Inhibition of Granule Cell Dispersion and Seizure Development by Astrocyte Elevated Gene-1 in a Mouse Model of Temporal Lobe Epilepsy.

Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy.

MiR129-5p-loaded exosomes suppress seizure-associated neurodegeneration in status epilepticus model mice by inhibiting HMGB1/TLR4-mediated neuroinflammation.

BACKGROUND: Neuroinflammation contributes to both epileptogenesis and the associated neurodegeneration, so regulation of inflammatory signaling is a potential strategy for suppressing epilepsy development and pathological progression. Exosomes are enriched in microRNAs (miRNAs), considered as vital communication tools between cells, which have been proven as potential therapeutic method for neurological disease. Here, we investigated the role of miR129-5p-loaded mesenchymal stem cell (MSC)-derived exosomes in status epilepticus (SE) mice model. METHODS: Mice were divided into four groups: untreated control (CON group), kainic acid (KA)-induced SE groups (KA group), control exosome injection (KA + Exo-con group), miR129-5p-loaded exosome injection (KA + Exo-miR129-5p group). Hippocampal expression levels of miR129-5p, HMGB1, and TLR4 were compared among groups. Nissl and Fluoro-jade B staining were conducted to evaluate neuronal damage. In addition, immunofluorescence staining for IBA-1 and GFAP was performed to assess glial cell activation, and inflammatory factor content was determined by ELISA. Hippocampal neurogenesis was assessed by BrdU staining. RESULTS: The expression of HMGB1 was increased after KA-induced SE and peaking at 48 h, while hippocampal miR129-5p expression decreased in SE mice. Exo-miR129-5p injection reversed KA-induced upregulation of hippocampal HMGB1 and TLR4, alleviated neuronal damage in the hippocampal CA3, reduced IBA-1 + and GFAP + staining intensity, suppressed SE-associated increases in inflammatory factors, and decreased BrdU + cell number in dentate gyrus. CONCLUSIONS: Exosomes loaded with miR129-5p can protect neurons against SE-mediated degeneration by inhibiting the pro-inflammatory HMGB1/TLR4 signaling axis.

Comparative Analysis of Anticonvulsant Activity of Trans and Cis 5,5'-Diphenylhydantoin Schiff Bases.

Recently, the four 5,5'-diphenylhydantoin Schiff bases, possessing different aromatic species (SB1-Ph, SB2-Ph, SB3-Ph and SB4-Ph) were synthesized, characterized, and evaluated for anticonvulsant activity in combination with phenytoin. In the present study, the SB1-Ph and SB4-Ph compounds were selected, based on their anticonvulsant potency, and compared with their cis isomers, prepared after a one-hour exposure to the UV source, for their anticonvulsant potency in the maximal electroshock (MES) test and the kainate (KA)-induced status epilepticus (SE) test in mice. In the MES test, the cisSB1-Ph compound exhibited superior to phenytoin and trans isomer activity in the three tested doses, while the cisSB4-Ph compound entirely suppressed the electroshock-induced seizure spread at the highest dose of 40 mg/kg. Pretreatment with the cisSB1-Ph compound and the cisSB4-Ph at the doses of 40 mg/kg, respectively, for seven days, significantly attenuated the severity of KA SE compared to the matched control group pretreated with a vehicle, while phenytoin was ineffective in this test. The cisSB4-Ph but not the cisSB1-Ph demonstrated an antioxidant effect against the KA-induced SE in the hippocampus. Our results suggest that trans-cis conversion of 5,5'-diphenylhydantoin Schiff bases has potential against seizure spread in the MES test and mitigated the KA-induced SE. The antioxidant potency of cisSB4-Ph might be associated with its efficacy in mitigating the SE.

Sex and gonadectomy modify behavioral seizure susceptibility and mortality in a repeated low-dose kainic acid systemic injection paradigm in mice.

OBJECTIVE: Sex differences in epilepsy appear driven in part due to effects of gonadal steroids, with varying results in experimental models based on species, strain, and method of seizure induction. Furthermore, removing the main source of these steroids via gonadectomy may impact seizure characteristics differently in males and females. Repeated low-dose kainic acid (RLDKA) systemic injection paradigms were recently shown to reliably induce status epilepticus (SE) and hippocampal histopathology in C57BL/6J mice. Here, we investigated whether seizure susceptibility in a RLDKA injection protocol exhibits a sex difference and whether gonadectomy differentially influences response to this seizure induction paradigm in males and females. METHODS: Adult C57BL/6J mice were left gonad-intact as controls or gonadectomized (females: ovariectomized, OVX; males: orchidectomized, ORX). At least 2 weeks later, KA was injected ip, every 30 minutes at 7.5 mg/kg or less until the animal reached SE, defined by at least 5 generalized seizures (GS, Racine stage 3 or higher). Parameters of susceptibility to GS induction, SE development, and mortality rates were quantified. RESULTS: No differences in seizure susceptibility or mortality were observed between control males and control females. Gonadectomized mice exhibited increased susceptibility and reduced latency to both GS and SE in comparison to corresponding controls of the same sex, but the effects were stronger in males. In addition, ORX males, but not OVX females, exhibited strongly increased seizure-induced mortality. SIGNIFICANCE: The RLDKA protocol is notable for its efficacy in inducing SE and seizure-induced histopathology in C57BL/6J mice, the background for many transgenic strains in current use in epilepsy research. The present results indicate that this protocol may be beneficial for investigating the effects of gonadal hormone replacement on seizure susceptibility, mortality, and seizure-induced histopathology, and that gonadectomy unmasks sex differences in susceptibility to seizures and mortality not observed in gonad-intact controls.

Spinal microRNA-134-5p targets glutamate receptor ionotropic kainate 3 to modulate opioid induced hyperalgesia in mice.

Background: Fentanyl and its analogs are extensively used for pain relief. However, their paradoxically pronociceptive effects often lead to increased opioids consumption and risk of chronic pain. Compared to other synthetic opioids, remifentanil has been strongly linked to acute opioid hyperalgesia after exposure [remifentanil-induced hyperalgesia (RIH)]. The epigenetic regulation of microRNAs (miRNAs) on targeted mRNAs has emerged as an important pathogenesis in pain. The current research aimed at exploring the significance and contributions of miR-134-5p to the development of RIH. Methods: Both the antinociceptive and pronociceptive effects of two commonly used opioids were assessed, and miRNA expression profiles in the spinal dorsal horn (SDH) of mice acutely exposed to remifentanil and remifentanil equianalgesic dose (RED) sufentanil were screened. Next, the candidate miRNA level, cellular distribution, and function were examined by qPCR, fluorescent in situ hybridization (FISH) and Argonaute-2 immunoprecipitation. Furthermore, bioinformatics analysis, luciferase assays, miRNA overexpression, behavioral tests, golgi staining, electron microscopy, whole-cell patch-clamp recording, and immunoblotting were employed to investigate the potential targets and mechanisms underlying RIH. Results: Remifentanil induced significant pronociceptive effects and a distinct miRNA-profile from sufentanil when compared to saline controls. Among top 30 differentially expressed miRNAs spectrum, spinal miR-134-5p was dramatically downregulated in RIH mice but remained comparative in mice subjected to sufentanil. Moreover, Glutamate Receptor Ionotropic Kainate 3 (Grik3) was a target of miR-134-5p. The overexpression of miR-134-5p attenuated the hyperalgesic phenotype, excessive dendritic spine remodeling, excitatory synaptic structural plasticity, and Kainate receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in SDH resulting from remifentanil exposure. Besides, intrathecal injection of selective KA-R antagonist was able to reverse the GRIK3 membrane trafficking and relieved RIH. Conclusion: The miR-134-5p contributes to remifentanil-induced pronociceptive features via directly targeting Grik3 to modulate dendritic spine morphology and synaptic plasticity in spinal neurons.

Inflachromene attenuates seizure severity in mouse epilepsy models via inhibiting HMGB1 translocation.

Epilepsy is not well controlled by current anti-seizure drugs (ASDs). High mobility group box 1 (HMGB1) is a DNA-binding protein in the nucleus regulating transcriptional activity and maintaining chromatin structure and DNA repair. In epileptic brains, HMGB1 is released by activated glia and neurons, interacting with various receptors like Toll-like receptor 4 (TLR4) and downstream glutamatergic NMDA receptor, thus enhancing neural excitability. But there is a lack of small-molecule drugs targeting the HMGB1-related pathways. In this study we evaluated the therapeutic potential of inflachromene (ICM), an HMGB-targeting small-molecule inhibitor, in mouse epilepsy models. Pentylenetetrazol-, kainic acid- and kindling-induced epilepsy models were established in mice. The mice were pre-treated with ICM (3, 10 mg/kg, i.p.). We showed that ICM pretreatment significantly reduced the severity of epileptic seizures in all the three epilepsy models. ICM (10 mg/kg) exerted the most apparent anti-seizure effect in kainic acid-induced epileptic status (SE) model. By immunohistochemical analysis of brain sections from kainic acid-induced SE mice, we found that kainic acid greatly enhanced HMGB1 translocation in the hippocampus, which was attenuated by ICM pretreatment in subregion- and cell type-dependent manners. Notably, in CA1 region, the seizure focus, ICM pretreatment mainly inhibited HMGB1 translocation in microglia. Furthermore, the anti-seizure effect of ICM was related to HMGB1 targeting, as pre-injection of anti-HMGB1 monoclonal antibody (5 mg/kg, i.p.) blocked the seizure-suppressing effect of ICM in kainic acid-induced SE model. In addition, ICM pretreatment significantly alleviated pyramidal neuronal loss and granule cell dispersion in kainic acid-induced SE model. These results demonstrate that ICM is an HMGB-targeting small molecule with anti-seizure potential, which may help develop a potential drug for treating epilepsy.

Toward Evidence-Based Severity Assessment in Mouse Models with Repeated Seizures: (II.) Impact of Surgery and Intrahippocampal Kainate.

INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation.

Rutin, a naturally derived flavonoid molecule with known neuroprotective properties, has been demonstrated to have anticonvulsive potential, but the mechanism of this effect is still unclear. The current study aimed to investigate the probable antiseizure mechanisms of rutin in rats using the kainic acid (KA) seizure model. Rutin (50 and 100 mg kg-1) and carbamazepine (100 mg kg-1) were administered daily by oral gavage for 7 days before KA (15 mg kg-1) intraperitoneal (i.p.) injection. Seizure behavior, neuronal cell death, glutamate concentration, excitatory amino acid transporters (EAATs), glutamine synthetase (GS), glutaminase, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B, activated astrocytes, and inflammatory and anti-inflammatory molecules in the hippocampus were evaluated. Supplementation with rutin attenuated seizure severity in KA-treated rats and reversed KA-induced neuronal loss and glutamate elevation in the hippocampus. Decreased glutaminase and GluN2B, and increased EAATs, GS, GluA1, GluA2 and GluN2A were observed with rutin administration. Rutin pretreatment also suppressed activated astrocytes, downregulated the protein levels of inflammatory molecules [interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4)] and upregulated anti-inflammatory molecule interleukin-10 (IL-10) protein expression. Taken together, the results indicate that the preventive treatment of rats with rutin attenuated KA-induced seizures and neuronal loss by decreasing glutamatergic hyperactivity and suppressing the IL-1R1/TLR4-related neuroinflammatory cascade.

The Clock gene regulates kainic acid-induced seizures through inhibiting ferroptosis in mice.

OBJECTIVES: Temporal lobe epilepsy (TLE) is a common and intractable form of epilepsy. There is a strong need to better understand molecular events underlying TLE and to find novel therapeutic agents. Here we aimed to investigate the role of Clock and ferroptosis in regulating TLE. METHODS: TLE model was established by treating mice with kainic acid (KA). Regulatory effects of the Clock gene on KA-induced seizures and ferroptosis were evaluated using Clock knockout (Clock-/-) mice. mRNA and protein levels were determined by quantitative real-time PCR and western blotting, respectively. Ferroptosis was assessed by measuring the levels of iron, GSH and ROS. Transcriptional regulation was studied using a combination of luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays. KEY FINDINGS: We found that Clock ablation exacerbated KA-induced seizures in mice, accompanied by enhanced ferroptosis in the hippocampus. Clock ablation reduced the hippocampal expression of GPX4 and PPAR-γ, two ferroptosis-inhibitory factors, in mice and in N2a cells. Moreover, Clock regulates diurnal expression of GPX4 and PPAR-γ in mouse hippocampus and rhythmicity in KA-induced seizures. Consistent with this finding, Clock overexpression up-regulated GPX4 and PPAR-γ and protected against ferroptosis in N2a cells. In addition, luciferase reporter, mobility shift and ChIP assays showed that CLOCK trans-activated Gpx4 and Ppar-γ through direct binding to the E-box elements in the gene promoters. CONCLUSION: CLOCK protects against KA-induced seizures through increased expression of GPX4 and PPAR-γ and inhibition of ferroptosis.

Neferine, an Alkaloid from Lotus Seed Embryos, Exerts Antiseizure and Neuroprotective Effects in a Kainic Acid-Induced Seizure Model in Rats.

Current anti-seizure drugs fail to control approximately 30% of epilepsies. Therefore, there is a need to develop more effective anti-seizure drugs, and medicinal plants provide an attractive source for new compounds. This study aimed to evaluate the possible anti-seizure and neuroprotective effects of neferine, an alkaloid from the lotus seed embryos of Nelumbo nucifera, in a kainic acid (KA)-induced seizure rat model and its underlying mechanisms. Rats were intraperitoneally (i.p.) administrated neferine (10 and 50 mg/kg) 30 min before KA injection (15 mg/kg, i.p.). Neferine pretreatment increased seizure latency and reduced seizure scores, prevented glutamate elevation and neuronal loss, and increased presynaptic protein synaptophysin and postsynaptic density protein 95 expression in the hippocampi of rats with KA. Neferine pretreatment also decreased glial cell activation and proinflammatory cytokine (interleukin-1ß, interleukin-6, tumor necrosis factor-α) expression in the hippocampi of rats with KA. In addition, NOD-like receptor 3 (NLRP3) inflammasome, caspase-1, and interleukin-18 expression levels were decreased in the hippocampi of seizure rats pretreated with neferine. These results indicated that neferine reduced seizure severity, exerted neuroprotective effects, and ameliorated neuroinflammation in the hippocampi of KA-treated rats, possibly by inhibiting NLRP3 inflammasome activation and decreasing inflammatory cytokine secretion. Our findings highlight the potential of neferine as a therapeutic option in the treatment of epilepsy.

G-alpha interacting protein interacting protein, C terminus 1 regulates epileptogenesis by increasing the expression of metabotropic glutamate receptor 7.

AIMS: It has been reported that the G-alpha interacting protein (GAIP) interacting protein, C terminus 1 (GIPC1/GIPC) engages in vesicular trafficking, receptor transport and expression, and endocytosis. However, its role in epilepsy is unclear. Therefore, in this study, we aimed to explore the role of GIPC1 in epilepsy and its possible underlying mechanism. METHODS: The expression patterns of GIPC1 in patients with temporal lobe epilepsy (TLE) and in mice with kainic acid (KA)-induced epilepsy were detected. Behavioral video monitoring and hippocampal local field potential (LFP) recordings were carried out to determine the role of GIPC1 in epileptogenesis after overexpression of GIPC1. Coimmunoprecipitation (Co-IP) assay and high-resolution immunofluorescence staining were conducted to investigate the relationship between GIPC1 and metabotropic glutamate receptor 7 (mGluR7). In addition, the expression of mGluR7 after overexpression of GIPC1 was measured, and behavioral video monitoring and LFP recordings after antagonism of mGluR7 were performed to explore the possible mechanism mediated by GIPC1. RESULTS: GIPC1 was downregulated in the brain tissues of patients with TLE and mice with KA-induced epilepsy. After overexpression of GIPC1, prolonged latency period, decreased epileptic seizures and reduced seizure severity in behavioral analyses, and fewer and shorter abnormal brain discharges in LFP recordings of KA-induced epileptic mice were observed. The result of the Co-IP assay showed the interaction between GIPC1 and mGluR7, and the high-resolution immunofluorescence staining also showed the colocalization of these two proteins. Additionally, along with GIPC1 overexpression, the total and cell membrane expression levels of mGluR7 were also increased. And after antagonism of mGluR7, increased epileptic seizures and aggravated seizure severity in behavioral analyses and more and longer abnormal brain discharges in LFP recordings were observed. CONCLUSION: GIPC1 regulates epileptogenesis by interacting with mGluR7 and increasing its expression.

Human Stem Cell-Derived GABAergic Interneurons Establish Efferent Synapses onto Host Neurons in Rat Epileptic Hippocampus and Inhibit Spontaneous Recurrent Seizures.

Epilepsy is a complex disorder affecting the central nervous system and is characterised by spontaneously recurring seizures (SRSs). Epileptic patients undergo symptomatic pharmacological treatments, however, in 30% of cases, they are ineffective, mostly in patients with temporal lobe epilepsy. Therefore, there is a need for developing novel treatment strategies. Transplantation of cells releasing γ-aminobutyric acid (GABA) could be used to counteract the imbalance between excitation and inhibition within epileptic neuronal networks. We generated GABAergic interneuron precursors from human embryonic stem cells (hESCs) and grafted them in the hippocampi of rats developing chronic SRSs after kainic acid-induced status epilepticus. Using whole-cell patch-clamp recordings, we characterised the maturation of the grafted cells into functional GABAergic interneurons in the host brain, and we confirmed the presence of functional inhibitory synaptic connections from grafted cells onto the host neurons. Moreover, optogenetic stimulation of grafted hESC-derived interneurons reduced the rate of epileptiform discharges in vitro. We also observed decreased SRS frequency and total time spent in SRSs in these animals in vivo as compared to non-grafted controls. These data represent a proof-of-concept that hESC-derived GABAergic neurons can exert a therapeutic effect on epileptic animals presumably through establishing inhibitory synapses with host neurons.

Diurnal burden of spontaneous seizures in early epileptogenesis in the post-kainic acid rat model of epilepsy.

Patients with epilepsy can experience diurnal seizure patterns. However, few studies in rodent models of temporal lobe epilepsy (TLE) routinely quantify the diurnal pattern of spontaneous recurrent seizures (SRS), and those that have conducted such assessments used small groups. This study thus aimed to define whether there was a diurnal pattern of SRS in the early phases of epileptogenesis in a large cohort (n = 40) of post-kainic acid (KA)-induced status epilepticus (SE) male Sprague Dawley rats. Rats were monitored by continuous 24/7 video-EEG in two-week epochs up to 6 weeks post-KA-induced SE. The total number of SRS by 6 weeks post-SE correlated to body weight at the time of SE insult (R2  = .1465, P = .0143). The total number of spontaneous behavioral and electrographic seizures, seizure severity, and seizure burden was recorded during lights ON (light) or lights OFF (dark) phases. All measures significantly increased with time post-SE; we detected significantly more seizures during the lights OFF phase of the post-SE monitoring periods. Moreover, a subset of rats demonstrated marked seizure preference in the lights OFF phase. Our study confirms that a diurnal pattern of SRS is variably detectable in early epileptogenesis in this model of TLE.

Public health risks associated with chronic, low-level domoic acid exposure: A review of the evidence.

Domoic acid (DA), the causative agent for the human syndrome Amnesic Shellfish Poisoning (ASP), is a potent, naturally occurring neurotoxin produced by common marine algae. DA accumulates in seafood, and humans and wildlife alike can subsequently be exposed when consuming DA-contaminated shellfish or finfish. While strong regulatory limits protect people from the acute effects associated with ASP, DA is an increasingly significant public health concern, particularly for coastal dwelling populations, and there is a growing body of evidence suggesting that there are significant health consequences following repeated exposures to levels of the toxin below current safety guidelines. However, gaps in scientific knowledge make it difficult to precisely determine the risks of contemporary low-level exposure scenarios. The present review characterizes the toxicokinetics and neurotoxicology of DA, discussing results from clinical and preclinical studies after both adult and developmental DA exposure. The review also highlights crucial areas for future DA research and makes the case that DA safety limits need to be reassessed to best protect public health from deleterious effects of this widespread marine toxin.

LncRNA UCA1 alleviates aberrant hippocampal neurogenesis through regulating miR-375/SFRP1-mediated WNT/ß-catenin pathway in kainic acid-induced epilepsy.

Temporal lobe epilepsy (TLE) is a chronic disease of the nervous system, associated with increased proliferation in the hippocampus. Urothcarcinoma associated 1 (UCA1) is a long long non-coding RNA that was shown to regulate proliferation and differentiation of neural progenitors in vitro. We hypothesised that TLE-associated abnormal proliferation is a consequence of the downregulation of UCA1. This hypothesis was tested in mice with kainic acid (KA)-induced seizures, and then the potential mechanism was explored in vitro and in vivo. Result showed that the expression of UCA1 and Secreted Frizzled Related Protein 1 (SFRP1) were significantly reduced in hippocampal tissues of epileptic mice, while miR-375 was increased compared with the control group. Pearson correlation analysis showed that UCA1 was positively correlated with SFRP1, while miR-375 was negatively correlated with UCA1 and SFRP1. Besides, UCA1 was overexpressed in mice and the overexpression of UCA1 significantly reversed the abnormal proliferation of hippocampal neurons in epilepsy mice. In vitro Luciferase assay showed that UCA1 and Sfrp1 are both the targets of miR-375, and UCA1 promotes the expression of Sfrp1 by competitively adsorbing miR-375, thereby inhibiting the activation of the WNT/ß-catenin pathway. The inactivation of the WNT/ß-catenin pathway prevented the abnormal proliferation of neural progenitors in the epileptic hippocampus. In conclusion, our findings provide a theoretical basis for the clinical application of UCA1.

Deviant reporter expression and P2X4 passenger gene overexpression in the soluble EGFP BAC transgenic P2X7 reporter mouse model.

The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. P2X7 effects were also described in neurons and astrocytes but its localisation and function in these cell types has been challenging to demonstrate in situ. BAC transgenic mouse lines have greatly advanced neuroscience research and two BAC-transgenic P2X7 reporter mouse models exist in which either a soluble EGFP (sEGFP) or an EGFP-tagged P2X7 receptor (P2X7-EGFP) is expressed under the control of a BAC-derived P2rx7 promoter. Here we evaluate both mouse models and find striking differences in both P2X expression levels and EGFP reporter expression patterns. Most remarkably, the sEGFP model overexpresses a P2X4 passenger gene and sEGFP shows clear neuronal localisation but appears to be absent in microglia. Preliminary functional analysis in a status epilepticus model suggests functional consequences of the observed P2X receptor overexpression. In summary, an aberrant EGFP reporter pattern and possible effects of P2X4 and/or P2X7 protein overexpression need to be considered when working with this model. We further discuss reasons for the observed differences and possible caveats in BAC transgenic approaches.

TCD, a triterpenoid isolated from wild bitter gourd, reduces synaptosomal release of glutamate and protects against kainic acid-induced neuronal death.

3ß,7ß,25-Trihydroxycucurbita-5,23(E)-dien-19-al (TCD) is a triterpenoid isolated from wild bitter gourd that is a common tropical vegetable with neuroprotective effects. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the aims of this study were to examine the effect of TCD on glutamate release in vitro and to examine the effect of TCD in vivo. In rat cerebrocortical synaptosomes, TCD reduced 4-aminopyridine (4-AP)-stimulated glutamate release and Ca2+ concentration elevation, but had no effect on plasma membrane potential. TCD-mediated inhibition of 4-AP-induced glutamate release was dependent on the presence of extracellular calcium; persisted in the presence of the glutamate transporter inhibitor dl-TBOA, P/Q-type Ca2+ channel blocker ω-agatoxin IVA, and intracellular Ca2+-releasing inhibitors dantrolene and CGP37157; and was blocked by the vesicular transporter inhibitor bafilomycin A1 and the N-type Ca2+ channel blocker ω-conotoxin GVIA. Molecular docking studies have demonstrated that TCD binds to N-type Ca2+ channels. TCD-mediated inhibition of 4-AP-induced glutamate release was abolished by the Ca2+-dependent protein kinase C (PKC) inhibitor Go6976, but was unaffected by the Ca2+-independent PKC inhibitor rottlerin. Furthermore, TCD considerably reduced the phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate, a major presynaptic substrate for PKC. In a rat model of kainic acid (KA)-induced excitotoxicity, TCD pretreatment substantially attenuated KA-induced neuronal death in the CA3 hippocampal region. These results suggest that TCD inhibits synaptosomal glutamate release by suppressing N-type Ca2+ channels and PKC activity and exerts protective effects against KA-induced excitotoxicity in vivo.

Cardiac dysregulation following intrahippocampal kainate-induced status epilepticus.

Status epilepticus (SE) is a prevalent disorder associated with significant morbidity, including the development of epilepsy and mortality. Cardiac arrhythmias (i.e. inappropriate sinus tachycardia and bradycardia, asystole, and atrioventricular blocks) are observed in patients following SE. We characterized ictal (during a seizure) and interictal (between seizure) cardiac arrhythmogenesis following SE using continuous electrocardiography and video electroencephalography (vEEG) recordings throughout a 14-day monitoring period in an intrahippocampal chemoconvulsant mouse model that develops epilepsy. We quantified heart rhythm abnormalities and examined whether the frequency of cardiac events correlated with epileptiform activity, circadian (light/dark) cycle, the presence of seizures, and survival during this period of early epileptogenesis (the development of epilepsy) following SE. Shortly following SE, mice developed an increased interictal heart rate and heart rhythm abnormalities (i.e. sinus pause and sinus arrhythmias) when compared to control mice. Heart rhythm abnormalities were more frequent during the light cycle and were not correlated with increased epileptiform activity or seizure frequency. Finally, SE animals had early mortality, and a death event captured during vEEG recording demonstrated severe bradycardia prior to death. These cardiac changes occurred within 14 days after SE and may represent an early risk factor for sudden death following SE.

Aberrant expression of PAR bZIP transcription factors is associated with epileptogenesis, focus on hepatic leukemia factor.

Epilepsy is a widespread neurological disease characterized by abnormal neuronal activity resulting in recurrent seizures. There is mounting evidence that a circadian system disruption, involving clock genes and their downstream transcriptional regulators, is associated with epilepsy. In this study, we characterized the hippocampal expression of clock genes and PAR bZIP transcription factors (TFs) in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainic acid (KA). The expression of PAR bZIP TFs was significantly altered following KA injection as well as in other rodent models of acquired epilepsy. Although the PAR bZIP TFs are regulated by proinflammatory cytokines in peripheral tissues, we discovered that the regulation of their expression is inflammation-independent in hippocampal tissue and rather mediated by clock genes and hyperexcitability. Furthermore, we report that hepatic leukemia factor (Hlf), a member of PAR bZIP TFs family, is invariably downregulated in animal models of acquired epilepsy, regulates neuronal activity in vitro and its overexpression in dentate gyrus neurons in vivo leads to altered expression of genes associated with seizures and epilepsy. Overall, our study provides further evidence of PAR bZIP TFs involvement in epileptogenesis and points to Hlf as the key player.

LncRNA-UCA1 inhibits the astrocyte activation in the temporal lobe epilepsy via regulating the JAK/STAT signaling pathway.

This article aimed to reveal the mechanism of long noncoding RNA (lncRNA) urothelial cancer-associated 1 (UCA1) regulated astrocyte activation in temporal lobe epilepsy (TLE) rats via mediating the activation of the JAK/STAT signaling pathway. A model of TLE was established based on rats via kainic acid (KA) injection. All rats were divided into the Sham group (without any treatments), KA group, normal control (NC; injection with empty vector) + KA group, and UCA1 + KA group. The Morris water maze was used to test the learning and memory ability of rats, and the expression of UCA1 in the hippocampus was determined by quantitative real time polymerase chain reaction (qRT-PCR). Surviving neurons were counted by Nissl staining, and expression levels of glial cells glial fibrillary acidic protein (GFAP), p-JAK1, and p-STAT3 and glutamate/aspartate transporter (GLAST) were analyzed by immunofluorescence and Western blot analysis. A rat model of TLE was established by intraperitoneal injection of KA. qRT-PCR and fluorescence analyses showed that UCA1 inhibited astrocyte activation in the hippocampus of epileptic rats. Meanwhile, the Morris water maze analysis indicated that UCA1 improved the learning and memory in epilepsy rats. Moreover, the Nissl staining showed that UCA1 might have a protective effect on neuronal injury induced by KA injection. Furthermore, the immunofluorescence and Western blot analysis revealed that the overexpression of UCA1 inhibited KA-induced abnormal elevation of GLAST, astrocyte activation of the JAK/STAT signaling pathway, as well as hippocampus of epilepsy rats. UCA1 inhibited hippocampal astrocyte activation and JAK/STAT/GLAST expression in TLE rats and improved the adverse reactions caused by epilepsy.