Beclin1 Deficiency Suppresses Epileptic Seizures.

Epilepsy is a common disease of the nervous system. Autophagy is a degradation process involved in epilepsy, and in turn, seizures can activate autophagy. Beclin1 plays a critical role in autophagy and participates in numerous physiological and pathological processes. However, the mechanism underlying the effect of Beclin1 on epilepsy remains unclear. In this study, we detected increased expression of Beclin1 in brain tissues from patients with temporal lobe epilepsy (TLE). Heterozygous disruption of beclin1 decreased susceptibility to epilepsy and suppressed seizure activity in two mouse epilepsy models. We further illustrated for the first time that heterozygous disruption of beclin1 suppresses excitatory synaptic transmission, which may be caused by a decreased dendritic spine density. These findings suggest for the first time that the regulation of Beclin1 may serve as a strategy for antiepileptic therapy. In addition, Beclin1 participates in synaptic transmission, and the development of dendritic spines may be a biological function of Beclin1 independent of its role in autophagy.

Intestinal Klebsiella pneumoniae infection enhances susceptibility to epileptic seizure which can be reduced by microglia activation.

Epilepsy is a common nervous system disease, and the existing theory does not fully clarify its pathogenesis. Recent research suggests that intestinal microbes may be involved in the development of epilepsy, but which microbe is involved remains unclear. We used 16s rRNA sequencing to identify the most relevant gut microbe. To determine the relationship between this microbe and epilepsy, we used an animal model. In addition, western blotting and immunofluorescence, as well as inhibitor studies, were used to evaluate and confirm the role of microglia in this process. In this study, we first report an increase in gut Klebsiella pneumoniae in patients with epilepsy. Subsequently, animal studies revealed that Klebsiella pneumoniae in the intestinal tract affects seizure susceptibility and activates microglial cells to release inflammatory factors. Furthermore, the inflammatory response of microglial cells plays a protective role in the seizure susceptibility caused by an increased abundance of Klebsiella pneumoniae. Our results suggest that gut disruption may be involved in seizure regulation and microglia protect the brain against seizure under this condition. These findings provide a new perspective for research on the pathogenesis and prevention of epilepsy.

HAP1 Modulates Epileptic Seizures by Regulating GABAAR Function in Patients with Temporal Lobe Epilepsy and in the PTZ-Induced Epileptic Model.

The number of γ-aminobutyric acid type A receptors (GABAARs) expressed on the surface membrane and at synaptic sites is implicated in the enhanced excitation of neuronal circuits and abnormal network oscillations in epilepsy. Huntingtin-associated protein 1 (HAP1), a key mediator of pathological alterations in protein trafficking, directly interacts with GABAARs and facilitates their recycling back to synapses after internalization from the surface; thus, HAP1 regulates the strength of inhibitory synaptic transmission. Here, we show that HAP1 modulates epileptic seizures by regulating GABAAR function in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We demonstrate that GABAARß2/3 and HAP1 expression are decreased and that the HAP1-GABAARß2/3 complex is disrupted in the epileptic rat brain. We found that HAP1 upregulation exerts antiepileptic activity in the PTZ-induced seizure and that these changes are associated with increased surface GABAARß2/3 expression and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). This study provides evidence that hippocampal HAP1 is linked to GABAARs in evoking seizures and suggests that this mechanism is involved in epileptic seizures in the brain, representing a potential therapeutic target for epilepsy.

SAD-B modulates epileptic seizure by regulating AMPA receptors in patients with temporal lobe epilepsy and in the PTZ-induced epileptic model.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.

Exposure to carbon black nanoparticles increases seizure susceptibility in male mice.

Carbon black nanoparticles (CBNPs) can enter the central nervous system through blood circulation and olfactory nerves, affecting brain development or increasing neurological disease susceptibility. However, whether CBNPs exposure affects seizure is unclear. Herein, mice were exposed to two different doses of CBNPs (21 and 103 µg/animal) based on previous studies and the maximum exposure limitation (4 mg/m3) in occupational workplaces set by the Chinese government. In the pentylenetetrazol (PTZ) and kainic acid (KA) seizure models, high-dose CBNPs exposure increased seizure susceptibility in both models and increased spontaneous recurrent seizure (SRS) frequency in the KA model. In vivo local field potential (LFP) recording in KA model mice revealed that both low-dose and high-dose CBNPs exposure increased seizure-like event (SLE) frequency in the SRS interval but shortened SLE duration. Intriguingly, H&E staining and Nissl staining on brain tissue revealed that CBNPs exposure did not cause significant brain tissue morphology or neuronal damage. Detection of inflammatory factors, such as TNF-α, TGF-ß1, IL-1ß, and IL-6, in brain tissue showed that only high dose of CBNPs exposure increased the expression of cortical TGF-ß1. By using the primary cultured neurons, we observed that CBNPs exposure not only significantly decreased the expression of the neuronal marker MAP2 but also enhanced the levels of action potential frequency in the neurons. In general, CBNPs exposure can affect abnormal epileptic discharges during the seizure interval and enhance susceptibility to frequent seizures. Our findings suggest that minimizing CBNPs exposure may be a potential way to prevent or ease seizure.

MicroRNA-25-3p suppresses epileptiform discharges through inhibiting oxidative stress and apoptosis via targeting OXSR1 in neurons.

MicroRNA-25-3p (miR-25-3p) has been reported to be closely related with oxidative stress and apoptosis. Here, we aimed to detect the effects of miR-25-3p in the primarily cultured hippocampal neurons. Kainic acid (KA) was used to induce epileptic seizures in the rats. We predicted that oxidative stress responsive 1 (OXSR1) might be a potential target of miR-25-3p with TargetScan prediction and luciferase assays, and the primarily cultured hippocampal neurons were exposed to Mg2+-free solution for 3 h to induce spontaneous recurrent epileptiform discharges (SREDs). Then, the expression of miR-25-3p and OXSR1 in the rats hippocampi and primarily cultured hippocampal neurons were detected. Those SREDs neurons were treated with miR-25-3p mimic, miR-25-3p inhibitor or/and OXSR1 over-expression vector, and SREDs, oxidative stress and apoptosis were observed. We found down-regulation of miRNA-25-3p and up-regulation of OXSR1 in hippocampi of KA-treated rats and Mg2+-free-treated neurons. MiRNA-25-3p mimic could down-regulate OXSR1 expression, inhibit SREDs, reduce oxidative stress and decrease apoptosis. Additionally, over-expression of OXSR1 weakened those effects of miR-25-3p mimic. Those data indicated that miR-25-3p had anti-epileptic, anti-oxidant and anti-apoptosis effects on the primarily cultured neurons through targeting OXSR1, which provided a novel target for the treatment of epilepsy.

SAD-B modulates epileptic seizure by regulating AMPA receptors in patients with temporal lobe epilepsy and in the PTZ-induced epileptic model

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.

Inhibition of Nwd1 activity attenuates neuronal hyperexcitability and GluN2B phosphorylation in the hippocampus.

BACKGROUND: NACHT and WD repeat domain-containing protein 1 (Nwd1) is a member of the innate immune protein subfamily. Nwd1 contributes to the androgen receptor signaling pathway and is involved in axonal growth. However, the mechanisms that underlie pathophysiological dysfunction in seizures remain unclear. METHODS: Biochemical methods were used to assess Nwd1 expression and localization in a mouse model of kainic acid (KA)-induced acute seizures and temporal lobe epilepsy (TLE) patients. Electrophysiological recordings were used to measure the role of Nwd1 in regulating synaptic transmission and neuronal hyperexcitability in a model of magnesium-free-induced seizure in vitro. Behavioral experiments were performed, and seizure-induced pathological changes were evaluated in a KA-induced seizure model in vivo. GluN2B expression was measured and its correlation with Tyr1472-GluN2B phosphorylation was analyzed in primary hippocampal neurons. FINDINGS: We demonstrated high protein levels of Nwd1 in brain tissues obtained from mice with acute seizures and TLE patients. Silencing Nwd1 in mice using an adeno-associated virus (AAV) profoundly suppressed neuronal hyperexcitability and the occurrence of acute seizures, which may have been caused by reducing GluN2B-containing NMDA receptor-dependent glutamatergic synaptic transmission. Moreover, the decreased activation of Nwd1 reduced GluN2B expression and the phosphorylation of the GluN2B subunit at Tyr1472. INTERPRETATION: Here, we report a previously unrecognized but important role of Nwd1 in seizure models in vitro and in vivo, i.e., modulating the phosphorylation of the GluN2B subunit at Tyr1472 and regulating neuronal hyperexcitability. Meanwhile, our findings may provide a therapeutic strategy for the treatment of epilepsy or other hyperexcitability-related neurological disorders. FUND: The funders have not participated in the study design, data collection, data analysis, interpretation, or writing of the report.

TMEM25 modulates neuronal excitability and NMDA receptor subunit NR2B degradation.

The expression of the transmembrane protein 25 gene (Tmem25) is strongly influenced by glutamate ionotropic receptor kainate type subunit 4, and its function remains unknown. Here, we showed that TMEM25 was primarily localized to late endosomes in neurons. Electrophysiological experiments suggested that the effects of TMEM25 on neuronal excitability were likely mediated by N-methyl-d-aspartate receptors. TMEM25 affected the expression of the N-methyl-d-aspartate receptor NR2B subunit and interacted with NR2B, and both were colocalized to late endosome compartments. TMEM25 induced acidification changes in lysosome compartments and accelerated the degradation of NR2B. Furthermore, TMEM25 expression was decreased in brain tissues from patients with epilepsy and epileptic mice. TMEM25 overexpression attenuated the behavioral phenotypes of epileptic seizures, whereas TMEM25 downregulation exerted the opposite effect. These results provide some insights into TMEM25 biology in the brain and the functional relationship between TMEM25 and epilepsy.

Transgenic overexpression of furin increases epileptic susceptibility.

The proprotein convertase Furin plays crucial roles in the pathology of many diseases. However, the specific role of furin in epilepsy remains unclear. In our study, furin protein was increased in the temporal neocortex of epileptic patients and in the hippocampus and cortex of epileptic mice. The furin transgenic (TG) mice showed increased susceptibility to epilepsy and heightened epileptic activity compared with wild-type (WT) mice. Conversely, lentivirus-mediated knockdown of furin restrained epileptic activity. Using whole-cell patch clamp, furin knockdown and overexpression influenced neuronal inhibitory by regulating postsynaptic gamma-aminobutyric acid A receptor (GABAAR)-mediated synaptic transmission. Importantly, furin influenced the expression of GABAAR ß2/3 membrane and total protein in epileptic mice by changing transcription level of GABAAR ß2/3, not the protein degradation. These results reveal that furin may regulate GABAAR-mediated inhibitory synaptic transmission by altering the transcription of GABAAR ß2/3 subunits in epilepsy; this finding could provide new insight into epilepsy prevention and treatment.

ZDHHC8 critically regulates seizure susceptibility in epilepsy.

Epilepsy is one of the most prevalent and drug-refractory neurological disorders. Zinc finger DHHC-type containing 8 (ZDHHC8) is a putative palmitoyltransferase that is highly expressed in the brain. However, the impact of ZDHHC8 on seizures remains unclear. We aimed to explore the association of ZDHHC8 with epilepsy and investigate its in epileptogenesis in in vivo and in vitro models through behavioral, electrophysiological, and pathological studies. We used kainic acid- and pilocarpine-induced C57BL/6 mice and magnesium-free-induced pyramidal neurons as experimental epileptic models in this study. We first found increased ZDHHC8 expression in the brains of temporal lobe epilepsy (TLE) patients, similar to that observed in chronic epileptic mice, strongly suggesting that ZDHHC8 is correlated with human epilepsy. In the in vitro seizure models, knocking down ZDHHC8 using recombinant adeno-associated virus (rAAV) delayed seizure precipitation and decreased chronic spontaneous recurrent seizures (SRSs) and epileptiform-like discharges, while ZDHHC8 overexpression had the opposite effect. ZDHHC8 levels were consistent with seizure susceptibility in induced mice with SRSs. In an in vitro magnesium-free model, neuronal hyperexcitability and hypersynchrony were reduced in ZDHHC8-knockdown neurons but were increased in ZDHHC8-overexpressing neurons. To further explore the potential mechanisms, we observed that ZDHHC8 had a significant modulatory effect on 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA) receptor-related excitatory, but not inhibitory, glutamatergic synaptic neurotransmission, further affecting the inward rectification of AMPA currents in acute hippocampal slices in whole-cell recordings. ZDHHC8 facilitated GluA1 trafficking to the neuronal surface in the hippocampus, as shown by immunoprecipitation and Western blotting. These results suggest that ZDHHC8 may promote the generation and propagation of seizures in humans and that knocking down ZDHHC8 might produce anti-epileptogenic effects in drug-resistant epilepsy. Our study provides evidence that may facilitate the development of an alternative approach for the treatment of epilepsy by modulating AMPA/GluA1-mediated neurotransmission.