Critical role of large-conductance calcium- and voltage-activated potassium channels in leptin-induced neuroprotection of N-methyl-d-aspartate-exposed cortical neurons

ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / lo c a t e / y p h r s

Critical

role

of

large-conductance

calcium-

and

voltage-activated

potassium

channels

in

leptin-induced

neuroprotection

of

N-methyl-d-aspartate-exposed

cortical

neurons

Maria

Mancini

_,

_Maria

_Virginia

_Soldovieri

_,

_Guido

_Gessner

_,

_Bianka

_Wissuwa

_,

Vincenzo

Barrese

_,

_Francesca

_Boscia

_,

_Agnese

_Secondo

_,

_Francesco

_Miceli

_,

Cristina

Franco

_,

_Paolo

_Ambrosino

_,

_Lorella

_Maria

_Teresa

_Canzoniero

_,

_Michael

_Bauer

_,

Toshinori

Hoshi

_,

_Stefan

_H.

_Heinemann

_,

_Maurizio

_Taglialatela

a_{DepartmentofMedicineandHealthScience,UniversityofMolise,Campobasso,Italy}

b_{DepartmentofScienceandTechnology,UniversityofSannio,Benevento,Italy}

c_{CenterforMolecularBiomedicine,DepartmentofBiophysics,FriedrichSchillerUniversityandJenaUniversityHospital,Jena,Germany}

d_{CenterforSepsisControlandCare,JenaUniversityHospital,Jena,Germany}

e_{DepartmentofNeuroscience,UniversityofNaples“FedericoII”,Naples,Italy}

f_{DepartmentofPhysiology,UniversityofPennsylvania,Philadelphia,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21May2014

Receivedinrevisedform17June2014

Accepted18June2014

Availableonline26June2014

Keywords:

Calcium-andvoltage-activatedpotassium

channels(BKchannels) Leptin N-methyl-d-aspartate(NMDA) Neuroprotection Paxilline Iberiotoxin Corticalneurons IntracellularCa2+_{concentration}

a

b

s

t

r

a

c

t

Inthepresentstudy,theneuroprotectiveeffectsoftheadipokineleptin,andthemolecularmechanism involved,havebeenstudiedinratandmicecorticalneuronsexposedtoN-methyl-d-aspartate(NMDA) invitro.Inratcorticalneurons,leptinelicitedneuroprotectiveeffectsagainstNMDA-inducedcelldeath, whichwereconcentration-dependent(10–100ng/ml)andlargestwhentheadipokinewaspreincubated for2hbeforetheneurotoxicstimulus.Inbothratandmousecorticalneurons,leptin-induced neuropro-tectionwasfullyantagonizedbypaxilline(Pax,0.01–1␮M)andiberiotoxin(Ibtx,1–100nM),withEC50sof 38±10nMand5±2nMforPaxandIbtx,respectively,closetothosereportedforPax-andIbtx-induced Ca2+_{-andvoltage-activatedK}+_{channels(Slo1BKchannels)blockade;theBKchannelopenerNS1619} (1–30␮M)inducedaconcentration-dependentprotectionagainstNMDA-inducedexcitotoxicity. More-over,corticalneuronsfrommicelackingoneorbothallelescodingforSlo1BKchannelpore-forming subunitswereinsensitivetoleptin-inducedneuroprotection.Finally,leptinexposuredose-dependently (10–100ng/ml)increasedintracellularCa2+_{levelsinratcorticalneurons.Inconclusion,ourresults} sug-gestthatSlo1BKchannelactivationfollowingincreasesinintracellularCa2+_{levelsisacriticalstepfor} leptin-inducedneuroprotectioninNMDA-exposedcorticalneuronsinvitro,thushighlighting leptin-basedinterventionviaBKchannelactivationasapotentialstrategytocounteractneurodegenerative diseases.

©2014ElsevierLtd.Allrightsreserved.

Abbreviations: NMDA,N-methyl-d-aspartate; BK channels,large-conductanceCa2+_{- andvoltage-activatedK}+ _{channels;Pax,paxilline; Ibtx, iberiotoxin; OGD,}

oxygen–glucosedeprivation;KATP, ATP-sensitiveK+ channels;E,embryonicage;HBSS,Hank’sBalanced SaltSolution;MEM,MinimumEssentialMedia;FBS,fetal

bovineserum;HS,horseserum;DIV,daysinvitro;HCSS,HEPEScontrolsaltsolution;MS,mediastock;HEK293Tcells,humanembryonickidney293Tcells;MTT,

3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazoliumbromide;DMEM,Dulbecco’smodifiedEagle’smedium;Fura2-AM,

1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(21-amino-51-methylphenoxy)-ethane-N,N,N1,N1-tetraaceticacidpenta-acetoxymethylester;EC50,effectiveconcentration50;[Ca2+]i,intracellularcalcium

concentration;PI3K,phosphatidylinositol3-kinase.

∗ Correspondingauthorat:DepartmentofMedicineandHealthSciences,UniversityofMolise,ViaDeSanctis,86100Campobasso,Italy.Tel.:+390874404851;

fax:+390874404778.

E-mailaddresses:m.taglialatela@unimol.it,mtaglial@unina.it(M.Taglialatela).

http://dx.doi.org/10.1016/j.phrs.2014.06.010

Introduction

Leptin, a 16-kDa peptide hormone produced by white adipocytes,controlsappetitivebehaviorsbyactingon hypothal-amic neurons involved in food intake and energy expenditure [1].Leptinreceptorsareexpressedindiversebrainregionssuch asthehippocampus,thecortex,andthecerebellum,beingoften locatedataxonalandsynapticsubcellularsites[2]. Extrahypotha-lamic actions of leptin are increasingly being recognized; for example,leptininfluencessynapticplasticityinhippocampal neu-rons[3,4]and inhibitsepileptiform-likeactivityinhippocampal [5–7]aswellasinneocorticalneurons[8].Severalstudieshave alsohighlighted theability of leptin toexertboth invitro and invivoneuroprotectiveeffectsagainstoxygen–glucosedeprivation, hypoxia,ischemia,neurotrophicfactorwithdrawal,andexcitotoxic oroxidativestimuli inneuronalpopulationsfromdistinctbrain areas[9–15].

Amongthemolecularmechanismsresponsiblefortheeffectsof leptin,neuronalsilencingviaactivationofpotassium(K+_)channels appearstoplayamajorrole.Amongleptin-sensitiveK+_channels, activationofATP-sensitiveK+_channels(K

ATP)hasbeenproposedto mediateleptin-inducedsuppressionofexcitabilityofhypothalamic neurons[16,17].Similarly,leptin-inducedinsulinrelease suppres-sionfrompancreatic␤cellsalsodependsonKATPactivation[18]. Morerecently,pharmacologicalevidencehassuggestedthat large-conductance, Ca2+_{- and voltage-activatedK}+ _{channels (Slo1 BK} channels), which are particularlyabundantin axons and nerve terminals[19,20],wheretheystabilizetheneuronal membrane potentialandregulateexcitatoryneurotransmitterrelease[21–26], mediateatleastpartofleptin’seffectsofneuronalexcitability.In fact,activationofBKchannelsmediatesleptin-inducedinhibition ofgastricmucosalvagalafferents[27],andhippocampalneuronal firing[5]andepileptiform-likeevents[6].Notably,BKchannel acti-vationexertsstrongneuroprotectiveeffectsinanimalmodelsof cerebralischemia[28],andattenuatedcerebraledemaand neuro-logicmotorimpairmentaftertraumaticbraininjury[29].Activation ofBKchannelsalsoseemstomediateleptineffectsonprimary hippocampalneuronalexcitabilityduringhypoxia[15].

Despite theseresults, directevidence forBKchannel activa-tionbythisadipokineduringneuroprotectionislacking.Therefore, inthepresentstudy,theneuroprotectivepotentialofBKchannel activationbyleptinandtheunderlyingmolecularmechanism(s) havebeenassessedincorticalneuronsexposedtotheionotropic glutamatereceptoragonistN-methyl-d-aspartate(NMDA),a clas-sicalexcitotoxicinsult.Theresultsobtainedindicatethat leptin is endowed withsignificantneuroprotective effects in both rat andmousecorticalneuronsexposedtoNMDA;the pharmacolog-icalblockadeofBKchannels,orthelackofone(Slo1+/−_mice)or both(Slo1−/−mice)Slo1allelesfullycounteractedleptin-mediated neuroprotection. Furthermore, intracellular Ca2+ _{concentration} ([Ca2+_]

i)monitoringinsinglemousecorticalneuronsrevealedthat leptin(10–100ng/ml)applicationpromptedanoscillatory behav-iorin[Ca2+_]

i.TheseresultsrevealthattheactivationofBKchannels is an obligatory step for leptin-induced neuroprotection, high-lightingleptin-basedinterventionviaBKchannelactivationasa potentialstrategytotreatneurodegenerativediseases.

Materialsandmethods

Animals

Animalswerekeptunderstandardconditionsoftemperature, humidityand light, and weresupplied withstandard foodand wateradlibitum.Animalswerehandledin accordancewiththe recommendationsof theNational Institutesof Health Guidefor

theCareandUseofLaboratoryAnimalsandinaccordancewitha protocolapprovedbytheinstitutionalanimalcarecommittees.All effortsweremadetominimizeanimalsufferingandtoreducethe numberofanimalsused.

PregnantWistarratswerepurchasedfromacommercialsource (CharlesRiver,Calco,Italy),whilewild-type,heterozygousandSlo1 knockoutFVB/NJmicewereobtainedfromProf.R.Aldrich (Univer-sityofTexas,Austin,TX,USA),andgenotypedaspreviousdescribed [30].Briefly,genomicDNAfromtailsnipswasamplifiedbyPCR usingprimersNeo5(5-ATAGCCTGAAGAACGAGATCAGC-3) andRA140253(5-CCTCAAGAAGGGGACTCTAAAC-3), amplify-ingtheSlo1−/−alleleproductof800bp,andtheexon15–3(5-TTC ATCATCTTGCTCTGGCGGACG-3)andWT3–2(5-CCATAGTCA CCAATAGCCC-3)amplifyingthewild-typeproductof332bp. Ratandmousecorticalcellcultures

Primaryculturesofratandmousecorticalneuronswere pre-paredfromembryosat15–17daysofgestation;embryonicage(E) wascalculatedbyconsideringE0.5thedaywhenavaginalplugwas detected.Briefly,pregnantanimalswereanesthetizedwithdiethyl ether(CarlRothGmbH&CoKG,Karlsruhe,Germany)and sacri-ficedbycervicaldislocation.Corticaltissuesfromembryoswere dissectedinice-coldmedium(HBSS,Hank’sBalancedSaltSolution, supplementedwith27mMglucose,20mMsucrose,4mMsodium bicarbonate),centrifuged,andtheresultingpelletwas mechani-callydissociatedwithaglasspipette.Cellswereresuspendedin platingmediumconsistingofEagle’sMEM(MEM,Earle’ssalts, sup-pliedbicarbonate-free)supplementedwith5%fetalbovineserum (FBS,BiochromAG,Berlin,Germany),5%horseserum(HS,Sigma Aldrich,Taufkirchen, Germany), 2mMl-glutamine, 20mM glu-cose,26mMbicarbonate,andplatedon24-wellplates(Thermo FisherScientific, Waltham, MA,USA) oron18mmglass cover-slips(GlaswarenfabrikKarlHechtKG,Sondheim,Germany)coated with 100_␮g/ml poly(d)-lysine (Sigma–Aldrich) at a density of four embryo cerebral hemispheres/10ml; when single-embryo dissectionswerenecessary,resuspensionvolumeswerechanged accordingly. Glial replicationwasinhibitedby24hexposureto 10␮Mcytosinearabinofuranoside(Sigma–Aldrich)after4daysin vitro(DIV).Afterthistreatment,themediumwassupplemented with10%HSandpartiallysubstitutedtwiceaweek.Allthe exper-imentswere performedat 12–16 DIV.HBSS, Eagle’sMEM, and glutaminewerepurchasedfromLifeTechnologies(Oslo,Norway). AllotherreagentswerefromSigma–Aldrich.

Cellulartreatmentsandassessmentofneuronalsurvival

Prior todrug exposure, corticalneurons werewashed thor-oughly to remove serum using HEPES control salt solution (HCSS,120mMNaCl,5.4mMKCl,0.8mMMgCl2,20mMHEPES, 15mM glucose, 1.8mM CaCl2, 10mM NaOH, pH 7.4). NMDA (Sigma–Aldrich)exposurewascarriedoutinHCSSfor15minat roomtemperature,followedbyNMDAwashoutinmediastock(MS, MEM supplemented with20mM glucose and 26mM bicarbon-ate).Recombinanthumanleptin(R&DSystemsInc.,Minneapolis, MN,USA;stocksolutionwas1mg/mldissolvedin20mMTris–HCl, pH8.0)wasusedformouseandratcorticalneurons,assimilar affinitieshavebeenmeasuredforhumanleptinathuman,mouse, andratleptinreceptors[31].Leptinwasaddedsimultaneouslyto NMDAexposure,aswellas15min,2h,or6hbeforeNMDA expo-sure,accordingtotheexperimentalprotocol.Paxilline,iberiotoxin, orNS1619wereaddedatthedesiredconcentrationsfromstock solutionsinDMSO(maximalfinalDMSOconcentrationwas<1%) 15minbeforeleptinapplicationandkeptthroughoutthe experi-ment.AfterNMDAexposure,cultureswerewashedseveraltimes withHCSSbufferandmaintainedinMSfor24h.

Neuronalmitochondrialfunctionwasassessedbythe 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay [32], a colorimetric assay which evaluates the ability of metabolicallyactivecellstocleaveatetrazoliumsaltsuchasMTT intoacoloredformazanproduct;tothisaim,neuronswere incu-batedinthedarkat37◦Cin a5%CO2 atmospherefor 1hwith 0.5mg/mlMTT (Sigma–Aldrich)and solubilizedwithacidic iso-propanol.Theabsorbancewasmeasuredat570nm(background subtractionat650nm).Datawereexpressedaspercentageofthe absorbancemeasuredinuntreatedcells.Neuronalcellviabilitywas evaluatedbypropidiumiodide(PI)fluorescence[33].PIisavery stableandhighlypolarcompoundthatonlyenterscellswith dam-agedorleakyplasmamembranes,bindstoDNA,andemitsabright redfluorescencewhenexposedtoblue-greenlight.Attheendofthe experiment,cellswereincubatedwith5␮g/mlPIfor1h.PIuptake wasrecordedbyadigitalcamera(MediaCybernetics,SilverSprings, MD,USA) mountedonaNikon Eclipse400fluorescence micro-scope(NikonInstruments,Florence,Italy;excitation510–560nm, emission590nm).Threephasecontrastdigitalimagesofrandomly selectedmicroscopicfieldswereusedfordataanalysisusingImageJ software(NIH,Bethesda,MD,USA).

Ca2+_imaging

For Ca2+_{-imaging experiments, the cells grown on glass} coverslips were loaded for 20–30min with 5␮M 1-[2-(5- carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(21-amino-51-methylphenoxy)-ethane-N,N,N1,N1-tetraacetic acid penta-acetoxymethyl ester (Fura-2 AM; Life Technologies) in control buffermedium containing(inmM):140NaCl,3 KCl,1CaCl2,1 MgCl2,10 HEPES,10 glucose,pH7.4withNaOH.Attheend of theFura-2AM-loadingperiod,thecoverslipwasintroducedinto a microscope chamber onthe stageof an inverted microscope (Axiovert 200, CarlZeiss, Jena, Germany) equippedwitha 20× or 40× objectives. Cells were washed twice with the control buffermedium,andmaintainedinthesamesolutionthroughout the experiment. Fluorescence measurements were performed at room temperature by means of a digital imaging system composedof MicroMax 512BFT cooled CCD camera (Princeton Instruments,Trenton,NJ,USA),LAMBDA10-2filterwheel(Sutter Instruments,Novato,CA,USA)connected toaxenonlamp, and Meta-Morph/MetaFluor Imaging System software (Universal Imaging,WestChester,PA,USA).Pairsofimages(excitationat340 and380nm)ofFura-2fluorescenceintensityweremeasuredevery 1s;backgroundfluorescencewassubtractedineachexperiment. Fluorescencevalueswereconvertedto[Ca2+_]

iusingacalibration curve,asdescribed[34].

Statisticalanalysis

Alldataareshownasmean±S.E.M.Unlessstatedotherwise, comparisonbetweengroupswasmadeusingtheStudent’spaired orunpairedt-test,asnecessary.Theanalysisofone-wayvariance (ANOVA)followedbytheNewman–Keulstestwasperformedto assesssignificanceamongmultipleexperimentalgroups.APvalue <0.05wasconsideredasstatisticallysignificant.

Results

LeptinprotectsratcorticalneuronsagainstNMDA-induced neurotoxicity

Ratprimarycorticalneuronsexposedfor15mintoincreasing concentrationsofNMDA(30–300_␮M;plus10_␮Mglycine)showed, overthenext24h,aconcentration-dependentreductionin mito-chondrialfunction,asmeasuredbytheMTTassay(Fig.1A).This

Fig.1.LeptinreducesNMDA-inducedneuronaldeathinratcorticalneuronsin

vitro.(A)Mitochondrialfunction,measuredasMTTabsorbance,wasassessed24h

aftera15-minexposuretotheindicatedconcentrationsofNMDAintheabsenceor

presenceoftheNMDA-antagonistMK801.(B)Timecourse(15–360min)and

con-centrationdependence(10,30,100ng/ml)ofleptineffectonneurotoxicity,induced

by100␮MNMDA.Dataareexpressedaspercentageofleptin-induced

neuroprotec-tionrelativetocontrols(100␮MNMDA,noleptintreatment).(C)Neuronaldeath

wasquantifiedbymeasuringtheuptakeofpropidiumiodide(PI)in12DIVneuronal

culturesafter24hexposuretothefollowingexperimentalconditions:control,

lep-tin(2hpreincubationat30ng/ml),NMDA(15minat100␮M),leptin(30ng/ml,

2hpreincubation)followedbyNMDA(15minat100␮M).PIfluorescencedataare

expressedaspercentofPI-positivecellsofthetotalcellpopulation(countedinthe

correspondingbrightfieldimages)ineachmicroscopicfield.Eachdatapointisthe

mean±S.E.M.ofatleastthreeseparateexperiments.Theasteriskdenotes

statisti-caldifferencewithP<0.05.(Forinterpretationofthereferencestocolorintext,the

readerisreferredtothewebversionofthearticle.)

effect wasentirely prevented upon co-exposure withthe non-competitiveNMDAreceptorantagonistMK-801(10␮M)(P=0.78 versuscontrol).Exposureto100␮MNMDAresultedin approxi-mately40%reductionin mitochondrialMTT-reducingpotential; thisconcentrationwasusedinallsubsequentexperiments.

Leptin(10–100ng/ml),whenappliedonlyduringthe15minof NMDAexposure,didnotsignificantlyenhancecellsurvival(Fig.1B, whitebars).Bycontrast,whenleptinexposurewasstartedbefore theNMDAinsultandkeptthroughout,significantneuroprotective effectsoccurred;inparticular,leptinpre-treatmentfor2hbefore NMDAapplicationelicitedrobust(about60%)anddose-dependent neuroprotection(Fig.1B,redbars).Shorter(15min,bluebars)or longer(6h,greenbars)pretreatmentdurationscausedmuchless intenseneuroprotectiveeffects(Fig.1B).Inneuronsnotexposedto

NMDA,leptinhadnosignificanteffectonMTTreduction(datanot shown).

Since 30ng/ml was the lowest leptin concetration exerting maximalcytoprotectiveeffects whenpre-incubated for2h, this adipokineconcentrationandthispreincubationtimewerechosen toassessleptin-inducedneuronalprotectionbyanindependent method,namelyPIincorporation.AsshowninFig.1C,the expo-sure of cortical neurons for 15minto 100␮M NMDA resulted inabout40%ofPI-positiveneuronsafter24h;2hofleptin pre-treatmentsignificantly reduced NMDA-induced neuronal death (P<0.05versusNMDAalone).AspreviouslydescribedfortheMTT assay,leptinfailedtomodifyPIincorporationintheabsenceofthe neurotoxicstimulus(Fig.1C).

ThepharmacologicalmodulationofBKchannelsinterfereswith leptin-inducedneuroprotection

ToinvestigatetheroleofBKchannelsinleptin-induced neu-roprotection during NMDA exposure, the effect of BK channel modulatorsinthisinvitromodelweretested.BothnaturalBK chan-nelblockerspaxilline(Pax; 0.01–1␮M;Fig.2A)and iberiotoxin (Ibtx;1–100nM;Fig.2B)reversedleptin-inducedneuroprotection inadose-dependentmanner;theEC50valueswere38±10nMand 5±2nM,respectively,forPaxandIbtx.Thesevaluesarecloseto thereportedEC50 valuesfor Pax- and Ibtx-inducedBKchannel blockade(about10nMforboth;[35]).Aconcentrationof100nM ofeitherdrugcompletelypreventedleptin-induced neuroprotec-tiveeffectsincorticalneuronsexposedto100␮MNMDA(P=0.25 for Pax; P=0.06 for Ibtx). These results strongly indicated that theactivationofBKchannelsmediatesleptin-induced neuropro-tectiveeffects againstNMDA-induced deathofcorticalneurons invitro.Consistentwiththis,pre-treatmentofthecells(2h)with theBKchannelopenerNS1619(1–30␮M)induceda concentration-dependentprotectionagainstNMDA-inducedexcitotoxicity,with 30_␮Mbeingtheconcentrationassociatedwiththehighestlevelof neuroprotection(Fig.2C);higherNS1619concentrationscouldnot beused,astheyprovedtobeneurotoxic(datanotshown). LeptinfailstoexertneuroprotectiveeffectsagainstNMDA-induced excitotoxicityinSlo1+/−andSlo1−/−mouseneuronalcultures

To provide furtherevidence for the role of BK channels in leptin-mediatedneuroprotection,theeffectsofthisadipokinewere studiedinprimarycorticalneuronsderivedfromBL6wild-type mice (Slo1+/+_{) and mice heterozygous (Slo1}+/−_{) or homozygous} (Slo1−/−)foradeletionoftheSlo1gene,encodingthepore-forming ␣subunitofBKchannels[30].Single-brainculturesfrommouse embryoswereexposedtoNMDAin thepresence orabsenceof leptin.NMDA(100␮M)exposuresimilarlyimpairedmitochondrial functionincorticalneuronsfromSlo1+/+_,Slo1+/−_,andSlo1−/−_mice (P=0.12;Fig.3).InNMDA-exposedcorticalculturesfromwild-type mice,leptin(30ng/ml;2hpreincubation)elicitedneuroprotective effectswhoseextent(about60%)wasidenticaltothatpreviously describedinratcorticalneurons(seeFig.1).Inaddition,as pre-viouslyshownforratneurons,leptin-inducedneuroprotectionin wild-typemousecorticalneuronswasfullyantagonized byPax (1␮M)orIbtx(100nM).Bycontrast,leptin(30ng/ml;2h preincu-bation)failedtodisplayanysignificantneuroprotectiveeffectsin corticalneuronsfromSlo1+/−_{(P=0.19versuscontrol,NMDA-only} exposedneurons)orSlo1−/−mice(P=0.70versuscontrol, NMDA-onlyexposedneurons).

Leptin-inducedincreasein[Ca2+_]

iinmousecorticalneurons

Toinvestigatethemechanism(s)bywhichleptinactivatedBK currents,[Ca2+_]

iweremonitoredinleptin-exposedmousecortical

Fig.2. EffectofBKchannelmodulatorsonleptin-inducedneuroprotectioninrat

corticalneurons.(AandB)Concentration-dependenteffectoftheBKblockers

pax-illine(A,Pax)andiberiotoxin(B,Ibtx)onleptin-inducedneuroprotectionof100␮М

NMDA-inducedneuronaldeath.(C)Concentration-dependenteffectoftheBK

chan-nelactivatorNS1619onNMDA-inducedneuronaldeath.Ineachpanel,thebarson

theleftindicatethedrugexposureprotocol.Neuronalviabilitywasmeasured24h

aftertheinsultwiththeMTTassay.Dataareexpressedaspercentageofneuronal

viabilityinuntreated,controlneurons.Eachdatapointisthemean±S.E.M.offive

separateexperiments,eachperformedintriplicate.Asterisksdenotevalues

statisti-callydifferentfromrespectivecontrols(NMDAalone;P<0.05).(Forinterpretation

ofthereferencestocolorintext,thereaderisreferredtothewebversionofthe

article.)

neurons.AsshowninFig.4A,leptin(30ng/ml)inducedtransient increasesin[Ca2+_]

iinabout70%ofcells,triggeringanoscillatory pattern.Noeffecton[Ca2+_]

iwasobserveduponvehicleperfusion. In most cells, [Ca2+_]

i oscillations appeared soon after leptin application,althoughinafewcellstransientincreasesin[Ca2+_]

i werespreadovertheentiredurationofleptinexposure(8min). Fig.4Bshowsasummaryofthedose-dependenteffectsofleptin (10–100ng/mL)on[Ca2+_]

i.

Discussion

Theadipokineleptinhasafirmlyestablishedroleinpromoting appetitesuppressionandenergyexpenditure.Inadditionto appeti-tivebehaviors,leptinisalsoknowntoexertneuroprotectiveeffects

Fig.3.Leptin-mediatedneuroprotectionfromNMDA-inducedinjuryisdependentonSlo1geneexpression.Viabilityofcorticalneuronsisolatedfromwild-type(Slo1+/+_;

red),heterozygous(Slo1+/−_{;blue)orhomozygous(Slo1}−/−_{;lightblue)Slo1knockoutmouseembryos;neuronswerepre-incubatedwith30ng/mlleptinfor2hbefore}

15-mintreatmentwith100␮MNMDA.NeuronalviabilitywasmeasuredwiththeMTTassay24haftertheinsult;neuronalviabilityisexpressedaspercentofthecontrols

(correspondingtountreatedneuronsfromeachexperimentalgroup).TheBKchannelblockerspaxilline(Pax,1␮M)andiberiotoxin(Ibtx,100nM)wereappliedtowild-type

neuronsasdescribedinFig.2.Dataaremean±S.E.M.ofthreeindependentexperiments,eachperformedintriplicate.Theasteriskdenotesavaluestatisticallydifferentfrom

therespectivecontrol(NMDAalone;P<0.05).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

inseveralinvitroandinvivomodelsofneurotoxicity[13].Inthe presentstudywedemonstratedthatleptinexertssignificant neu-roprotectioninaninvitromodelofratandmousecorticalneurons excitotoxicitytriggeredbyexposuretoNMDA.Leptin-induced neu-roprotectionappearedtobestronglydependentonthe concentra-tionandthetimingofexposure:significantneuroprotectiveactions wereonly observedwhentheadipokinewasapplied2hbefore

Fig.4.Effectofleptinon[Ca2+_]

iincorticalneurons.(A)Vehicle-(blacktrace)or

leptin(30ng/ml;bluetrace)-inducedchangesinFura-2measured[Ca2+_]

iin12

DIVmousecorticalneurons.Thebarontopofthegraphcorrespondstothe

dura-tionofvehicleorleptinexposure.(B)Concentrationdependenceofleptin-induced

enhancementofpeak[Ca2+_]

i.Eachbaristhemean±S.E.M.ofthedatafrom65cells

recordedinthreedifferentexperimentalsessions.Theincreaseof[Ca2+_]

iwas

cal-culatedas%ofincreaseofmeanpeakamplitudeoverbasallevel.*P<0.05versus

vehicle-exposedneurons;**P<0.05versusvehicle-exposedneuronsandversus

10ng/mlleptin-exposedneurons.(Forinterpretationofthereferencestocolorin

thisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

thetoxicinsult,whileleptinwaslargelyineffectiveaftershorteror longerincubationtimes.Bycontrast,leptinachievedsimilar neuro-protectiveeffectsincorticalneuronsexposedinvitrotooxygenand glucosedeprivation(OGD)whenapplied15minbeforeor180min aftertheneurotoxicstimulus[14],suggestingthatthe neuropro-tectivemechanismstriggeredbyleptinduring OGDandNMDA displayeda differentialtime dependence.Interestingly,Pax and Ibtx,twowell-knownBKchannelinhibitors,completely counter-actedleptin-inducedneuroprotection;bothdrugsdisplayedEC50s inthelownanomolarconcentrationrange,consistentwiththeir BKchannelblockingactions[35],andsuggestingthatBKchannel openingisacrucialmechanismforleptin-inducedneuroprotection duringNMDAexposure.Consistentwiththis,theprototypicalBK channelopenerNS1619,similarlytoleptin,alsopromoted neuro-protectiveeffects;however,theselectivityofthiscompoundforBK channelshasbeenquestioned[36],andadditionalmolecular mech-anismssuchastheinhibitionofvoltage-dependentCa2+_channels

[37],voltage-dependentK+_{channels,andK}

ATPchannels[38]have beencalledintoplaytoexplainNS1619-inducedneuroprotection. BMS-204352,anopenerofBKandKv7potassiumchannels[39–41], alsoshowedstrongneuroprotectiveeffects inanimalmodelsof cerebralischemia[28],andattenuatedcerebraledemaand neuro-logicmotorimpairmentaftertraumaticbraininjury[29],although itfailedtoshowsuperiorefficacycomparedtoplaceboinacute strokepatients[42].Inkeepingwiththeneuroprotectiveroleof BKchannels,BKchannelblockersaggravatedneuronaldamagein ischemichippocampalorganotypiccultures[43].

BKchannels are tetramers of pore-forming Slo1␣ subunits encodedbytheKcnma1gene.Slo1subunitscanassemblewith dis-tinctauxiliary␤subunits(␤1–␤4),inatissue-specificmanner;such anassociationsignificantlymodifiesthebiophysicaland pharma-cologicalpropertiesofBKchannels[44–46].Tofurthersubstantiate theinvolvement of BKchannels in leptin-mediated effects, we investigatedtheneuroprotectiveactionsofthisadipokinein neu-rons from mice carrying a deletion of exon 1 of the Kcnma1 locus[30].Theresultsobtainedshowedthatleptinaffordedstrong neuroprotectiononlyincorticalneuronsfromwild-typeanimals, whereashomozygousknockoutmicefortheSlo1genewerefully resistanttoleptineffects;corticalneuronsfromheterozygousSlo1 knockoutmicewerealsoinsensitivetoleptin.Thesedataconfirm thatBKchannelsarecrucialmediatorsofleptin-induced neuro-protection; moreover,the fact that even a partial reduction in functionalBKchannels(suchasthatpredictedtooccurinSlo1+/− heterozygousanimals)ablatesleptineffects,suggeststhatatight

controlofBKchanneldensityisrequiredforneuronalprotection bythisadipokine.Noteworthy,intheseexperiments,despitethe dramaticdifferenceinleptinsensitivity,NMDAtreatmentfailedto inducegreatertoxicityincorticalneuronsfromSlo1−/−orSlo1+/− animalswhencomparedtoSlo1+/+_{neurons,aresultconsistentwith} thelackofpotentiationofNMDA-inducedtoxicityinSlo1+/+ neu-ronsbyBKblockersfoundinthepresentexperiments.Theseresults suggestthatBKchannels,thoughinvolvedinleptin-induced pro-tection,donotmediateNMDA-induceddamage.Bycontrast,Liao etal.[47]showedgreaterneurotoxicitybyintra-cerebralinjection ofNMDAinSlo1–/–_{micewhencomparedtoSlo1}+/+_{mice;however,} itshouldbeemphasizedthat,inourinvitromodel,theextracellular concentrationsofendogenousglutamateareunlikelytoachievethe highneurotoxiclevelsobservedintheintacttissueinvivoduring NMDA-inducedneurodegeneration.

Inattempttoinvestigatethemechanism bywhichBK chan-nels participatedin leptin-inducedneuroprotection, westudied bysingle-cell[Ca2+_]

iimagingwhetherleptininducedchangesin [Ca2+_]

i. When used at concentrations similar to those trigger-ingneuroprotection(30–100ng/ml),leptinwasfoundtoinduce transient increases in [Ca2+_]

i in cortical neurons, in most cells appearingshortlyafterleptinexposure.Althoughthisresultclearly suggeststhatacuteleptinapplicationenhances[Ca2+_]

i,the molec-ular mechanism by which leptin-mediated increase in [Ca2+_]i affects BKchannelproperties is yetunknown.Directactivation seemsunlikely,giventhat[Ca2+_]

ichangeslargerthanthoseherein observed are required to directlyenhance BK currents [48]. In support of this view, patch-clamp experiments performed in bothmousecorticalneuronsandinHEK293-Tcellsconstitutively expressingleptinreceptors[49]andtransfectedwithSlo1 chan-nels failed to demonstrate a direct current enhancement after few(10–15)minofleptin(30ng/ml)application(datanotshown). Thus,alsoconsideringthe2htimelagbetweenleptinexposureand neuroprotection,itseemslikelythatleptin-inducedsub-threshold [Ca2+_]

ichallengemayupregulateBKcurrentactivityviaanindirect mechanism.

Leptin-inducedBKchannelactivationinneuronshasbeenfirst proposedbyShanleyetal.[5,6]toexplaintheabilityofthis hor-monetoinhibithippocampalneuronexcitability.However,inthese studies, despite similar slow kineticsand apparentincrease in [Ca2+_]

isensitivityofBKchannels,leptinfailedtoincrease[Ca2+]i; rather, it wassubsequently proposed that phosphatidylinositol 3-kinase(PI3K)wasinvolvedinBKcurrentactivationbyleptin, aneffectconsequenttotheabilityofthis enzymetoalter actin dynamics,andtoclusterandactivateBKchannelsintheneuronal membrane[7].However,leptin-inducedchangesin[Ca2+_]

ihave beendescribedinporcinesomatotropes[50]andinmouse hypo-thalamicastrocytes[51].Moreover,bothstimulatoryandinhibitory effects of leptinon [Ca2+_]

i have been reportedin hippocampal neurons [3] and in cerebellar granular cells;while stimulatory effectshavebeenrelatedtotheabilityofleptintofacilitateNMDA receptor-mediatedCa2+_{influx[52],inhibitoryactionshavebeen} largelyascribedtotheinhibitionofvoltage-gatedCa2+_channels

[53].Leptin-inducedPI3Kactivationand [Ca2+_]

ichanges donot appearasmutually-exclusivephenomena;inmousemacrophages, leptintriggersmigrationbypromoting[Ca2+_]

imobilizationandby activatingjanuskinase/signaltransducersandactivatorsof trans-duction(JAK/STAT),mitogen-activatedproteinkinase(MAPK),and PI3Kpathways[54];bycontrast,inHEK293cellsstablyexpressing full-lengthleptinreceptor(OB-Rb),leptininhibitslysophosphatidic acid-inducedintracellularCa2+_{mobilization,aneffectabrogatedby} PI3Kinhibitors[55].

In conclusion, our resultssuggest that leptin-dependent BK channelactivationisa fundamentalstepintheneuroprotective effects triggered by this adipokine against NMDA-induced cor-tical neuronal degeneration. This cellularmechanism seemsto

render cortical neuronsmore “tolerant” to a neurotoxic insult. Thus,despiteafairlyrigidtimeframeforleptin-induced neuro-protection,onecanspeculatethatleptin-inducedcellulareffects, suchasanenhanced[Ca2+_]

iavailability,couldactasalong-lasting preconditioningevent.Furtherworkwillbeneededtodecipher themolecularpathwaysbywhichtheobservedoscillatory[Ca2+_]

i behaviortriggeredbyleptinrendersneuronsresistanttothe sub-sequentneurotoxic stimulus,and to setonfirmer grounds the potentialutilityofleptininthepreventionand/ortreatmentof neurodegenerativedisorders.

Conflictofinterest

Theauthorsdeclarethattheyhavenoconflictofinterest.

Acknowledgements

The present study was supported by grants from Telethon (GGP07125),theFondazioneSanPaolo–IMI(ProjectNeuroscience 2008.1155),RegioneMolise(ConvenzioneAIFA/RegioneMolise), andPRIN2009(MT),andbytheGermanResearchFoundation,DFG FOR1738(toSHH,TH,MB).

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