Research ArticleMicroRNA-132 Interact with p250GAPCdc42 Pathway inthe Hippocampal Neuronal Culture Model ofAcquired Epilepsy and Associated with Epileptogenesis Process

Jinxian Yuan12 Hao Huang12 Xin Zhou12 Xi Liu12 Shu Ou12 Tao Xu12

Ruohan Li12 Limin Ma12 and Yangmei Chen12

1Department of Neurology Second Affiliated Hospital of Chongqing Medical University Chongqing 400016 China2Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology Chongqing Medical UniversityChongqing 400016 China

Correspondence should be addressed to Yangmei Chen 489455407qqcom

Received 5 March 2016 Revised 11 May 2016 Accepted 26 May 2016

Academic Editor Clive R Bramham

Copyright copy 2016 Jinxian Yuan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increasing evidence suggests that epilepsy is the result of synaptic reorganization and pathological excitatory loop formationin the central nervous system however the mechanisms that regulate this process are not well understood We proposed thatmicroRNA-132 (miR-132) and p250GAP might play important roles in this process by activating the downstream Rho GTPasefamily We tested this hypothesis using a magnesium-free medium-induced epileptic model of cultured hippocampal neuronsWe investigated whether miR-132 regulates GTPase activity through p250GAP and found that Cdc42 was significantly activated inour experimental model SilencingmiR-132 inhibited the electrical excitability level of cultured epileptic neurons whereas silencingp250GAP had an opposite effect In addition we verified the effect ofmiR-132 in vivo and found that silencingmiR-132 inhibited theaberrant formation of dendritic spines and chronic spontaneous seizure in a lithium-pilocarpine-induced epileptic mouse modelFinally we confirmed that silencing miR-132 has a neuroprotective effect on cultured epileptic neurons however this effect did notoccur through the p250GAP pathway Generally silencing miR-132 may suppress spontaneous seizure activity through the miR-132p250GAPCdc42 pathway by regulating the morphology and electrophysiology of dendritic spines therefore miR-132 mayserve as a potential target for the development of antiepileptic drugs

1 Introduction

Epilepsy is a neurological disorder that is characterizedby recurrent seizures that result from abnormal and syn-chronous firing of neurons in the brain Approximatelyone-third of the patients with epilepsy do not respond todrugs and are said to have intractable epilepsy Althoughthe precise mechanism of seizure recurrence remains elusiveelucidation of themechanisms involved in the transformationof a normal brain into one capable of producing recurrentseizures and of maintaining an epileptic state is essentialfor understanding epileptogenesis and for developing newtreatments for epilepsy

MicroRNAs as posttranscriptional regulators for up to60 of proteins are a major determinant of protein levels in

cells [1]MicroRNA-132 (miR-132) is significantly upregulatedduring active synaptogenesis and plays important roles inspine formation andmaturation [2ndash5] miR-132 also regulatesthe inflammatory response and neuronal apoptosis afteracute brain injury [6ndash8] Several studies have shown thatmiR-132 is persistently upregulated during epileptogenesisafter acute brain injury [9ndash13] Because synaptic dysfunctionand reorganization are the most important histopathologicalchanges in epileptic foci [14] we aimed to investigate whethermiR-132 plays a role in epileptogenesis by regulating synapticreorganization

p250GAP is a target of miR-132 and is enriched in theNMDA receptor complex of neuronal synapses [2] p250GAPis a Rho family GTPase-activating protein that can interactwith a variety of synaptic proteins by inhibiting the activity

Hindawi Publishing CorporationNeural PlasticityVolume 2016 Article ID 5108489 14 pageshttpdxdoiorg10115520165108489

2 Neural Plasticity

of downstream Rho family GTPases including RhoA Rac1and Cdc42 [2 15 16] It is an important cytoskeletal regulatorthat is regulated by neuronal activity-related signaling path-ways that result in the depolymerization of the cytoskeletonand a reduction in the density and volume of dendriticspines In the central nervous system (CNS) p250GAP hasbeen reported to mainly regulate the activity of Rac1 andCdc42 This study aimed to explore the possible molecularmechanisms of miR-132 and its target p250GAP duringepileptogenesis We also aimed to determine how GTPasesare regulated by p250GAP in the pathological process ofepilepsy

2 Materials and Methods

21 Animals Adult male (8ndash12 weeks) C57BL6 mice wereused in this study The mice were kept in an animal room ata constant temperature (22 plusmn 1∘C) and a 12-h lightdark cyclewith free access to food and water All experimental proce-dures were performed in accordance with the internationalguidelines for the use of animals and the guidelines of theAnimal Care Committee of Chongqing Medical UniversityChina

22 Hippocampal Neuron Culture Hippocampal neuronsfrom 17- to 19-day-old embryonic mice were cultured(5 times 105 cells per square centimeter) on plates coatedwith poly-L-lysine (Catalog number P1399 Sigma USA) asdescribed previously [17]The neurons were then maintainedin neurobasal medium (Catalog number 21103-049 GibcoUSA) supplemented with B27 (Catalog number 17504-044Gibco) and 05mM L-glutamine (Catalog number G3126Sigma) Approximately 13 to 12 of the culture mediumwas changed every 3-4 days Ten micromolar cytosine 120573-D-arabinofuranoside (Catalog number C1768 Sigma) wasadded to the culture medium at 3 days in vitro (DIV3) toinhibit the growth of gliocytes The cultured neurons werestained at DIV7 with a neuron-specific marker microtubule-associated protein 2 (MAP2) (Catalog number 11267 AbcamUSA) to evaluate the purity of the cultured neurons Only thecultured cellswhose puritywas higher than 98were used forthe following experiment

23 Induction of Spontaneous Recurrent Epileptiform Dis-charges (SREDs) of Cultured Hippocampal Neurons AtDIV10 SREDs were induced in the neuronal cultures byexposing the neurons to magnesium-free (MGF) medium(145mM NaCl 10mM HEPES 25mM KCl 2mM CaCl

2

10mM glucose and 0001mM glycine with the pH adjustedto 73 with NaOH and the osmolarity adjusted to 280ndash320mOsm with sucrose) for 3 h The sham controls were treatedwith nonmagnesium-free medium (non-MGF) which isMGF medium supplemented with 1mM MgCl

2 SREDs are

typically observed within 12ndash24 h using patch clamp record-ings and can last for the life of the neurons in culture Thishippocampal neuronal culture model of status epilepticus(SE) has been well characterized as a useful in vitro modelof refractory SE [18]

24 Cell Transfection A miR-132 antagomir (ant-132) wasused to silence the expression level of miR-132 A nontarget-ing scrambled sequence (Scr) was used as a control (Cata-log number miR30000067-1-10 RiboBio China) p250GAPexpression was silenced using a lentivirus (LV-shp250GAP)and the same lentivirus vector expressing GFP alone (LV-GFP) was chosen as a control (Catalog number LVCON077GeneChem China) Neuronal transfection was conductedaccording to the manufacturerrsquos instructions and the culturemedium was completely changed 10 h after transfection

25 Patch Clamp Recordings The membrane potentials ofneurons were measured with whole-cell current-clamprecordings using a patch clamp amplifier A cell culture dishwas mounted on the stage of an inverted microscope (IX-51 Olympus Japan) and patch pipettes were filled with anintracellular solution containing 110mM KAsp 30mM KCl10mM EGTA 10mM HEPES 5mM Na-ATP 1mM CaCl

2

2mMMgCl2 and 10mM TEACl with the pH adjusted to 73

with CsOH and the osmolarity adjusted to 280ndash300 mOsmwith sucrose The experiments were performed at room tem-perature (22ndash24∘C)The pipette resistance in the intracellularsolution was 2ndash4MΩ The pipette resistance and capacitancewere compensated electronically after the establishment ofa gigaseal After the whole-cell capacitance was compen-sated recordings were made only when the series resistancewas lt20MΩ To optimize the success of recording frompyramidal neurons phase-bright cells were selected basedon both size and pyramidal soma Cultured neurons withsmall dendritic arborizations long axons and soma diam-eters of 20ndash26120583m were selected for the electrophysiologicalrecordings to avoid space clamp artifacts Routinely 60ndash80series resistance compensation was employed continuallymonitored and adjusted as required Whole-cell resistanceand resting membrane potential were also monitored beforeand during the experiments and a cell was accepted forstudy only if these parameters remained stable Whole-cell recordings were performed using an EPC-10 amplifier(HEKA Germany) in the current-clamp mode Data werecollected and analyzed using Clamp-fit 100 software (AxonUSA)

26 SYBR-Green Quantitative Real-Time PCR (qRT-PCR)Reverse transcription (RT) reactions were performed usinga PrimeScript RT Reagent Kit (Catalog number RR047ATaKaRa China) miRNA-specific stem-loop primers wereused for RT of miR-132 Samples were run at 37∘C for15min and 85∘C for 5 sec followed by a hold at 4∘C RTproducts were stored undiluted at minus20∘C prior to runningreal-time PCR Real-time PCR was carried out on a Bio-Rad real-time PCR system SYBR Premix Ex Taq II(TaKaRa China) was used The PCR mixture contained125 120583L of SYBR Premix Ex Taq II 1 120583L of 10 120583MPCR forwardprimer 1 120583L of 10 120583M PCR reverse primer 2120583L of the RTreaction solution and 85 120583L of ddH

2O Real-time PCR was

performed under the following conditions stage 1 95∘C for30 s stage 2 40 cycles at 95∘C for 5 s and 60∘C for 30 s andstage 3 dissociation The primers used were (miR-132 RT)

Neural Plasticity 3

51015840-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCA-CTGGATACGACCGACCA-31015840 (miR-132 forward primer)51015840-GCGGCGGTAACAGTCTACAGCC-31015840 and (miR-132 re-verse primer) 51015840-ATCCAGTGCAGGGTCCGAGG-31015840 Thedata were analyzed with Bio-Rad CFXManager software thedata are represented as the mean 2minusΔΔCTplusmn standard deviation(SD)

27 Western Blot (WB) Total proteins were extracted usinga whole protein extraction kit (Catalog number P0013Beyotime China) The total protein concentrations weredetermined using an Enhanced Bicinchoninic Acid Protein(BCA) Assay Kit (Catalog number P0012s Beyotime China)and the samples were stored at minus20∘C until use WB analysiswas performed as described previously [8] The primaryantibodies used were goat anti-p250GAP (1 1000 Catalognumber 138167 Santa Cruz USA) and rabbit anti-cleavedcaspase-3 (1 150 Catalog number 9661 CST USA)

28 Measurements of Rac1 and Cdc42 Activation The activa-tion of Rac1 and Cdc42 was measured using a Rac1Cdc42Activation Assay Kit (Catalog number 17-441 MilliporeUSA) according to the manufacturerrsquos protocol This assayuses the downstream effector of RacCdc42 p21-activatedprotein kinase (PAK1) to isolate the active GTP-bound formof RacCdc42 from the sample The p21-binding domain(PBD) of PAK1 is expressed as a GST fusion protein and cou-pled to agarose beads After the proteins were precipitatedan immunoblot was performed and the activated Rac1 andCdc42 were detected with specific monoclonal antibodiesfollowed by an HRP-conjugated secondary antibody

29 TdT-Mediated dUTP Nick-End Labeling (TUNEL) AssayA TUNEL assay Roche Kit (Catalog number 11684817910Roche Switzerland) was used to detect the apoptosis levelof cultured hippocampal neurons according to the manufac-turerrsquos instructions Briefly after cortical neurons were fixedin freshly prepared 4 formaldehyde solution in phosphate-buffered saline (PBS) for 20min at room temperature andpermeabilized with 02 Triton X-100 for 5min they wereincubated with 50120583L of a TUNEL reaction mixture for60min at 37∘C in the dark and then rinsed with PBS (pH74) 3 times for 5min each To detect the nuclei the slideswere incubated with DAPI for 5min at room temperature inthe dark and observed with a fluorescence microscope Theapoptotic index was expressed as the ratio of the number ofTUNEL-positive neurons to the total number of neurons

210 Temporal Lobe Epilepsy (TLE) Experimental MouseModels and Ant-132 Intervention The pilocarpine model hasbeen widely used to simulate human TLE For SE inductionall the mice were intraperitoneally (ip) injected with pilo-carpine (300mgkg 01mL10 g ip Sigma) Atropine sulfate(1mgkg ip) was administered 30min prior to the first doseof pilocarpine The mice that developed a stage 4 or 5 seizure(Racinersquos scale) [19] within 2 h after pilocarpine adminis-tration were considered kindled in our study Diazepam(10mgkg ip) was given to terminate the convulsions 1 h

after SE onset All of the mice were allowed to recover for48 h after SE and were then intracerebroventricularly (icv)injected with 2 120583L of saline (TLE group) Scr-132 (TLE+Scr-132 group) or ant-132 (TLE+ant-132 group) The dose of ant-132 was 1 nmol which was achieved by dilution in double-distilled water

211 Analysis of Spontaneous Seizures by Continuous VideoMonitoring Animals in the chronic periodwith spontaneousrecurrent seizures (SRS) associated with pilocarpine epilepsywere recorded every day for 24 h using a closed circuit videosystem to detect the class 4 and class 5 seizures at the 6thweek An observer who was blinded to the study reviewedthe videos Seizures were counted using a modified six-point Racine scale Clinical events with a score below 2 wereexcluded

212 Golgi-Cox Staining A Hito Golgi-Cox OptimStain Kit(Catalog number HTKNS1125 Hitobiotec Inc USA) wasused to visualize the dendritic spines Brain tissues obtainedfrom all the mice in the ant-132-treated and Scr-132 con-trol groups in our experiment were sectioned into 100-120583mcoronal sections on a cryostat (Leica Germany) and thestaining process was conducted following the manufacturerrsquosinstructions

213 Morphological Analysis and Density Quantification ofDendritic Spines For morphometric measurements at least15 neurons were analyzed for each data point reportedand 3 times 15 120583m of dendrites in each neuron was chosenThe IMARIS FilamentTracer module (Andor TechnologyBelfast Northern Ireland) was used to detect quantifyand characterize spine structures Filopodia were defined asdendritic protrusions with mean head width le mean neckwidth Thin spines were defined as dendritic protrusionswith two times mean neck width lt spine length and withmean neck width le maximum head width Stubby spineswere defined as dendritic protrusions with length lt 1 120583mMushroom spines were defined as dendritic protrusions withmean head width gtmean neck width To assess the reliabilityof the counting of dendritic protrusions a blind study wasinitially performed [20] Each experiment was repeated atleast three times using independent preparations

214 Data Analysis All data are expressed as the means plusmnSD Statistical analyses were performed with SPSS Statisticsfor Windows Version 200 (Armonk NY USA) Differencesbetween the experimental groups were compared using one-way analysis of variance (ANOVA) and Studentrsquos t-test wasused for comparisons 119901 lt 005 was considered statisticallysignificant

3 Results

31 Expression of miR-132 and p250GAP Is Associated withSynaptogenesis and Electrophysiological Activity It has beenreported that the expression level of mature miR-132 islow during the first week in the neonatal rat hippocampuswith a significant increase in miR-132 levels during weeks

4 Neural Plasticity

Day 1 Day 7 Day 12 Day 19 Day 25 Day 30

Day 1 Day 7 Day 13 Day 19 Day 25 Day 30

miR-132

120573-actin

0

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

p250GAP

p250GAP

252KD

42KD

(a)

120573-actin

DIV5 DIV7 DIV9 DIV11 DIV13 DIV150

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

252KD

42KD

p250GAP

miR-132p250GAP

Day 5 Day 7 Day 9 Day 11 Day 15 Day 15

(b)

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)

ControlMGF

lowast

lowast

0

1

2

3

lowastlowast

3d 5d 7d6h

(c)

120573-actin

3d 5d 7d6h00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

p250GAP

3d 5d 7d6hCC C CMGF MGF MGF MGF

252KD

42KD

lowastlowast

lowast

lowast

ControlMGF

(d)

Figure 1 (a) The expression levels of miR-132 and p250GAP in C57BL6 mice during postnatal d 1 to d 30 were evaluated using quantitativereverse transcription PCR and Western blot analyses (b) The expression levels of miR-132 and p250GAP protein during DIV5ndashDIV15 incultured hippocampal neurons (c d) Expression level of miR-132 and p250GAP 6 h to 7 d after MGF treatment as compared to controlgroup The data are presented as the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 eachdata point represents mean plusmn SD of 5 experiments)

2ndash4 which is also a critical period for the developmentand maturation of spines in rodents [15] In our studythe expression levels of miR-132 and p250GAP in C57BL6mice during postnatal d 1 to d 30 were similar to those ofprevious studies (Figure 1(a)) We evaluated the chronolog-ical expression level of miR-132 and p250GAP during the

maturation process of cultured hippocampal neurons Thelevel of miR-132 increased from DIV5 to DIV13 and wasmaintained at a relatively high level whereas the expressionof p250GAP protein was high at DIV5 but decreased atDIV15 (Figure 1(b)) This result indicated that the expres-sion of miR-132 and p250GAP might correlate with the

Neural Plasticity 5

process of physiological spine maturation and synaptogen-esis

The expression levels of miR-132 and p250GAP at 6 h 3 d5 d and 7 d afterMGF treatment were evaluated to determinewhether the levels of miR-132 and p250GAP were influencedby the electrophysiological activity of cultured neurons Theresults showed that miR-132 was upregulated 6 h to 7 dfollowingmagnesium-freemedium treatment with statisticalsignificance at 6 h 3 d and 7 d while p250GAP protein wasdownregulated from 6 h to 7 d following magnesium-freemedium treatment with statistical significance at 3 d 5 d and7 d (Figures 1(c) and 1(d))

The results suggest that miR-132 expression is increasedduring the active synaptogenesis periods of the immaturebrain Moreover the upregulation of miR-132 in the SREDmodel of hippocampal neurons suggests that the pathologicalelectrical excitability may also influence miR-132 expressionwhich may correlate with the pathological synaptogenesis ofthe CNS during epileptogenesis

32 miR-132 Regulates the Activation of Cdc42 via p250GAPin SRED Model of Hippocampal Neurons We used a specificantagonist of miR-132 and RNA interference to knock downthe expression of miR-132 and p250GAP (Figures 2(a) and2(b)) The transfection efficiency was confirmed by qRT-CPR for miR-132 (Figure 2(c)) and by WB for p250GAP(Figure 2(d)) The expression level of p250GAP has signif-icantly upregulated after ant-132 treatment which indicatedthat the expression level of p250GAPwas negatively regulatedby miR1-132 in our cultured epileptic hippocampal neurons(Figure 2(e))

Rac1 and Cdc42 are considered two important promotorsof dendritic branching and synaptic plasticity while RhoAacts in the oppositemanner [16] p250GAP has been reportedto mainly regulate the activation of Rac1 and Cdc42 in theCNS [16] The pathological plasticity of neuronal spines andsynapses is important in the pathological process of epilepsythus we studied the activation levels of Rac1 and Cdc42to explore whether Rac1 and Cdc42 are regulated by themiR-132p250GAP pathway in our SRED model of culturedhippocampal neuronsThe activation level of Rac1 andCdc42was tested

First we found that the activation levels of both Rac1and Cdc42 were significantly elevated in MGF-treated hip-pocampal neurons compared to control hippocampal neu-rons (Figures 3(a1) and 3(a2)) To determine whether andhow Rac1 and Cdc42 are regulated by the miR-132p250GAPpathway in our cultured hippocampal neurons we evaluatedthe activation levels of Rac1 and Cdc42 in cultured hip-pocampal neurons when miR-132 or p250GAP expressionwas inhibitedThe results showed that transfection of ant-132or LV-shp250GAP did not significantly affect the activity ofRac1 (Figures 3(b1) and 3(b2)) while the activity ofCdc42wassignificantly inhibited after ant-132 transfection and elevatedafter LV-shp250GAP transfection (Figures 3(c1) and 3(c2))Furthermore we obtained similar results in the MGF-treatedSRED model of cultured hippocampal neurons while theexpression of miR-132 or p250GAP was inhibited (Figures3(d1) 3(d2) 3(e1) and 3(e2))

Our data suggest that p250GAP may primarily functionas a GAP for Cdc42 in this epileptic model of culturedneurons

33 miR-132 Influences Neuronal Electrical Excitabilitythrough p250GAP in SRED Model of Hippocampal NeuronsTo clarify the effect of miR-132 and p250GAP on neuronalexcitability we performed electrophysiological evaluationsof the MGF medium-treated cultured hippocampal neuronsOur experiment showed that ant-132 treatment significantlydecreased the AP frequency suggesting that ant-132 cansignificantly inhibited neuronal electrical excitability in ourSRED model of cultured hippocampal neurons (Figures4(a2)ndash4(a4)) In contrast ant-132 and LV-shp250GAPcotreatment significantly increased the AP frequency(Figures 4(a5) and 4(a6))

34 Neuroprotective Effect of the miR-132p250GAP Pathwayon Cultured Neurons Silencing the overexpression of miR-132 is known to have a neuroprotective effect after acutebrain injury [8 20] therefore we asked whether miR-132regulates neuronal apoptosis via the p250GAP pathway Wefirst transected the cultured neurons with ant-132 at DIV7and then induced the SRED model at DIV10 Next wesilenced the expression levels of both miR-132 and p250GAPto investigatewhether p250GAPwas involved in the neuronalapoptosis effect of miR-132 TUNEL staining (Figures 5(a)and 5(b)) and WB detection of cleaved caspase-3 levels(Figure 5(c)) were performed 24 h later to investigate thelevel of apoptosis Our results showed that regardless ofwhether the expression level of p250GAP was suppressedmiR-132 silencing decreased neuronal apoptosisThis findingindicates that miR-132 silencing has a neuroprotective effectbut that this effect may occur through pathways other thanthe p250GAP pathway

35 miR-132 Silencing Decreases SRS in a Lithium-PilocarpineModel In Vivo We then investigated whether miR-132 inhi-bition could suppress chronic seizures in vivo using a lithium-pilocarpine model The mice were injected with ant-132 icv48 h after SE Continuous video monitoring was performedin the 6th week for 7 d The frequency of chronic seizurewas quantified and statistically analyzed by comparing theScr-132 control group (Figure 6(a)) and the ant-132 group(Figure 6(b)) Statistical analysis of our behavioral investiga-tion indicated that the ant-132 treatment can protect exper-imental mice against developing chronic recurrent seizuresand can significantly decrease spontaneous seizure frequency(Figure 6(c))

36 miR-132 Silencing Decreases the Remodeling of Spines inMice with Chronic Seizures In general mushroom-shapedand stubby spines represent more mature and stable spineswhile thin spines and filopodia tend to be more plasticand immature (Figure 7(b)) [21] Disrupted maintenanceof the dendritic tree and spinogenesis are two importantneurophysiological features of epilepsy After the behavioral

6 Neural Plasticity

Ant-132

(a)

LV-shp250GAP

(b)

lowastlowast

00

05

10

15

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)Scr-132Control Ant-132

(c)

p250GAP

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

LV-GFP LV-shp250GAPControl

120573-actin

252KD

42KD

lowastlowast

(d)

p250GAP

lowastlowast

120573-actin

252KD

42KD

0

1

2

3

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

EP+Scr-132 EP+ant-132EP(e)

Figure 2 Knockdown of miR-132 and p250GAP in cultured neurons (a) Cells were transfected with ant-132 at DIV7 and observed using afluorescence microscope 48 h after transfection Transfection efficiency was measured by RFP fluorescence (b) Cells were transfected withLV-shp250GAP at DIV7 and observed 72 h after transfection Transfection efficiency was measured by GFP fluorescence Scale bar is 50120583m(c d) Measurement of knocked down expression in transfected neurons was performed by qRT-PCR for miR-132 and by WB for p250GAP(e) Expression level of p250GAP 72 h after ant-132 treatment in cultured epileptic hippocampal neurons lowastlowast119901 lt 001 (119899 = 10 the data arerepresentative of 5 experiments)

observations were completed all the brain tissues of the ant-132-treated (EP+Ant-132) Scr-132-treated (EP+Scr-132) andnonintervention control (EP) groups were excised andGolgi-stained to further investigate the morphological effects of

ant-132 treatment on the dendritic spine (Figure 7(a)) Thetotal number and different forms of spines in the hippocam-pal dentate gyrus (DG) and CA1 regions were quantified andstatistically analyzed to investigate the differences in spine

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

2 Neural Plasticity

of downstream Rho family GTPases including RhoA Rac1and Cdc42 [2 15 16] It is an important cytoskeletal regulatorthat is regulated by neuronal activity-related signaling path-ways that result in the depolymerization of the cytoskeletonand a reduction in the density and volume of dendriticspines In the central nervous system (CNS) p250GAP hasbeen reported to mainly regulate the activity of Rac1 andCdc42 This study aimed to explore the possible molecularmechanisms of miR-132 and its target p250GAP duringepileptogenesis We also aimed to determine how GTPasesare regulated by p250GAP in the pathological process ofepilepsy

2 Materials and Methods

21 Animals Adult male (8ndash12 weeks) C57BL6 mice wereused in this study The mice were kept in an animal room ata constant temperature (22 plusmn 1∘C) and a 12-h lightdark cyclewith free access to food and water All experimental proce-dures were performed in accordance with the internationalguidelines for the use of animals and the guidelines of theAnimal Care Committee of Chongqing Medical UniversityChina

22 Hippocampal Neuron Culture Hippocampal neuronsfrom 17- to 19-day-old embryonic mice were cultured(5 times 105 cells per square centimeter) on plates coatedwith poly-L-lysine (Catalog number P1399 Sigma USA) asdescribed previously [17]The neurons were then maintainedin neurobasal medium (Catalog number 21103-049 GibcoUSA) supplemented with B27 (Catalog number 17504-044Gibco) and 05mM L-glutamine (Catalog number G3126Sigma) Approximately 13 to 12 of the culture mediumwas changed every 3-4 days Ten micromolar cytosine 120573-D-arabinofuranoside (Catalog number C1768 Sigma) wasadded to the culture medium at 3 days in vitro (DIV3) toinhibit the growth of gliocytes The cultured neurons werestained at DIV7 with a neuron-specific marker microtubule-associated protein 2 (MAP2) (Catalog number 11267 AbcamUSA) to evaluate the purity of the cultured neurons Only thecultured cellswhose puritywas higher than 98were used forthe following experiment

23 Induction of Spontaneous Recurrent Epileptiform Dis-charges (SREDs) of Cultured Hippocampal Neurons AtDIV10 SREDs were induced in the neuronal cultures byexposing the neurons to magnesium-free (MGF) medium(145mM NaCl 10mM HEPES 25mM KCl 2mM CaCl

2

10mM glucose and 0001mM glycine with the pH adjustedto 73 with NaOH and the osmolarity adjusted to 280ndash320mOsm with sucrose) for 3 h The sham controls were treatedwith nonmagnesium-free medium (non-MGF) which isMGF medium supplemented with 1mM MgCl

2 SREDs are

typically observed within 12ndash24 h using patch clamp record-ings and can last for the life of the neurons in culture Thishippocampal neuronal culture model of status epilepticus(SE) has been well characterized as a useful in vitro modelof refractory SE [18]

24 Cell Transfection A miR-132 antagomir (ant-132) wasused to silence the expression level of miR-132 A nontarget-ing scrambled sequence (Scr) was used as a control (Cata-log number miR30000067-1-10 RiboBio China) p250GAPexpression was silenced using a lentivirus (LV-shp250GAP)and the same lentivirus vector expressing GFP alone (LV-GFP) was chosen as a control (Catalog number LVCON077GeneChem China) Neuronal transfection was conductedaccording to the manufacturerrsquos instructions and the culturemedium was completely changed 10 h after transfection

25 Patch Clamp Recordings The membrane potentials ofneurons were measured with whole-cell current-clamprecordings using a patch clamp amplifier A cell culture dishwas mounted on the stage of an inverted microscope (IX-51 Olympus Japan) and patch pipettes were filled with anintracellular solution containing 110mM KAsp 30mM KCl10mM EGTA 10mM HEPES 5mM Na-ATP 1mM CaCl

2

2mMMgCl2 and 10mM TEACl with the pH adjusted to 73

with CsOH and the osmolarity adjusted to 280ndash300 mOsmwith sucrose The experiments were performed at room tem-perature (22ndash24∘C)The pipette resistance in the intracellularsolution was 2ndash4MΩ The pipette resistance and capacitancewere compensated electronically after the establishment ofa gigaseal After the whole-cell capacitance was compen-sated recordings were made only when the series resistancewas lt20MΩ To optimize the success of recording frompyramidal neurons phase-bright cells were selected basedon both size and pyramidal soma Cultured neurons withsmall dendritic arborizations long axons and soma diam-eters of 20ndash26120583m were selected for the electrophysiologicalrecordings to avoid space clamp artifacts Routinely 60ndash80series resistance compensation was employed continuallymonitored and adjusted as required Whole-cell resistanceand resting membrane potential were also monitored beforeand during the experiments and a cell was accepted forstudy only if these parameters remained stable Whole-cell recordings were performed using an EPC-10 amplifier(HEKA Germany) in the current-clamp mode Data werecollected and analyzed using Clamp-fit 100 software (AxonUSA)

26 SYBR-Green Quantitative Real-Time PCR (qRT-PCR)Reverse transcription (RT) reactions were performed usinga PrimeScript RT Reagent Kit (Catalog number RR047ATaKaRa China) miRNA-specific stem-loop primers wereused for RT of miR-132 Samples were run at 37∘C for15min and 85∘C for 5 sec followed by a hold at 4∘C RTproducts were stored undiluted at minus20∘C prior to runningreal-time PCR Real-time PCR was carried out on a Bio-Rad real-time PCR system SYBR Premix Ex Taq II(TaKaRa China) was used The PCR mixture contained125 120583L of SYBR Premix Ex Taq II 1 120583L of 10 120583MPCR forwardprimer 1 120583L of 10 120583M PCR reverse primer 2120583L of the RTreaction solution and 85 120583L of ddH

2O Real-time PCR was

performed under the following conditions stage 1 95∘C for30 s stage 2 40 cycles at 95∘C for 5 s and 60∘C for 30 s andstage 3 dissociation The primers used were (miR-132 RT)

Neural Plasticity 3

51015840-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCA-CTGGATACGACCGACCA-31015840 (miR-132 forward primer)51015840-GCGGCGGTAACAGTCTACAGCC-31015840 and (miR-132 re-verse primer) 51015840-ATCCAGTGCAGGGTCCGAGG-31015840 Thedata were analyzed with Bio-Rad CFXManager software thedata are represented as the mean 2minusΔΔCTplusmn standard deviation(SD)

27 Western Blot (WB) Total proteins were extracted usinga whole protein extraction kit (Catalog number P0013Beyotime China) The total protein concentrations weredetermined using an Enhanced Bicinchoninic Acid Protein(BCA) Assay Kit (Catalog number P0012s Beyotime China)and the samples were stored at minus20∘C until use WB analysiswas performed as described previously [8] The primaryantibodies used were goat anti-p250GAP (1 1000 Catalognumber 138167 Santa Cruz USA) and rabbit anti-cleavedcaspase-3 (1 150 Catalog number 9661 CST USA)

28 Measurements of Rac1 and Cdc42 Activation The activa-tion of Rac1 and Cdc42 was measured using a Rac1Cdc42Activation Assay Kit (Catalog number 17-441 MilliporeUSA) according to the manufacturerrsquos protocol This assayuses the downstream effector of RacCdc42 p21-activatedprotein kinase (PAK1) to isolate the active GTP-bound formof RacCdc42 from the sample The p21-binding domain(PBD) of PAK1 is expressed as a GST fusion protein and cou-pled to agarose beads After the proteins were precipitatedan immunoblot was performed and the activated Rac1 andCdc42 were detected with specific monoclonal antibodiesfollowed by an HRP-conjugated secondary antibody

29 TdT-Mediated dUTP Nick-End Labeling (TUNEL) AssayA TUNEL assay Roche Kit (Catalog number 11684817910Roche Switzerland) was used to detect the apoptosis levelof cultured hippocampal neurons according to the manufac-turerrsquos instructions Briefly after cortical neurons were fixedin freshly prepared 4 formaldehyde solution in phosphate-buffered saline (PBS) for 20min at room temperature andpermeabilized with 02 Triton X-100 for 5min they wereincubated with 50120583L of a TUNEL reaction mixture for60min at 37∘C in the dark and then rinsed with PBS (pH74) 3 times for 5min each To detect the nuclei the slideswere incubated with DAPI for 5min at room temperature inthe dark and observed with a fluorescence microscope Theapoptotic index was expressed as the ratio of the number ofTUNEL-positive neurons to the total number of neurons

210 Temporal Lobe Epilepsy (TLE) Experimental MouseModels and Ant-132 Intervention The pilocarpine model hasbeen widely used to simulate human TLE For SE inductionall the mice were intraperitoneally (ip) injected with pilo-carpine (300mgkg 01mL10 g ip Sigma) Atropine sulfate(1mgkg ip) was administered 30min prior to the first doseof pilocarpine The mice that developed a stage 4 or 5 seizure(Racinersquos scale) [19] within 2 h after pilocarpine adminis-tration were considered kindled in our study Diazepam(10mgkg ip) was given to terminate the convulsions 1 h

after SE onset All of the mice were allowed to recover for48 h after SE and were then intracerebroventricularly (icv)injected with 2 120583L of saline (TLE group) Scr-132 (TLE+Scr-132 group) or ant-132 (TLE+ant-132 group) The dose of ant-132 was 1 nmol which was achieved by dilution in double-distilled water

211 Analysis of Spontaneous Seizures by Continuous VideoMonitoring Animals in the chronic periodwith spontaneousrecurrent seizures (SRS) associated with pilocarpine epilepsywere recorded every day for 24 h using a closed circuit videosystem to detect the class 4 and class 5 seizures at the 6thweek An observer who was blinded to the study reviewedthe videos Seizures were counted using a modified six-point Racine scale Clinical events with a score below 2 wereexcluded

212 Golgi-Cox Staining A Hito Golgi-Cox OptimStain Kit(Catalog number HTKNS1125 Hitobiotec Inc USA) wasused to visualize the dendritic spines Brain tissues obtainedfrom all the mice in the ant-132-treated and Scr-132 con-trol groups in our experiment were sectioned into 100-120583mcoronal sections on a cryostat (Leica Germany) and thestaining process was conducted following the manufacturerrsquosinstructions

213 Morphological Analysis and Density Quantification ofDendritic Spines For morphometric measurements at least15 neurons were analyzed for each data point reportedand 3 times 15 120583m of dendrites in each neuron was chosenThe IMARIS FilamentTracer module (Andor TechnologyBelfast Northern Ireland) was used to detect quantifyand characterize spine structures Filopodia were defined asdendritic protrusions with mean head width le mean neckwidth Thin spines were defined as dendritic protrusionswith two times mean neck width lt spine length and withmean neck width le maximum head width Stubby spineswere defined as dendritic protrusions with length lt 1 120583mMushroom spines were defined as dendritic protrusions withmean head width gtmean neck width To assess the reliabilityof the counting of dendritic protrusions a blind study wasinitially performed [20] Each experiment was repeated atleast three times using independent preparations

214 Data Analysis All data are expressed as the means plusmnSD Statistical analyses were performed with SPSS Statisticsfor Windows Version 200 (Armonk NY USA) Differencesbetween the experimental groups were compared using one-way analysis of variance (ANOVA) and Studentrsquos t-test wasused for comparisons 119901 lt 005 was considered statisticallysignificant

3 Results

31 Expression of miR-132 and p250GAP Is Associated withSynaptogenesis and Electrophysiological Activity It has beenreported that the expression level of mature miR-132 islow during the first week in the neonatal rat hippocampuswith a significant increase in miR-132 levels during weeks

4 Neural Plasticity

Day 1 Day 7 Day 12 Day 19 Day 25 Day 30

Day 1 Day 7 Day 13 Day 19 Day 25 Day 30

miR-132

120573-actin

0

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

p250GAP

p250GAP

252KD

42KD

(a)

120573-actin

DIV5 DIV7 DIV9 DIV11 DIV13 DIV150

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

252KD

42KD

p250GAP

miR-132p250GAP

Day 5 Day 7 Day 9 Day 11 Day 15 Day 15

(b)

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)

ControlMGF

lowast

lowast

0

1

2

3

lowastlowast

3d 5d 7d6h

(c)

120573-actin

3d 5d 7d6h00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

p250GAP

3d 5d 7d6hCC C CMGF MGF MGF MGF

252KD

42KD

lowastlowast

lowast

lowast

ControlMGF

(d)

Figure 1 (a) The expression levels of miR-132 and p250GAP in C57BL6 mice during postnatal d 1 to d 30 were evaluated using quantitativereverse transcription PCR and Western blot analyses (b) The expression levels of miR-132 and p250GAP protein during DIV5ndashDIV15 incultured hippocampal neurons (c d) Expression level of miR-132 and p250GAP 6 h to 7 d after MGF treatment as compared to controlgroup The data are presented as the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 eachdata point represents mean plusmn SD of 5 experiments)

2ndash4 which is also a critical period for the developmentand maturation of spines in rodents [15] In our studythe expression levels of miR-132 and p250GAP in C57BL6mice during postnatal d 1 to d 30 were similar to those ofprevious studies (Figure 1(a)) We evaluated the chronolog-ical expression level of miR-132 and p250GAP during the

maturation process of cultured hippocampal neurons Thelevel of miR-132 increased from DIV5 to DIV13 and wasmaintained at a relatively high level whereas the expressionof p250GAP protein was high at DIV5 but decreased atDIV15 (Figure 1(b)) This result indicated that the expres-sion of miR-132 and p250GAP might correlate with the

Neural Plasticity 5

process of physiological spine maturation and synaptogen-esis

The expression levels of miR-132 and p250GAP at 6 h 3 d5 d and 7 d afterMGF treatment were evaluated to determinewhether the levels of miR-132 and p250GAP were influencedby the electrophysiological activity of cultured neurons Theresults showed that miR-132 was upregulated 6 h to 7 dfollowingmagnesium-freemedium treatment with statisticalsignificance at 6 h 3 d and 7 d while p250GAP protein wasdownregulated from 6 h to 7 d following magnesium-freemedium treatment with statistical significance at 3 d 5 d and7 d (Figures 1(c) and 1(d))

The results suggest that miR-132 expression is increasedduring the active synaptogenesis periods of the immaturebrain Moreover the upregulation of miR-132 in the SREDmodel of hippocampal neurons suggests that the pathologicalelectrical excitability may also influence miR-132 expressionwhich may correlate with the pathological synaptogenesis ofthe CNS during epileptogenesis

32 miR-132 Regulates the Activation of Cdc42 via p250GAPin SRED Model of Hippocampal Neurons We used a specificantagonist of miR-132 and RNA interference to knock downthe expression of miR-132 and p250GAP (Figures 2(a) and2(b)) The transfection efficiency was confirmed by qRT-CPR for miR-132 (Figure 2(c)) and by WB for p250GAP(Figure 2(d)) The expression level of p250GAP has signif-icantly upregulated after ant-132 treatment which indicatedthat the expression level of p250GAPwas negatively regulatedby miR1-132 in our cultured epileptic hippocampal neurons(Figure 2(e))

Rac1 and Cdc42 are considered two important promotorsof dendritic branching and synaptic plasticity while RhoAacts in the oppositemanner [16] p250GAP has been reportedto mainly regulate the activation of Rac1 and Cdc42 in theCNS [16] The pathological plasticity of neuronal spines andsynapses is important in the pathological process of epilepsythus we studied the activation levels of Rac1 and Cdc42to explore whether Rac1 and Cdc42 are regulated by themiR-132p250GAP pathway in our SRED model of culturedhippocampal neuronsThe activation level of Rac1 andCdc42was tested

First we found that the activation levels of both Rac1and Cdc42 were significantly elevated in MGF-treated hip-pocampal neurons compared to control hippocampal neu-rons (Figures 3(a1) and 3(a2)) To determine whether andhow Rac1 and Cdc42 are regulated by the miR-132p250GAPpathway in our cultured hippocampal neurons we evaluatedthe activation levels of Rac1 and Cdc42 in cultured hip-pocampal neurons when miR-132 or p250GAP expressionwas inhibitedThe results showed that transfection of ant-132or LV-shp250GAP did not significantly affect the activity ofRac1 (Figures 3(b1) and 3(b2)) while the activity ofCdc42wassignificantly inhibited after ant-132 transfection and elevatedafter LV-shp250GAP transfection (Figures 3(c1) and 3(c2))Furthermore we obtained similar results in the MGF-treatedSRED model of cultured hippocampal neurons while theexpression of miR-132 or p250GAP was inhibited (Figures3(d1) 3(d2) 3(e1) and 3(e2))

Our data suggest that p250GAP may primarily functionas a GAP for Cdc42 in this epileptic model of culturedneurons

33 miR-132 Influences Neuronal Electrical Excitabilitythrough p250GAP in SRED Model of Hippocampal NeuronsTo clarify the effect of miR-132 and p250GAP on neuronalexcitability we performed electrophysiological evaluationsof the MGF medium-treated cultured hippocampal neuronsOur experiment showed that ant-132 treatment significantlydecreased the AP frequency suggesting that ant-132 cansignificantly inhibited neuronal electrical excitability in ourSRED model of cultured hippocampal neurons (Figures4(a2)ndash4(a4)) In contrast ant-132 and LV-shp250GAPcotreatment significantly increased the AP frequency(Figures 4(a5) and 4(a6))

34 Neuroprotective Effect of the miR-132p250GAP Pathwayon Cultured Neurons Silencing the overexpression of miR-132 is known to have a neuroprotective effect after acutebrain injury [8 20] therefore we asked whether miR-132regulates neuronal apoptosis via the p250GAP pathway Wefirst transected the cultured neurons with ant-132 at DIV7and then induced the SRED model at DIV10 Next wesilenced the expression levels of both miR-132 and p250GAPto investigatewhether p250GAPwas involved in the neuronalapoptosis effect of miR-132 TUNEL staining (Figures 5(a)and 5(b)) and WB detection of cleaved caspase-3 levels(Figure 5(c)) were performed 24 h later to investigate thelevel of apoptosis Our results showed that regardless ofwhether the expression level of p250GAP was suppressedmiR-132 silencing decreased neuronal apoptosisThis findingindicates that miR-132 silencing has a neuroprotective effectbut that this effect may occur through pathways other thanthe p250GAP pathway

35 miR-132 Silencing Decreases SRS in a Lithium-PilocarpineModel In Vivo We then investigated whether miR-132 inhi-bition could suppress chronic seizures in vivo using a lithium-pilocarpine model The mice were injected with ant-132 icv48 h after SE Continuous video monitoring was performedin the 6th week for 7 d The frequency of chronic seizurewas quantified and statistically analyzed by comparing theScr-132 control group (Figure 6(a)) and the ant-132 group(Figure 6(b)) Statistical analysis of our behavioral investiga-tion indicated that the ant-132 treatment can protect exper-imental mice against developing chronic recurrent seizuresand can significantly decrease spontaneous seizure frequency(Figure 6(c))

36 miR-132 Silencing Decreases the Remodeling of Spines inMice with Chronic Seizures In general mushroom-shapedand stubby spines represent more mature and stable spineswhile thin spines and filopodia tend to be more plasticand immature (Figure 7(b)) [21] Disrupted maintenanceof the dendritic tree and spinogenesis are two importantneurophysiological features of epilepsy After the behavioral

6 Neural Plasticity

Ant-132

(a)

LV-shp250GAP

(b)

lowastlowast

00

05

10

15

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)Scr-132Control Ant-132

(c)

p250GAP

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

LV-GFP LV-shp250GAPControl

120573-actin

252KD

42KD

lowastlowast

(d)

p250GAP

lowastlowast

120573-actin

252KD

42KD

0

1

2

3

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

EP+Scr-132 EP+ant-132EP(e)

Figure 2 Knockdown of miR-132 and p250GAP in cultured neurons (a) Cells were transfected with ant-132 at DIV7 and observed using afluorescence microscope 48 h after transfection Transfection efficiency was measured by RFP fluorescence (b) Cells were transfected withLV-shp250GAP at DIV7 and observed 72 h after transfection Transfection efficiency was measured by GFP fluorescence Scale bar is 50120583m(c d) Measurement of knocked down expression in transfected neurons was performed by qRT-PCR for miR-132 and by WB for p250GAP(e) Expression level of p250GAP 72 h after ant-132 treatment in cultured epileptic hippocampal neurons lowastlowast119901 lt 001 (119899 = 10 the data arerepresentative of 5 experiments)

observations were completed all the brain tissues of the ant-132-treated (EP+Ant-132) Scr-132-treated (EP+Scr-132) andnonintervention control (EP) groups were excised andGolgi-stained to further investigate the morphological effects of

ant-132 treatment on the dendritic spine (Figure 7(a)) Thetotal number and different forms of spines in the hippocam-pal dentate gyrus (DG) and CA1 regions were quantified andstatistically analyzed to investigate the differences in spine

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 3

51015840-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCA-CTGGATACGACCGACCA-31015840 (miR-132 forward primer)51015840-GCGGCGGTAACAGTCTACAGCC-31015840 and (miR-132 re-verse primer) 51015840-ATCCAGTGCAGGGTCCGAGG-31015840 Thedata were analyzed with Bio-Rad CFXManager software thedata are represented as the mean 2minusΔΔCTplusmn standard deviation(SD)

27 Western Blot (WB) Total proteins were extracted usinga whole protein extraction kit (Catalog number P0013Beyotime China) The total protein concentrations weredetermined using an Enhanced Bicinchoninic Acid Protein(BCA) Assay Kit (Catalog number P0012s Beyotime China)and the samples were stored at minus20∘C until use WB analysiswas performed as described previously [8] The primaryantibodies used were goat anti-p250GAP (1 1000 Catalognumber 138167 Santa Cruz USA) and rabbit anti-cleavedcaspase-3 (1 150 Catalog number 9661 CST USA)

28 Measurements of Rac1 and Cdc42 Activation The activa-tion of Rac1 and Cdc42 was measured using a Rac1Cdc42Activation Assay Kit (Catalog number 17-441 MilliporeUSA) according to the manufacturerrsquos protocol This assayuses the downstream effector of RacCdc42 p21-activatedprotein kinase (PAK1) to isolate the active GTP-bound formof RacCdc42 from the sample The p21-binding domain(PBD) of PAK1 is expressed as a GST fusion protein and cou-pled to agarose beads After the proteins were precipitatedan immunoblot was performed and the activated Rac1 andCdc42 were detected with specific monoclonal antibodiesfollowed by an HRP-conjugated secondary antibody

29 TdT-Mediated dUTP Nick-End Labeling (TUNEL) AssayA TUNEL assay Roche Kit (Catalog number 11684817910Roche Switzerland) was used to detect the apoptosis levelof cultured hippocampal neurons according to the manufac-turerrsquos instructions Briefly after cortical neurons were fixedin freshly prepared 4 formaldehyde solution in phosphate-buffered saline (PBS) for 20min at room temperature andpermeabilized with 02 Triton X-100 for 5min they wereincubated with 50120583L of a TUNEL reaction mixture for60min at 37∘C in the dark and then rinsed with PBS (pH74) 3 times for 5min each To detect the nuclei the slideswere incubated with DAPI for 5min at room temperature inthe dark and observed with a fluorescence microscope Theapoptotic index was expressed as the ratio of the number ofTUNEL-positive neurons to the total number of neurons

210 Temporal Lobe Epilepsy (TLE) Experimental MouseModels and Ant-132 Intervention The pilocarpine model hasbeen widely used to simulate human TLE For SE inductionall the mice were intraperitoneally (ip) injected with pilo-carpine (300mgkg 01mL10 g ip Sigma) Atropine sulfate(1mgkg ip) was administered 30min prior to the first doseof pilocarpine The mice that developed a stage 4 or 5 seizure(Racinersquos scale) [19] within 2 h after pilocarpine adminis-tration were considered kindled in our study Diazepam(10mgkg ip) was given to terminate the convulsions 1 h

after SE onset All of the mice were allowed to recover for48 h after SE and were then intracerebroventricularly (icv)injected with 2 120583L of saline (TLE group) Scr-132 (TLE+Scr-132 group) or ant-132 (TLE+ant-132 group) The dose of ant-132 was 1 nmol which was achieved by dilution in double-distilled water

211 Analysis of Spontaneous Seizures by Continuous VideoMonitoring Animals in the chronic periodwith spontaneousrecurrent seizures (SRS) associated with pilocarpine epilepsywere recorded every day for 24 h using a closed circuit videosystem to detect the class 4 and class 5 seizures at the 6thweek An observer who was blinded to the study reviewedthe videos Seizures were counted using a modified six-point Racine scale Clinical events with a score below 2 wereexcluded

212 Golgi-Cox Staining A Hito Golgi-Cox OptimStain Kit(Catalog number HTKNS1125 Hitobiotec Inc USA) wasused to visualize the dendritic spines Brain tissues obtainedfrom all the mice in the ant-132-treated and Scr-132 con-trol groups in our experiment were sectioned into 100-120583mcoronal sections on a cryostat (Leica Germany) and thestaining process was conducted following the manufacturerrsquosinstructions

213 Morphological Analysis and Density Quantification ofDendritic Spines For morphometric measurements at least15 neurons were analyzed for each data point reportedand 3 times 15 120583m of dendrites in each neuron was chosenThe IMARIS FilamentTracer module (Andor TechnologyBelfast Northern Ireland) was used to detect quantifyand characterize spine structures Filopodia were defined asdendritic protrusions with mean head width le mean neckwidth Thin spines were defined as dendritic protrusionswith two times mean neck width lt spine length and withmean neck width le maximum head width Stubby spineswere defined as dendritic protrusions with length lt 1 120583mMushroom spines were defined as dendritic protrusions withmean head width gtmean neck width To assess the reliabilityof the counting of dendritic protrusions a blind study wasinitially performed [20] Each experiment was repeated atleast three times using independent preparations

214 Data Analysis All data are expressed as the means plusmnSD Statistical analyses were performed with SPSS Statisticsfor Windows Version 200 (Armonk NY USA) Differencesbetween the experimental groups were compared using one-way analysis of variance (ANOVA) and Studentrsquos t-test wasused for comparisons 119901 lt 005 was considered statisticallysignificant

3 Results

31 Expression of miR-132 and p250GAP Is Associated withSynaptogenesis and Electrophysiological Activity It has beenreported that the expression level of mature miR-132 islow during the first week in the neonatal rat hippocampuswith a significant increase in miR-132 levels during weeks

4 Neural Plasticity

Day 1 Day 7 Day 12 Day 19 Day 25 Day 30

Day 1 Day 7 Day 13 Day 19 Day 25 Day 30

miR-132

120573-actin

0

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

p250GAP

p250GAP

252KD

42KD

(a)

120573-actin

DIV5 DIV7 DIV9 DIV11 DIV13 DIV150

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

252KD

42KD

p250GAP

miR-132p250GAP

Day 5 Day 7 Day 9 Day 11 Day 15 Day 15

(b)

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)

ControlMGF

lowast

lowast

0

1

2

3

lowastlowast

3d 5d 7d6h

(c)

120573-actin

3d 5d 7d6h00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

p250GAP

3d 5d 7d6hCC C CMGF MGF MGF MGF

252KD

42KD

lowastlowast

lowast

lowast

ControlMGF

(d)

Figure 1 (a) The expression levels of miR-132 and p250GAP in C57BL6 mice during postnatal d 1 to d 30 were evaluated using quantitativereverse transcription PCR and Western blot analyses (b) The expression levels of miR-132 and p250GAP protein during DIV5ndashDIV15 incultured hippocampal neurons (c d) Expression level of miR-132 and p250GAP 6 h to 7 d after MGF treatment as compared to controlgroup The data are presented as the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 eachdata point represents mean plusmn SD of 5 experiments)

2ndash4 which is also a critical period for the developmentand maturation of spines in rodents [15] In our studythe expression levels of miR-132 and p250GAP in C57BL6mice during postnatal d 1 to d 30 were similar to those ofprevious studies (Figure 1(a)) We evaluated the chronolog-ical expression level of miR-132 and p250GAP during the

maturation process of cultured hippocampal neurons Thelevel of miR-132 increased from DIV5 to DIV13 and wasmaintained at a relatively high level whereas the expressionof p250GAP protein was high at DIV5 but decreased atDIV15 (Figure 1(b)) This result indicated that the expres-sion of miR-132 and p250GAP might correlate with the

Neural Plasticity 5

process of physiological spine maturation and synaptogen-esis

The expression levels of miR-132 and p250GAP at 6 h 3 d5 d and 7 d afterMGF treatment were evaluated to determinewhether the levels of miR-132 and p250GAP were influencedby the electrophysiological activity of cultured neurons Theresults showed that miR-132 was upregulated 6 h to 7 dfollowingmagnesium-freemedium treatment with statisticalsignificance at 6 h 3 d and 7 d while p250GAP protein wasdownregulated from 6 h to 7 d following magnesium-freemedium treatment with statistical significance at 3 d 5 d and7 d (Figures 1(c) and 1(d))

The results suggest that miR-132 expression is increasedduring the active synaptogenesis periods of the immaturebrain Moreover the upregulation of miR-132 in the SREDmodel of hippocampal neurons suggests that the pathologicalelectrical excitability may also influence miR-132 expressionwhich may correlate with the pathological synaptogenesis ofthe CNS during epileptogenesis

32 miR-132 Regulates the Activation of Cdc42 via p250GAPin SRED Model of Hippocampal Neurons We used a specificantagonist of miR-132 and RNA interference to knock downthe expression of miR-132 and p250GAP (Figures 2(a) and2(b)) The transfection efficiency was confirmed by qRT-CPR for miR-132 (Figure 2(c)) and by WB for p250GAP(Figure 2(d)) The expression level of p250GAP has signif-icantly upregulated after ant-132 treatment which indicatedthat the expression level of p250GAPwas negatively regulatedby miR1-132 in our cultured epileptic hippocampal neurons(Figure 2(e))

Rac1 and Cdc42 are considered two important promotorsof dendritic branching and synaptic plasticity while RhoAacts in the oppositemanner [16] p250GAP has been reportedto mainly regulate the activation of Rac1 and Cdc42 in theCNS [16] The pathological plasticity of neuronal spines andsynapses is important in the pathological process of epilepsythus we studied the activation levels of Rac1 and Cdc42to explore whether Rac1 and Cdc42 are regulated by themiR-132p250GAP pathway in our SRED model of culturedhippocampal neuronsThe activation level of Rac1 andCdc42was tested

First we found that the activation levels of both Rac1and Cdc42 were significantly elevated in MGF-treated hip-pocampal neurons compared to control hippocampal neu-rons (Figures 3(a1) and 3(a2)) To determine whether andhow Rac1 and Cdc42 are regulated by the miR-132p250GAPpathway in our cultured hippocampal neurons we evaluatedthe activation levels of Rac1 and Cdc42 in cultured hip-pocampal neurons when miR-132 or p250GAP expressionwas inhibitedThe results showed that transfection of ant-132or LV-shp250GAP did not significantly affect the activity ofRac1 (Figures 3(b1) and 3(b2)) while the activity ofCdc42wassignificantly inhibited after ant-132 transfection and elevatedafter LV-shp250GAP transfection (Figures 3(c1) and 3(c2))Furthermore we obtained similar results in the MGF-treatedSRED model of cultured hippocampal neurons while theexpression of miR-132 or p250GAP was inhibited (Figures3(d1) 3(d2) 3(e1) and 3(e2))

Our data suggest that p250GAP may primarily functionas a GAP for Cdc42 in this epileptic model of culturedneurons

33 miR-132 Influences Neuronal Electrical Excitabilitythrough p250GAP in SRED Model of Hippocampal NeuronsTo clarify the effect of miR-132 and p250GAP on neuronalexcitability we performed electrophysiological evaluationsof the MGF medium-treated cultured hippocampal neuronsOur experiment showed that ant-132 treatment significantlydecreased the AP frequency suggesting that ant-132 cansignificantly inhibited neuronal electrical excitability in ourSRED model of cultured hippocampal neurons (Figures4(a2)ndash4(a4)) In contrast ant-132 and LV-shp250GAPcotreatment significantly increased the AP frequency(Figures 4(a5) and 4(a6))

34 Neuroprotective Effect of the miR-132p250GAP Pathwayon Cultured Neurons Silencing the overexpression of miR-132 is known to have a neuroprotective effect after acutebrain injury [8 20] therefore we asked whether miR-132regulates neuronal apoptosis via the p250GAP pathway Wefirst transected the cultured neurons with ant-132 at DIV7and then induced the SRED model at DIV10 Next wesilenced the expression levels of both miR-132 and p250GAPto investigatewhether p250GAPwas involved in the neuronalapoptosis effect of miR-132 TUNEL staining (Figures 5(a)and 5(b)) and WB detection of cleaved caspase-3 levels(Figure 5(c)) were performed 24 h later to investigate thelevel of apoptosis Our results showed that regardless ofwhether the expression level of p250GAP was suppressedmiR-132 silencing decreased neuronal apoptosisThis findingindicates that miR-132 silencing has a neuroprotective effectbut that this effect may occur through pathways other thanthe p250GAP pathway

35 miR-132 Silencing Decreases SRS in a Lithium-PilocarpineModel In Vivo We then investigated whether miR-132 inhi-bition could suppress chronic seizures in vivo using a lithium-pilocarpine model The mice were injected with ant-132 icv48 h after SE Continuous video monitoring was performedin the 6th week for 7 d The frequency of chronic seizurewas quantified and statistically analyzed by comparing theScr-132 control group (Figure 6(a)) and the ant-132 group(Figure 6(b)) Statistical analysis of our behavioral investiga-tion indicated that the ant-132 treatment can protect exper-imental mice against developing chronic recurrent seizuresand can significantly decrease spontaneous seizure frequency(Figure 6(c))

36 miR-132 Silencing Decreases the Remodeling of Spines inMice with Chronic Seizures In general mushroom-shapedand stubby spines represent more mature and stable spineswhile thin spines and filopodia tend to be more plasticand immature (Figure 7(b)) [21] Disrupted maintenanceof the dendritic tree and spinogenesis are two importantneurophysiological features of epilepsy After the behavioral

6 Neural Plasticity

Ant-132

(a)

LV-shp250GAP

(b)

lowastlowast

00

05

10

15

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)Scr-132Control Ant-132

(c)

p250GAP

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

LV-GFP LV-shp250GAPControl

120573-actin

252KD

42KD

lowastlowast

(d)

p250GAP

lowastlowast

120573-actin

252KD

42KD

0

1

2

3

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

EP+Scr-132 EP+ant-132EP(e)

Figure 2 Knockdown of miR-132 and p250GAP in cultured neurons (a) Cells were transfected with ant-132 at DIV7 and observed using afluorescence microscope 48 h after transfection Transfection efficiency was measured by RFP fluorescence (b) Cells were transfected withLV-shp250GAP at DIV7 and observed 72 h after transfection Transfection efficiency was measured by GFP fluorescence Scale bar is 50120583m(c d) Measurement of knocked down expression in transfected neurons was performed by qRT-PCR for miR-132 and by WB for p250GAP(e) Expression level of p250GAP 72 h after ant-132 treatment in cultured epileptic hippocampal neurons lowastlowast119901 lt 001 (119899 = 10 the data arerepresentative of 5 experiments)

observations were completed all the brain tissues of the ant-132-treated (EP+Ant-132) Scr-132-treated (EP+Scr-132) andnonintervention control (EP) groups were excised andGolgi-stained to further investigate the morphological effects of

ant-132 treatment on the dendritic spine (Figure 7(a)) Thetotal number and different forms of spines in the hippocam-pal dentate gyrus (DG) and CA1 regions were quantified andstatistically analyzed to investigate the differences in spine

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

4 Neural Plasticity

Day 1 Day 7 Day 12 Day 19 Day 25 Day 30

Day 1 Day 7 Day 13 Day 19 Day 25 Day 30

miR-132

120573-actin

0

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

p250GAP

p250GAP

252KD

42KD

(a)

120573-actin

DIV5 DIV7 DIV9 DIV11 DIV13 DIV150

2

4

6

8

10

Nor

mal

ised

miR

-132

leve

ls (fo

ld D

IV7)

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

DIV

7)

252KD

42KD

p250GAP

miR-132p250GAP

Day 5 Day 7 Day 9 Day 11 Day 15 Day 15

(b)

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)

ControlMGF

lowast

lowast

0

1

2

3

lowastlowast

3d 5d 7d6h

(c)

120573-actin

3d 5d 7d6h00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

p250GAP

3d 5d 7d6hCC C CMGF MGF MGF MGF

252KD

42KD

lowastlowast

lowast

lowast

ControlMGF

(d)

Figure 1 (a) The expression levels of miR-132 and p250GAP in C57BL6 mice during postnatal d 1 to d 30 were evaluated using quantitativereverse transcription PCR and Western blot analyses (b) The expression levels of miR-132 and p250GAP protein during DIV5ndashDIV15 incultured hippocampal neurons (c d) Expression level of miR-132 and p250GAP 6 h to 7 d after MGF treatment as compared to controlgroup The data are presented as the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 eachdata point represents mean plusmn SD of 5 experiments)

2ndash4 which is also a critical period for the developmentand maturation of spines in rodents [15] In our studythe expression levels of miR-132 and p250GAP in C57BL6mice during postnatal d 1 to d 30 were similar to those ofprevious studies (Figure 1(a)) We evaluated the chronolog-ical expression level of miR-132 and p250GAP during the

maturation process of cultured hippocampal neurons Thelevel of miR-132 increased from DIV5 to DIV13 and wasmaintained at a relatively high level whereas the expressionof p250GAP protein was high at DIV5 but decreased atDIV15 (Figure 1(b)) This result indicated that the expres-sion of miR-132 and p250GAP might correlate with the

Neural Plasticity 5

process of physiological spine maturation and synaptogen-esis

The expression levels of miR-132 and p250GAP at 6 h 3 d5 d and 7 d afterMGF treatment were evaluated to determinewhether the levels of miR-132 and p250GAP were influencedby the electrophysiological activity of cultured neurons Theresults showed that miR-132 was upregulated 6 h to 7 dfollowingmagnesium-freemedium treatment with statisticalsignificance at 6 h 3 d and 7 d while p250GAP protein wasdownregulated from 6 h to 7 d following magnesium-freemedium treatment with statistical significance at 3 d 5 d and7 d (Figures 1(c) and 1(d))

The results suggest that miR-132 expression is increasedduring the active synaptogenesis periods of the immaturebrain Moreover the upregulation of miR-132 in the SREDmodel of hippocampal neurons suggests that the pathologicalelectrical excitability may also influence miR-132 expressionwhich may correlate with the pathological synaptogenesis ofthe CNS during epileptogenesis

32 miR-132 Regulates the Activation of Cdc42 via p250GAPin SRED Model of Hippocampal Neurons We used a specificantagonist of miR-132 and RNA interference to knock downthe expression of miR-132 and p250GAP (Figures 2(a) and2(b)) The transfection efficiency was confirmed by qRT-CPR for miR-132 (Figure 2(c)) and by WB for p250GAP(Figure 2(d)) The expression level of p250GAP has signif-icantly upregulated after ant-132 treatment which indicatedthat the expression level of p250GAPwas negatively regulatedby miR1-132 in our cultured epileptic hippocampal neurons(Figure 2(e))

Rac1 and Cdc42 are considered two important promotorsof dendritic branching and synaptic plasticity while RhoAacts in the oppositemanner [16] p250GAP has been reportedto mainly regulate the activation of Rac1 and Cdc42 in theCNS [16] The pathological plasticity of neuronal spines andsynapses is important in the pathological process of epilepsythus we studied the activation levels of Rac1 and Cdc42to explore whether Rac1 and Cdc42 are regulated by themiR-132p250GAP pathway in our SRED model of culturedhippocampal neuronsThe activation level of Rac1 andCdc42was tested

First we found that the activation levels of both Rac1and Cdc42 were significantly elevated in MGF-treated hip-pocampal neurons compared to control hippocampal neu-rons (Figures 3(a1) and 3(a2)) To determine whether andhow Rac1 and Cdc42 are regulated by the miR-132p250GAPpathway in our cultured hippocampal neurons we evaluatedthe activation levels of Rac1 and Cdc42 in cultured hip-pocampal neurons when miR-132 or p250GAP expressionwas inhibitedThe results showed that transfection of ant-132or LV-shp250GAP did not significantly affect the activity ofRac1 (Figures 3(b1) and 3(b2)) while the activity ofCdc42wassignificantly inhibited after ant-132 transfection and elevatedafter LV-shp250GAP transfection (Figures 3(c1) and 3(c2))Furthermore we obtained similar results in the MGF-treatedSRED model of cultured hippocampal neurons while theexpression of miR-132 or p250GAP was inhibited (Figures3(d1) 3(d2) 3(e1) and 3(e2))

Our data suggest that p250GAP may primarily functionas a GAP for Cdc42 in this epileptic model of culturedneurons

33 miR-132 Influences Neuronal Electrical Excitabilitythrough p250GAP in SRED Model of Hippocampal NeuronsTo clarify the effect of miR-132 and p250GAP on neuronalexcitability we performed electrophysiological evaluationsof the MGF medium-treated cultured hippocampal neuronsOur experiment showed that ant-132 treatment significantlydecreased the AP frequency suggesting that ant-132 cansignificantly inhibited neuronal electrical excitability in ourSRED model of cultured hippocampal neurons (Figures4(a2)ndash4(a4)) In contrast ant-132 and LV-shp250GAPcotreatment significantly increased the AP frequency(Figures 4(a5) and 4(a6))

34 Neuroprotective Effect of the miR-132p250GAP Pathwayon Cultured Neurons Silencing the overexpression of miR-132 is known to have a neuroprotective effect after acutebrain injury [8 20] therefore we asked whether miR-132regulates neuronal apoptosis via the p250GAP pathway Wefirst transected the cultured neurons with ant-132 at DIV7and then induced the SRED model at DIV10 Next wesilenced the expression levels of both miR-132 and p250GAPto investigatewhether p250GAPwas involved in the neuronalapoptosis effect of miR-132 TUNEL staining (Figures 5(a)and 5(b)) and WB detection of cleaved caspase-3 levels(Figure 5(c)) were performed 24 h later to investigate thelevel of apoptosis Our results showed that regardless ofwhether the expression level of p250GAP was suppressedmiR-132 silencing decreased neuronal apoptosisThis findingindicates that miR-132 silencing has a neuroprotective effectbut that this effect may occur through pathways other thanthe p250GAP pathway

35 miR-132 Silencing Decreases SRS in a Lithium-PilocarpineModel In Vivo We then investigated whether miR-132 inhi-bition could suppress chronic seizures in vivo using a lithium-pilocarpine model The mice were injected with ant-132 icv48 h after SE Continuous video monitoring was performedin the 6th week for 7 d The frequency of chronic seizurewas quantified and statistically analyzed by comparing theScr-132 control group (Figure 6(a)) and the ant-132 group(Figure 6(b)) Statistical analysis of our behavioral investiga-tion indicated that the ant-132 treatment can protect exper-imental mice against developing chronic recurrent seizuresand can significantly decrease spontaneous seizure frequency(Figure 6(c))

36 miR-132 Silencing Decreases the Remodeling of Spines inMice with Chronic Seizures In general mushroom-shapedand stubby spines represent more mature and stable spineswhile thin spines and filopodia tend to be more plasticand immature (Figure 7(b)) [21] Disrupted maintenanceof the dendritic tree and spinogenesis are two importantneurophysiological features of epilepsy After the behavioral

6 Neural Plasticity

Ant-132

(a)

LV-shp250GAP

(b)

lowastlowast

00

05

10

15

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)Scr-132Control Ant-132

(c)

p250GAP

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

LV-GFP LV-shp250GAPControl

120573-actin

252KD

42KD

lowastlowast

(d)

p250GAP

lowastlowast

120573-actin

252KD

42KD

0

1

2

3

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

EP+Scr-132 EP+ant-132EP(e)

Figure 2 Knockdown of miR-132 and p250GAP in cultured neurons (a) Cells were transfected with ant-132 at DIV7 and observed using afluorescence microscope 48 h after transfection Transfection efficiency was measured by RFP fluorescence (b) Cells were transfected withLV-shp250GAP at DIV7 and observed 72 h after transfection Transfection efficiency was measured by GFP fluorescence Scale bar is 50120583m(c d) Measurement of knocked down expression in transfected neurons was performed by qRT-PCR for miR-132 and by WB for p250GAP(e) Expression level of p250GAP 72 h after ant-132 treatment in cultured epileptic hippocampal neurons lowastlowast119901 lt 001 (119899 = 10 the data arerepresentative of 5 experiments)

observations were completed all the brain tissues of the ant-132-treated (EP+Ant-132) Scr-132-treated (EP+Scr-132) andnonintervention control (EP) groups were excised andGolgi-stained to further investigate the morphological effects of

ant-132 treatment on the dendritic spine (Figure 7(a)) Thetotal number and different forms of spines in the hippocam-pal dentate gyrus (DG) and CA1 regions were quantified andstatistically analyzed to investigate the differences in spine

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 5

process of physiological spine maturation and synaptogen-esis

The expression levels of miR-132 and p250GAP at 6 h 3 d5 d and 7 d afterMGF treatment were evaluated to determinewhether the levels of miR-132 and p250GAP were influencedby the electrophysiological activity of cultured neurons Theresults showed that miR-132 was upregulated 6 h to 7 dfollowingmagnesium-freemedium treatment with statisticalsignificance at 6 h 3 d and 7 d while p250GAP protein wasdownregulated from 6 h to 7 d following magnesium-freemedium treatment with statistical significance at 3 d 5 d and7 d (Figures 1(c) and 1(d))

The results suggest that miR-132 expression is increasedduring the active synaptogenesis periods of the immaturebrain Moreover the upregulation of miR-132 in the SREDmodel of hippocampal neurons suggests that the pathologicalelectrical excitability may also influence miR-132 expressionwhich may correlate with the pathological synaptogenesis ofthe CNS during epileptogenesis

32 miR-132 Regulates the Activation of Cdc42 via p250GAPin SRED Model of Hippocampal Neurons We used a specificantagonist of miR-132 and RNA interference to knock downthe expression of miR-132 and p250GAP (Figures 2(a) and2(b)) The transfection efficiency was confirmed by qRT-CPR for miR-132 (Figure 2(c)) and by WB for p250GAP(Figure 2(d)) The expression level of p250GAP has signif-icantly upregulated after ant-132 treatment which indicatedthat the expression level of p250GAPwas negatively regulatedby miR1-132 in our cultured epileptic hippocampal neurons(Figure 2(e))

Rac1 and Cdc42 are considered two important promotorsof dendritic branching and synaptic plasticity while RhoAacts in the oppositemanner [16] p250GAP has been reportedto mainly regulate the activation of Rac1 and Cdc42 in theCNS [16] The pathological plasticity of neuronal spines andsynapses is important in the pathological process of epilepsythus we studied the activation levels of Rac1 and Cdc42to explore whether Rac1 and Cdc42 are regulated by themiR-132p250GAP pathway in our SRED model of culturedhippocampal neuronsThe activation level of Rac1 andCdc42was tested

First we found that the activation levels of both Rac1and Cdc42 were significantly elevated in MGF-treated hip-pocampal neurons compared to control hippocampal neu-rons (Figures 3(a1) and 3(a2)) To determine whether andhow Rac1 and Cdc42 are regulated by the miR-132p250GAPpathway in our cultured hippocampal neurons we evaluatedthe activation levels of Rac1 and Cdc42 in cultured hip-pocampal neurons when miR-132 or p250GAP expressionwas inhibitedThe results showed that transfection of ant-132or LV-shp250GAP did not significantly affect the activity ofRac1 (Figures 3(b1) and 3(b2)) while the activity ofCdc42wassignificantly inhibited after ant-132 transfection and elevatedafter LV-shp250GAP transfection (Figures 3(c1) and 3(c2))Furthermore we obtained similar results in the MGF-treatedSRED model of cultured hippocampal neurons while theexpression of miR-132 or p250GAP was inhibited (Figures3(d1) 3(d2) 3(e1) and 3(e2))

Our data suggest that p250GAP may primarily functionas a GAP for Cdc42 in this epileptic model of culturedneurons

33 miR-132 Influences Neuronal Electrical Excitabilitythrough p250GAP in SRED Model of Hippocampal NeuronsTo clarify the effect of miR-132 and p250GAP on neuronalexcitability we performed electrophysiological evaluationsof the MGF medium-treated cultured hippocampal neuronsOur experiment showed that ant-132 treatment significantlydecreased the AP frequency suggesting that ant-132 cansignificantly inhibited neuronal electrical excitability in ourSRED model of cultured hippocampal neurons (Figures4(a2)ndash4(a4)) In contrast ant-132 and LV-shp250GAPcotreatment significantly increased the AP frequency(Figures 4(a5) and 4(a6))

34 Neuroprotective Effect of the miR-132p250GAP Pathwayon Cultured Neurons Silencing the overexpression of miR-132 is known to have a neuroprotective effect after acutebrain injury [8 20] therefore we asked whether miR-132regulates neuronal apoptosis via the p250GAP pathway Wefirst transected the cultured neurons with ant-132 at DIV7and then induced the SRED model at DIV10 Next wesilenced the expression levels of both miR-132 and p250GAPto investigatewhether p250GAPwas involved in the neuronalapoptosis effect of miR-132 TUNEL staining (Figures 5(a)and 5(b)) and WB detection of cleaved caspase-3 levels(Figure 5(c)) were performed 24 h later to investigate thelevel of apoptosis Our results showed that regardless ofwhether the expression level of p250GAP was suppressedmiR-132 silencing decreased neuronal apoptosisThis findingindicates that miR-132 silencing has a neuroprotective effectbut that this effect may occur through pathways other thanthe p250GAP pathway

35 miR-132 Silencing Decreases SRS in a Lithium-PilocarpineModel In Vivo We then investigated whether miR-132 inhi-bition could suppress chronic seizures in vivo using a lithium-pilocarpine model The mice were injected with ant-132 icv48 h after SE Continuous video monitoring was performedin the 6th week for 7 d The frequency of chronic seizurewas quantified and statistically analyzed by comparing theScr-132 control group (Figure 6(a)) and the ant-132 group(Figure 6(b)) Statistical analysis of our behavioral investiga-tion indicated that the ant-132 treatment can protect exper-imental mice against developing chronic recurrent seizuresand can significantly decrease spontaneous seizure frequency(Figure 6(c))

36 miR-132 Silencing Decreases the Remodeling of Spines inMice with Chronic Seizures In general mushroom-shapedand stubby spines represent more mature and stable spineswhile thin spines and filopodia tend to be more plasticand immature (Figure 7(b)) [21] Disrupted maintenanceof the dendritic tree and spinogenesis are two importantneurophysiological features of epilepsy After the behavioral

6 Neural Plasticity

Ant-132

(a)

LV-shp250GAP

(b)

lowastlowast

00

05

10

15

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)Scr-132Control Ant-132

(c)

p250GAP

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

LV-GFP LV-shp250GAPControl

120573-actin

252KD

42KD

lowastlowast

(d)

p250GAP

lowastlowast

120573-actin

252KD

42KD

0

1

2

3

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

EP+Scr-132 EP+ant-132EP(e)

Figure 2 Knockdown of miR-132 and p250GAP in cultured neurons (a) Cells were transfected with ant-132 at DIV7 and observed using afluorescence microscope 48 h after transfection Transfection efficiency was measured by RFP fluorescence (b) Cells were transfected withLV-shp250GAP at DIV7 and observed 72 h after transfection Transfection efficiency was measured by GFP fluorescence Scale bar is 50120583m(c d) Measurement of knocked down expression in transfected neurons was performed by qRT-PCR for miR-132 and by WB for p250GAP(e) Expression level of p250GAP 72 h after ant-132 treatment in cultured epileptic hippocampal neurons lowastlowast119901 lt 001 (119899 = 10 the data arerepresentative of 5 experiments)

observations were completed all the brain tissues of the ant-132-treated (EP+Ant-132) Scr-132-treated (EP+Scr-132) andnonintervention control (EP) groups were excised andGolgi-stained to further investigate the morphological effects of

ant-132 treatment on the dendritic spine (Figure 7(a)) Thetotal number and different forms of spines in the hippocam-pal dentate gyrus (DG) and CA1 regions were quantified andstatistically analyzed to investigate the differences in spine

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

6 Neural Plasticity

Ant-132

(a)

LV-shp250GAP

(b)

lowastlowast

00

05

10

15

Nor

mal

ised

miR

-132

leve

ls (fo

ld co

ntro

l)Scr-132Control Ant-132

(c)

p250GAP

00

05

10

15

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

LV-GFP LV-shp250GAPControl

120573-actin

252KD

42KD

lowastlowast

(d)

p250GAP

lowastlowast

120573-actin

252KD

42KD

0

1

2

3

Nor

mal

ised

p250

GA

P le

vels

(fold

cont

rol)

EP+Scr-132 EP+ant-132EP(e)

Figure 2 Knockdown of miR-132 and p250GAP in cultured neurons (a) Cells were transfected with ant-132 at DIV7 and observed using afluorescence microscope 48 h after transfection Transfection efficiency was measured by RFP fluorescence (b) Cells were transfected withLV-shp250GAP at DIV7 and observed 72 h after transfection Transfection efficiency was measured by GFP fluorescence Scale bar is 50120583m(c d) Measurement of knocked down expression in transfected neurons was performed by qRT-PCR for miR-132 and by WB for p250GAP(e) Expression level of p250GAP 72 h after ant-132 treatment in cultured epileptic hippocampal neurons lowastlowast119901 lt 001 (119899 = 10 the data arerepresentative of 5 experiments)

observations were completed all the brain tissues of the ant-132-treated (EP+Ant-132) Scr-132-treated (EP+Scr-132) andnonintervention control (EP) groups were excised andGolgi-stained to further investigate the morphological effects of

ant-132 treatment on the dendritic spine (Figure 7(a)) Thetotal number and different forms of spines in the hippocam-pal dentate gyrus (DG) and CA1 regions were quantified andstatistically analyzed to investigate the differences in spine

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 7

(a1) (a2)

Active Cdc42

Total Cdc42

lowastlowast

lowast

50

0

100

150

200

Activ

e Cdc

42 (

of c

ont)

MGFControl MGFControl

MGFControl MGFControl

Active Rac1

Total Rac1

0

50

100

150

200

Activ

e Rac

1 (

of c

ont)

Active Rac1

Total Rac1

Control LV-shp250GAPLV-GFPScr-132 Ant-132Control

Active Rac1

Total Rac1

(b1) (b2)

(c1) (c2)

Active Cdc42

NS

NSNS

NS

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

Scr-132 Ant-132Control Control LV-shp250GAPLV-GFP

Control LV-GFP LV-shp250GAP

Control LV-GFP LV-shp250GAP

lowastlowastlowastlowast

lowastlowastlowastlowast

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

Ant-132Control Scr-132

Ant-132Control Scr-132

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

Total Cdc42

Active Cdc42

Total Cdc42

Figure 3 Continued

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

8 Neural Plasticity

MGF MGF LV-shp250GAPLV-GFPScr-132 Ant-132

Active Rac1

Total Rac1

Active Rac1

Total Rac1

0

50

100

150

Activ

e Rac

1 (

of c

ont)

0

50

100

150

Activ

e Rac

1 (

of c

ont)

LV-GFPMGF LV-shp250GAPMGF Scr-132 Ant-132

MGF Scr-132 Ant-132

MGF Scr-132 Ant-132

LV-GFPMGF LV-shp250GAP

LV-GFPMGF LV-shp250GAP

Active Cdc42

Total Cdc42

Active Cdc42

Total Cdc42

0

50

100

150

Activ

e Cdc

42 (

of c

ont)

0

50

100

150

200

Activ

e Cdc

42 (

of c

ont)

lowast

lowast

lowast

(d1) (d2)

(e1) (e2)

NS

NS

NS

NS

Figure 3 (a1-a2)The levels of activeCdc42 andRac1were significantly elevated inMGF-treated neurons compared to control neurons (b1-b2)Effect of ant-132 and LV-shp250GAP transfection on active Rac1 levels Cultured hippocampal neurons were transfected at DIV7 with ant-132and LV-shp250GAP and the level of active Rac1 was detected at DIV13 Transfection of ant-132 or LV-shp250GAP did not significantly affectRac1 activity (c1-c2) Cdc42 activationwas significantly inhibited after ant-132 transfection and elevated after LV-shp250GAP transfection (d1-d2) Effect of ant-132 and LV-shp250GAP transfection on Rac1 activation level in the MGF medium-treated hippocampal neurons Culturedhippocampal neurons were transfected at DIV7 with ant-132 and LV-shp250GAP and then treated with MGF medium at DIV10 The levelof active Rac1 was detected at DIV13 Rac1 activation level in the MGF medium-treated hippocampal neurons was not affected by ant-132and LV-shp250GAP (e1-e2)The activation level of Cdc42 in theMGFmedium-treated hippocampal neurons was significantly affected by theinhibition of miR-132 and p250GAP expression lowast119901 lt 005 lowastlowast119901 lt 001 (119899 = 5 the data are representative of 5ndash7 experiments)

density and morphology in the hippocampus of the experi-mental miceWe found that ant-132 treatment could decreasethe spine density and elevate the proportion of stable spines(Figure 7(c)) indicating that the ant-132 treatment mighthelp to suppress spine remodeling under the pathologicalcondition of epilepsy

4 Discussion

Four major findings were obtained in this study First thealtered expression of miR-132 and p250GAP in vivo andin vitro indicated that miR-132-mediated p250GAP activitymight be related to the remodeling process of spines Second

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 9

MGFminus50mv

20mv

MGF+ant-132

MGF+ant-132+LV-GFP

Control

MGF+ant -132+LV-shp250GAP

(a1) (a2)

(a3) (a4)

(a5) (a6)

NS

NS

lowastlowast

lowastlowast

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FPCon

trol

MG

F+Sc

r-132

0

5

10

15

AP

frequ

ency

(HZ)

(b)

MGFminus50mv

MGF+ant-132

Control

(a1) (a2)

(a3) (a4)MGF+Scr-132

MG

F+an

t-132+

LV-s

hp250

GA

P

Figure 4 Representative trace of AP in cultured hippocampal neurons Cultured hippocampal neurons were separately transfected at DIV7with Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP and then treated with MGFmedium at DIV10 AP frequency was tested24 h after theMGF treatment (a1) Representative recordings of non-MGFmedium-treated neurons (control) (a2) Representative recordingsof MGF medium-treated neurons (a3ndasha6) Representative recordings of MGF medium-treated neurons separately after Scr-132 ant-132 ant-132+LV-GFP and ant-132+LV-shp250GAP transfection (b) Statistical analysis of AP frequencies NS

119901 gt 005 lowast119901 lt 005 and lowastlowast119901 lt 001(119899 = 24 the data are representative of 5 experiments)

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

10 Neural Plasticity

Control MGF MGF+ant-132 MGF+ant-132+LV-GFP

MGF+ant-132+LV-shp250GAPMGF+Scr-132

(A) (D) (G) (J) (M) (P)

(B) (E) (H) (K) (N) (Q)

(C) (F) (I) (L) (O) (R)

50120583m50120583m50120583m50120583m50120583m50120583m

(a)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

lowastlowast

lowast

lowast

lowastlowast

0

10

20

30

40

Tune

l pos

itive

cells

( o

f tot

al)

(b)

MG

F

MG

F+an

t-132

MG

F+an

t-132+

LV-G

FP

Con

trol

MG

F+an

t-132+

LV-s

hp250

GA

P

MG

F+Sc

r-132

Cleaved caspase-3

120573-actin 42KD

17KD

(c)Figure 5 (a) Neuroprotective effect of ant-132 in cultured MGF-treated hippocampal neurons TUNEL assay was used to evaluate theapoptosis level Apoptotic cells in the non-MGF-treated control group (AndashC)MGFmedium-treated control group (DndashF) Neurons pretreatedwith Scr-132 (GndashI) Neurons pretreated with ant-132 (JndashL) Neurons pretreated with ant-132+LV-GFP (MndashO) Neurons pretreated with ant-132+LV-shp250GAP (PndashR) The white arrow indicates the apoptotic cell Scale bar is 50120583m (b) Histogram showing the percentage of cellswith condensed nuclei in each groupThe data are presented the mean plusmn SD values and subjected to ANOVA and Tukeyrsquos posttest lowast119901 lt 005lowastlowast

119901 lt 001 (119899 = 6 the data are representative of 5 experiments)

miR-132p250GAP regulates the activity of Cdc42 in a hip-pocampal neuronal culturemodel of acquired epilepsy whichis possibly the reason for dendritic spine remodeling duringepileptogenesis because Cdc42 is an important cytoskeletalregulator that has been reported to trigger the outgrowth of

peripheral spike-like protrusions called filopodia [22] Thirdant-132 can decrease neuronal electrical excitability in vitrothrough p250GAP and ant-132 can decrease chronic recur-rent seizure development in vivo Finally the neuroprotectiveeffect of ant-132 was once again verified in our experimental

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 11

Scr-132-1Scr-132-2Scr-132-3

Scr-132-4Scr-132-5

0

2

4

6

8N

umbe

r of s

eizu

res

6 74 52 31Recording days

(a)

ant-132-1ant-132-2ant-132-3

ant-132-4ant-132-5

3 4 52 6 71Recording days

0

2

4

6

8

Num

ber o

f sei

zure

s

(b)

Ant-132Scr-132

lowastlowast

0

1

2

3

4

5

Num

ber o

f sei

zure

s per

day

(c)Figure 6 The number of recurrent spontaneous seizures in the epileptic chronic phase (a) The number of spontaneous seizures per day foreach mouse in the Scr-132 control group (b) The number of seizures in the ant-132 group (c) The mean number of epileptic seizures per dayin the two groups lowastlowast119901 lt 001

model of cultured neurons and we revealed that this effectdoes not occur through the p250GAP pathway

miR-132 is an activity-dependent gene that is implicatedin synapse formation through regulating the translation ofits target p250GAP [2] Physiological synapse formation inthe immature brain andor a bicuculline-induced increasedin synaptic activity can change the expression levels of miR-132 and p250GAP Conversely altered expression of miR-132 and p250GAP can significantly affect dendritic spinedensity and head size [2] Epilepsy is a pathological processof synaptic plasticity and excitatory loop formation thatfollows many types of acute brain injury and that canchange neuronal activity [14 15] We proposed that miR-132-regulated p250GAP also participates in the pathologicalprocess of epilepsy Although the upregulation ofmiR-132 hasbeen observed in several epileptic models in vivo [2ndash5] nostudy has clarified the molecular mechanism of miR-132 inthe epileptic process

We have demonstrated the role of miR-132 and itstarget p250GAP in an epileptic model of cultured hip-pocampal neurons The p250GAP protein can regulate Rho

GTPase family members including RhoA Rac1 and Cdc42and mediates actin cytoskeleton organization Rac1Cdc42promote dendritic branching and growth previous studiesperformed in vivo had inconsistent results about the abilityof p250GAP to regulate the GTP-loaded state of Rac1 andCdc42 [16 22] Here we investigated the activation levelsof the dendritic growth promoting factors Rac1Cdc42 andfurther demonstrated that under the condition of aber-rant neuronal activity of epileptic discharge miR-132 andp250GAP principally regulated the activation level of Cdc42which indicated that Cdc42 might be a more appropriatetarget for further study aboutmiR-132 and epilepsy AlthoughsilencingmiR-132 or p250GAPhas no significant effect on theactivation level of Rac1 the activation level of Rac1 has alsoupregulated in our cultured epileptic hippocampal neuronsfurther studies are needed to reveal if Rac1 has taken part inthe epileptogenesis process

Several studies have shown that miR-132 may regulatea significant increase in the density of spines and length ofdendrites under the condition of aberrant neuronal activityin vivo [2 15] In our study we attempted to detect the effect

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

12 Neural Plasticity

10120583m

10120583m

EP (CA1) EP+Scr-132 (CA1) EP+ant-132 (CA1)

EP (DG) EP+Scr-132 (DG) EP+ant-132 (DG)

(a)

Filopodia (F) Thin spine (T) Stubby spine (S) Mushroom-like spine (M)

(b)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

EPEP+Scr-132EP+ant-132

EPEP+Scr-132EP+ant-132

0

5

10

15

Den

driti

c spi

nes (

CA1

)15120583

m

0

5

10

15

Den

driti

c spi

nes (

DG

)15120583

m

Filopodia TotalMushroom-likeStubby ThinFilopodia TotalMushroom-likeStubby Thin

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

NS NS NS

(c)

Figure 7 Golgi-Cox staining of hippocampal brain tissues for visualization of the dendritic spines (a) Alteration of dendritic spines in theDG and CA1 region of hippocampal neurons visualized using Golgi staining Scale bar is 10 120583mThe red arrows indicate the mushroom-likeor stubby spines black arrows indicate the filopodia or thin spines (b) Schematic representation of spine classes (c) Statistical analysis ofthe total number and different forms of spines between the EP group EP+ant-132 group and EP+Scr-132 group NS

119901 gt 005 lowast119901 lt 005 andlowastlowast

119901 lt 001

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity 13

of miR-132 on spinous remodeling during epileptogenesisthrough morphological analysis of the dendritic spines ofant-132-treated pilocarpine-induced chronic epileptic miceTreatment with ant-132 significantly increased the proportionof mushroom-shaped dendritic spines in the DG and CA1regions of the hippocampus which are two critical areasof pathological synaptic formation that leads to epilepsyBecause mushroom-shaped stubby dendritic spines are con-sidered more mature and stable while dendritic spines withother shapes such as filopodia and thin spines are immatureand more plastic [23 24] our experimental results indicatethat ant-132 could be helpful for stabilize andmaintaining theproper functioning of the dendritic spines of the hippocam-pus in the epileptogenesis process

Treatment with ant-132 can decrease SRS in vivo Ourelectrophysiological study of cultured neurons also con-firmed that ant-132 can inhibit the AP frequency of magne-sium-free medium-treated neurons and that this effect canbe reversed if p250GAP is silencedThese results suggest thatant-132 may regulate neuronal electrical excitation which isan important pathological change during epileptic formationvia the p250GAP pathway

Several studies have reported that miR-132 overex-pression can inhibit cell proliferation and promote cellu-lar apoptosis [8 21 25] however the underlying molec-ular mechanism is still not clear Our study verified thatmiR-132 upregulation promotes neuronal apoptosis in anepileptic model of cultured hippocampal neurons and thatthe neuroprotective effect of ant-132 still occurs even ifp250GAP has been inhibited thus indicating that otherpathways are involved in the neuroprotective effect ofant-132

5 Conclusion

In summary we found that the expression of miR-132and its target p250GAP are relevant to epileptogenesisSilencing miR-132 can decrease chronic recurrent seizuresmiR-132 likely functions through regulating the expressionof p250GAP the activation of downstream Cdc42 andthe maintenance of the proper form and function of thedendritic spines which may affect neural electrical activityof neurons and epileptogenesis Treatment with ant-132 hasa neuroprotective effect however this effect is not mediatedthrough the p250GAP pathway Further research is needed toreveal the detailed mechanism

Competing Interests

The authors have declared that no competing interests exist

Authorsrsquo Contributions

(1) Jinxian Yuan contributedmostly to this study (2) YangmeiChen conceived and designed the experiments (3) JinxianYuanHaoHuang Xin Zhou Xi Liu ShuOu TaoXu RuohanLi and Limin Ma performed the experiments (4) JinxianYuan analyzed the data and wrote the paper

Acknowledgments

This work was sponsored by research grants from theNational Natural Science Foundation of China (no 81171225and no 81571259 to Yangmei Chen)

References

[1] R C Friedman K K-H Farh C B Burge and D P BartelldquoMost mammalian mRNAs are conserved targets of microR-NAsrdquo Genome Research vol 19 no 1 pp 92ndash105 2009

[2] GAWaymanMDavareHAndo et al ldquoAn activity-regulatedmicroRNA controls dendritic plasticity by down-regulatingp250GAPrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 26 pp 9093ndash90982008

[3] N Vo M E Klein O Varlamova et al ldquoA cAMP-response ele-ment binding protein-induced microRNA regulates neuronalmorphogenesisrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 45 pp 16426ndash16431 2005

[4] H Kawashima T Numakawa E Kumamaru et al ldquoGlucocor-ticoid attenuates brain-derived neurotrophic factor-dependentupregulation of glutamate receptors via the suppression ofmicroRNA-132 expressionrdquo Neuroscience vol 165 no 4 pp1301ndash1311 2010

[5] K F Hansen K Sakamoto G A Wayman S Impey and KObrietan ldquoTransgenicmiR132 alters neuronal spine density andimpairs novel object recognition memoryrdquo PLoS ONE vol 5no 11 article e15497 2010

[6] H Kong F Yin F He et al ldquoThe effect of miR-132 miR-146a and miR-155 on MRP8TLR4-induced astrocyte-relatedinflammationrdquo Journal of Molecular Neuroscience vol 57 no 1pp 28ndash37 2015

[7] M A Nahid B Yao P R Dominguez-Gutierrez L KesavaluM Satoh and E K L Chan ldquoRegulation of TLR2-mediatedtolerance and cross-tolerance through irak4 modulation bymir-132 and mir-212rdquo The Journal of Immunology vol 190 no3 pp 1250ndash1263 2013

[8] YHuang J Guo QWang andY Chen ldquoMicroRNA-132 silenc-ing decreases the spontaneous recurrent seizuresrdquo InternationalJournal of Clinical and Experimental Medicine vol 7 no 7 pp1639ndash1649 2014

[9] J Peng A Omran M U Ashhab et al ldquoExpression patternsof miR-124 miR-134 miR-132 and miR-21 in an immature ratmodel and childrenwithmesial temporal lobe epilepsyrdquo Journalof Molecular Neuroscience vol 50 no 2 pp 291ndash297 2013

[10] K Wibrand D Panja A Tiron et al ldquoDifferential regulationof mature and precursor microRNA expression by NMDA andmetabotropic glutamate receptor activation during LTP in theadult dentate gyrus in vivordquo European Journal of Neurosciencevol 31 no 4 pp 636ndash645 2010

[11] J A Gorter A Iyer I White et al ldquoHippocampal subregion-specific microRNA expression during epileptogenesis in exper-imental temporal lobe epilepsyrdquoNeurobiology ofDisease vol 62pp 508ndash520 2014

[12] P Roncon M Soukupova A Binaschi et al ldquoMicroRNAprofiles in hippocampal granule cells and plasma of rats withpilocarpine-induced epilepsymdashcomparison with human epi-leptic samplesrdquo Scientific Reports vol 5 Article ID 14143 2015

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

14 Neural Plasticity

[13] D C Henshall ldquoMicroRNAs in the pathophysiology and treat-ment of status epilepticusrdquo Frontiers in Molecular Neurosciencevol 6 article 37 2013

[14] M Kusmierczak F Lajeunesse L Grand and I TimofeevldquoChanges in long-range connectivity and neuronal reorganiza-tion in partial cortical deafferentation model of epileptogene-sisrdquo Neuroscience vol 284 pp 153ndash164 2015

[15] S Impey M Davare A Lasiek et al ldquoAn activity-inducedmicroRNA controls dendritic spine formation by regulatingRac1-PAK signalingrdquo Molecular and Cellular Neuroscience vol43 no 1 pp 146ndash156 2010

[16] H Long X Zhu P Yang Q Gao Y Chen and L Ma ldquoMyo9band RICS modulate dendritic morphology of cortical neuronsrdquoCerebral Cortex vol 23 no 1 pp 71ndash79 2013

[17] C Viesselmann J Ballweg D Lumbard and E W DentldquoNucleofection and primary culture of embryonic mouse hip-pocampal and cortical neuronsrdquo Journal of Visualized Experi-ments no 47 article e2373 2011

[18] R J DeLorenzo D A Sun and L S Deshpande ldquoErratumto lsquoCellular mechanisms underlying acquired epilepsy the cal-cium hypothesis of the induction and maintenance of epilepsyrsquo[Pharmacology and Therapeutics vol 105 no 3 2005 pp 229ndash266]rdquo Pharmacology and Therapeutics vol 111 no 1 pp 288ndash325 2006

[19] R J Racine ldquoModification of seizure activity by electricalstimulation II Motor seizurerdquo Electroencephalography andClinical Neurophysiology vol 32 no 3 pp 281ndash294 1972

[20] I Gonzalez-Burgos ldquoDentritic spine plasticity and learningmemory processes theory evidence and perspectivesrdquo in Den-tritic Spines BiochemistryModeling and Properties L R BaylogEd pp 163ndash186 Nova Science New York NY USA 2009

[21] E M Jimenez-Mateos I Bray A Sanz-Rodriguez et alldquoMiRNA expression profile after status epilepticus and hip-pocampal neuroprotection by targeting miR-132rdquo AmericanJournal of Pathology vol 179 no 5 pp 2519ndash2532 2011

[22] T Nakazawa A M Watabe T Tezuka et al ldquop250GAP anovel brain-enriched GTPase-activating protein for Rho familyGTPases is involved in the N-methyl-D-aspartate receptorsignalingrdquoMolecular Biology of the Cell vol 14 no 7 pp 2921ndash2934 2003

[23] J C Fiala J Spacek and K M Harris ldquoDendritic spine pathol-ogy cause or consequence of neurological disordersrdquo BrainResearch Reviews vol 39 no 1 pp 29ndash54 2002

[24] M Wong and D Guo ldquoDendritic spine pathology in epilepsycause or consequencerdquoNeuroscience vol 251 pp 141ndash150 2013

[25] A Mehta M Mann J L Zhao et al ldquoThe microRNA-212132cluster regulates B cell development by targeting Sox4rdquo Journalof Experimental Medicine vol 212 no 10 pp 1679ndash1692 2015

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neural Plasticity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Psychiatry Journal

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014