Down-Regulation of CRMP-1 in Patients with Epilepsy and a Rat Model

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein identified as a signaling molecule of Semaphorin-3A and act as axon repellent guidance factor in nervous system. Recent studies indicated that axon guidance molecules may play a role in synaptic reorganization in the adult brain and thereby promote epileptogenesis. This study aimed to investigate expression pattern of CRMP-1 in epileptogenesis. Using double immunofluorescence labeling, immunohistochemistry and western blot analysis, we looked into the CRMP-1 expression in temporal neocortex from patients with temporal lobe epilepsy (TLE) and histological normal temporal neocortex from the controls. We also studied the expression pattern of CRMP-1 in hippocampus and adjacent cortex of a TLE rat model on 6, 24, 72 h, 1, 2 weeks, 1 month, and 2 months post-seizure, and from control rats. CRMP-1 was mainly expressed in the neuronal cytoplasm in the temporal lobe of intractable TLE patients, which was co-expressed with -2. CRMP-1 expression was downregulated in temporal neocortical of TLE patients. In addition, in pilocarpine-induced animal model of epilepsy, CRMP-1 dynamically decreased in a range of 2 months. Thus, our results indicate that CRMP-1 may be involved in the development of TLE.     

GPER1 Modulates Synaptic Plasticity During the Development of Temporal Lobe Epilepsy in Rats

G-protein coupled estrogen receptor 1 (GPER1) is a novel type of estrogen receptor. Several studies have shown that it has an anti-inflammatory action,which plays an important role in remyelination and cognitive ability adjustment. However, whether it is involved in the development of temporal lobe epilepsy (TLE) is still unknown. The present study established a TLE model by intraperitoneal injection of lithium chloride (3 mmol/kg) and pilocarpine (50 mg/kg) in rats to study the effect of GPER1 in the synaptic plasticity during the development of temporal lobe epilepsy. A microinjection cannula was implanted into the lateral ventricle region of rats via a stereotaxic instrument. G-1 is the specific GPER1 agonist and G15 is the specific GPER1 antagonist. The G1 or G15 and Dimethyl sulfoxide were injected into the rat brains in the intervention groups and control group, respectively. After G1 intervention, the learning and memory abilities and hippocampal neuron damage in epileptic rats were significantly improved, while G15 weakened the neuroprotective effect of GPER1. Meanwhile, G1 controlled the abnormal formation of hippocampal mossy fiber sprouting caused by seizures, and participated in the regulation of synaptic plasticity by reducing the expression of Synapsin I and increasing the expression of gephyrin. Inhibitory synapse gephyrin may play a significant role in synaptic plasticity.

     

Decreased Astroglial Monocarboxylate Transporter 4 Expression in Temporal Lobe Epilepsy

Efflux of monocaroxylates like lactate, pyruvate, and ketone bodies from astrocytes through monocarboxylate transporter 4 (MCT4) supplies the local neuron population with metabolic intermediates to meet energy requirements under conditions of increased demand. Disruption of this astroglial-neuron metabolic coupling pathway may contribute to epileptogenesis. We measured MCT4 expression in temporal lobe epileptic foci excised from patients with intractable epilepsy and in rats injected with pilocarpine, an animal model of temporal lobe epilepsy (TLE). Cortical MCT4 expression levels were significantly lower in TLE patients compared with controls, due at least partially to MCT4 promoter methylation. Expression of MCT4 also decreased progressively in pilocarpine-treated rats from 12 h to 14 days post-administration. Underexpression of MCT4 in cultured astrocytes induced by a short hairpin RNA promoted apoptosis. Knockdown of astrocyte MCT4 also suppressed excitatory amino acid transporter 1 (EAAT1) expression. Reduced MCT4 and EAAT1 expression by astrocytes may lead to neuronal hyperexcitability and epileptogenesis in the temporal lobe by reducing the supply of metabolic intermediates and by allowing accumulation of extracellular glutamate.     

Upregulated P2X3 Receptor Expression in Patients with Intractable Temporal Lobe Epilepsy and in a Rat Model of Epilepsy

Purinergic P2X3 receptors (P2X3Rs) play extensive roles in nerve cells in the central nervous system, particularly in hyperexcitability and calcium (Ca²⁺) influx. However, the role of P2X3Rs in epilepsy has not been previously investigated. To determine the relationship between P2X3Rs and epilepsy, the expression and cellular location of P2X3Rs in patients with intractable temporal lobe epilepsy (TLE) and in a lithium chloride-pilocarpine-induced chronic rat model of epilepsy were assessed. Furthermore, the function of P2X3Rs was assessed in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to evaluate the expression levels of P2X3Rs in brain tissues from TLE patients and an epileptic rat model, whereas immunofluorescence labeling was applied to determine the distribution of target proteins. Whole-cell recording was subsequently performed to identify the influence of P2X3Rs on seizure-like discharges. P2X3Rs were located at the cell bodies and dendrites of neurons with significantly increased expression in the TLE patients and epileptic rat model. In vitro, P2X3R activation accelerated sustained repetitive firing, whereas P2X3R inhibition led to relatively low-frequency discharges. To the best of our knowledge, this is the first study provide evidence that upregulated P2X3R expression exists in both epileptic humans and rats and may aggravate the epileptic state in vitro. Thus, P2X3Rs may represent a novel therapeutic target for antiepileptic drugs.     

Protective Effect of Resveratrol against Kainate-induced Temporal Lobe Epilepsy in Rats

Resveratrol (Res) is a phytoalexin produced naturally by several plants, which has multi functional effects such as neuroprotection, anti-inflammatory, and anti-cancer. The present study was to evaluate a possible anti-epileptic effect of Res against kainate-induced temporal lobe epilepsy (TLE) in rat. We performed behavior monitoring, intracranial electroencepholography (IEEG) recording, histological analysis, and Western blotting to evaluate the anti-epilepsy effect of Res in kainate-induced epileptic rats. Res decreased the frequency of spontaneous seizures and inhibited the epileptiform discharges. Moreover, Res could protect neurons against kainate-induced neuronal cell death in CA1 and CA3a regions and depressed mossy fiber sprouting, which are general histological characteristics both in TLE patients and animal models. Western blot revealed that the expression level of kainate receptors (KARs) in hippocampus was reduced in Res-administrated rats compared to that in epileptic ones. These results suggest that Res is a potent anti-epilepsy agent, which protects against epileptogenesis and progression of the kainate-induced TLE animal. The authors Z. Wu and Q. Xu contributed equally to this work.     

Abnormal Expression of FBXL20 in Refractory Epilepsy Patients and a Pilocarpine-Induced Rat Model

E3 ubiquitin ligases are important protein-modifying enzymes involved in the pathogenesis of a variety of neurodegenerative diseases. F-box and leucine-rich repeat protein 20 (FBXL20), an E3 ubiquitin ligase widely expressed in the central nervous system, plays an important role in the ubiquitin-dependent degradation of regulating synaptic membrane exocytosis 1 (RIM1), which is an important factor in the release of synaptic vesicles. FBXL20 has been associated with a variety of neurodegenerative diseases; thus, we hypothesized that FBXL20 is involved in the development of epilepsy. Herein, we used immunofluorescence staining, immunohistochemistry and western blotting to determine the expression pattern of FBXL20 in temporal lobe epilepsy patients and pilocarpine-induced epilepsy animal models. We also injected SD rats with lentivirus-vector mediated overexpression of FBXL20. The results showed that FBXL20 is expressed in the membrane and the cytoplasm of cortical neurons, and overexpression of FBXL20 decreased the onset level of spontaneous seizure, the frequency and duration of seizures. Additionally, FBXL20 protein level was decreased but RIM1 protein level was increased in the epileptic group compared with the LV-FBXL20 and LV-GFP group. These findings in humans were consistent with the results from a pilocarpine-induced animal model of chronic epilepsy. Thus, abnormal expression of FBXL20 might play an important role in the development of epilepsy.     

Increased Expression of Rac1 in Epilepsy Patients and Animal Models

The mechanisms of epilepsy remain incompletely understood. Rac1 (ras-related C3 botulinum toxin substrate 1) belongs to the Rho family of small GTPases. Rac1 play important roles in cytoskeleton rearrangement and neuronal synaptic plasticity, which had also been implicated in epilepsy. However, little is known regarding the expression of Rac1 in the epileptic brain or whether Rac1-targeted interventions affect the progression of epilepsy. The aim of this study was to investigate the expression profile of Rac1 in brain tissues from patients suffering from temporal lobe epilepsy (TLE) and experimental epileptic rats and determine the possible role of Rac1 in epilepsy. We demonstrated that the expression of Rac1 is significantly increased in TLE patients and in lithium-pilocarpine epilepsy model animals compared to the corresponding controls. Rac1 inhibitor NSC23766 reduced the severity of status epilepticus during the acute stage in a lithium-pilocarpine animal model. Consistent with these results, the latent period of a PTZ kindling animal model also increased. Our results demonstrated that the increased expression of Rac1 may contribute to pathophysiology of epilepsy.

     

SAPAP3 regulates epileptic seizures involving GluN2A in post-synaptic densities

Aberrantly synchronized neuronal discharges in the brain lead to epilepsy, a devastating neurological disease whose pathogenesis and mechanism are unclear. SAPAP3, a cytoskeletal protein expressed at high levels in the postsynaptic density (PSD) of excitatory synapses, has been well studied in the striatum, but the role of SAPAP3 in epilepsy remains elusive. In this study, we sought to investigate the molecular, cellular, electrophysiological and behavioral consequences of SAPAP3 perturbations in the mouse hippocampus. We identified a significant increase in the SAPAP3 levels in patients with temporal lobe epilepsy (TLE) and in mouse models of epilepsy. In addition, behavioral studies showed that the downregulation of SAPAP3 by shRNA decreased the seizure severity and that the overexpression of SAPAP3 by recombinant SAPAP3 yielded the opposite effect. Moreover, SAPAP3 affected action potentials (APs), miniature excitatory postsynaptic currents (mEPSCs) and N-methyl-D-aspartate receptor (NMDAR)-mediated currents in the CA1 region, which indicated that SAPAP3 plays an important role in excitatory synaptic transmission. Additionally, the levels of the GluN2A protein, which is involved in synaptic function, were perturbed in the hippocampal PSD, and this perturbation was accompanied by ultrastructural morphological changes. These results revealed a previously unknown function of SAPAP3 in epileptogenesis and showed that SAPAP3 may represent a novel target for the treatment of epilepsy.Subject terms: Diagnostic markers, Epilepsy     

Rosiglitazone Suppresses In Vitro Seizures in Hippocampal Slice by Inhibiting Presynaptic Glutamate Release in a Model of Temporal Lobe Epilepsy

Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg²⁺ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg²⁺ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE.     

Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.     

伴海马硬化的颞叶癫痫患者海马组织内BDNF表达变化

目的：研究伴海马硬化的难治性颞叶癫痫（TLE）患者海马组织内脑源性神经营养因子（brain derivedneurotrophic factor,BDNF）的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法：采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应（RT—PCR）法检测BDNFmRNA表达,并与3例非海马硬化TLE患者对照。结果：与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNFmRNA表达明显增加（P〈0．01）。结论：伴海马硬化的难治性TLE患者海马组织中BDNFmRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

Long-Term Effects of Temporal Lobe Epilepsy on Local Neural Networks: A Graph Theoretical Analysis of Corticography Recordings

Purpose

Pharmaco-resistant temporal lobe epilepsy (TLE) is often treated with surgical intervention at some point. As epilepsy surgery is considered a last resort by most physicians, a long history of epileptic seizures prior to surgery is not uncommon. Little is known about the effects of ongoing TLE on neural functioning. A better understanding of these effects might influence the moment of surgical intervention. Functional connectivity (interaction between spatially distributed brain areas) and network structure (integration and segregation of information processing) are thought to be essential for optimal brain functioning. We report on the impact of TLE duration on temporal lobe functional connectivity and network characteristics.

Methods

Functional connectivity of the temporal lobe at the time of surgery was assessed by means of interictal electrocorticography (ECoG) recordings of 27 TLE patients by using the phase lag index (PLI). Graphs (abstract network representations) were reconstructed from the PLI matrix and characterized by the clustering coefficient C (local clustering), the path length L (overall network interconnectedness), and the “small world index” S (network configuration).

Results

Functional connectivity (average PLI), clustering coefficients, and the small world index were negatively correlated with TLE duration in the broad frequency band (0.5–48 Hz).

Discussion

Temporal lobe functional connectivity is lower in patients with longer TLE history, and longer TLE duration is correlated with more random network configuration. Our findings suggest that the neural networks of TLE patients become more pathological over time, possibly due to temporal lobe changes associated with long-standing lesional epilepsy.     

Increased Metallothionein I/II Expression in Patients with Temporal Lobe Epilepsy

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and patients with TLE associated with tumor or dysplasia (TLE-TD) were evaluated for expression of MT-I/II, for the vesicular zinc levels, and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis, microgliosis and reduced neuronal population. In TLE-TD, the same changes were observed, except that were mainly confined to fascia dentata. Increased vesicular zinc was observed only in the inner molecular layer of MTLE patients, when compared to control cases. Correlation and linear regression analyses indicated an association between increased MT-I/II and increased astrogliosis in TLE. MT-I/II levels did not correlate with any clinical variables, but MTLE patients with secondary generalized seizures (SGS) had less MT-I/II than MTLE patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from TLE patients and our data suggest that it is associated with astrogliosis and may be associated with different seizure spread patterns.     

伴海马硬化的颞叶癫痫患者海马组织内BDNF 表达变化

目的:研究伴海马硬化的难治性颞叶癫痫(TLE)患者海马组织内脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法:采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应(RT-PCR)法检测BDNF mRNA表达,并与3例非海马硬化TLE患者对照。结果:与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNF mRNA表达明显增加(P<0.01)。结论:伴海马硬化的难治性TLE患者海马组织中BDNF mRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

颞叶癫痫脑“默认模式”的fMRI研究

功能磁共振成像(functional magnetic resonance imaging,fMRI)被用于检测静息时脑功能神经网络.作者运用静息fMRI检测海马硬化颞叶癫痫(temporal lobe epilepsy,TLE)脑"默认模式",采用感兴趣区域功能连接分析检测16例TLE患者和16名正常对照静息时脑的"默认模式",并进行组内和组间分析.研究发现,与正常对照相比,TLE静息时海马、颞极、额叶、颞叶、壳核及楔前叶等脑区与后扣带回的功能连接增强.研究结果表明TLE患者的固有脑功能组织模式有可能出现紊乱.这一研究将有助于从脑功能的角度了解癫痫患者某些临床症状的发病机理,为今后癫痫诊治的发展提供一定的帮助.     

Reduced D2/D3 Receptor Binding of Extrastriatal and Striatal Regions in Temporal Lobe Epilepsy

Objective

Dopamine is an endogenous neuromodulator in cortical circuits and the basal ganglia. In animal models of temporal lobe epilepsy (TLE), seizure threshold is modulated to some extent by dopamine, with D1-receptors having a pro- and D2-receptors an anticonvulsant effect. We aimed to extend our previously reported results on decreased D2/D3 receptor binding in the lateral epileptogenic temporal lobe and to correlate them with demographic and seizure variables to gain a more comprehensive understanding of the underlying involvement of the dopaminergic system in the epileptogenesis of TLE.

Methods

To quantify D2/D3 receptor binding, we studied 21 patients with TLE and hippocampal sclerosis (13 left- and eight right-sided) and 18 controls using PET with the high-affinity dopamine D2/D3-receptor ligand ¹⁸F-Fallypride to image striatal and extrastriatal binding. TLE was defined by interictal and ictal video-EEG, MRI and ¹⁸F-Fluorodeoxyglucose PET. Voxel-based statistical and regions-of-interest analyses were performed.

Results

¹⁸F-Fallypride binding potential was significantly reduced in the affected temporal lobe and bilateral putamen. A positive correlation between age at onset of epilepsy and [¹⁸F]FP BP_nd (binding potential non-displaceable) in temporal regions on the epileptogenic side was found, as well as a negative correlation between epilepsy duration and [¹⁸F]FP BP_nd in the temporal pole on the epileptogenic side and a positive correlation between the estimated number of lifetime GTCS and [¹⁸F]FP BP_nd in the hippocampus on the epileptogenic side.

Significance

The areas of reduced D2/D3 receptor availability correspond to “the irritative zone” surrounding the epileptogenic area. Moreover, reduced D2/D3 receptor availability was detectable in the basal ganglia, which are suspected to be involved in a control circuit for epileptic seizures. The correlational analysis additionally suggests that increased epilepsy duration leads to increasing impairment of the dopaminergic system.     

Exposure to Mozart music reduces cognitive impairment in pilocarpine-induced status epilepticus rats

Patients with temporal lobe epilepsy (TLE) often display cognitive deficits. However, current epilepsy therapeutic interventions mainly aim at how to reduce the frequency and degree of epileptic seizures. Recovery of cognitive impairment is not attended enough, resulting in the lack of effective approaches in this respect. In the pilocarpine-induced temporal lobe epilepsy rat model, memory impairment has been classically reported. Here we evaluated spatial cognition changes at different epileptogenesis stages in rats of this model and explored the effects of long-term Mozart music exposure on the recovery of cognitive ability. Our results showed that pilocarpine rats suffered persisting cognitive impairment during epileptogenesis. Interestingly, we found that Mozart music exposure can significantly enhance cognitive ability in epileptic rats, and music intervention may be more effective for improving cognitive function during the early stages after Status epilepticus. These findings strongly suggest that Mozart music may help to promote the recovery of cognitive damage due to seizure activities, which provides a novel intervention strategy to diminish cognitive deficits in TLE patients.     

Increased Intrinsic Connectivity of the Default Mode Network in Temporal Lobe Epilepsy: Evidence from Resting-State MEG Recordings

The electrophysiological signature of resting state oscillatory functional connectivity within the default mode network (DMN) during spike-free periods in temporal lobe epilepsy (TLE) remains unclear. Using magnetoencephalographic (MEG) recordings, this study investigated how the connectivity within the DMN was altered in TLE, and we examined the effect of lateralized TLE on functional connectivity. Sixteen medically intractable TLE patients and 22 controls participated in this study. Whole-scalp 306-channel MEG epochs without interictal spikes generated from both MEG and EEG data were analyzed using a minimum norm estimate (MNE) and source-based imaginary coherence analysis. With this processing, we obtained the cortical activation and functional connectivity within the DMN. The functional connectivity was increased between DMN and the right medial temporal (MT) region at the delta band and between DMN and the bilateral anterior cingulate cortex (ACC) regions at the theta band. The functional change was associated with the lateralization of TLE. The right TLE showed enhanced DMN connectivity with the right MT while the left TLE demonstrated increased DMN connectivity with the bilateral MT. There was no lateralization effect of TLE upon the DMN connectivity with ACC. These findings suggest that the resting-state functional connectivity within the DMN is reinforced in temporal lobe epilepsy during spike-free periods. Future studies are needed to examine if the altered functional connectivity can be used as a biomarker for treatment responses, cognitive dysfunction and prognosis in patients with TLE.