Yanke Zhang

Plic-1, a new target in repressing epileptic seizure by regulation of GABAAR function in patients and a rat model of epilepsy

Dysfunction of γ-aminobutyric acid A (GABAA) receptors (GABAARs) is a prominent factor affecting intractable epilepsy. Plic-1, an ubiquitin-like protein enriched in the inhibitory synapses connecting GABAARs and the ubiquitin protease system (UPS), plays a key role in the modification of GABAAR functions. However, the relationship between Plic-1 and epileptogenesis is not known. In the present study, we aimed to investigate Plic-1 levels in patients with temporal lobe epilepsy, as well as the role of Plic-1 in regulating onset and progression of epilepsy in animal models. We found that Plic-1 expression was significantly decreased in patients with epilepsy as well as pilocarpine- and pentylenetetrazol (PTZ)-induced rat epileptic models. Intrahippocampal injection of the PePα peptide, which disrupts Plic-1 binding to GABAARs, significantly shortened the latency of seizure onset, and increased the seizure severity and duration in these two epileptic models. Overexpressed Plic-1 through lentivirus transfection into a PTZ model resulted in a reduction in both seizure severity and generalized tonic-clonic seizure duration. Whole-cell clamp recordings revealed that the PePα peptide decreased miniature inhibitory postsynaptic currents (mIPSCs) whereas overexpressed Plic-1 increased mIPSCs in the pyramidal neurons of the hippocampus. These effects can be blocked by picrotoxin, a GABAAR inhibitor. Our results indicate that Plic-1 plays an important role in managing epileptic seizures by enhancing seizure inhibition through regulation of GABAARs at synaptic sites.

ENT1 Inhibition Attenuates Epileptic Seizure Severity Via Regulation of Glutamatergic Neurotransmission

Type 1 equilibrative nucleoside transporter (ENT1) promotes glutamate release by inhibition of adenosine signaling. However, whether ENT1 plays a role in epileptic seizure that involves elevated glutamatergic neurotransmission is unknown. Here, we report that both seizure rats and patients show increased expression of ENT1. Intrahippocampal injection of a specific inhibitor of ENT1, nitrobenzylthioinosine (NBTI), attenuates seizure severity and prolongs onset latency. In order to examine whether NBTI would be effective as antiepileptic after peripheral application, we injected NBTI intraperitoneally, and the results were similar to those obtained after intrahippocampal injection. NBTI administration leads to suppressed neuronal firing in seizure rats. In addition, increased mEPSC in seizure are inhibited by NBTI. Finally, NBTI results in deactivation of phosphorylated cAMP-response element-binding protein in the seizure rats. These results indicate that ENT1 plays an important role in the development of seizure. Inhibition of ENT1 might provide a novel therapeutic approach toward the control of epileptic seizure.

Expression of Glypican-4 in the brains of epileptic patients and epileptic animals and its effects on epileptic seizures.

Glypican-4 (Gpc4) has been found to play an important role in enhancing miniature excitatory postsynaptic currents (mEPSCs). But, the relationship between Gpc4 and epilepsy is still a mystery. In this study, we investigated the expression patterns of Gpc4 in patients with epilepsy and in a pilocarpine-induced rat model of epilepsy. We also determined if altered Gpc4 expression resulted in increased susceptibility to seizures. Western blotting and immunofluorescent methods were utilized. Gpc4 was significantly increased in patients and epileptic rats induced by pilocarpine injection. According to behavioral studies, downregulation of Gpc4 by Gpc4 siRNA decreased spontaneous seizure frequency, while upregulation of Gpc4 by recombinant Gpc4 overexpression led to a converse result. These findings support the hypothesis that increased expression of Gpc4 in the brain is associated with epileptic seizures.

Down-regulation of Pin1 in Temporal Lobe Epilepsy Patients and Mouse Model

Peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (Pin1) is a unique PPIase belonging to the parvulin family, and it isomerizes peptide bond between phospho-(Ser/Thr) and Pro. Pin1 has been linked to the pathogenesis of various human diseases; however, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of Pin1 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic mouse model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that Pin1 protein was mainly distributed in neurons, demonstrated by colocalization with the dendritic marker, MAP2. However, the expression of Pin1 decreased remarkably in epileptic patients and experimental mice. Furthermore, the reciprocal coimmunoprecipitation analysis showed that Pin1 interacted with NR2A and NR2B-containing NMDA receptors not AMPA receptors in epileptic mouse models. Our results are the first to indicate that the expression of Pin1 in epileptic brain tissue could play important roles in epilepsy.

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.

Inhibition of Cgkii Suppresses Seizure Activity and Hippocampal Excitation by Regulating the Postsynaptic Delivery of Glua1

Background/Aims: The imbalance between excitation and inhibition is a defining feature of epilepsy. GluA1 is an AMPA receptor subunit that can strengthen excitatory synaptic transmission when upregulated in the postsynaptic membrane, which has been implicated in the pathogenesis of epilepsy. cGKII, a cGMP-dependent protein kinase, regulates the GluA1 levels at the plasma membrane. Methods: To explore the role of cGKII in epilepsy, we investigated the expression of cGKII in patients with temporal lobe epilepsy (TLE) and in a pilocarpine-induced rat model and then performed behavioral, histological, and electrophysiological analyses by applying either a cGKII agonist or inhibitor in the hippocampus of the animal model. Results: cGKII expression was upregulated in the epileptogenic brain tissues of both humans and rats. Pharmacological activation or inhibition of cGKII induced changes in epileptic behaviors in vivo and epileptic discharges in vitro. Further studies indicated that cGKII activation disrupted the balance of excitation and inhibition due to strengthened AMPAR-mediated excitatory synaptic transmission. Moreover, cGKII regulated epileptic seizures by phosphorylating GluA1 at Ser845 to modulate the expression and function of GluA1 in the postsynaptic membrane. Conclusion: These results suggest that cGKII plays a key role in seizure activity and could be a potential therapeutic target for epilepsy.

Tubulin β‐III modulates seizure activity in epilepsy

Tubulin β‐III (TUBB3) is the most dynamic β‐tubulin isoform expressed in neurons, and is highly expressed in the central nervous system. However, the relationship between TUBB3 and epileptic seizures has not been thoroughly investigated. The aims of this study were to investigate the expression of TUBB3 in patients with temporal lobe epilepsy and two different rat models of chronic epilepsy, and to determine the specific roles of TUBB3 in epilepsy. TUBB3 expression was upregulated in human and rat epileptic tissue. Moreover, TUBB3 expression was associated with inhibitory GABAergic neurons and the inhibitory postsynaptic scaffold protein gephyrin. TUBB3 downregulation attenuated the behavioural phenotypes of epileptic seizures during the pilocarpine‐induced chronic phase of epileptic seizures and the pentylenetetrazole kindling process, whereas TUBB3 overexpression had the opposite effect. Whole‐cell clamp recordings and western blotting revealed that the amplitude of GABA‐A receptor‐mediated miniature inhibitory postsynaptic currents and the surface expression of the GABA‐A receptor were increased in rats in which TUBB3 expression was downregulated. Importantly, TUBB3 interacted with GABA‐A receptor‐associated protein, which is known to be involved in GABA‐A receptor trafficking. These results indicate that TUBB3 plays a critical role in the regulation of epileptic seizures via GABA‐A receptor trafficking, suggesting a molecular mechanism for new therapeutic strategies. Copyright

POSH participates in epileptogenesis by increasing the surface expression of the NMDA receptor: a promising therapeutic target for epilepsy.

ABSTRACT Objectives: Plenty of SH3 (POSH) was originally found to be a key regulator of neuronal apoptosis, axon outgrowth, and neuronal migration. However, the role of POSH in epilepsy has not been reported. Methods: We investigated the expression of POSH in patients with intractable temporal epilepsy (TLE) and in a kainic acid (KA)-induced mouse model, and then we performed behavioral, electrophysiological and biochemical analyses after the lentivirus (LV)-mediated knockdown or overexpression of POSH in the KA-induced model. Results: POSH overexpression shortened the latency of seizure onset, increased the frequency of spontaneous recurrent seizures, and increased the frequency of electrical epileptic discharges, while POSH knockdown had contrasting effects. Whole-cell patch-clamp recordings confirmed that POSH overexpression and knockdown were associated with increased and decreased miniature excitatory postsynaptic currents (mEPSCs) and N-methyl-D-aspartate receptor (NMDAR)-mediated currents, respectively. Finally, co-immunoprecipitation showed that POSH and NMDA receptor subunit 1 (NMDAR1) precipitated with each other, and western blot analysis revealed that the surface expression of NMDAR1 was altered in the hippocampus of epileptic mice. Conclusion: These results show that POSH plays a critical role in the progression of epileptic seizures via NMDAR trafficking and suggest that the protein is a novel target for the treatment of human epilepsy.

Myeloid differentiation factor 88 is up-regulated in epileptic brain and contributes to experimental seizures in rats

Abstract Accumulating evidence supports that activation of inflammatory pathways is a crucial factor contributing to the pathogenesis of seizures. In particular, the activation of interleukin‐1 beta (IL‐1&bgr;) system exerts proconvulsant effects in a large variety of seizure models. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein in the signaling cascade elicited by IL‐1&bgr;. The present study aimed to investigate the expression pattern of MyD88 in rat models of seizures and in patients with refractory temporal lobe epilepsy (TLE), and to study the role of MyD88 in epileptic seizures. Our results revealed that MyD88 was up‐regulated in the hippocampus of rats in the lithium‐pilocarpine model of acute seizures. Importantly, MyD88 overexpression was also significantly present in the brain from chronic epileptic rats and the temporal neocortex specimens from drug‐resistant TLE patients. In the acute seizure model, both the behavioral and electrographic seizure activities were record and analyzed in rats for 90 min, starting immediately after pilocarpine injection. ST2825, a synthetic MyD88 inhibitor, was administered intracerebroventricularly (2.5–5.0–10 &mgr;g in 2 &mgr;l) 20 min before pilocarpine injection. We found that ST2825 at doses of 5 &mgr;g and 10 &mgr;g significantly inhibited the pilocarpine‐induced behavioral and electrographic seizures. Moreover, 10 &mgr;g ST2825 prevented the proconvulsant actions of IL‐1&bgr;. As previous evidence suggested that IL‐1&bgr; proconvulsant effects was mediated by enhancing the phosphorylation level of the NR2B subunit of N‐methyl‐d‐aspartate (NMDA) receptor, we then probed whether this molecular was involved in the effect of the pharmacological inhibition. Our results revealed that 10 &mgr;g ST2825 markedly reversed the increased Tyr1472‐phosphorylation of the NR2B subunit of NMDA receptor observed in the proconvulsant conditions of IL‐1&bgr; and in seizures induced by pilocarpine alone. These findings indicate that altered expression of MyD88 might contribute to the pathogenesis of seizures and targeting of this adaptor protein might represent a novel therapeutic strategy to suppress seizure activities. HighlightsUp‐regulation of MyD88 was found in acute and chronic epileptic seizures in rats.Up‐regulation of MyD88 was found in patients with intractable epilepsy.Inhibition of MyD88 by ST2825 exerted anticonvulsant effects.Inhibition of MyD88 by ST2825 prevented the proconvulsant activities of IL‐1&bgr;.ST2825 reduced Tyr1472‐phosphorylation of the NR2B subunit in epileptic seizures.

GPR40 modulates epileptic seizure and NMDA receptor function

GPR40 modulates epileptic seizure and NMDA receptor function through the regulation of NR2A and NR2B surface expression. Epilepsy is a common neurological disease, and approximately 30% of patients do not respond adequately to antiepileptic drug treatment. Recent studies suggest that G protein–coupled receptor 40 (GPR40) is expressed in the central nervous system and is involved in the regulation of neurological function. However, the impact of GPR40 on epileptic seizures remains unclear. In this study, we first reported that GPR40 expression was increased in epileptic brains. In the kainic acid–induced epilepsy model, GPR40 activation after status epilepticus alleviated epileptic activity, whereas GPR40 inhibition showed the opposite effect. In the pentylenetetrazole-induced kindling model, susceptibility to epilepsy was reduced with GPR40 activation and increased with GPR40 inhibition. Whole-cell patch-clamp recordings demonstrated that GPR40 affected N-methyl-d-aspartate (NMDA) receptor–mediated synaptic transmission. Moreover, GPR40 regulated NR2A and NR2B expression on the surface of neurons. In addition, endocytosis of NMDA receptors and binding of GPR40 with NR2A and NR2B can be regulated by GPR40. Together, our findings indicate that GPR40 modulates epileptic seizures, providing a novel antiepileptic target.