GD, and behavior was evaluated up to 18days later. Activity was studied using the Open Field, anxiety was assessed in the Zero Maze, and spatial learning and memory were evaluated with the Barnes Maze. The animals were euthanized at 24hours and 18days to determine neuropathology in the piriform cortex, amygdala, thalamus, and CA1, CA2/3, and CA4 regions of the hippocampus. Unlike the IL-1Ra KO, the IL-1R1 KO showed less neuropathology compared to WT at 24hours, but moderate to severe injury was found in all strains at 18days. Compared to their saline controls, the exposed WT mice were significantly more active in the Open Field, and the IL-1R1 KO strain showed reduced anxiety in the Zero Maze Test. Compared to WT mice, IL-1R1 and IL-1Ra KO mice had spatial learning and memory impairments in the Barnes Maze. Therefore, the IL-1 signaling pathway affects neurodegeneration and behavior after GD-induced convulsions., (Published by Elsevier B.V.)

This study aims to investigate the role of zolpidem in lithium-pilocarpine induced status epilepticus (SE) and probable mechanisms involved in seizure threshold alteration. In the present study, lithium chloride (127mg/kg) was administered 20h prior to pilocarpine (60mg/kg) to induce SE in adult male Wistar rats. Different doses of zolpidem (0.1, 1, 2, 5, 10mg/kg) were injected 30min before pilocarpine administration. Furthermore, to find out whether nitric oxide (NO) plays a role in the observed effect, L-arginine and L-NAME were injected 15min before zolpidem. Afterward, we identified the particular NO isoform mediating the effect of zolpidem by injecting aminoguanidine (AG) and 7-Nitroindazole (7-NI) 15min prior to zolpidem. Moreover, in both 6 and 24h after pilocarpine injection, experimental groups underwent hippocampectomy to evaluate cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes expression by quantitative reverse transcription-PCR (qRT-PCR). Pre-treatment with zolpidem significantly prevented the onset of SE in a dose-dependent manner. AG and L-NAME significantly potentiated the anticonvulsant effect of zolpidem while L-arginine inverted this effect. Our qRT-PCR exerted that there was a continuous elevation of iNOS and COX-2 genes expression over 6 and 24h after pilocarpine administration in SE and L-arginine+Zolpidem groups while in AG/L-NAME+Zolpidem and zolpidem groups this upregulation was prevented. Our study indicates that zolpidem prevents the onset of SE through inhibition of iNOS/COX-2 genes upregulation following lithium-pilocarpine administration. Consistent with our results, we suggest that iNOS activation could be probably upstream of COX-2 gene expression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mitogen-activated protein kinase (MAPK) signaling has been implicated in a wide range of neuronal processes, including development, plasticity, and viability. One of the principal downstream targets of both the extracellular signal-regulated kinase/MAPK pathway and the p38 MAPK pathway is Mitogen- and Stress-activated protein Kinase 1 (MSK1). Here, we sought to understand the role that MSK1 plays in neuroprotection against excitotoxic stimulation in the hippocampus. To this end, we utilized immunohistochemical labeling, a MSK1 null mouse line, cell viability assays, and array-based profiling approaches. Initially, we show that MSK1 is broadly expressed within the major neuronal cell layers of the hippocampus and that status epilepticus drives acute induction of MSK1 activation. In response to the status epilepticus paradigm, MSK1 KO mice exhibited a striking increase in vulnerability to pilocarpine-evoked cell death within the CA1 and CA3 cell layers. Further, cultured MSK1 null neurons exhibited a heighted level of N-methyl-D-aspartate-evoked excitotoxicity relative to wild-type neurons, as assessed using the lactate dehydrogenase assay. Given these findings, we examined the hippocampal transcriptional profile of MSK1 null mice. Affymetrix array profiling revealed that MSK1 deletion led to the significant (>1.25-fold) downregulation of 130 genes and an upregulation of 145 genes. Notably, functional analysis indicated that a subset of these genes contribute to neuroprotective signaling networks. Together, these data provide important new insights into the mechanism by which the MAPK/MSK1 signaling cassette confers neuroprotection against excitotoxic insults. Approaches designed to upregulate or mimic the functional effects of MSK1 may prove beneficial against an array of degenerative processes resulting from excitotoxic insults.

Status epilepticus (SE) can cause brain damage and lead to neural dysfunction. Developing novel targets for SE therapy and diagnosis is important and necessary. Previously, we found several differentially expressed microRNAs (miRNAs) in the developing hippocampus following SE, including the autophagy-related miR-96. In the present study, we employed immunofluorescence staining and Western blot analysis to assess the expression of autophagy-related 7 (Atg7) and Atg16L1 and the status of autophagosome formation in the hippocampus of immature rats with SE. Additional in vivo intervention was also performed to investigate the potential therapeutic function of miR-96 in developing rats with SE. We found that Atg7 and Atg16L1 were up-regulated in the neurons after SE, together with an increase in autophagosome formation. Meanwhile, overexpression of miR-96 significantly prevented brain damage in SE rats by inhibiting Atg7 and Atg16L1 expression and autophagosome formation in the hippocampus. Furthermore, Rapamycin negated miR-96 mediated brain injury attenuation through inducing autophagosome formation. Our study indicates that miR-96 might be a potential target for therapy of pediatric SE.

Overexpression of P-glycoprotein (P-gp) in the brain is an important mechanism involved in drug‑resistant epilepsy (DRE). High-mobility group box 1 (HMGB1), an inflammatory cytokine, significantly increases following seizures and may be involved in upregulation of P‑gp. However, the underlying mechanisms remain elusive. The aim of the present study was to evaluate the role of HMGB1 and its downstream signaling components, receptor for advanced glycation end‑product (RAGE) and nuclear factor‑κB (NF‑κB), on P‑gp expression in rat brains during status epilepticus (SE). Small interfering RNA (siRNA) was administered to rats prior to induction of SE by pilocarpine, to block transcription of the genes encoding HMGB1 and RAGE, respectively. An inhibitor of NF‑κB, pyrrolidinedithiocarbamic acid (PDTC), was utilized to inhibit activation of NF‑κB. The expression levels of HMGB1, RAGE, phosphorylated‑NF‑κB p65 (p‑p65) and P‑gp were detected by western blotting. The relative mRNA expression levels of the genes encoding these proteins were measured using reverse transcription‑quantitative polymerase chain reaction and the cellular localization of the proteins was determined by immunofluorescence. Pre‑treatment with HMGB1 siRNA reduced the expression levels of RAGE, p‑p65 and P‑gp. PDTC reduced the expression levels of P‑gp. These findings suggested that overexpression of P‑gp during seizures may be regulated by HMGB1 via the RAGE/NF‑κB signaling pathway, and may be a novel target for treating DRE.

Brief, non-harmful seizures (preconditioning) can temporarily protect the brain against prolonged, otherwise injurious seizures. Following focal-onset status epilepticus (SE) in preconditioned (tolerance) and sham-preconditioned (injury) mice, we screened for protein changes using a proteomic approach and identified several putative candidates of epileptic tolerance. Among SE-induced changes to both proteomic screens, proteins clustered in key regulatory pathways, including protein trafficking and cytoskeletal regulation. Downregulation of one such protein, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), was unique to injury and not evident in tolerance. UCHL1 inhibition decreased hippocampal ubiquitin, disrupted UPS function, interfered with seizure termination and exacerbated seizure-induced cell death. Though UCHL1 transcription was maintained after SE, we observed downregulation of the pro-translational antisense Uchl1 (AsUchl1) and confirmed that both AsUchl1 and rapamycin can increase UCHL1 expression in vivo. These data indicate that the post-transcriptional loss of UCHL1 following SE is deleterious to neuronal survival and may contribute to hyperexcitability, and are suggestive of a novel modality of rapamycin therapy.

There is a need for diagnostic biomarkers of epilepsy and status epilepticus to support clinical examination, electroencephalography and neuroimaging. Extracellular microRNAs may be potentially ideal biomarkers since some are expressed uniquely within specific brain regions and cell types. Cerebrospinal fluid offers a source of microRNA biomarkers with the advantage of being in close contact with the target tissue and sites of pathology. Here we profiled microRNA levels in cerebrospinal fluid from patients with temporal lobe epilepsy or status epilepticus, and compared findings to matched controls. Differential expression of 20 microRNAs was detected between patient groups and controls. A validation phase included an expanded cohort and samples from patients with other neurological diseases. This identified lower levels of miR-19b in temporal lobe epilepsy compared to controls, status epilepticus and other neurological diseases. Levels of miR-451a were higher in status epilepticus compared to other groups whereas miR-21-5p differed in status epilepticus compared to temporal lobe epilepsy but not to other neurological diseases. Targets of these microRNAs include proteins regulating neuronal death, tissue remodelling, gliosis and inflammation. The present study indicates cerebrospinal fluid contains microRNAs that can support differential diagnosis of temporal lobe epilepsy and status epilepticus from other neurological and non-neurological diseases.

This article describes the recent advances in epileptogenesis and novel therapeutic approaches for the prevention of epilepsy, with a special emphasis on the pharmacological basis of disease-modification of epileptogenesis for curing epilepsy. Here we assess animal studies and human clinical trials of epilepsy spanning 1982-2016. Epilepsy arises from a number of neuronal factors that trigger epileptogenesis, which is the process by which a brain shifts from a normal physiologic state to an epileptic condition. The events precipitating these changes can be of diverse origin, including traumatic brain injury, cerebrovascular damage, infections, chemical neurotoxicity, and emergency seizure conditions such as status epilepticus. Expectedly, the molecular and system mechanisms responsible for epileptogenesis are not well defined or understood. To date, there is no approved therapy for the prevention of epilepsy. Epigenetic dysregulation, neuroinflammation, and neurodegeneration appear to trigger epileptogenesis. Targeted drugs are being identified that can truly prevent the development of epilepsy in at-risk people. The promising agents include rapamycin, COX-2 inhibitors, TRK inhibitors, epigenetic modulators, JAK-STAT inhibitors, and neurosteroids. Recent evidence suggests that neurosteroids may play a role in modulating epileptogenesis. A number of promising drugs are under investigation for the prevention or modification of epileptogenesis to halt the development of epilepsy. Some drugs in development appear rational for preventing epilepsy because they target the initial trigger or related signaling pathways as the brain becomes progressively more prone to seizures. Additional research into the target validity and clinical investigation is essential to make new frontiers in curing epilepsy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Global expression profiling of neurologic or psychiatric disorders has been confounded by variability among laboratories, animal models, tissues sampled, and experimental platforms, with the result being that few genes demonstrate consistent expression changes. We attempted to minimize these confounds by pooling dentate granule cell transcriptional profiles from 164 rats in seven laboratories, using three status epilepticus (SE) epilepsy models (pilocarpine, kainate, self-sustained SE), plus amygdala kindling. In each epilepsy model, RNA was harvested from laser-captured dentate granule cells from six rats at four time points early in the process of developing epilepsy, and data were collected from two independent laboratories in each rodent model except SSSE. Hierarchical clustering of differentially-expressed transcripts in the three SE models revealed complete separation between controls and SE rats isolated 1 day after SE. However, concordance of gene expression changes in the SE models was only 26-38% between laboratories, and 4.5% among models, validating the consortium approach. Transcripts with unusually highly variable control expression across laboratories provide a 'red herring' list for low-powered studies.

The current knowledge about neuroprotective mechanisms in humans after status epilepticus is scarce. One reason is the difficulty to measure possible mediators of these neuroprotective mechanisms. The dawn of microRNA detection in the cerebrospinal fluid (CSF) and the recent advancements in measuring proteins in the CSF such as progranulin, which is, e.g., responsible for neurite outgrowth and limiting exceeding neuroinflammatory responses, have given us new insights into putative neuroprotective mechanisms following status epilepticus. This should complement the animal data. In this review, we cover what is known about the role of progranulin as well as the links between microRNA changes and the progranulin pathway following status epilepticus in humans and animals hypothesizing neuroprotective and neurorehabilitative effects. Progranulin has also been found to feature prominently in the neuroprotective processes under hypoxic conditions and initiating neurorehabilitative processes. These properties may be used therapeutically, e.g., through drugs that raise the progranulin levels and therefore the cerebral progranulin levels as well with the goal of improving the outcome after status epilepticus.

Objective: Canonical transient receptor potential (TRPC) channels constitute a family of cation channels that exhibit a regional and cell-specific expression pattern throughout the brain. It has been reported previously that TRPC3 channels are effectors of the brain-derived neurotrophic factor (BDNF)/trkB signaling pathway. Given the long postulated role of BDNF in epileptogenesis, TRPC3 channels may be a critical component in the underlying pathophysiology of seizure and epilepsy. In this study, we investigated the precise role of TRPC3 channels in pilocarpine-induced status epilepticus (SE)., Methods: The role of TRPC3 channels was investigated using TRPC3 knockout (KO) mice and TRPC3-selective inhibitor Pyr3. Video and electroencephalography (EEG) recording of pilocarpine-induced seizures were performed., Results: We found that genetic ablation of TRPC3 channels reduces behavioral manifestations of seizures and the root-mean-square (RMS) power of SE, indicating a significant contribution of TRPC3 channels to pilocarpine-induced SE. Furthermore, the reduction in SE in TRPC3KO mice is caused by a selective attenuation of pilocarpine-induced theta activity, which dominates both the preictal phase and SE phase. Pyr3 also caused a reduction in the overall RMS power of pilocarpine-induced SE and a selective reduction in the theta activity during SE., Significance: Our results demonstrate that TRPC3 channels unequivocally contribute to pilocarpine-induced SE and could be a novel molecular target for new anticonvulsive drugs., Competing Interests: of Conflict of Interest: None of the authors has any conflict of interest to disclose., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

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., Competing Interests: The authors declare no competing financial interests.

Several members of the Bcl-2 gene family are dysregulated in human temporal lobe epilepsy and animal studies show that genetic deletion of some of these proteins influence electrographic seizure responses to chemoconvulsants and associated brain damage. The BH3-only proteins form a subgroup comprising direct activators of Bax-Bak that are potently proapoptotic and a number of weaker proapoptotic BH3-only proteins that act as sensitizers by neutralization of antiapoptotic Bcl-2 family members. Noxa was originally characterized as a weaker proapoptotic, 'sensitizer' BH3-only protein, although recent evidence suggests it too may be potently proapoptotic. Expression of Noxa is under p53 control, a known seizure-activated pathway, although Noxa has been linked to energetic stress and autophagy. Here we characterized the response of Noxa to prolonged seizures and the phenotype of mice lacking Noxa. Status epilepticus induced by intra-amygdala kainic acid caused a rapid increase in expression of noxa in the damaged CA3 subfield of the hippocampus but not undamaged CA1 region. In vivo upregulation of noxa was reduced by pifithrin-α, suggesting transcription may be partly p53-dependent. Mice lacking noxa developed less severe electrographic seizures during status epilepticus in the model but, surprisingly, displayed equivalent hippocampal damage to wild-type animals. The present findings indicate Noxa does not serve as a proapoptotic BH3-only protein during seizure-induced neuronal death in vivo. This study extends the comprehensive phenotyping of seizure and damage responses in mice lacking specific Bcl-2 gene family members and provides further evidence that these proteins may serve roles beyond control of cell death in the brain.

We report a female patient with Dravet syndrome (DS) with erratic segmental myoclonus, the origin of which was first identified in the cerebral cortex by the detection of myoclonus-associated cortical discharges. The discharges were disclosed through jerk-locked back-averaging of electroencephalogram (EEG) data using the muscle activity of myoclonus as triggers. The detected spikes on the contralateral parieto-central region preceded myoclonic muscle activity in the forearms by 28-46ms. The patient was six months old at the time of examination, and was developing normally before seizure onset at two months of age. She suffered from recurrent afebrile or febrile generalized tonic-clonic seizures that often developed into status epilepticus. Interictal EEG and brain magnetic resonance imaging (MRI) showed no significant findings. The amplitudes of the somatosensory-evoked potentials were not extremely large. She has a chromosomal microdeletion involving SCN1A and adjacent genes., (Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Accumulating evidence suggest that TRPC channels play critical roles in various aspects of epileptogenesis. TRPC1/4 channels are major contributors to nonsynaptically derived epileptiform burst firing in the CA1 and the lateral septum. TRPC7 channels play a critical role in synaptically derived epileptiform burst firing. The reduction of spontaneous epileptiform bursting in the CA3 is correlated to a reduction in pilocarpine-induced SE in vivo in TRPC7 knockout mice. TRPC channels are also significant contributors to SE-induced neuronal cell death. Although the pilocarpine-induced SE itself is not significantly reduced, the SE-induced neuronal cell death is significantly reduced in the CA1 and the lateral septum, indicating that TRPC1/4 channels directly contribute to SE-induced neuronal cell death. Genetic ablation of TRPC5 also reduces SE-induced neuronal cell death in the CA1 and CA3 areas of the hippocampus.

Background: MiR-134 is enriched in dendrites of hippocampal neurons and plays crucial roles in the progress of epilepsy. The present study aims to investigate the effects of antagomirs targeting miroRNA-134 (Ant-134) on limk1 expression and the binding of miR-134 and limk1 in experimental seizure., Methods: Status epilepticus (SE) rat model was established by lithium chloride-pilocarpine injection and was treated with Ant-134 by intracerebroventricular injection. Low Mg2+-exposed primary neurons were used as an in vitro model of SE. The expression of miR-134 was determined using real-time PCR. Protein expressions of limk1 and cofilin were determined by Western blotting. Luciferase reporter assay was used to examine the binding between miR-134 and limk1 3'-untranslated region., Results: The expression of miR-134 was markedly enhanced in hippocampus of the SE rats and low Mg2+-exposed neurons. Ant-134 increased the expression of limk1 and reduced the expression of cofilin in the SE hippocampus and Low Mg2+-exposed neurons. In addition, luciferase reporter assay confirmed that miR-134 bound limk1 3'-UTR. MiR-134 overexpression inhibited limk1 mRNA and protein expressions in neurons., Conclusion: Blockage of miR-134 upregulates limk1 expression and downregulated cofilin expression in hippocampus of the SE rats. This mechanism may contribute to the neuroprotective effects of Ant-134., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine-induced status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3'UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-β receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naïve group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective To observe the expression of snail in mouse cortex in the model of status epilepticus (SE) induced by kainic acid (KA) and explore the role of snail in SE. Methods Sixty-four adult male C57/BL6 mice were randomly divided into control group and SE group, 32 mice in each group. Intraperitoneal injection of normal saline was performed in control group; instead, KA was injected intraperitoneally in SE group to induce SE. The organs were harvested 3, 8, 24 and 72 hours post the cessation of each group. Reverse transcription-PCR was used to detect the expression of snail mRNA; the expression of snail protein was tested by Western blotting; the distribution of snail was determined using immunohistochemistry; snail expression in cortex was located by immunofluorescence technique. Results Compared with the control group, mRNA and protein expression of snail in SE group were significant higher, and they reached the peak at 24 hours. Immunohistochemistry indicated weak positive expression of snail in control group. In SE group, it was widely distributed in different cortical neurons and glial cells. Immunofluorescence histochemistry further proved that snail was localized in neurons and astrocytes. Conclusion Snail is strongly expressed in neuron and astrocyte of mouse cortex at KA-induced SE, which indicates snail is possibly related to the attack of SE.

Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant-induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild-type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild-type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV-1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild-type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV-2959 in both models. This finding indicates that JMV-1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

Ring chromosome 20 [r(20)] syndrome is a rare chromosomal disorder that is characterized by the development of refractory epilepsy during childhood with gradual declines in cognitive performance and behavior. Although the prognoses of seizures and intellectual disability associated with this condition are poor, life-threatening complications have rarely been described. We herein presented a case of a 17-year-old female with [r(20)] syndrome who developed recurrent status epilepticus (SE) at 14years of age that evolved into unremitting SE in spite of vigorous antiepileptic treatments. She was administered thiopental anesthesia for 1year, and was subsequently left in severe neurological sequelae. It is important to note that patients with this syndrome not only have severe epileptic encephalopathy persisting into adulthood, but are also at risk of fatal SE., (Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

We evaluated seizure profile, Toll-like receptor (TLR)-4 polymorphisms, and serum matrix metalloproteinases (MMPs) in patients with calcified neurocysticercosis.One-hundred nine patients with calcified neurocysticercosis with newly diagnosed seizures and 109 control subjects were enrolled. TLR-4 Asp299Gly and Thr399Ile polymorphisms and serum MMP-9 levels were evaluated. The patients were followed for 1 year.Asp/Gly (P = 0.012) and Thr/Ile (P = 0.002), Gly (Asp/Gly plus Gly/Gly) (P = 0.008) and Ile (Thr/Ile plus Ile/Ile) (P = 0.003) genotypes were significantly associated with calcified neurocysticercosis compared with controls. Gly/Gly and Ile/Ile genotypes were not significantly associated (P = 0.529 for Gly/Gly, P = 0.798 for Ile/Ile) with either group. The levels of MMP-9 were higher in calcified neurocysticercosis (P =  < 0.001). The levels of MMP-9 were higher in patients with multiple calcified neurocysticercosis compared with single calcified neurocysticercosis (P =  < 0.001).Headache (P = 0.031), status epilepticus (P = 0.029), Todd paralysis (P = 0.039), lesion size >10 mm (P = 0.001), and perilesional edema (P =  < 0.001) were significantly associated with seizure recurrence. Heterozygous form Asp/Gly (P =  < 0.001) and heterozygous form Thr/Ile (P =  < 0.001) were significantly associated with seizure recurrence. The Gly (Asp/Gly plus Gly/Gly) (P =  < 0.001) and Ile (Thr/Ile plus Ile/Ile) (P =  < 0.001) genotypes were also significantly associated with seizure recurrence. Higher serum MMP-9 levels were significantly associated with seizure recurrence (P =  < 0.001).The TLR-4 gene abnormalities may trigger inflammation around calcified neurocysticercosis leading to an increase in perilesional edema and provocation of seizures., Competing Interests: The authors have no conflicts of interest to disclose.

Insult-provoked transformation of neuronal networks into epileptic ones involves multiple mechanisms. Intervention studies have identified both dysregulated inflammatory pathways and NRSF-mediated repression of crucial neuronal genes as contributors to epileptogenesis. However, it remains unclear how epilepsy-provoking insults (e.g., prolonged seizures) induce both inflammation and NRSF and whether common mechanisms exist. We examined miR-124 as a candidate dual regulator of NRSF and inflammatory pathways. Status epilepticus (SE) led to reduced miR-124 expression via SIRT1--and, in turn, miR-124 repression--via C/EBPα upregulated NRSF. We tested whether augmenting miR-124 after SE would abort epileptogenesis by preventing inflammation and NRSF upregulation. SE-sustaining animals developed epilepsy, but supplementing miR-124 did not modify epileptogenesis. Examining this result further, we found that synthetic miR-124 not only effectively blocked NRSF upregulation and rescued NRSF target genes, but also augmented microglia activation and inflammatory cytokines. Thus, miR-124 attenuates epileptogenesis via NRSF while promoting epilepsy via inflammation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

Epileptogenesis in the temporal lobe elicits regulation of gene expression and protein translation, leading to reorganization of neuronal networks. In this process, miRNAs were described as being regulated in a cell-specific manner, although mechanistics of miRNAs activity are poorly understood. The specificity of miRNAs on their target genes depends on their intracellular concentration, reflecting the balance of biosynthesis and degradation. Herein, we confirmed that pilocarpine application promptly (<30 min) induces status epilepticus (SE) as revealed by changes in rat electrocorticogram particularly in fast-beta range (21-30 Hz). SE simultaneously upregulated XRN2 and downregulated PAPD4 gene expression in the hippocampus, two genes related to miRNA degradation and stability, respectively. Moreover, SE decreased the number of XRN2-positive cells in the hilus, while reduced the number of PAPD4-positive cells in CA1. XRN2 and PAPD4 levels did not change in calretinin- and CamKII-positive cells, although it was possible to determine that PAPD4, but not XRN2, was upregulated in parvalbumin-positive cells, revealing that SE induction unbalances the accumulation of these functional-opposed proteins in inhibitory interneurons that directly innervate distinct domains of pyramidal cells. Therefore, we were able to disclose a possible mechanism underlying the differential regulation of miRNAs in specific neurons during epileptogenesis.

It is widely known that there is an increase in the inflammatory responses and oxidative stress in temporal lobe epilepsy (TLE). Further, the seizures follow a circadian rhythmicity. Retinoic acid receptor-related orphan receptor alpha (RORα) is related to anti-inflammatory and antioxidant enzyme expression and is part of the machinery of the biological clock and circadian rhythms. However, the participation of RORα in this neurological disorder has not been studied. The aim of this study was to evaluate the RORα mRNA and protein content profiles in the hippocampus of rats submitted to a pilocarpine-induced epilepsy model at different time points throughout the 24-h light-dark cycle analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases of the experimental model. Real-time PCR and immunohistochemistry results showed that RORα mRNA and protein expressions were globally reduced in both acute and silent phases of the pilocarpine model. However, 60days after the pilocarpine-induced status epilepticus (chronic phase), the mRNA expression was similar to the control except for the time point 3h after the lights were turned off, and no differences were found in immunohistochemistry. Our results indicate that the status epilepticus induced by pilocarpine is able to change the expression and daily variation of RORα in the rat hippocampal area during the acute and silent phases. These findings enhance our understanding of the circadian pattern present in seizures as well as facilitate strategies for the treatment of seizures., (Copyright © 2015 Elsevier Inc. All rights reserved.)