Protective Effect And Mechanism Of GluR2/GAPDH Interfering Peptide On Hippocampus Of Epileptic Rats Induced By Lithium Pilocarpine

ObjectiveEpilepsy is a common nervous system disease.There are about 30 million people in the world with epilepsy,1 million 500 thousand new epileptic patients are added each year,and about 8 million of the epileptic patients in China,of which 25%-40%is refractory epilepsy.Temporal lobe epilepsy(TLE)is the most common type of refractory epilepsy which is characterized a progressive expansion of complex partial seizures arising from the limbic system regions and Hippocampal sclerosis.The main pathological changes of HS were the damage of neurons in pyramidal cell layer and dentate gyrus of hippocampal CA1 and CA3 regions.Hippocampal injury is not only the result of epilepsy but also the cause of temporal lobe epilepsy.Neuron injury in acute phase gradually develops into chronic temporal lobe epilepsy characterized by SRS in chronic phase,and then manifests learning and cognitive impairment.The lithium chloride-pilocarpine kindling model is the most commonly used and ideal epilepsy model which well simulates the whole process of the occurrence,progress and formation of human epilepsy.It is one of the most commonly used models for studying the status of status epilepticus(SE)and(spontaneous recurrent epileptic seizure,SRS),which is similar to the pathophysiological mechanism of TLE.According to the pathological manifestations of the model,it can be divided into acute phase 0-72 hours after SE,persistent toxicity phase 4-7 days,incubation period lasting 14-15 days and chronic phase lasting several weeks or even months.The central nervous system excitatory eurotransmitter balance disorder is a classic mechanism leading to epilepsy.The excessive release of glutamateneurotransmitters plays a key role in the occurrence and development of epilepsy.Its neuronal cell injury is closely related to epilepsy.As the main glutamate receptor in the nervous system,(a-amino-3hydroxy-5-methylisoxazole-4-propionic acid,AMPA)receptor-mediated excitotoxicity has been implicated inthe pathogenesis of epilepsy.The accumulation of glutamate results in excessive stimulation of AMPA receptors and leads to neurotoxicity.The main pathological mechanism is likely mediated by Ca2+ influx with subsequent activation of diverse downstream cell death signals.In neurons,mature AMPA receptors are found as tetramersconsisting of various combinations of GluR1 toGluR4subunits,each of which has the same topology:threetransmembrane domains and one membrain re-entrantloop.All subunits are permeable to Ca2+ ionswith the exception of GluR2,which is uniquelyimpermeable to Ca2+.GluR1-GluR3 was mainly expressed in the forebrain,while the granulosa cells in the hippocampus and dentate gyrus showed high expression of GluR2 subunit.GIuR1/GluR2 heteromer formed in 80%of the vertebral neurons in the hippocampal CA1 region,A variety of epilepsy animal models indicate that:the expression level of GluR2 is increased during automatic epilepsy.it is still unclear how activation of theGluR2-containing AMPAR leads to neuronal cell death.In the present study,wehave identified a new AMPAR-interacting partner,GAPDH(Glyceraldehyde-3-phosphate dehydrogenase).GAPDH is a multifunction protein which plays an important role in cell apoptosis.We show that secreted GAPDH binds specifically to Y142-K172 site of the extracellular NT domain of the GluR2 subunit,a processwhich is promoted by AMPAR activation.The result of interaction of GluR2/GPADH will induce neurotoxicity which is implicated in neuropathological disorders such as stroke and epilepsy.To study the function of AMPAR/GAPDH,we havedeveloped an interfering peptide that can disrupt theGluR2/GAPDH interaction to prevent AMPAR-mediated excitotoxicity.Disruption of this complex with a competitive protein peptide(G-Gpep)could rescue glutamate induced excitatory neuronal death in primary neuronal culture and animal models of ischemic stroke with neuron death in CA1.Finally,based on the forementioned analysis,the present study aims to provide new idea for treatment of epilepsy targeting AMPA receptors.In present article,pilocarpine(PILO)has been used to cause epilepsy of rats by inducing death of hippocampal neuron.Dynamic changes of hippocampal neuronal damage and astrocytes in the hippocampus were investigated,spatial learning and memory were tested by Morris water maze.In this reserch,we investigated the potential protective effects of G-Gpep in pilocarpine model of temporal lobe dpilepsy.Meanwhile this study provides experimental basis and theoretical basis for the development of antiepileptic drugs.This study is divided into three parts:Part 1 Effect of G-Gpep pretreatment on hippocampal injury of rats in actue stage with seizures induced by lithium-pilocarpineObjective:Toestablisha PILO-induced SE rat model and evaluate the efficacy of G-Gpep preconditioningfor preventing SE-associated death of hippocampal neurons and to evaluate the effects of the rat behavior,the morphology of hippocampal neurons and the histochemistry of the hippocampal neurons.Methods:1、Peptide synthesis:Treatment peptide was synthesized by Biomatik Corporation(Cambridge,USA).Tofacilitate the intracellular delivery of the peptide,GluR2NT1-3-2 peptide was fused into cell membrane transduction domain of the HIV-1 TATprotein[YGRKKRRQRRR]to generate TAT-GluR2NT1-3-2 peptide,and the sequence of TAT-GluR2NT1-3-2 peptide was TYGRKKRRQRRR-YYQWDKFAYLYDSDRG-LSTLQAVLDSAAEK.The peptide was purifiedby HPLC and the purity was at least 90%,The peptide was dissolvedin saline and aliquot accordingly prior to use and kept at-80℃。2、Male Wistar rats were divided into three groups including control group,Pilo group and G-Gpep group.Of these,G-Gpep group was subdivided into TAT 5nmol group and TAT 10nmol groups.Before 60 minutes,rats in these two sub-groups were respectively injected with G-Gpep 5nmol and 10nmol in the lateral ventricle by brain stereotaxic device respectively.3、Racine score criteria was used to determine the level of seizures.Rats in each group were fed under the same experimental conditions.4、The rats were sacrificed by intraperitoneal injection of 10%chloral hydrate(3ml/kg)at 6h,24h and 72h after anesthesia.Fluoro-jade B staining and TUNEL staining were used to determine neuronal cell degeneration and apoptosis in hippocampal neurons.Results:1、In this experiment,rats in Pilo group was found to be seizures-like seizures after injection of pilocarpine from 5 minutes to 10 minutes.Grade Ⅳ is considered to successfully induce epilepsy,and success rate of grade IV is 62.5%.The mortality after SE induction was 41.2%in Pilo group,27.3%in TAT 5 nmol group and 16.7%in TAT 10 nmol group.Seizure incubation period of rats in G-Gpep group(62.62±6.64 min)was significantly longer than that in Pilo group(18.43±8.86min).In addition,seizure incubation period of rats in TAT10nmol group(70.9±8.12min)was longer than that in TAT5nmol group,and the success rate of grade V was much lower(19%).The control group did not show any behavior of epileptic seizures.2、Each slice randomly selected 3 vision to take pictures.The application software of Image-Pro Plus 6.0 converted the fluorescent photos into black and white pictures,each photo was analyzed for the positive cumulative light density(IOD).FJB-positive neurons were found in Pilo group and G-Gpep groupwhich were mainly distributed in hippocampal CA1 and CA3 regions 6 h after SE.The number of FJB-positive neurons in CA1 and CA3 of Pilo group were significantly increased at 24 h(4827.52 ±105.87,4633.26±45.39).FJB-positive neurons in CA1 and CA3 of Pilo group further increased at 72 hours after epileptic seizure(6238.73 ± 159.89,6065.46 ±91.23).However,FJB-positive neurons in hippocampal CA1 and CA3 regions of G-Gpep group(4935.84 ±51.14,4403.26 ± 89.27),and FJB-positive neurons in TATA10nmol group(2834.53±35.10,2608.17±76.28)was less in comparison with TAT5nmol group.3、Each slice randomly selected 3 vision to take pictures.The application software of Image-Pro Plus6.0 was used to analyse the results.The percentage of positive cells is the rate of apoptosis.There was no TUNEL positive cells or only micro-expression in hippocampus in control group.After 24h,TUNEL positive cells in CA1 and CA3 regions of the hippocampus in Pilo group were significantly increased and reached the peak(34.97±2.26%,39.36±2.15%)at 72 h after SE.but the number of TUNEL positive cells in the hippocampus of G-Gpep group(17.81 ± 1.09%,8.53 ±0.78%)was significantly less than that in the Pilo group.Meanwhile the number of TUNEL positive cells in TAT10nmol group(8.91 ±0.39%,6.41 ±0.43%)was less than that in TAT5nmol group.Conclusion:1、G-Gpep has protective effect on hippocampal neurons in PILO-induced SE rat model,2、G-Gpep has antiepileptic effect.3、G-Gpep might be dose-dependent,the effect of 10nmol is stronger than that of 5nmol.Part 2 TheProtective mechanism of G-Gpep pretreatment on hippocampal neurons in pilocarpine induced epileptic ratsObjective:To establish a PILO-induced SE rat model and evaluate the role of GluR2/GAPDH complex in rat hippocampus in the process of neuron injury which mediated by AMPAR receptor,the nuclear translocation of GAPDH and the possible protective mechanism of G-Gpep were discussed.Methods:1、Male Wistar rats were divided into three groups including control group,Pilo group and G-Gpep group.The synthesis of the G-Gpep is the same as the first part,Of these,G-Gpep group was subdivided into TAT 5nmol group and TAT 10nmol group.Before 60 minutes,rats in these two sub-groups were respectively injected with G-Gpep 5nmol and 10nmol in the lateral ventricle by brain stereotaxic device respectively.2、At 24 h after SE,5-6 rats were randomly selected,total protein of the rat hippocampus was extracted and the formation of GluR2/GAPDH complex in hippocampus of rats was detected by Co-immunoprecipitation assay.3、At 24 h after SE,5-6 rats were randomly selected,total protein of the rat hippocampus was extracted and the expression of GluR2 and GAPDH in hippocampus of rats was detected by Western blot..4、At 24 h after SE,five-six surviving rats in each group were randomly selected.The nucleoproteins and cytoplasmic proteins of hippocampal cells were extracted by nucleoprotein extraction kit,and the nuclear translocation of GAPDH was detected by Western blot.Results:1、The results of immunoprecipitation showed that the interaction between GIuR2/GAPDH in Pilo group was significantly increased(170800±7984.97).Further experiments showed that the interaction of GluR2/GAPDH could be blocked by G-Gpep(143500±7546.57).There was no significant difference in the total expression of protein GAPDH among each group and we found that the blocking effect of TAT10nmol group(135415±8782.85)on GluR2/GAPDH was higher than that of 5nmol pretreatment group.2、The expression of GluR2 in the hippocampus of the control group was 0.59 ±0.03,Pilo group was 0.86 ± 0.02,TAT 5 nmol group was 0.88±0.03 and TAT 10 nmol group was 0.88 ±0.06.Compared with the control group,the expression of GluR2 increased 24 hours after SE in Pilo group,TAT 5 nmol group and TAT 10 nmol group,but there was no significant difference in the expression of GluR2 among Pilo group,TAT 5 nmol group and TAT 10 nmol group.3、The results of GAPDH nuclear translocation experiment showed that the expression of GAPDH in nucleoprotein of Pilo group was increased(0.74± 0.07)and the GAPDH in cytoplasm was decreased compared with the control group(0.13 ±0.02).These results suggest that GAPDH nuclear translocation in SE rats is increased,and further experimental results show that GAPDH nuclear translocation can be blocked by G-Gpep(0.6 ±0.05).The blocking effect of 10 nmol pretreatment group(0.48± 0.06)is higher than that of 5 nmol pretreatment group.Conclusion:1、The interaction of GluR2/GAPDH in hippocampus of Pilo induced SE rats is enhanced,and this effect can be blocked by G-Gpep.2、Compared with Pilo group,G-Gpep and different doses of G-Gpep pretreatment had no effect on the expression of GluR2 in SE rats.3、The expression of nuclear protein GAPDH in hippocampus of SE rats induced by Pilo was significantly increased,while G-Gpep could reduce the expression of nuclear protein GAPDH,and then block the cytotoxic effect of GAPDH nuclear transposition.4、In this study,we found that G-Gpep might be dose-dependent.The effect of 10nmol is stronger than that of 5nmol.Part 3 Effect of G-Gpep pretreatment on cognitive function and astrocytes at chronic stage inpilocarpine-induced rat epilepsyObjective:Toestablisha PILO-induced SE rat model and evaluate the efficacy of G-Gpep preconditioning on epileptic seizure,cognitive function and astrocytes at chronic stage.Methods:1、Male Wistar rats were divided into four groups including controlgroup,Epilepsymodel group(EM),TAT-GluR2NT1-3-2-pretreated epilepsy-model(TPEM)and GluR2NT-scrampretreated epilepsy-modelgroup(FEM).The synthesis of the G-Gpep is the same as the first part,The sequence of TAT-GluR2NT-scram was:YGRKKRRQRRR-AFDLSQYDLKWQVDYLKYDYGTASELRASA.Before60 minutes,rats in G-Gpep group were injected with G1uR2NT1-3-2 ingterfering peptide and GluR2NT-scram interfering peptide 10nmol in the lateral ventricle by brain stereotaxic device respectively.2、At 15 days after SE,5-6 rats were randomly selected.Times of epilepsy seizures,Racine scale and epilepsy duration were recorded.To study animals’ spatial learning and memory,at 30 days after SE.Morris water maze testing was performed 30 days after SE.3、Nissl staining:5-6 rats in each group were randomly selected and the surviving neurons in CA1 and CA3 areas of hippocampus were counted by Nissl staining 7 days after SE.4、To investigate the dynamic changes of astrocytes,5-6 rats were randomly selected in each group at 3 day,7 days,15 days and 30 days after SE.Results:1、According to video recording,the rats in control group were in good state without epilepsy seizure;the rats in EM groupwere attacked by epilepsy seizures for 8.53 ± 0.45times/day,epilepsy scale was:3.48±0.16,epilepsy duration was:62.21 ±7.49s.Epilepsyseizures(2.45±0.77),Racinescale(1.82±0.18)and epilepsy duration(36.47±7.22s)in TPEM group were significantly lower than EM group.There’s no statistical difference in epilepsy seizures,Racine scaleand epilepsy duration between EM group(8.13±0.46,3.85±0.22,61.44±7.28s)and FEM group(P>0.05).2、Morris water maze testing result:During the three continuous 5 days for recording,escape latency in all groups got shorter with time,indicating that the rats in all group remembered the position of platform.At first day and second day,escape latency of There was no difference among each group(P>0.05).From the third day,the escape latency of each group was statistically different at each time point;Compared with Control group,the escape latency of EM group and FEM group was significantly longer(P<0.05),while that of TPEM group was significantly shorter(P<0.05).The day after Positioning navigation experiment,spatial learning was assessed.In control group,the swimming time at the platform significantly longer than EM group(P<0.05);TPEM group was shorter than EM group(P<0.05).FEMgroup was similar to EM group(P>0.05).The times of crossing the platform in control group,EM group,TPEM group and FEM group were:11.2±1.48,4.2±1.3,7.4±1.14,3.8±0.84.The times of crossing the platform inTPEM group was more than EM group P<0.05.3、Nissl staining results:The number of pyramidal cells with intact membrane and clear nucleoli in each 1 mm segment of CA1 and CA3 region of hippocampus was counted under optical microscope,and the average number of pyramidal cells in each section of bilateral hippocampus was counted.The results showed that the neurons in CA1 and CA3 regions of the hippocampus in the control group were intact without loss of neurons;the neurons in CA1 and CA3 regions of the hippocampus in the EM group and FEM group were obviously lost at7 days after SE.The cell structure of equine neurons was partially intact,only a small amount of chromatin agglutination and the number of Nissl bodies increased significantly in TPEM group.Neuronal survival in each group:CA1:Control group:123.8 + 5.36;EM group:45.8 + 3.11;TPEM group:77 + 5.71;FEM group:46 + 2.73;CA3:Control group:128.4 + 5.85;EM group:48.6+ 2.41;TPEM group:81.8 + 2.77;FEM group:50.4 + 2.88.Compared with Control group,the number of neurons in CA1 and CA3 areas of hippocampus in EM group decreased significantly(P<0.05),while the number of neurons in hippocampus in TPEM group increased significantly after G-Gpep pretreatment(P<0.05).There was no significant difference between group EM and group FEM(P>0.05).4、Each slice randomly selected 3 vision to take pictures.The application software of Image-Pro Plus 6.0 was used to analyze the positive cumulative light density(IOD).Astrocytes of CA1 in Control group had few processes,and were lightly-stained,while in EM group they showed many big and thick processes with deep staining.There were significantly more astrocytes in EM group than in control group.In EM group,GFAP IOD was:2618.09±4.88 at 72 h after SE,6014.92±4.39 at 7 d,3213.91±2.69 at 15 d,1251.00±2.36 at 30 d.They reached the peak at 7d after SE,and then decreased but were still more than control group.GFAP IOD of TPEM group reached the peak at 15 d after SE(675.15 ±2.77),and had significant reduction at 30 d(453.41±2.58),but had significantly less astrocytes than EM group.There was no significant difference between FEMgroup and EMgroup.Those results suggested that pretreatment of G-Gpep could inhibit proliferation and activation of astrocytes.Conclusion:1、In chronic stage,Epilepsyseizures,Racinescaleand epilepsy duration in G-Gpep group were significantly lower thanPilo group which suggested the antiepileptic effectof G-Gpep.2、At the chronic stage,pilocarpine-induced rat epilepsy models presented with SRS and obvious cognitive dysfunction.The pretreatment of G-Gpep could improve epilepsy-induced cognitive disorders.3、The pretreatment of G-Gpep could improve the survival rate of hippocampal neurons in the toxic phase and inhibit the proliferation of astrocytes in the latent and chronic phase.