The_role_of_ubiquitinNedd4-2_in_the_pathogenesis_of_mesial_temporal_lobe_epilepsy1

The role of ubiquitin/Nedd4-2in the pathogenesis of mesial temporal lobe epilepsy

Liwen Wu,Jing Peng,Huimin Kong,Ping Yang,Fang He,Xiaolu Deng,Na Gan,Fei Yin ?

Department of Pediatrics,Xiangya Hospital,Central South University,Changsha 410008,Hunan,PR China

H I G H L I G H T S ?We explored that ubiquitin and Nedd4-2differentiated as expressed in MTLE rats.?Ubiquitin and Nedd4-2co-localized during epileptogenesis both in vivo and in vitro.?Inhibition of UPS could aggravate the epileptogenesis of MTLE.

?

Nedd4-2was a critical E3ligase involved in the epileptogenesis of MTLE.

a b s t r a c t

a r t i c l e i n f o Article history:

Received 4June 2014

Received in revised form 13February 2015Accepted 16February 2015

Available online 17February 2015Keywords:

Mesial temporal lobe epilepsy Ubiquitin –proteasome system Ubiquitin Nedd4-2

Although the pathogenesis and epileptogenesis of mesial temporal lobe epilepsy (MTLE)have been studied for years,many questions remain.The ubiquitin –proteasome system (UPS)is one factor that might regulate ion channels,in ?ammation and neuron excitability.Nedd4-2is an E3ubiquitin ligase linked with ion channels and synaptic vesicle recycling.Here,we explore the role of the UPS and its E3ligase Nedd4-2in the pathogenesis of MTLE.Our western blot results revealed that ubiquitin and Nedd4-2were expressed differentially in different stages of MTLE.Co-immunoprecipitation and double immunostaining results indicated that Nedd4-2was the substrate protein of ubiquitin both in vivo and in vitro.Inhibition of the UPS aggravated the epileptogenesis of MTLE,causing early and frequent spontaneous seizures,more obvious neuron loss and aberrant mossy ?ber sprouting.Inhibition of ubiquitin also enhanced the activation of Nedd4-2,and switched ion channel α-ENaC downstream.Our study is the ?rst to report that the UPS participates in the pathogenesis of MTLE,inhibition of UPS could aggravate the epileptogenesis,and that Nedd4-2is a critical E3ligase involved in this process.

?2015Elsevier Inc.All rights reserved.

1.Introduction

Mesial temporal lobe epilepsy (MTLE)is a common,medically intractable syndrome.It arises from limbic structures,most notably the hippocampus,that is highly sensitive to the effects of stress.Hippo-campal sclerosis with prominent neuronal loss and gliosis,as well as increased hyperexcitability and recurrent excitation associated with aberrant mossy ?ber sprouting (MFS)in the hippocampus,are the most common pathologies in MTLE [1].Although the pathogenesis and epileptogenesis of MTLE have been studied for years,many ques-tions remain.Existing research has focused on genetics,ion channels,neurodevelopmental factors,endocytosis and exocytosis,autoimmuni-ty and in ?ammation,hypoxia ischemic injury,stress,and so on [2,3].The ubiquitin –proteasome system (UPS)is one factor that could regu-late ion channels,in ?ammation and neuron excitability.

The UPS is the main stay of protein quality control in eukaryotic cells.It governs a wide variety of regulatory pathways,from cell-cycle control and transcription to development.The timely and selective degradation of surplus and/or aberrant proteins by the UPS is essential for normal cellular physiology.Any disturbance,delay or exaggeration in the pro-cess of selection,sequestration,labeling for degradation and degrada-tion of target proteins by the UPS will compromise cellular and tissue homeostasis [4].Previous studies have shown that alterations in the ubiquitin –proteasome pathway (UPP)can contribute to the develop-ment and progression of various human diseases,such as Parkinson's disease,Alzheimer's disease,Angelman syndrome,ischemic heart dis-ease,and other neurodegenerative,autoimmune,and in ?ammatory diseases [5].Also,studies about UPS involvement in long-term synaptic plasticity and epileptic diseases such as Lafora disease have recently emerged [6].It is important to note that,although there is ample evidence implicating the UPS in neurological diseases,the molecular identity of the ligases and proteins associated with it remains largely unknown.

Physiology &Behavior 143(2015)104–112

?Corresponding author.

E-mail address:yf3079@https://www.wendangku.net/doc/4216048314.html, (F.

Yin).https://www.wendangku.net/doc/4216048314.html,/10.1016/j.physbeh.2015.02.0260031-9384/?2015Elsevier Inc.All rights

reserved.

Contents lists available at ScienceDirect

Physiology &Behavior

j o u r na l ho m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p h b

The UPS is made up of six components:ubiquitin(Ub),the Ub-activating enzyme(E1),a group of Ub-conjugating enzymes(E2s),a larger group of Ub ligases(E3),the proteasome and the deubiquitinases (DUBs)[7].During conventional ubiquitination,Ub is covalently conju-gated to any target protein.Besides regulating the target protein turn-over or clearance by the proteasomes,ubiquitination of some proteins regulates non-proteolytic processes such as endocytosis,protein locali-zation/targeting,complex assembly and regulation of the duration and intensity of signaling by effector molecules.In our previous study [1,8],we found that ubiquitin was differentially expressed in the hippocampus of MTLE rats,suggesting that the UPS affects the patho-genesis of MTLE.

Nedd4-2is an E3ubiquitin ligase previously shown to regulate ion transport by controlling cellular traf?cking/endocytosis and lysosomal degradation of ion channels and transporters[9].It was recently shown to decrease in rat dorsal root ganglion in the spared nerve injury model of traumatic nerve injury-induced neuropathic pain[10].NEDD4-2can negatively regulate the epithelial Na+chan-nel(ENaC)and voltage-gated sodium channels(Navs)[11].As we know,the electrical excitability of neurons is mediated

primarily

Fig.2.Cytoarchitecture visualization and neuron density in the CA1region.A:Nissl staining in the CA1region.AS,LS,and CS show the neurons in the hippocampal regions of MTLE rats in the acute,latent and chronic stages.M-AS,M-LS,and M-CS show the same areas from MTLE rats after MG-132pre-treatment.AC,LC,and CC show the same areas from control rats.B:The histogram shows neuron density in the CA1region at different stages.Neuron loss was signi?cant in MTLE rats compared with control rats.In the acute and chronic stages of MTLE rats pre-treated with MG-132,neuron loss and decreased nissl bodies were more obvious compared with MTLE rats.In the latent stage,no statistical differences were observed between MTLE rats and MG-132pre-treated MTLE rats(#p b0.05,MTLE vs control;*p b0.05,MTLE vs MG-132pre-treatment MTLE;Δp N0.05,LS vs

M-LS).

Fig.1.Dynamic expression of ubiquitin and Nedd4-2by western blot analysis in MTLE rats and controls.A:Gray value rate of ubiquitin,Nedd4-2andβ-actin expression in the three stages of MTLE development compared with control groups.B and C:ubiquitin signi?cantly down-regulated in the acute stage(AS)and latent stage(LS)of MTLE rats,and up-regulated in the chronic stage(CS).Nedd4-2signi?cantly down-regulated in AS and CS,and showed no statistical signi?cance in the latent stage,compared to the control groups(#p b0.05,MTLE vs con-trol;*p N0.05,LC vs LS).

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by voltage-gated ion channels,particularly voltage-gated Na(+) (Na(v)),K(+)(K(v))and Cl(?)(ClC)channels.Alterations in voltage-gated ion channel activity,composition and distribution can contribute to the pathophysiology of epilepsy,hypertension,and neuropathic and in?ammatory pain.One mechanism for retrieval is ubiquitination,when speci?c ubiquitin ligases bind to membrane pro-teins to modulate and regulate their cellular fate[12].Here,we focus on the UPS,ubiquitin and its E3ubiquitin ligase Nedd4-2.We demonstrate that they are involved in the mechanisms of MTLE,and describe a novel paradigm of the pathogenesis of the aberrant UPP in epilepsy.

2.Materials and methods

2.1.Materials and chemicals

Chemicals were mainly obtained from Amresco(Solon,OH,USA). Pilocarpine,MG-132and ubiquitin aldehyde were obtained from Sigma(St.Louis,USA).All other chemicals were of analytical reagent grade.PVDF blotting membranes,ECL and Hyper?lm,rabbit polyclonal antibody against ubiquitin,α-ENaC,Nedd4-2,and phosphorylation-Nedd4-2(P-Nedd4-2)were from Abcam,Inc.The laser confocal micro-scope was purchased from Olympus.

2.2.Animals

25-day-old Sprague–Dawley rats of either sex(obtained from the Experimental Animal Center,Central South University)were used in this study.Following arrival,animals were maintained in a room with a controlled light–dark cycle(lights on from07:00–19:00h)and a con-stant temperature(20±2°C).They were allowed to adapt to laborato-ry conditions for at least one week before starting the experiments.All procedures were approved by the Institutional Animal Care and Use Committee of Central South University.2.3.Epilepsy induction and MG-132treatment

On postnatal day25,the rats(n=155)were injected with lithium chloride(3mEq/kg,i.p.)18–20h before pilocarpine treatment. Methylscopolamine(1mg/kg,i.p.),a muscarinic antagonist that does not cross the blood–brain barrier,was administered15min prior to pilocarpine treatment to reduce the peripheral effects of the convulsant and thus enhance survival.Pilocarpine hydrochloride(30mg/kg,i.p.) was then injected to induce status epilepticus(SE).Diazepam (10mg/kg,i.p.)was administered90min after the onset of SE to termi-nate the seizure.MG-132(proteasome inhibitor,0.5mg/kg,i.p.)was peritoneally injected30min prior to pilocarpine treatment and then once a day for8weeks after SE induction.Following pilocarpine treat-ment,the rats were video-monitored for8weeks.All rats were divided into9groups based on different stages of epilepsy development:acute control group(AC,n=15,control rats2h after pilocarpine administra-tion),acute seizure group(AS,n=15,induced rats2h after pilocarpine administration),latent control group(LC,n=15,control rats3weeks after pilocarpine administration),latent seizure group(LS,n=15, induced rats3weeks after pilocarpine administration),chronic control group(CC,n=15,control rats8weeks after pilocarpine administra-tion),chronic seizure group(CS,n=15,induced rats8weeks after pilocarpine administration),MG-132treated rats in the acute seizure group(M-AS,n=15),MG-132treated rats in the latent seizure group(M-LS,n=15),and MG-132treated rats in the chronic seizure group(M-CS,n=15).The rats(n=20)that didn't reach SE after pilo-carpine administration were executed by euthanasia and rejected from the experiment.

2.4.Morphological observation

Nine groups of rats(n=5in each group)subjected to pilocarpine-induced SE were perfused at2h,3weeks and8weeks

after

Fig.3.Cytoarchitecture visualization and neuron density in the CA3region.A:Nissl staining in the CA3region.AS,LS,and CS show the neurons in the hippocampal regions of MTLE rats in the acute,latent and chronic stages.M-AS,M-LS,and M-CS show the same areas from MTLE rats after MG-132pre-treatment.AC,LC,and CC show the same areas from control rats.B:The histogram shows neuron density in the CA3region at different stages.Neuron loss was signi?cant in MTLE rats compared with control rats,and was most severe after MG-132treatment (#p b0.05,MTLE vs control;*p b0.05,MTLE vs MG-132pre-treatment MTLE).

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pilocarpine-induced SE and processed for nissl and neo-Timm staining. Rats(5from each group)were deeply anesthetized with thionembutal and sequentially perfused through the heart.Frozen coronal sections were then processed for nissl staining(6μm)and neo-Timm staining (30μm).Nissl staining was applied to observe the extent of neuron loss.Cresyl violet was used as the nissl stain substance.Timm

staining

Fig.5.Distribution patterns of Timm granules in the hippocampus of the CA3region.A:Timm staining in the CA3region.AS,LS,and CS show the Timm granules in the hippocampal regions of MTLE rats in the acute,latent and chronic stages.M-AS,M-LS,and M-CS show the same areas from MTLE rats after MG-132pre-treatment.B:The histogram shows a signi?cant increase of Timm granules in the acute,latent and chronic stages of MG-132pre-treated MTLE rats compared with MTLE rats.In the LS and CS groups,the increase in Timm granules was signi?cant compared with controls.There was no statistical difference in the acute stage(#p b0.05,MTLE vs control;*p b0.05,MTLE vs MG-132pre-treatment MTLE;Δp N0.05,AC vs

AS).

Fig.4.Cytoarchitecture visualization and neuron density in the DG region.A:Nissl staining in the DG region.AS,LS,and CS show the neurons in the hippocampal regions of MTLE rats in the acute,latent and chronic stages.M-AS,M-LS,and M-CS show the same areas from MTLE rats after MG-132pre-treatment.AC,LC,and CC show the same areas from control rats.B:The histogram shows neuron density in the DG region at different stages.Neuron loss was signi?cant in MTLE rats compared with control rats,and was most severe after MG-132pre-treatment (#p b0.05,MTLE vs control;*p b0.05,MTLE vs MG-132pre-treatment MTLE).

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was used to label the zinc-rich axon terminals of granule cells according to a modi?ed protocol.

2.5.The hippocampal neuronal culture(HNC)model of epilepsy and ubiq-uitin aldehyde(UBA)treatment

Primary hippocampal neuron cultures were prepared from newborn (b24h)Sprague–Dawley rats according to the method described by Jo-seph and Stefanie[13],with slight modi?cations.After sterilizing with 75%medical alcohol,the hippocampi were dissected and trypsinized. Planting medium was added to terminate the trypsinization.Cells were plated at a density of105/cm2with the planting medium on either 6cm petri dishes or12-well plates with glass coverslips.The dishes and coverslips were coated with poly-L-lysine(0.2mg/ml,Sigma)for2h and washed three times with PBS,then air-dried before plating.After culturing for4–6h,the medium was completely replaced with mainte-nance medium supplemented with2%B27,1mM L-glutamine,100U/ml penicillin and100μg/ml streptomycin.The neurons were cultured at 37°C in an atmosphere of5%CO2/95%air.From that time on,half the medium was renewed twice a week.The cells were used for studies on days7–14.Mg2+-free culture was exposed to the recording solu-tion without adding MgCl2;after3h,the HNC model of epilepsy in vitro was acquired.

The growth medium of primary hippocampal neuron cultures in24-well culture plates was replaced with fresh?lter-sterilized DMEM-F12 containing up to10lm UBA(an inhibitor of ubiquitin)for a maximum of48h.Thus the activity of ubiquitin was inhibited.Then the following experiments could be administered.

2.6.Cell transfection of shRNA

Ubiquitin gene targeting(GenBank accession XM005189083)du-plex components were obtained from Shanghai GeneChem Co.,Ltd. (China).The shRNA was transfected into neurons with Lipofectamine 2000solution(Invitrogen,Carlsbad,CA,USA)as recommended by the manufacturer.Brie?y,the neurons were inoculated in a6-well plate at 5×?105cells/well and incubated at37°C for24h.In serum-free me-dium,10μl of40μM shRNA duplex in a total volume of250μl of Opti-MEMI was mixed with10μl of Lipofectamine2000for20min at RT.Li-pofectamine–shRNA complex was added to each well of neurons and the solution was mixed gently by rocking the plate for6h at37°C in a humidi?ed5%CO2incubator.After24h of further incubation in serum-free medium at37°C in a humidi?ed5%CO2atmosphere, ubiquitin–shRNA was transfected and further treatment performed. Anon-targeting sequence(con-shRNA)was used as a lentivirus nega-tive control.

2.7.Western blot analysis and co-immunoprecipitation

An individual protein sample from each group was chosen randomly for western blot.In total,30μg of total protein was run on a10%sodium dodecyl sulfate-polyacrylamide gel;the PVDF membranes were incu-bated overnight at4°C with monoclonal antibodies against ubiquitin,α-ENaC,Nedd4-2,and P-Nedd4-2at a1:1000dilution,respectively. They were subsequently incubated with HRP-conjugated sheep anti-rabbit IgG at a1:2000dilution.β-Actin was used as loading control. The reactions were visualized using an enhanced chemilumines-cence(ECL)detection system.Signals on the blots were visualized by autography.The?lm signals were digitally scanned and then quanti-?ed using FluorChem software.The experiments were performed in triplicate.

Co-immunoprecipitations were performed with at least400μg total protein.Antibody against ubiquitin at a1:1000dilution was added to the lysate and incubated for2.5h at4°C with rotation.Twenty microli-ters of protein G-Sepharose beads was added to the protein–antibody mix and incubated for1.25h at4°with rotation.Beads were pelleted at3000g,washed4×at4°C with1ml of wash buffer,and resuspended in40μl of2×sample buffer.Samples were fractionated by

SDS-PAGE,

Fig.6.Distribution patterns of Timm granules in the hippocampus of the supragranular layer.A:Timm staining of the supragranular layer.AS,LS,and CS show the Timm granules in the hippocampal regions of MTLE rats in the acute,latent and chronic stages.M-AS,M-LS,and M-CS show the same areas from MTLE rats after MG-132pre-treatment.B:The histogram shows a signi?cant increase of Timm granules in MTLE rats compared with controls,which was most severe after MG-132pre-treatment.In the acute stage,there was no statistical difference among the three groups(#p b0.05,MTLE vs control;*p b0.05,MTLE vs MG-132pre-treatment MTLE;Δp N0.05,AC vs AS;□p N0.05,M-AS vs AS).

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transferred to nitrocellulose membranes,and analyzed by western blot-ting as described.Anti-Nedd4-2monoclonal antibody was used at a dilution of1:1000.

2.8.Double immunostaining

Double immunostaining of ubiquitin and Nedd4-2was performed in both hippocampal slides and cultured neurons.

Frozen sections of hippocampus were?xed with4%paraformalde-hyde.Neurons were seeded onto cover slips in12-well tissue culture plates.After treatment,neurons were?xed with4%paraformaldehyde for15min.Then hippocampal slides and cultured neurons were used for immuno?uorescence analysis.For cytoplasmic staining,samples were incubated with0.25%Trixton for10min and blocked with5% BSA in PBS.The samples were then incubated with primary antibodies ubiquitin(1:100)and Nedd4-2(1:200)together overnight at4°C, then incubated again with goat anti-rabbit and anti-mouse IgG(FITC)-conjugated secondary antibodies(1:500).Cellular DNA was stained with4′,6-diamidino-2-phenylindole(DAPI,1.5μg/ml).After antibody labeling,cells were viewed with an Olympus BX51laser confocal micro-scope and cell imaging software(Olympus,Hamburg,Germany). Colocalization studies were carried out to identify ubiquitin and Nedd4-2expression in neurons.

2.9.Statistics

The results are the means±standard deviation(SD)of at least three independent experiments.Statistical analyses were carried out with Student's t test when comparing two variables and one-way ANOVA when comparing multiple variables.Values were considered signi?cant when p b0.05.

3.Results

3.1.Abnormal behaviors in rat models

For each pilocarpine-treated animal,clinical signs of seizure activity were observed.All rats exhibited a well-de?ned pattern of behavior after pilocarpine treatment,such as akinesia,ataxic lurching,tremor, head bobbing,masticatory automatisms with myoclonus of facial mus-cles and wet dog shakes at onset.Then87.1%of the rats(n=135) progressed to SE with bilateral limb clonus,rearing,and falling around 15–35min after pilocarpine injection.Spontaneous seizures usually oc-curred3weeks after SE and tended to be stable8weeks after SE.Spon-taneous seizures were generally characterized by a focal onset (immobility,mechanical mutation,mouth clonus,forelimb clonus),oc-casionally culminating into a generalized convulsive stage lasting about 30s to1.5min,one to several times per day.These behavioral changes were quite consistent with the features of human MTLE.

The MG-132pre-treated rats were more irritable and susceptible to SE in the acute stage.In the latent and chronic stages,they were easily frightened and exhibited bellicose behavior,and spontaneous seizures occurred early and frequently(7–53days after SE).These

results

Fig.8.Double immunostaining of ubiquitin and Nedd4-2in vivo.A:Ubiquitin and Nedd4-2co-localized in the cytoplasm of neurons in the hippocampus of MTLE rats.B:The?uorescence intensity of Nedd4-2weakened after MG-132

pre-treatment.

Fig.7.Co-immunoprecipitation of ubiquitin and Nedd4-2.In the hippocampal extracts of

both MTLE rats and the HNC model of epilepsy,western blot of Nedd4-2in wash buffer

with anti-ubiquitin G-Sepharose beads was positive.Furthermore,Nedd4-2down-regulated

in MTLE rats;in the HNC model of epilepsy,the expression levels of Nedd4-2signi?cantly

decreased after UBA administration.

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indicate that inhibition of the UPS aggravated the clinical manifestation of MTLE.

3.2.Dynamic expression of ubiquitin and Nedd4-2in MTLE models

The expression levels of ubiquitin and Nedd4-2in the hippocampus of MTLE rats were detected by western blot(Fig.1).The results indicate that ubiquitin down-regulated in the AS and LS groups(both p b0.01), but up-regulated in the SS group(p b0.01).Nedd4-2showed no signif-icant difference in expression(p N0.05)in the latent groups,while it down-regulated in the AS and SS groups(p b0.01).These results indicate that,in the acute stage of seizure attack,the down-regulated expression of UPS proteins was one of the factors causing epileptogenesis, and in the chronic stage,the decreased expression of Nedd4-2was closely related with spontaneous seizures.

3.3.Neuron loss and MFS in MTLE models

The hippocampal cytoarchitecture of the CA1region(Fig.2),CA3 region(Fig.3)and dentate gyrus(Fig.4)was evaluated with Nissl stain-ing at different stages(acute,latent,and chronic)after pilocarpine or MG-132pre-treatment by intraperitoneal injection.The results revealed neurons with regular shape and abundant Nissl bodies in the CA1region,CA3region and dentate gyrus in the control groups.Cell swelling and lysis,as well as extensive cytoplasmic vacuolization, appeared in AS.Gradually,nissl bodies decreased or disappeared,neuronal nuclear stains deepened,and cell body shrinkage and glial cell hyperplasia were observed in LS and CS.These symptoms were most severe after MG-132treatment.

Mossy?bers from granule cells in the CA3(Fig.5)and DG areas (Fig.6)undergo reorganization of their terminal projections during the latent and chronic stages of MTLE in our animal model.Timm-staining revealed a relatively normal pattern,with few Timm particles in both the CA3sub?eld and supragranular region in the AC,LC and CC control groups,as well as AS.A few Timm particles emerged in LS and increased signi?cantly in CS.In the MG-132treatment groups, Timm particles were observed at an early time point in M-AS,more obvious than the corresponding stages of LS and CS.

As mentioned above,morphological observation by using Nissl and Timm staining indicated that inhibition of the UPS leads to more obvi-ous neuron loss and abnormal MFS in MTLE models.This suggests that inhibition of the UPS could aggravate the epileptogenesis of MTLE.

3.4.Ubiquitin and Nedd4-2co-localization work together in the pathogenesis of MTLE

Co-immunoprecipitation results(Fig.7)con?rmed the interactive relationship of ubiquitin and Nedd4-2in our MTLE rat model and the neuron model of epilepsy.The expression of Nedd4-2down-regulated in our MTLE model,and decreased after UBA was administered in the neuron model.Double immunostaining results(Figs.8,9)indicate that ubiquitin and Nedd4-2co-localized in the cytoplasm of

neurons.

Fig.9.Double immunostaining of ubiquitin and Nedd4-2in vitro.A:Ubiquitin and Nedd4-2co-localized in the cytoplasm of neurons in the HNC model of epilepsy.B:The?uorescence intensity of Nedd4-2weakened after UBA pre-treatment.

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The?uorescence intensity of Nedd4-2weakened after MG-132pre-treatment(Fig.8)and decreased after UBA was administered(Fig.9), coinciding with the co-immunoprecipitation results.These results fur-ther show that inhibition of Nedd4-2could aggravate epileptogenesis.

3.5.Ubiquitin affects the HNC model of epilepsy by regulating the phosphorylation of Nedd4-2

In the HNC model of epilepsy,we inhibited the function of ubiquitin with UBA adoption or shRNA–ubiquitin transfection.Thus we could ob-serve that inhibition of ubiquitin leads to down-regulation of Nedd4-2 and up-regulation of P-Nedd4-2.Meanwhile,α-ENaC,the downstream substrate of Nedd4-2,up-regulated(Fig.10).These results suggest that inhibition of ubiquitin enhances the phosphorylation of Nedd4-2, switching of ion channels in the downstream and regulation of neuron electric discharge.

4.Discussion

To date,more and more papers have focused on the important role of the UPS in the pathogenesis of epilepsy[14,15].Ubiquitin and the ubiquitin–proteasome system regulate transmission at inhibitory syn-apses and have implications for understanding nervous system pathol-ogies,such as epilepsy,that are characterized by misregulated GABA signaling[16].In order to explore the pathogenesis of MTLE,this study screened protein expression consecutively from the acute to the latent to the chronic stages of disease in an MTLE rat model.Ubiquitin was revealed to be one of the crucial proteins.Furthermore,after pre-treatment with proteasome inhibitor MG-132,we observed that the rats were more irritable and experienced early and frequent spontane-ous seizures.Accordingly,neuron loss and aberrant MFS were more obvious in this group than in the untreated MTLE rats.These results in-dicate that the UPS is involved in the pathogenesis of MTLE,as inhibition of UPS very likely aggravated the epileptogenesis in these rats.

The UPS,evolutionarily conserved for the regulation of protein turnover,targets proteins for degradation via a complex,temporally regulated process.These highly speci?c protein–protein interactions ensure ubiquitin-targeting speci?city and regulate many aspects of protein turnover housekeeping and cellular maintenance[17].Studies on UPS function have centered mainly on de?ning the role of E3ubiqui-tin ligases,which are the substrate-recruiting component of the ubiquitination pathway[18].Speci?c E3ubiquitin ligases play critical roles in different pathological and physiological diseases.In this study, both co-immunoprecipitation and double immunostaining results indi-cated that ubiquitin and Nedd4-2were co-localized in the cytoplasm of neurons.Inhibition of the UPS or ubiquitin weakened the?uorescence intensity of Nedd4-2.And the expression levels of Nedd4-2changed signi?cantly in the different stages of MTLE.In the chronic stage,the expression of Nedd4-2was remarkably inhibited when spontaneous seizures occurred.Therefore,we regard Nedd4-2as a critical E3ubiqui-tin ligase participating in the pathogenesis of MTLE.

The ubiquitin ligases Nedd4and Nedd4-2regulate voltage-gated ion channels and underpin aberrant ion channel function in neurological disorders[12].Dibbens[19]found that the NEDD4-2gene encodes a ubiquitin protein ligase that may regulate the cell surface levels of several ion channels,receptors and transporters involved in regulat-ing neuronal excitability,including voltage-gated sodium channels (VGSCs),the most clinically relevant of the epilepsy genes.The arti-cle named NEDD4-2as a potential candidate susceptibility gene for epileptic photosensitivity.In addition,Nedd4-2has previously been demonstrated to regulate ENaC and other ion channels and transporters.It has also been shown that loss of Nedd4-2leads to excessive ENaC function[20].In our study,using the HNC model of epilepsy,we inhibited the function of ubiquitin via UBA adoption or shRNA-ubiquitin transfection,and found that Nedd4-2down-regulated,while P-Nedd4-2andα-ENaC up-regulated.These results suggest that inhibition of ubiquitin enhances the phosphorylation of Nedd4-2,switching of ion channels in the downstream and regulation of the electric discharge of neurons.

The important roles of the UPS and Nedd4-2in oncology and neuro-degenerative disease have been widely studied.Indeed,several studies now show that ubiquitin and its E3ligase Nedd4-2are

inextricably

Fig.10.Western blot analysis of Nedd4-2,P-Nedd4-2andα-ENaC.A:Gray value rate of Nedd4-2,P-Nedd4-2,α-ENaC andβ-actin expression,in the following order:HNC model of epilepsy,UBA pre-treatment,shRNA–ubiquitin transfection and con-ubiquitin transfection.B,C and D:The histogram of Nedd4-2,P-Nedd4-2andα-ENaC.After UBA or shRNA–ubiquitin intervention,Nedd4-2down-regulated while P-Nedd4-2andα-ENaC up-regulated,compared to the control groups(#p b0.05,+UBA vs control(HNC model of epilepsy);*p b0.05, +shRNA–ubiquitin vs+con-ubiquitin).

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linked with voltage-gated sodium channels in primary cortical neurons [21],regulating ion transports[22]and synaptic vesicle recycling[23], which are all likely involved in the mechanism of epilepsy.Our study is the?rst to report that the UPS participates in the pathogenesis of MTLE,that inhibition of the UPS can aggravate epileptogenesis,and that Nedd4-2is a critical E3ligase involved in this pathogenesis process. Acknowledgments

This work was supported by the Young Teachers of Central South University Booster Project(2011QNZT152)and The National Natural Science Foundation of China Youth Fund Project(81100846).The authors declare that there is no con?ict of interest with either?nancial support or relationships.

Appendix A.Supplementary data

Supplementary data to this article can be found online at http://dx. https://www.wendangku.net/doc/4216048314.html,/10.1016/j.physbeh.2015.02.026.

References

[1]L.Wu,J.Peng,C.Wei,G.Liu,G.Wang,K.Li,F.Yin,Characterization,using compar-

ative proteomics,of differentially expressed proteins in the hippocampus of the mesial temporal lobe of epileptic rats following treatment with valproate,Amino Acids40(1)(2011)221–238.

[2]M.Thom,Review:Hippocampal sclerosis in epilepsy:a neuropathology review,

Neuropathol Appl Neurobiol.40(5)(2014)520–543.

[3] F.Cendes,A.C.Sakamoto,R.Sprea?co,W.Bingaman,A.J.Becker,Epilepsies associat-

ed with hippocampal sclerosis,Acta Neuropathol128(1)(2014)21–37.

[4]S.Y.Aghdam,N.Sheibani,The ubiquitin–proteasome system and microvascular

complications of diabetes,J.Ophthalmic Vis.Res.8(3)(2013)244–256.

[5]Z.Zemeckien?, A.Vitkauskien?,T.Sjakste, B.Sitkauskien?,R.Sakalauskas,

Proteasomes and proteasomal gene polymorphism in association with in?amma-tion and various diseases,Medicina(Kaunas)49(5)(2013)207–213.

[6]P.Garyali,P.Siwach,P.K.Singh,R.Puri,S.Mittal,S.Sengupta,R.Parihar,S.Ganesh,

The malin–laforin complex suppresses the cellular toxicity of misfolded proteins by promoting their degradation through the ubiquitin–proteasome system,Hum.Mol.

Genet.18(4)(2009)688–700.

[7]M.Shen,S.Schmitt,D.Buac,Q.P.Dou,Targeting the ubiquitin–proteasome system

for cancer therapy,Expert Opin.Ther.Targets17(9)(2013)1091–1108.

[8]L.Wu,C.Zhang,F.Yin,Dynamic changes in the proteomic pro?le of mesial temporal

lobe epilepsy at different disease stages in an immature rat model,Protein Pept.Lett.

22(2)(Oct.2015)180–192.

[9] D.Rotin,O.Staub,Nedd4-2and the regulation of epithelial sodium transport,Front.

Physiol.3(2012)212.

[10] https://www.wendangku.net/doc/4216048314.html,edermann,M.Cachemaille,G.Kirschmann,M.Pertin,R.D.Gosselin,I.Chang,

M.Albesa,C.Towne,B.L.Schneider,S.Kellenberger,H.Abriel,I.Decosterd,Dysreg-ulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2in neuro-pathic pain,J.Clin.Invest.123(7)(2013)3002–3013.

[11] A.B.Fotia,J.Ekberg,D.J.Adams,D.I.Cook,P.Poronnik,S.Kumar,Regulation of neu-

ronal voltage-gated sodium channels by the ubiquitin–protein ligases Nedd4and Nedd4-2,J.Biol.Chem.279(28)(2004)28930–28935.

[12] D.Bongiorno,F.Schuetz,P.Poronnik,D.J.Adams,Regulation of voltage-gated ion

channels in excitable cells by the ubiquitin ligases Nedd4and Nedd4-2,Channels (Austin)5(1)(2011)79–88.

[13]R.E.Blair,L.S.Deshpande,S.Sombati,K.W.Falenski,B.R.Martin,R.J.DeLorenzo,

Activation of the cannabinoid type-1receptor mediates the anticonvulsant proper-ties of cannabinoids in the hippocampal neuronal culture models of acquired epilepsy and status epilepticus,J.Pharmacol.Exp.Ther.317(3)(2006)1072–1078.

[14]J.M.Girard,J.Turnbull,N.Ramachandran,B.A.Minassian,Progressive myoclonus

epilepsy,Handb.Clin.Neurol.113(2013)1731–1736.

[15]P.X.He,Y.L.Tang,W.L.Cai,Y.H.Huang,C.Fang,H.L.Jing,Morphology changes

of ubiquitin–proteasome system in traumatic epilepsy,Fa Yi Xue Za Zhi26(1) (2010)10–14.

[16]J.R.Kowalski,H.Dube,D.Touroutine,K.M.Rush,P.R.Goodwin,M.Carozza,Z.Didier,

M.M.Francis,P.Juo,The anaphase-promoting complex(APC)ubiquitin ligase regu-lates GABA transmission at the C.elegans neuromuscular junction,Mol.Cell.

Neurosci.58(2014)62–75.

[17] C.T.Kuo,S.Zhu,S.Younger,L.Y.Jan,Y.N.Jan,Identi?cation of E2/E3ubiquitinating

enzymes and caspase activity regulating Drosophila sensory neuron dendrite prun-ing,Neuron51(3)(2006)283–290.

[18]S.L.Stone,The role of ubiquitin and the26S proteasome in plant abiotic stress sig-

naling,Front.Plant Sci.5(2014)135.

[19]L.M.Dibbens,J.Ekberg,I.Taylor,B.L.Hodgson,S.J.Conroy,I.L.Lensink,S.Kumar,M.A.

Zielinski,L.A.Harkin,G.R.Sutherland,D.J.Adams,S.F.Berkovic,I.E.Scheffer,J.C.

Mulley,P.Poronnik,NEDD4-2as a potential candidate susceptibility gene for epilep-tic photosensitivity,Genes Brain Behav.6(8)(2007)750–755.

[20] D.Rotin,O.Staub,Nedd4-2and the regulation of epithelial sodium transport,Front.

Physiol.3(2012)212.

[21] https://www.wendangku.net/doc/4216048314.html,edermann,I.Decosterd,H.Abriel,Ubiquitylation of voltage-gated sodium

channels,Handb.Exp.Pharmacol.221(2014)231–250.

[22] D.Rotin,O.Staub,Role of the ubiquitin system in regulating ion transport,P?ugers

Arch.461(1)(2011)1–21.

[23] D.H.Hryciw,J.Ekberg,A.Lee,I.L.Lensink,S.Kumar,W.B.Guggino,D.I.Cook,C.A.

Pollock,P.Poronnik,Nedd4-2functionally interacts with ClC-5:involvement in con-stitutive albumin endocytosis in proximal tubule cells,J.Biol.Chem.279(53) (2004)54996–55007.

112L.Wu et al./Physiology&Behavior143(2015)104–112