Hypothalamic Tumor Necrosis Factor-αConverting Enzyme Mediates Excitatory Amino Acid ...

12 Pages·2005·0.72 MB·English

TheJournalofNeuroscience,January4,2006•26(1):51–62•51
Development/Plasticity/Repair
(cid:1)
Hypothalamic Tumor Necrosis Factor- Converting Enzyme
Mediates Excitatory Amino Acid-Dependent Neuron-to-Glia
Signaling in the Neuroendocrine Brain
AlejandroLomniczi,AndaCornea,MariaE.Costa,andSergioR.Ojeda
DivisionofNeuroscience,OregonNationalPrimateResearchCenter/OregonHealthandScienceUniversity,Beaverton,Oregon97006
GlialerbB1receptorsplayasignificantroleinthehypothalamiccontroloffemalepuberty.Activationofthesereceptorsbytransforming
growth factor (cid:1)(TGF(cid:1)) results in production of prostaglandin E , which then stimulates luteinizing hormone releasing hormone
2
(LHRH)neuronstosecreteLHRH,theneuropeptidecontrollingsexualdevelopment.Glutamatergicneuronssetinmotionthisglia-to-
neuronsignalingpathwaybytransactivatingerbB1receptorsviacoactivationofAMPAreceptors(AMPARs)andmetabotropicgluta-
matereceptors(mGluRs).Becausethemetalloproteinasetumornecrosisfactor(cid:1)convertingenzyme(TACE)releasesTGF(cid:1)fromits
transmembraneprecursorbeforeTGF(cid:1)canbindtoerbB1receptors,wesoughttodeterminewhetherTACEisrequiredforexcitatory
aminoacidstoactivatetheTGF(cid:1)–erbB1signalingmoduleinhypothalamicastrocytes,andthusfacilitatetheadventofpuberty.Coacti-
vationofastrocyticAMPARsandmGluRscausedextracellularCa2(cid:1)influx,aCa2(cid:1)/proteinkinaseC-dependentincreaseinTACE-like
activity,andenhancedreleaseofTGF(cid:1).Withinthehypothalamus,TACEismostabundantlyexpressedinastrocytesofthemedian
eminence(ME),anditsenzymaticactivityincreasesselectivelyinthisregionatthetimeofthefirstpreovulatorysurgeofgonadotropins.
MEexplantsrespondtostimulationofAMPARsandmGluRswithLHRHrelease,andthisresponseispreventedbyblockingTACE
activity.InvivoinhibitionofTACEactivitytargetedtotheMEdelayedtheageatfirstovulation,indicatingthatME-specificchangesin
TACEactivityarerequiredforthenormaltimingofpuberty.TheseresultssuggestthatTACEisacomponentoftheneuron-to-glia
signalingprocessusedbyglutamatergicneuronstocontrolfemalesexualdevelopment.
Keywords:TGF(cid:1);astrocytes;ectodomainshedding;proteolyticcleavage;femalepuberty;hypothalamus
Introduction (Plant et al., 1989; Urbanski and Ojeda, 1990). They act via
Theinitiationofmammalianpubertyrequiresanincreaseinhy- NMDAandAMPA/kainatereceptorsexpressedinbothLHRH
pothalamic luteinizing hormone release hormone (LHRH) se- neuronsandinterneuronsconnectedtotheLHRHneuronalnet-
cretion(OjedaandTerasawa,2002).LHRHsecretionissubjected work (Gore et al., 1996; Eyigor and Jennes, 1997; Eyigor and
to both neuron-to-neuron (Kordon et al., 1994) and glia-to- Jennes, 2000; Ottem et al., 2002). Notwithstanding the impor-
neuron(Ojedaetal.,2000)regulatoryinputs.Asignificantpartof tance of this trans-synaptic control, it recently became evident
thelattercontrolisexertedatthelevelofthemedianeminence thatglutamatergicneuronscanalsocontrolastrocyticfunction,
(ME),acircumventricularorgan(McKinleyetal.,1990)devoid andthereforeLHRHrelease,viafacilitationoferbB-dependent
ofneuronalcellbodiesbutrichinastrocytes(Kobayashietal., glia-to-neuronsignalingpathways(Dziedzicetal.,2003).Oneof
1972;Ru¨tzelandSchiebler,1980)andmodifiedependymoglial these pathways is initiated by transforming growth factor (cid:1)
cellsknownastanycytes(Knowles,1972;KozlowskiandCoates, (TGF(cid:1)), which in both hypothalamic astrocytes (Ma et al.,
1985). Within the ME, glial–neuronal contacts (Kordon et al., 1994c)andtanycytes(Prevotetal.,2003b)signalsviaerbB1re-
1994;Silvermanetal.,1994)providetheanatomicalsubstratefor ceptors. The integrity of erbB1 signaling is important for the
theoperationofseveralglia-to-neuronsignalingpathwayscon- timingofpuberty,becausefemalesexualdevelopmentisdelayed
trollingLHRHrelease(Garcia-Seguraetal.,1996;Galbiatietal., inmicecarryingmutatederbB1receptorsoracombinederbB1/
2003;Ojedaetal.,2003). erbB4 receptor defect (Apostolakis et al., 2000; Prevot et al.,
A key trans-synaptic input required for the activation of 2003a,2005).
LHRHneuronsatpubertyisprovidedbyglutamatergicneurons Glutamate activates both erbB1- and erbB4-mediated path-
waysbyconcomitantlystimulatingAMPAreceptors(AMPARs)
andmetabotropicglutamatereceptors(mGluRs)locatedonas-
ReceivedJuly15,2005;revisedOct.28,2005;acceptedNov.3,2005.
ThisworkwassupportedbyNationalInstitutesofHealthGrantsHD25123,RR00163,andU54HD18185through troglialcells(Dziedzicetal.,2003).Stimulationofthesegluta-
acooperativeagreementaspartoftheSpecializedCooperativeCenter’sPrograminReproductionResearch. matereceptorsresultsinrecruitmentoferbB1anderbB4recep-
CorrespondenceshouldbeaddressedtoAlejandroLomnicziorSergioR.Ojeda,DivisionofNeuroscience,Oregon tors and their ligands to the glial cell membrane and causes
NationalPrimateResearchCenter,505Northwest185thAvenue,Beaverton,OR97006.E-mail:[email protected].
phosphorylation of both receptors. This transactivation is pre-
DOI:10.1523/JNEUROSCI.2939-05.2006
Copyright©2006SocietyforNeuroscience 0270-6474/06/260051-12$15.00/0 ventedbyinhibitionofmetalloproteinase(MMP)activity(Dz-
52•J.Neurosci.,January4,2006•26(1):51–62 Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling
iedzic et al., 2003). Because MMP-mediated processing of incubation period, both the medium and the cells were collected for
membrane-bound erbB ligands is required for the ligand- TGF(cid:1)measurement.Themediumwastransferredtotubeskeptonice
dependentactivationoferbBreceptorsbetweencellsincontact andcontaining(finalconcentration)1mMphenylmethylsulfonylfluo-
(Dongetal.,1999;Prenzeletal.,1999;Gschwindetal.,2001), ride,leupeptinat10(cid:2)g/ml,and3mMaprotinin.Thereafter,thetubes
werecentrifugedtoremovecelldebris,andthesupernatantsweresaved
AMPAR/mGluR-dependent transactivation of astrocytic erbB
at(cid:2)85°CuntilassayedforTGF(cid:1).Thecellswererapidlyfrozenondryice,
receptorsmayinvolveectodomainsheddingoferbBligands.
Wenowreportthattumornecrosisfactor(cid:1)convertingen- extractedwith25mMTrisbuffer,pH7.4,containing1%TritonX-100,
andthesameproteaseinhibitorsmentionedabove.Thecellextractswere
zyme(TACE),amemberofthedisintegrinandmetalloproteases thenfrozenat(cid:2)85°Cuntilassayed.
(ADAMs) family of metalloproteases (Schlo¨ndorff and Blobel, Determinationofintracellularcalciumlevels.Astrocyteswereseededat
1999),andthemajormetalloproteinasemediatingTGF(cid:1)ectodo- 50,000cellsperwellinblack96-wellplates(Corning,Acton,MA)and
mainprocessing(Peschonetal.,1998;Sahinetal.,2004),isre- grownfor48hinserum-freeADM(Maetal.,1999).Attheendofthis
quired for AMPAR/mGluR-dependent activation of TGF(cid:1)re- period,theywerewashedonceinHBSScontaining130mMNaCl,5mM
lease from hypothalamic astrocytes and for LHRH release. We KCl,1.5mMCaCl2,1mMMgCl2,5.5mMglucose,25mMHEPES,pH7.4,
alsoshowthatastrocytesoftheMEarerichinTACEandthat and0.1%BSAandloadedwith2(cid:2)Mfura-2AMinHBSS(Invitrogen,
Carlsbad,CA)for60minatroomtemperature.Afterthreewashesof10
blockadeofTACEactivitytargetedtotheMEdelaystheonsetof
mineachwithHBSS,cellularfluorescencewasdeterminedusingafluo-
female puberty. These findings suggest that TACE-mediated
ectodomainsheddingoferbBligands,epitomizedbyTGF(cid:1),isa rescenceplatereaderFlexstationII(MolecularDevices,Sunnyvale,CA)
atanexcitationof335nmandemissionof505nm.Measurementswere
keycomponentoftheneuron-to-gliasignalingmechanismused
made every 1.5 s for a total of 80 s. After 20 s of basal fluorescence
by excitatory amino acids to facilitate the advent of female determination,20(cid:2)lofAMPA(500(cid:2)MinHBSS),tACPD(500(cid:2)M),or
puberty. bothwereautomaticallydeliveredtothewells.Theresultsareexpressed
asF/F ,i.e.,theratioofthefluorescencedetectedatanygiventimepoint
o
MaterialsandMethods dividedbytheinitialfluorescenceinthatwell;valuesareexpressedas
Animals. Immature Sprague Dawley rats were obtained from Harlan mean(cid:4)SEMofeightwellspergroup.
SpragueDawley(Indianapolis,IN).Theywerehousedinaroomwith Westernblots.CellculturesortissueswerelysedinTris-bufferedsaline
controlledphotoperiod(14/10hlight/darkcycle)andtemperature(23– (TBS)lysisbuffercontaining1%(v/v)NonidetP-40andtheprotease
25°C)andwereallowedaccesstotapwaterandpelletedratchowad inhibitors 1,10-phenantroline (10 mM; Sigma) and Iomastat (1 (cid:2)M;
libitum. Animals younger than 21 d of age were housed with their Chemicon,Temecula,CA)toblockTACEautodigestion(Schlo¨ndorffet
mothers. al.,2000).Thelysateswerecentrifugedat12,000(cid:3)gfor10min,and500
Tissuedissection.FormeasurementofTACEmRNAorenzymeactiv- (cid:2)gofthesupernatantproteinswereincubatedwith20(cid:2)lofconcanavalin
ity, the hypothalamus was extracted as a whole including the region A-Sepharosebeads(Sigma)for2hat4°C.Thebeadswerewashedthree
abovetheopticchiasm,whichcontainsthepreopticareaandsuprachi- times with lysis buffer and boiled for 5 min in nonreducing sample
asmaticnucleus,andthemedialbasalhypothalamus(MBH),whichcon- buffer,sizedfractionatedbySDS-PAGEusinga8–16%gel,andtrans-
tains the ME, arcuate nucleus, and ventromedial nucleus. The lateral ferred to polyvinylidene difluoride membranes (Millipore, Bedford,
basalhypothalamus(betweentheMEandthehypothalamicsulci)was MA).Westernblotswereperformedasdescribedpreviously(Schlo¨n-
dissectedawayfromtheMEandwastermedMBH((cid:2)).Aftercollection, dorffetal.,2000).Rabbitpolyclonalantibodiesagainstthecytoplasmic
thetissueswerefrozenondryiceandstoredat(cid:2)85°CuntilRNAor tailofTACE(kindlyprovidedbyDr.CarlP.Blobel,HospitalforSpecial
proteinextraction. SurgeryatWeillMedicalCollegeofCornellUniversity,NewYork,NY)
ForexperimentsinvolvingincubationofMEfragmentswithexcita- wereusedata1:1000dilution(overnightat4°C),followedbyananti-
toryaminoacidreceptoragonists(seebelow),theMEwasmicrodis- rabbitHRPantibody(1h;1:50,000dilution).Thesignalwasdeveloped
sectedunderastereomicroscopeasdescribedpreviously(Negro-Vilaret byenhancedchemiluminescenceusingtheWesternlightningchemilu-
al.,1979). minescencesubstrate(PerkinElmerLifeSciences,Boston,MA).
Cellculture.Astrocyteswereisolatedfromthehypothalamiof1-to Reversetranscription-PCR.ADNAfragmentderivedfromthecoding
2-d-oldratsandculturedasdescribedpreviously(Maetal.,1994a,1999). regionofratTACEmRNA(GenBankaccessionnumberNM_020306)
After a growth period of 8–10 d in 75 cm culture flasks containing wasgeneratedbyreversetranscription(RT)-PCRoftotalRNAextracted
DMEM/F-12 medium supplemented with 10% calf serum (HyClone, fromprepubertalrathypothalami,culturedastrocytes,andculturedta-
Logan,UT),astrocyteswereisolatedfromothercelltypesbyovernight nycytes.Then,500ngoftotalRNAweretranscribedintocDNAina
shakingat250rpmandreplatedoneither10cmdishes(2(cid:3)106cellsper volumeof20(cid:2)lusing4UOmniscriptreversetranscriptase(Qiagen,
dish)forTACEactivityor12-wellplatesat300,000cellsperwellfor Valencia,CA),20UofRNaseinhibitor(Promega,Madison,WI),and25
TGF(cid:1)releaseexperiments.Afterreaching80–90%confluence,theme- pmolofanoligo-dTprimer.After1hofincubationat37°C,thereaction
diumwasreplacedwithaserum-free,astrocyte-definedmedium(ADM) wasterminatedbyheatingat95°Cfor5min.PCRswereperformedby
consistingofDMEMdevoidofphenolred,supplementedwith2mM using2(cid:2)lofeachreversetranscriptionreactionandTaqDNApolymer-
L-glutamine,15mMHEPES,5(cid:2)g/mlinsulin,and100(cid:2)Mputrescine(Ma ase(Promega)inavolumeof25(cid:2)l.Thethermocyclingconditionswere
et al., 1999). The cells were exposed 4 d later for the experiments to 95°Cfor4min,followedby35cyclesof94°Cfor15s,65°Cfor30s,and
determinetheeffectoftheexcitatoryaminoacidagonistsAMPA(100 72°Cfor30s,followedbyafinalextensionperiodof7minat72°C.A415
(cid:2)M)andtACPD[(1S,3R)trans-1-aminocyclopentane-1,3-dicarboxylic bpDNAfragmentwasamplifiedwiththeoligodeoxynucleotideprimers
acid](100(cid:2)M)(Sigma,St.Louis,MO)ortheselectiveTACEinhibitor (sense,5(cid:5)-CGTTGGGTCTGTTCTGGTTTTCTC-3(cid:5);andantisense,5(cid:5)-
(Arribas et al., 1996; Karkkainen et al., 2000) TAPI-2 [N-(R)-(2- GGCGGCCTTCTCACTCCTG-3(cid:5)) complementary to nucleotides
(hydroxyaminocarbolyl)methyl)-4-methylpentanoyl-L-t-butyl-glycine- 2016–2040 and 2412–2431, respectively, in the coding region of rat
L-alanine2-aminoethylamide][agiftfromImmmunex(Seattle,WA)or TACEmRNA.AllcDNAsgeneratedbyRT-PCRwereclonedintothe
purchasedfromBiomol(PlymouthMeeting,PA)].Theastrocyteswere riboprobevectorpGEM-T(Promega),andtheiridentitieswereverified
incubatedinthepresenceoftheseagentsfordifferentperiodsoftimeat bysequencing.
37°C,washedwithice-coldPBS,andfrozenindryicebeforeprotein Real-Time PCR. Primers were selected to amplify a 90 bp segment
extraction.TostudythereleaseofTGF(cid:1),theastrocytes(300,000astro- fromratTACEmRNA.The5(cid:5)forwardprimercorrespondstonucleo-
cytesperwell)werepreincubatedinthepresenceorabsenceofTAPI-2 tides 1945–1965 (5(cid:5)-ATTGAGCGGTTTTGGGATTTC-3(cid:5)), and the 3(cid:5)
(50(cid:2)Min500(cid:2)lofADM).After30minofincubationat37°C,thecells reverse primer is complementary to nucleotides 2015–2035 (5(cid:5)-
wereexposedfor4htoAMPAplustACPD(100(cid:2)Meach)orvehicle AAACCAGAACAGACCCAACGA-3(cid:5)) in the TACE mRNA sequence.
(DMEM medium) alone, with or without TAPI-2. At the end of this The internal fluorescent 29-nt-long oligodeoxynucleotide probe (5(cid:5)-
Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling J.Neurosci.,January4,2006•26(1):51–62•53
CCAGCTGAGCATCAACACTTTTGGGAAGT-3(cid:5); PerkinElmer Ap- posedtotheemulsionfor3weeksat4°C.Atthistime,theslideswere
pliedBiosystems,FosterCity,CA),complementarytonucleotides1971– developed,stainedwith0.1%thionin,quicklydehydratedinascending
1999,wascovalentlylinkedtothefluorescentdyeFAMatthe5(cid:5)endand alcoholconcentrations,dried,andcoverslippedformicroscopicexami-
thequencherdyeTAMRAatthe3(cid:5)end.TonormalizethemRNAvalues nation.ControlsectionswerehybridizedtoasenseRNAprobetran-
obtained, 18S ribosomal RNA was simultaneously measured in each scribedfromthesameTACEcDNAtemplatebutlinearizedatthe3(cid:5)end
sampleusingasetofprimerspurchasedasakit(TaqManRibosomal totranscribethesensestrandofthetemplate.
RNAControlReagentskit;PerkinElmerAppliedBiosystems).Otherde- Immunohistochemistry confocal microscopy. To detect TACE-
tailsofthereal-timePCRprocedureinourhandshavebeendescribed immunoreactiveproteininculturedastrocytesandbrainsections,we
previously(Romeroetal.,2002;Shahabetal.,2005). usedaproceduredescribedpreviously(Jungetal.,1997;Dziedzicetal.,
Determination of TACE-like activity. Tissues or cells were homoge- 2003)andrabbitpolyclonalantibodiestotheintracellulardomainofrat
nizedin25mMTrisbuffer,pH7.4,containing1%TritonX-100,1mM TACE(Schlo¨ndorffetal.,2000)generouslyprovidedbyDr.CarlP.Blo-
phenylmethylsulfonylfluoride,leupeptinat10(cid:2)g/ml,and3mMaproti- belorobtainedfromacommercialsource(C-15,sc-6416;SantaCruz
nin.Thehomogenateswerecentrifugedat12,000rpmfor20min,and Biotechnology,SantaCruz,CA).Inthecaseofastrocytes,thecultures
thesupernatantswerestoredat(cid:2)85°Cafterdeterminingtheirprotein werewashedtwicewithPBS,pH7.4,andfixedin4%paraformaldehyde
concentration using the Bradford reagent (Bio-Rad, Hercules, CA). for30minatroomtemperature.Afterextensivewashesin0.02Mpotas-
TACEactivitywasdeterminedinafinalvolumeof100(cid:2)l,using20(cid:2)gof sium phosphate buffer containing 0.9% NaCl (KPBS), the cells were
proteinextractperwell.Thereactionwasinitiatedwiththeadditionof10 incubatedfor15mininKPBS-A(0.02MKPBScontaining0.5%Triton
(cid:2)MMca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH2FluorogenicPeptideSub- X-100),followedby15mininavidinblockingsolution(avidin/biotin
strateIII(R&DSystems,Minneapolis,MN)andperformedat37°C. blockingkit;VectorLaboratories,Burlingame,CA)and15mininbiotin
TACEcleavesthispeptidesequencebetweenAlaandVal,separatingthe blockingsolution.AfterthreewashesinLKPBS(KPBScontaining2%
fluorochrome from the quencher, thus allowing the detection of the normaldonkeyserumand0.5%TritonX-100),thecultureswereincu-
fluorescence (Jin et al., 2002). Fluorescence intensity was determined
bated for 24 h at 4°C with the TACE antibodies (diluted 1:3000) in
withthefluorescenceplatereaderFlexStation(MolecularDevices)using
LKPBS.Theimmunoreactionwasdevelopedusingthebiotinylatedtyra-
anexcitationwavelengthat320nmandemissionat405nmevery10min
mineenhancementmethodasdescribedpreviously(Jungetal.,1997),
for4h.
withabiotinylateddonkeyanti-rabbitIgG(JacksonImmunoResearch
TGF(cid:1)ELISA.ATGF(cid:1)sandwichELISAwasdevelopedusinga“cap-
Laboratories, West Grove, PA) at 1:5000 dilution and an Alexa 488-
ture”andabiotinylatedgoathTGF(cid:1)antibodythatrecognizethemature
streptavidinfluorophore(Invitrogen)at1:1000.Othercultureswerein-
formofhumanTGF(cid:1)(R&DSystems).Briefly,a96-wellplate(Corning,
cubatedovernightat4°CwithamonoclonalantibodytoGFAP(1:5000;
Corning,NY)wascoatedovernightat4°Cwiththecaptureantibodyata
Sigma),followedby1hincubationwithfluorescein-conjugatedhorse
concentrationof0.4(cid:2)g/ml,dilutedincarbonatecoatingbuffer,pH9.7.
anti-mouseIg(1:250;JacksonImmunoResearch)todeveloptheimmu-
Thenextday,andafterasinglewashwithPBS,thenonspecificsiteswere
noreaction.WeperformedseparatereactionsforTACEandGFAPbe-
blockedwith300(cid:2)lofPBScontaining1%BSAand5%sucroseperwell
cause,ininitialculturesthatweredoublestainedforGFAPandTACE,we
for1hatroomtemperature,followedbythreewasheswithwashbuffer
noticedthatTACEwaspresentinGFAP-negativeastrocyticprocesses.
(PBSand0.05%Tween20).SerialdilutionsofaTGF(cid:1)standard(Pepro-
BecauseGFAPhasbeenreportedtobeabsentinfineastrocyticprocesses
tech,RockyHill,NJ)weremadeusingarangeofconcentrationsvarying
(DerouicheandFrotscher,2001;Chaoetal.,2002),wevisualizedthese
from 10 ng/ml to 10 pg/ml in ADM. Duplicates of each standard or
unknownwerethenpipettedintothewellsat100(cid:2)l/well,andthereac- processesbyexposingtheculturestophallotoxinconjugatedtoTexas
Red(1:150;Invitrogen)tolabelF-actinfilaments.Thecellswereincu-
tionwasallowedtoproceedfor24hat4°C.Afterthreewasheswithwash
buffer,biotinylatedanti-hTGF(cid:1)(300ng/ml)dilutedinPBSand1%BSA batedwithphallotoxinfor45minatroomtemperatureaftercompletion
wasaddedtoeachwell(100(cid:2)l/well)andallowedtoreactfor2hatroom oftheTACEorGFAPimmunoreactions.
temperature.Afterthreewashes,eachwellreceived100(cid:2)lofstrepta- For detection of TACE in brain sections, the brains were fixed by
transcardiacperfusionof4%paraformaldehyde–PBS,immersedin20%
vidin–horseradish peroxidase (Amersham Biosciences, Buckingham-
sucrose–PBS for 24–48 h, blocked, frozen on dry ice, and stored at
shire,UK)diluted1:500inTris-bufferedsalineand0.2%TritonX-100,
(cid:2)85°Cuntilsectionedat30(cid:2)musingafrozenslidingmicrotome.Float-
andthemixturewasincubatedfor30minatroomtemperature.Atthe
ing sections were incubated overnight with the TACE antibodies at a
endofthisperiod,thewellswerewashedagainthreetimesbeforedevel-
opingthereactionwith100(cid:2)lofUltra-TMB-ELISA(Pierce,Rockford, 1:2000dilution,andthereactionsweredevelopedasoutlinedabove.To
identifyastrocytes,westainedthesections(overnightat4°C)withmono-
IL)for30minatroomtemperature.Thereactionwasthenterminatedby
adding50(cid:2)lof2MHCl.Theabsorbancewasdeterminedonamicroplate clonalantibodiestoGFAP(1:5000;Sigma)anddevelopedthereaction
reader(Bio-TekInstruments,Winooski,VT)at450nm. thenextdaywithTexasRedgoatanti-mouseIgG(1:250;JacksonImmu-
Hybridizationhistochemistry.TolocalizeTACEmRNAintherathy- noResearch). Tanycytes were identified using monoclonal antibodies
pothalamus,weuseda415ntcRNAprobetranscribedfromthePCR- againstvimentin(1:600;Neomarkers,UnionCity,CA),acytoskeletal
generatedcDNAtemplatedescribedabove.Theprocedureusedfortis- proteinusedasamarkerforthesecells(Chauvetetal.,1998;DeSeranno
suepreparationandhybridizationhasbeenreportedpreviously(Maet etal.,2004).WhenimmunostainingforTGF(cid:1)andTACE,thegrowth
al.,1992;Berg-vonderEmdeetal.,1995).Inbrief,thebrainswerefixed factorwasidentifiedusingrabbitpolyclonalantibodies(1:200;Pepro-
bytranscardiacperfusionof4%paraformaldehyde–boratebuffer,pH tech),andthereactionwasdevelopedtoagreencolorusingbiotinylated
9.5.BothsolutionsweremadewithDEPC-treatedwater(0.1%dieth- donkey anti-rabbit Ig (1:250; Jackson ImmunoResearch), followed by
ylpyrocarbonate)tominimizeRNasecontamination.Afterdissectionof fluorescein-conjugatedstreptavidin(1:250;JacksonImmunoResearch).
the brain, tissue blocks were postfixed overnight in the same fixative TACEwasidentifiedwithgoatpolyclonalantibodiesdirectedagainstthe
containing20%sucrose,blockedandfrozenondryice,andstoredat C-terminusoftheprotein(sc-6316,1:200;SantaCruzBiotechnology),
(cid:2)85°Cuntilsectioning,asreportedpreviously(Maetal.,1992;Berg-von andtheimmunoreactionwasdevelopedtoaredcolorusingAlexa568
derEmdeetal.,1995).Thesections(30(cid:2)m)werecutonaslidingmic- donkeyanti-goatIg(1:250;Invitrogen).Cellnucleiwerestainedwith
rotome, mounted on Superfrost Plus slides (Fisher Scientific, Santa Hoescht33258(Invitrogen)at0.1(cid:2)g/mlKPBSfor1minaftercomple-
Clara, CA), dehydrated under vacuum overnight, and then frozen at tionoftheimmunoreactions.Controlsconsistedofsectionsincubated
(cid:2)85°Cuntilprocessingforhybridization.Thehybridizationprocedure withoutTACEantibodiesbutinthepresenceofeithervimentinorGFAP
wasthatrecommendedbySimmonsetal.(1989),asdescribedpreviously antibodies or sections incubated without TGF(cid:1)or TACE antibodies.
by us (Ma et al., 1994b; Berg-von der Emde et al., 1995), using a FluorescentimageswereacquiredwitheitheraLeica(Nussloch,Ger-
[35S]UTP-labeled cRNA probe. After an overnight hybridization at many)TCSSPconfocalmicroscopeasdescribedpreviously(Maetal.,
55°C,theslideswerewashedandprocessedforcRNAdetection.After 1999;DeFazioetal.,2002)oraMariannasimagingworkstation(Intelli-
dehydration,theslidesweredippedinNTB-2emulsionandwereex- gentImagingInnovations,Denver,CO).Slidebook4.1wasusedtocon-
54•J.Neurosci.,January4,2006•26(1):51–62 Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling
struct a montage of large fields of view and for nearest neighbors or
constraintiterativedeconvolutionandthree-dimensionalrendering.
IncubationofMEexplantsandmeasurementofLHRHrelease.During
microdissection, the MEs were placed in small plastic vials precoated
with0.1%gelatin(oneMEpervial).Thereafter,theywerepreincubated
for30mininKrebs’–Ringer’sbicarbonatebuffer,pH7.4,containing4.5
mg/mlD-dextroseat37°Cunderanatmosphereof95%O2,5%CO2with
shaking (60 cycles per minute), as described previously (Ojeda et al.,
1986b,1990).Attheendofthisperiod,themediumwasdiscardedand
replaced with fresh medium containing either TAPI-2 (50 (cid:2)M) or its
diluent(PBS).After1hofincubation,themediumwasreplacedagain,
thistimewithmediumcontainingAMPAandtACPD(500(cid:2)Meach)in
thepresenceorabsenceofTAPI-2,andtheincubationwascontinuedfor
another hour before collecting the medium for LHRH measurement.
LHRHreleasedtotheincubationmediumwasdetectedbyradioimmu-
noassay, as described previously (Ojeda et al., 1986a,b), using 125I-
labeledLHRHandtherabbitpolyclonalantibodyHU60,whichrecog-
nizes the fully processed, mature decapeptide (Urbanski and Ojeda,
1990),ata1:25,000dilution.
Intrahypothalamicimplants.TospecificallyblockendogenousTACE
activityintheME,TAPI-2wasdeliveredtothisregionofthehypothal-
amusviaastereotaxicallyimplantedcannula.TAPI-2(5mMinDMSO)
wasmixedwithcocoabutterina1:2(w/w)ratioandtappedintotheopen
endofastainlesssteel23-gaugecannula,asdescribedpreviously(Ojeda
andRamirez,1969;Maetal.,1992).Controlcannulascontainedonly
DMSO/cocoabutter.Thecannulaswerestereotaxicallyimplantedinto
theMEormammillarybodiesoflatejuvenile(LJ)28-to29-d-oldrats
accordingtothefollowingcoordinates:ME,(cid:2)0.0mmcaudaltobregma,
0.0mmlateralfrommidline,and8.5mmverticalfromthesurfaceofthe
brain;mammillarybodies,(cid:2)0.7mmcaudaltobregma,0.0mmlateral
fromthemidline,and8.5mmvertical.Afterimplantation,thecannulas
were fixed with dental cement to the skull, and the skin wound was
suturedwithsurgicalclips.
Phasesofsexualdevelopment.ChangesinhypothalamicTACEgene
expressionandenzymeactivityweredeterminedatthedifferentstagesof
puberty described previously (Ojeda and Urbanski, 1994; Ojeda and
Skinner,2005).Ifthevaginawasnotpatentandtheuterusweightwas60
mgorless,theanimalwasconsideredtobeintheLJphaseofpuberty[a
stagepreviouslytermedanestrus(OjedaandUrbanski,1994)].Whenthe
uteruswasenlargedandhaddetectablefluid(anindexofestradiolsecre-
tion),theanimalswereclassifiedasbeingintheearlyproestrusphase.
Animalsshowingauterineweightof200mgormoreandauterus“bal-
looned”withfluidwereconsideredaslateproestrus,i.e.,thedayofthe
firstpreovulatorysurgeofLHRHandgonadotropins.Thenextdayisthe
dayoffirstestrus.Ovulationtakesplaceintheearlymorninghoursof Figure1. TheTACEgeneisexpressedinbothisolatedhypothalamicglialcellsandtheintact
thisday.Thevaginaisopen,vaginalcytologyshowsapredominanceof rathypothalamus.A,DetectionbyRT-PCRofTACEmRNAinculturesofpurifiedastrocytesand
cornified cells, and the ovaries contain fresh corpora lutea. The first ependymoglialcells(tanycytes)andintheMBHofimmaturefemalerats.MM,Molecular
diestrusfollows;atthistime,leukocytesarethepredominantcelltype marker.B,CellularlocalizationofTACEmRNAinthehypothalamusofprepubertal(30-d-old)
foundinvaginallavages. femaleratsasdetectedbyinsituhybridizationusinga[35S]UTP-labeledcRNAprobe.Although
Statisticalanalysis.Thedifferencesbetweenseveralgroupswereana- TACEmRNA-containingcellsarepresentthroughoutthehypothalamus,includingependymal
lyzed by one-way ANOVA, followed by the Student–Newman–Keuls cellsliningthewallofthethirdventricle(3V;arrowheads),themostintensehybridization
multiplecomparisontestforunequalreplications. signalisobservedintheME(arrows).Scalebar,200(cid:2)m.C,Higher-magnificationimageillus-
tratingtheabundanceofTACEmRNAinglialcellsoftheME.Scalebar,50(cid:2)m.
Results
TACEisexpressedinhypothalamicastrocytesandtanycytes type known as astrocytic tanycytes (Za´borszky and Schiebler,
TACEmRNA,detectedbyRT-PCR,wasdistinctlyexpressedin 1978;Ru¨tzelandSchiebler,1980).
thehypothalamusofprepubertalfemaleratsasawholeandin Westernblotanalysisofproteinsextractedfromeitherhypo-
bothastrocytesandtanycytesisolatedfromthisregionandcul- thalamicastrocytesortheMEof30-d-oldratsdemonstratedthe
turedinserum-containingmedium(Fig.1A).Insituhybridiza- presenceofatripletof(cid:6)110kDaundernonreducingconditions
tionofbrainsectionsfrom30-to32-d-oldlatejuvenilefemale identicalinsizetoaproteinspeciesdetectedinCOScells(Fig.
rats demonstrated that, although TACE mRNA is expressed 2A)andthatwaspreviouslydemonstratedtocorrespondtothe
throughout the hypothalamus, it is also present in ependymal full-length TACE form containing the protein prodomain
cellsliningthewallofthethirdventricle(Fig.1B,arrowheads).A (Schlo¨ndorffetal.,2000).Abandof(cid:6)75–80kDadetectedinthe
prominentsiteofexpressionistheME,inwhichTACEmRNA ME,astrocytes,andCOS-7cellslikelyrepresentsmatureTACE
appearstobemostabundantinsubependymalcellsoftheinter- lackingtheprodomain(Schlo¨ndorffetal.,2000).Whenthepro-
mediate portion of the ME (Fig. 1B, arrows, C). This region teins are size fractionated under reducing conditions, the 100
contains astrocytes (Ma et al., 1999) and the intermediate cell kDaproteinmigratesasa120kDaspeciesandthe75–80kDaas
Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling J.Neurosci.,January4,2006•26(1):51–62•55
a 100 kDa form (data not shown), as described previously
(Schlo¨ndorffetal.,2000).
Initialexaminationofculturedhypothalamicastrocytesusing
immunofluorescenceconfocalmicroscopyshowedthepresence
of TACE in GFAP-positive astrocytic cell bodies (data not
shown). However, there was also TACE immunoreactivity not
associatedwithGFAP,suggestingthatTACEmightbeabundant
in astrocytic processes devoid of GFAP (Derouiche and
Frotscher, 2001; Chao et al., 2002). To test this possibility, we
stainedtheastrocyteswithphallotoxin,amushroomproteinthat
bindstoactinfilamentsthatcanrecognizemostastrocyticpro-
cesseslackingdetectableGFAP(Chaoetal.,2002).Asshownin
Figure2B,astrocyticprocessespositiveforactinareindeedGFAP
negative (arrowheads). Conversely, immunoreactive TACE is
present not only in astrocytic cell bodies in which it exhibits a
perinuclear localization (Fig. 2D, long arrows) but also in fine
astrocytic processes (C, D). Interestingly, TACE appears to be
moreabundantontheleadingedgeoftheprocesses(C,D,arrow-
heads)andatthebranchingpointsinwhichnewprocessesorig-
inate(D,shortarrows).Hypothalamicsectionsstainedwiththe
sameTACEantibodies(whichrecognizetheintracellulardomain
of the protein) demonstrated that, as was the case of TACE
mRNA,TACEimmunoreactivityispresentthroughoutthehy-
pothalamus (Fig. 2E). Its abundance, however, appeared to be
greaterinependymalcellsliningthethirdventricle(arrowheads)
andparticularlyintheME(arrows).Three-dimensionalrecon-
structions of higher-magnification images demonstrate that
TACEisindeedpresentinvimentin-positivetanycytes(Fig.2F,
arrowheads). In addition to tanycytes (Fig. 2F, arrowheads, G,
greenstaining),TACEisalsodetectedinsubependymalGFAP-
positiveastrocytes(Fig.2G,arrowheads)andothercellsdevoidof
GFAPimmunoreactivity,probablyneurons.IntheME,theas-
troglialexpressionofTACEisremarkablystrong,becausemost
oftheimmunoreactivityislocalizedtotheintermediateregionof
theMEinwhichastrocytesaremoredenselypacked(Fig.2H,
arrows).ConsistentwiththepresenceofTACEinGFAP-negative
astrocyticprocesses,adistinctfractionofTACE-immunoreactive
material in the ME did not appear to overlap with GFAP (Fig.
2H).
Double immunofluorescence for TGF(cid:1)and TACE, using a
differentTACEantiserum(SantaCruzBiotechnology)alsodi-
rectedagainsttheTACEintracellulardomainverifiedtheafore-
mentioned localizations and demonstrated that both TGF(cid:1)-
positiveependymoglialcellsliningthewallofthethirdventricle
and subependymal cells expressing TGF(cid:1)also contain TACE
Figure2. TheTACEproteinispresentinhypothalamicglialcellsandintheintactrathypo- (Fig.3A,B).AlthoughthepresenceofTACEinependymalcells
thalamus.A,ImmunoblottingofSepharose–concanavalinA-enrichedcelllysatesderivedfrom wasunambiguous(arrowheads),itsabundanceinTGF(cid:1)-positive
hypothalamicastrocytes(A),theMEofthehypothalamus,andCOS-7cells(positivecontrol)
subependymal cells (presumably astrocytes) was remarkable
demonstratesthepresenceofamajorimmunoreactivespeciesmigratingat(cid:6)110kDaunder
(Fig.3A,B,arrows).IntheME(Fig.3C),TACEimmunoreactiv-
thenonreducingconditionsusedandthatrepresentthefull-length,unprocessedTACE.A
smallerspeciesof(cid:6)75–80kDacorrespondingtoTACElackingtheprodomain(Schlo¨ndorffet
al.,2000)wasalsodetectedintheMEandCOScellsandtoalesserextentinastrocytes.Protein
4
sampleswereboiledinsamplebufferintheabsenceofdithiothreitolandsizefractionatedona
8–16%polyacrylamidegelbeforeimmunoblotting(seeMaterialandMethods).B,Detectionof withmonoclonalantibodiestoGFAP.Noticethat,inthiscase,theimmunoreactionwasdevel-
astrocyticprocessesnotstainedbyGFAPantibodies,usingphallotoxinconjugatedtoTexasRed. opedtoaredcolorusingTexasRedgoatanti-mouseIgG.E,TACEimmunoreactivity(green)is
GFAPstainingisshownasagreencolor;arrowheadspointtosomeoftheprocessesdevoidof presentincellsscatteredthroughoutthehypothalamus,butitisparticularlyevidentinependy-
GFAP.C,D,LocalizationofTACEinculturedhypothalamicastrocytes.TACEwasidentifiedwith moglialcells(tanycytes)liningthethirdventricle(3V;arrowheads)andtheME(arrows).F,
rabbitpolyclonalantibodiestoitscytoplasmictaildomain,andthereactionwasdevelopedtoa Three-dimensionalrenderingofastackofimagesoftheperiventricularregionshowingthe
greencolorwithAlexa488streptavidin.Thereafter,thecellswerestainedwithphallotoxin presenceofTACEinependymoglialcells(arrowheads)identifiedastanycytesbyvimentinim-
conjugatedtoTexasRedtovisualizeactininastrocyticprocessesthataredevoidofGFAPstain- munostaining(red).NoticethatTACEisalsopresentinependymoglialprocesses.G,Inaddition
ing.NoticethatTACEimmunoreactivityispresentatthetipofastrocyticprocesses(C,arrow- toependymoglialcells(green),TACEisalsopresentinsubependymalastrocytes(arrowheads).
heads),inadditiontohavingaperinuclearlocalization(D,longarrows).TACEisalsodetected H,TACE(green)isabundantinastrocytes(red)oftheME(arrows),butasubstantialfractionof
alongfineprocessesinwhichitappearstobemostabundantatthetipoftheprocesses(D, theTACE-immunoreactivematerialappearnottooverlapwiththeGFAPstaining,indicatingthe
arrowhead)andatbifurcationpoints(D,shortarrows).E–H,DetectionofTACEimmunoreac- presenceofTACEinGFAP-negativeastrocyticprocesses.Blue,Hoechstnuclearstain.Scalebars:
tivityinthehypothalamusofimmature30-to32-d-oldrats.Astrocyteswereidentifiedassuch B,D,F,20(cid:2)m;C,10(cid:2)m;E,100(cid:2)m;G,15(cid:2)m;H,40(cid:2)m.
56•J.Neurosci.,January4,2006•26(1):51–62 Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling
ity was detected in TGF(cid:1)-positive ta-
nycytes (arrowheads) but much more
abundantly in subependymal cells (ar-
rows),showninFigure2tobeastrocytes.
In the absence of TACE and TGF(cid:1)anti-
bodies, no immunostaining was detected
(Fig.3D).
Hypothalamicastrocytesdisplay
TACE-likeactivity
Variousconcentrationsofproteinextracts
from cultured hypothalamic astrocytes
wereincubatedwithaTACE-selectiveflu-
orescent substrate for 4 h, and the accu-
mulation of fluorescent product was de-
termined every 10 min (Fig. 4A). As
showninFigure4B,theastrocyteextracts
cleaved the fluorometric substrate in a
dose-andtime-dependentmanner.Tode-
termine the specificity of this TACE-like
enzymaticactivity,thecelllysateswerein-
cubatedwiththefluorometricsubstratein
thepresenceofdifferentdosesofTAPI-2,a
Figure3. ColocalizationofTGF(cid:1)andTACEinependymoglialandsubependymalcellsofthethirdventricle(3V)andMEof
selectiveTACEinhibitor(Ohtaetal.,2001;
immaturefemalerats.TACEimmunoreactivitywasdetectedwithgoatpolyclonalantibodies,andthereactionwasdevelopedto
Neumannetal.,2004;Juanesetal.,2005) aredcolorwithAlexa568donkeyanti-goatIg;TGF(cid:1)wasdetectedwithrabbitpolyclonalantibodies,andthereactionwas
(Fig. 4C). The inhibitor reduced basal developedtoagreencolorwithFITC–streptavidin.A,TACEispresentinbothTGF(cid:1)-positivetanycytes(arrowheads)andin
TACEactivityto33%ofcontrollevelsat subependymalTGF(cid:1)-positivecells(arrows).B,Higher-magnificationviewofAshowingtheabundanceofTACEinTGF(cid:1)-positive
thehighestdosetested(50(cid:2)M)(Fig.4D). subependymalcells(arrows).Thesecellsarepresumedtobeastrocytes(seeFig.2).C,IntheME,TACEispredominantlylocalized
Theresidualactivityseeninthepresence tosubependymalcells(arrows)andtoalesserextenttoTGF(cid:1)-positivetanycytes(arrowheads).D,Noimmunostainingisdetected
of TAPI-2 likely reflects the involvement intheMEofasectionprocessedintheabsenceofbothTACEandTGF(cid:1)antibodies.Scalebars:A,40(cid:2)m;B–D,20(cid:2)m.
of TAPI-2-insensitive proteases because,
atthisconcentration,TAPI-2completelyblocksTGF(cid:1)ectodo- concomitantapplicationoftheagonists,TACEactivityincreased
main shedding (Arribas et al., 1996, 1997; Juanes et al., 2005). rapidly(in(cid:7)5min),remainingelevatedforthedurationofthe
Accordingly,allsubsequentexperimentalvaluesarereportedaf- experiment(30min)(Fig.5B).Theincreasewasabolishedinthe
tersubtractionofthisbasal,TACE-independentactivity.Because presenceofTAPI-2.Theseresultsareinharmonywithaprevious
TACE-likeactivitywaslinearduringthefirst60minoftheincu- study showing that coactivation of AMPARs and mGluRs, but
bationperiodandtheenzymaticactivitywasmaximallyblocked nottheindividualstimulationofeachreceptor,resultsinastro-
byTAPI-2at50(cid:2)M,wechosethistimeandblockerconcentra- cyticprostaglandinE2(PGE2)release(Bezzietal.,1998).Theyare
tionforalladditionalexperiments. alsoconsistentwithourpreviousobservationsshowingthatonly
costimulationofbothreceptorsubtypescausescellularredistri-
ActivationofastrocyticAMPARsandmGluRsstimulates butionoferbBreceptorsandtheirligandsandtransactivationof
TACE-likeactivityinaCa2(cid:1)andproteinkinase erbBreceptorsinastrocytes(Dziedzicetal.,2003).
C-dependentmanner To determine whether the changes in intracellular calcium
Inapreviousstudy,weshowedthatcoactivationofmGluRsand levels and the increase in TACE activity induced by AMPA/
AMPARsinhypothalamicastrocytesleadstoametalloproteinase- tACPDstimulationarefunctionallyrelated,westudiedthede-
dependent increase in erbB1 phosphorylation (Dziedzic et al., pendenceofAMPAandtACPD-inducedTACEactivityonthe
2003).TACEisrequiredforthereleaseofmatureTGF(cid:1)fromits availabilityofextracellularandintracellularCa2(cid:1).Becauseithas
membrane-anchored precursor (Borrell-Page`s et al., 2003; beenshowninothercellularsystemsthatincreasesinTACEac-
Hinkleetal.,2003),andthisprocessisCa2(cid:1)andproteinkinaseC tivityrequireactivationofPKC(Monteroetal.,2002),wealso
(PKC)dependent(PandiellaandMassague,1991a,b;Haranoand determinedtheconsequencesofblockingproteinkinaseCactiv-
Mizuno,1994;Baselgaetal.,1996).Wethereforehypothesized ity on the AMPARs and mGluRs-dependent increase in TACE
that the increase in TACE elicited by coactivation of astrocytic activity. Hypothalamic astrocytes were stimulated with AMPA
mGluRsandAMPARsmightrequireanelevationinintracellular andtACPDfor15mininthepresenceoftheextracellularCa2(cid:1)
Ca2(cid:1)levels(Innocentietal.,2000). chelatingagentEGTA,themembrane-permeableCa2(cid:1)chelating
Totestthishypothesis,wefirstdeterminedtheeffectofAMPA agentBAPTA-AM,ortheproteinkinaseCblockerH7.Whenthe
andtACPDonintracellularCa2(cid:1)levelsusingastrocytesloaded extracellular Ca2(cid:1) was sequestered with 1 (cid:2)M EGTA, AMPA/
withfura-2.Althougheachreceptoragonistappliedindividually tACPD-inducedTACEactivitywasreducedby50%(Fig.5C).A
inducedanincreaseinintracellularCa2(cid:1),tACPDcausedafaster morepronouncedreductionwasobservedinthepresenceof1
increase(Fig.5A).Whenusedtogether,theagonistsinducedan (cid:2)MBAPTA-AM,indicatingthatinductionofTACEactivityre-
evenlargerandmoresustainedchangeinintracellularCa2(cid:1)lev- quiresanincreaseincytosolicfreeCa2(cid:1)concentrationsandthat
elsthanaftertheirindividualapplication.AstrocyticTACEactiv- thisincreaseisdependentonbothaninfluxofextracellularCa2(cid:1)
ity,determinedundersimilarconditions,wasaffectedbyneither and a redistribution of intracellular Ca2(cid:1) from microsomal
AMPAnortACPDappliedindividually(Fig.5B).However,after stores.BlockadeofproteinkinaseCactivitywith10(cid:2)MH7abol-
Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling J.Neurosci.,January4,2006•26(1):51–62•57
in the culture medium was accompanied
byasignificantdecreaseinTGF(cid:1)cellcon-
tent,andagainthisdecreasewasprevented
byblockageofTACEactivitywithTAPI-2
(Fig.6B).
TACEactivityincreasesinthemedian
eminenceatthetimeoffirstproestrus
To determine whether TACE activity
changesinthehypothalamusatthetimeof
femalepuberty,wemeasuredthisactivity
at different stages of pubertal develop-
ment.AsseeninFigure7,TACEactivity
selectivelyincreasedintheME(Fig.7A),at
the developmental stage (late proestrus)
when the first preovulatory surge of go-
nadotropinstakesplace.Theincreasewas
stage and region specific because the en-
zyme activity remained unchanged both
before and after late proestrus (Fig. 7A),
andwasnotseenintherestofthehypo-
thalamus(Fig.7B).BecauseTACEactivity
iselevatedatthetimewhencirculatinges-
tradiol levels are maximally increased
(OjedaandTerasawa,2002),wesoughtto
determine whether estradiol at preovula-
torylevelscouldincreaseTACEactivityin
Figure4. AssayofTACE-likeactivityusingtheTACEfluorescentsubstrateFluorogenicPeptideSubstrateIII.A,Serialdilutions
ofproteinextractsfromhypothalamicastrocytesincubatedwithasingleconcentration(10(cid:2)g)offluorescentsubstrate.The hypothalamicastrocytesinvitro.Asseenin
Figure7C,estradiolinducedTACEactiv-
reactionswereperformedat37°C,andthefluorescencewasdeterminedat10minintervals.B,Linearcorrelationbetween
theintensityofthefluorescentreaction,determinedafter2hofincubation,andtheamountofproteinaddedtothereaction.C, ity already at the concentration of 1 nM,
ThespecificityofthereactionwasdeterminedbyincubatingtheproteinextractswithincreasingdosesofTAPI-2(aselectiveTACE which is similar to the concentration of
inhibitor).TheTACEactivitymeasuredafter2hofincubationwasdosedependentlyinhibitedbyTAPI-2,withamaximumof65% estradiol in plasma at the time of first
inhibitionata50(cid:2)Mdose(D).**p(cid:7)0.01and***p(cid:7)0.001versuscontrolgroup(n(cid:8)7–8independentobservationsper proestrus(Andrewsetal.,1981).
group).RFU,Relativefluorescenceunits. To determine whether changes in hy-
pothalamic TACE gene expression may
ishedtheAMPA/tACPD-inducedchangesinTACEactivity,in- alsooccurinassociationwithfemalesexualmaturation,thecon-
dicatingthatproteinkinaseCisanessentialcomponentofthe tentofTACEmRNAintheMEwasdeterminedbyreal-timePCR
signalingcascadesetinmotionbythecoactivationofAMPARs atdifferentphasesofpuberty.NochangesinTACEmRNAabun-
andmGluRstostimulateTACEactivity(Fig.5C).BasalTACE dancewereseenatanystageofthepubertalprocess(Fig.7D).
activity remained unchanged in the presence of each of these Thus, puberty-related changes in hypothalamic TACE activity
blockersalone(datanotshown). appeartooccurselectivelyintheME,intheabsenceofchangesin
geneexpression.
TACEisrequiredforglutamatereceptor-dependent
astrocyticTGF(cid:2)release ActivationofAMPARsandmGluRsinisolatedMEscausesa
It has been shown previously by several authors that TACE is TACE-dependentincreaseinLHRHrelease
requiredfortheectodomainsheddingofTGF(cid:1)indifferentcell BecauseTGF(cid:1)-dependentactivationoferbB-1receptorsinthe
types(Arribasetal.,1997;Peschonetal.,1998;Borrell-Page`set MEcausesLHRHrelease(Ojedaetal.,1990;Prevotetal.,2003a),
al., 2003; Hinkle et al., 2003; Sahin et al., 2004). However, we sought to determine whether stimulation of the ME with
whetherneurotransmitterscaninducereleaseofmatureTGF(cid:1) AMPAandtACPDelicitsLHRHreleaseandwhetherthiseffectis
fromeitherneuronsorglialcellsandwhethertheyuseTACEin affected by blocking TACE enzymatic activity. Microdissected
the process is unknown. To determine whether the glutamate MEexplantsexposedfor1htoAMPAplustACPD(500(cid:2)Meach)
receptor-dependenttransactivationofastrocyticerbB-mediated respondedwithasignificantincreaseinLHRHrelease,whichwas
signalingwereportedpreviously(Dziedzicetal.,2003)involves preventedbyTAPI-2(Fig.8).Thus,concomitantstimulationof
TACE-dependentactivationofTGF(cid:1)release,wetreatedhypo- AMPARs and mGluRs in the ME leads to a TACE-dependent
thalamic astrocytes with AMPA/tACPD for several lengths of increaseinLHRHrelease,suggestingthatthisincreaserequires
time. Because the sensitivity of the TGF(cid:1)assay allows one to activationofaglia-dependentpathway.
reliablymeasuretheendogenouspeptideonlyafteritshasaccu-
mulatedintheculturemedium,wefirstmeasuredbasalTGF(cid:1) InvivoblockadeofTACEactivitytargetedtotheMEdelays
levelsatseveralintervalsafteramockstimulationinADMand femalepuberty
selected 4 h as the best poststimulation interval. As shown in In view of the aforementioned results, we next determined
Figure 6A, AMPA plus tACPD (at 100 (cid:2)M each) significantly whether blockade of TACE activity targeted to the ME would
increased TGF(cid:1) release, and this increase was prevented by delay the onset of female puberty. TAPI-2 administrated via a
blockingTACEactivitywithTAPI-2.TheincreaseinTGF(cid:1)levels stereotaxicallyimplantedcannulaintotheME(Fig.9A,B)mark-
58•J.Neurosci.,January4,2006•26(1):51–62 Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling
Figure5. ConcomitantactivationofastrocyticAMPARsandmGluRsresultsinaCa2(cid:1)andproteinkinaseC-dependentincreaseinTACE-likeactivity.A,CoactivationofAMPARsandmGluRswith
AMPAplustACPD(at100(cid:2)Meach)resultsinasustainedincreaseinintracellularCa2(cid:1)levels,determinedinmonolayersofhypothalamicastrocytesloadedwithfura-2AM.B,TACE-likeactivityis
alsoincreasedwhenAMPAandACPDareadministratedtogether,andthisincreaseispreventedbyTAPI-2,aselectiveTACEinhibitor.C,TheincreaseinastrocyticTACE-likeactivityinducedbyAMPA
plustACPDisinhibitedbysequesteringeitherextracellularorintracellularCa2(cid:1)withthechelatingagentsEGTAandBAPTA-AM,respectively(eachagentat1(cid:2)M);glutamatereceptoragonist-
inducedTACE-likeactivityispreventedbyinhibitingproteinkinaseCwithH7.InB,p(cid:7)0.001untreatedandAMPAortACPDonlyversusAMPAplusACPDandAMPAplusACPDplusTAPI.InC,**p(cid:7)
0.01versuscontrolgroup;#p(cid:7)0.05and##p(cid:7)0.01versusAMPAplusACPDgroup(n(cid:8)6–8independentobservationspergroup).RFU,Relativefluorescenceunits.
intracellularCa2(cid:1)levels,andproteinkinaseCactivity.Thephys-
iological importance of this excitatory amino acid receptor-
dependentincreaseinextracellularproteaseactivityisevidenced
by both its conspicuousness in the ME of the hypothalamus, a
regionenrichedinglialcells,andbythedelayinpubertyresulting
fromtheblockageofTACEactivitytargetedtothisregionofthe
brain.
Glutamatergic neurons communicate with associated astro-
cyticnetworksviaionotropic(AMPA/kainate)andmGlurecep-
tors(Stellaetal.,1994;Steinha¨userandGallo,1996;Bezzietal.,
Figure6. SimultaneousstimulationofAMPARsandmGluRselicitsaTACE-dependentin- 1998;Dziedzicetal.,2003;Matthiasetal.,2003).Coactivationof
creaseinTGF(cid:1)releasefromhypothalamicastrocytes.A,AccumulationofTGF(cid:1)intheculture thesetworeceptorssubtypesonastrocytes,butnotactivationof
mediumincreasesafterexposinghypothalamicastrocytestotheglutamatereceptoragonists eachreceptoralone,resultsinglutamaterelease,aprocessthat
AMPAplustACPD(100(cid:2)Meach),andthisincreaseispreventedbyblockadeofTACEactivity requiresbothCa2(cid:1)mobilizationandformationofprostaglan-
withTAPI-2.B,ThecellularcontentofTGF(cid:1)decreasesinAMPAplustACPD-treatedastrocytes,
dins(Bezzietal.,1998).Astrocytesoftheneuroendocrinebrain
concomitantlywiththeenhancedreleaseofthematurepeptideintotheculturemedium,and
display AMPAR containing the subunits 2 and 3 and mGluR
thedecreaseispreventedbyblockingTACEactivitywithTAPI-2.Allmeasurementsweremade
aftera4hincubationperiodwiththereceptoragonists.TGF(cid:1)wasmeasuredwithanELISAthat subtype5(Dziedzicetal.,2003).Coactivationofthesereceptors
usesantibodiesdirectedagainsttheTGF(cid:1)maturepeptideandthatisabletodetectboththe resultsinacascadeofeventsthatincluderecruitmentoferbB1
cleavedpeptideandthepeptidestillattachedtoitsprecursor.*p(cid:7)0.05versusallothergroups anderbB4receptorsandtheirligands,TGF(cid:1)andneuregulin-(cid:3)1
(n(cid:8)8–12independentobservationspergroup). (NRG-(cid:3)1), to the cell membrane and transphosphorylation of
bothreceptors(Dziedzicetal.,2003).Ageneralinhibitorofmet-
alloproteaseactivitypreventederbBreceptortransphosphoryla-
edlydelayedtheonsetofpuberty,asassessedbytheageatfirst
tion,suggestingthatregulationofligandavailabilitytoerbBre-
ovulation(Fig.9C,D).Bythetimeallcontrolanimalsreceiving
ceptors might be a step required for these receptors to be
thevehiclealoneandallanimalsinwhichtheTAPI-2implants
activatedinresponsetoglutamatergicstimulation.Thepresent
wereplacedinthemammillarybodieshadovulated,onlyoneof
six rats receiving TAPI-2 in the ME had reached puberty (Fig. resultsofferexperimentalevidenceinsupportofthisviewand
9C).Themeanageatfirstovulationwassimilarinthetwocon- identifyforthefirsttimearoleformetalloproteasesinneuron–
trols groups but significantly delayed in rats implanted with gliacommunication.
TAPI-2intotheME(Fig.9D).Thus,activationofTACEactivity It is rather recently that roles for metalloproteases and
in the ME is required for the timely initiation of the pubertal ADAMsinneuron-to-neuroncommunicationhavebeenidenti-
process. fied(McFarlane,2003).Forinstance,MMPscontributetoregu-
lating axonal guidance (Galko and Tessier-Lavigne, 2000),
Discussion ADAM23mightplayaroleinregulatingneuronalconnectivity
Thepresentresultsidentifyaproteolyticprocessinthecell–cell (Leighton et al., 2001), and MMP9 is involved in transmitter-
signalingmechanismusedbyglutamatergicneuronstocommu- dependentdendriticremodelingofthemammalianhippocam-
nicatewithhypothalamicastrocytes.ByrapidlyactivatingTACE, pus(Kaczmareketal.,2002).Tothebestofourknowledge,no
theproteaseresponsiblefortheproteolyticsheddingofproTGF(cid:1) reports have appeared demonstrating an involvement of either
(Arribasetal.,1997;Peschonetal.,1998),stimulationofastro- MMPs or ADAMs in neuron–glia communication, although
cyticglutamatereceptorscausesTGF(cid:1)release,therebysettingin sucharolewassuggestedbythepresenceoftheDm1-MMPinthe
motionaTGF(cid:1)/erbB1receptor-dependentpathwaythatiscru- Drosophilamidlineglia(Llanoetal.,2000).Ourresultsindicate
cialforastrocyticback-signalingtothoseneuroendocrineneu- thatTACEisexpressedinastroglialcellsofthehypothalamusand
rons that control sexual development (Ojeda and Terasawa, ependymoglial cells of the third ventricle and ME. A more de-
2002).WefurthershowthatTACEactivationrequiresthecon- tailedexaminationofastrocytesinculturedemonstratedthat,in
comitant stimulation of AMPARs and mGluRs, an increase in thesecells,TACEislocatedbothperinuclearly(presumablyasso-
Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling J.Neurosci.,January4,2006•26(1):51–62•59
Figure8. ConcomitantstimulationofAMPARsandmGluRswithAMPAplustACPD(500(cid:2)M
each)elicitsaTACE-dependentLHRHreleasefromMEexplantsincubatedinvitrowiththe
agonists.TheincreaseinLHRHreleasewaspreventedbyblockingTACEactivitywithTAPI-2.
**p(cid:7)0.01versusallothergroups(n(cid:8)6independentobservationspergroup).
ciatedwiththeendoplasmicreticulum)and,surprisingly,atboth
thetipofastrocyticprocessesandinbranchingpointsinwhich
newprocessesoriginate.SuchTACE“hotspots”mayhavecon-
siderable physiological significance, because TACE might act
withinthesemicrodomainstoprocesstheectodomainofgrowth
factorreceptorsandligandsinvolvedinglia-to-neuroncommu-
nication, such as the TGF(cid:1)–erbB1 signaling complex. The
emergingroleofMMPsinaxonalguidance(McFarlane,2003),
andremodelingofbothneuronalconnections(Kaczmareketal.,
2002)andtheextracellularmatrix(Llanoetal.,2000),raisesad-
ditionalpossibilities.
InadditiontoTGF(cid:1),TACEisrequiredfortheectodomain
releaseofothercomponentsoftheligand–erbBreceptorcom-
plexinvolvedintheglialcontrolofLHRHsecretion,including
NRGs (Montero et al., 2000) and erbB-4 receptors (Rio et al.,
2002). Thus, the effects of inhibiting TACE activity on LHRH
secretion and the onset of puberty cannot be attributed exclu-
sively to a defect in TGF(cid:1)-dependent activation of erbB1. In-
stead,itismostlikelythattheseeffectsreflectthecombinedloss
ofTGF(cid:1)andNRGsignalingcapabilitiesbecause,intheabsence
of ectodomain shedding, neither TGF(cid:1)(Peschon et al., 1998;
Borrell-Page`setal.,2003)norNRGs(Liuetal.,1998)arecapable
ofactivatingtheirrespectivereceptors.Itisalsounlikelythatthe
inhibitionofTACE-likeenzymaticactivitydetectedwiththeflu-
orescence substrate we used reflects an exclusive loss of TACE
activity, because ADAM-10 activity (which is inhibited by
TAPI-2) is also detected using this substrate (Neumann et al.,
2004).TheimportancethatADAM-10mighthaveinneuroen-
docrinefunctionis,however,unclear.Basedontheseconsider-
ations,itwouldappearreasonabletoconcludethat,amongthe
erbB-dependent signaling complexes requiring ligand ectodo-
main shedding for activation, those mediated by TGF(cid:1)and
NRGsarethemostrelevanttothecontrolofLHRHreleaseand,
4
surge.*p(cid:7)0.05versusallothergroups(n(cid:8)7–8ratspergroup).B,Thisincreaseisnot
detectedintherestofthehypothalamus.C,Hypothalamicastrocytesrespondtopreovulatory
levelsofestradiol(similartothoseseenonthedayoffirstproestrus,LP)withincreasedTACE
activity.**p(cid:7)0.01,***p(cid:7)0.001versusbasalgroup;n(cid:8)6wellspergroup.D,Theincrease
inTACE-likeactivityintheMEisnotaccompaniedbycorrespondingchangesinmRNAexpres-
sion.LJ,Latejuvenile,prepubertalrats;EP,earlyproestrus,thephaseofpubertywhenestradiol
secretionbeginstoincreasedrivenbyanenhancedpulsatileLHrelease;LP,lateproestrus,this
stagecorrespondstothefirstproestruswhenthefirstpreovulatorysurgeofgonadotropins
Figure7. TheonsetofpubertyinfemaleratsisaccompaniedbyME-specificchangesin takesplace;E,thefirstestrus,bothvaginalopeningandthefirstovulationtakesplaceonthis
TACE-likeactivityintheabsenceofchangesinTACEmRNAexpression.A,TACE-likeactivity day;D,thedayofthefirstdiestrus,correspondingtothephaseofpubertywhenthefirst
increasesselectivelyintheMEofperipubertalfemaleratsonthedayofthefirstpreovulatoryLH functionalcorporaluteaareformed.RFU,Relativefluorescenceunits.
60•J.Neurosci.,January4,2006•26(1):51–62 Lomniczietal.•TACEMediatesNeuron-to-GliaSignaling
hence, sexual development. Of these two
systems, we selected TGF(cid:1)as the proto-
type for this study not only because glial
cells of the ME (including astrocytes and
tanycytes)containTGF(cid:1)anditserbB1re-
ceptor (Ma et al., 1992, 1994c) but also
because the targeted manipulation of
erbB1receptorfunctionintheMEaffects
the onset of female puberty (Ma et al.,
1992).Whereasblockingthereceptorsde-
layspuberty(Maetal.,1992),theiractiva-
tionadvancessexualdevelopment(Rageet
al.,1997).
The present results indicate that the
MEmaybeamajorsiteofglutamate/erbB-
dependent neuron–glia bidirectional
communication.Relevanttothisconcept
are the findings that astrocytic processes
arerichinglutamatereceptors(Matuteet
al., 1994; Eyigor and Jennes, 1998;
Kawakami,2000),thatstimulationofneu- Figure9. InvivoblockadeofTACEactivitytargetedtotheMEofthehypothalamusdelaystheonsetofpubertyinfemalerats,
ronal fibers results in astrocytic calcium asassessedbythetimeatfirstovulation.A,VentralviewofthehypothalamusillustratingtheMElocalizationofastainlesssteel
waves confined to specific glial microdo- cannulacarryinginitstiptheTACEinhibitorTAPI-2.B,Aschematicrepresentationmappingtheanatomicallocalizationofthe
mains(Groscheetal.,1999),andthatglu- implants.Theblocker(TAPI-2;blackcircles)orvehicle(DMSO/cocoabutter;whitecircles)weredeliveredtotheMEwithinthetip
tamatereleasedbyastrocytescanenhance ofastereotaxicallyimplantedstainlesssteelcannula;someimplantswerelocatedtoocaudally(withinthemammillarybodies;
neuronal activity by acting on axonal re- graysquares)andthusserveasanadditionalcontrolgroup.C,Thenumberofratsreachingpubertyatagivenpostnatalage
(expressedaspercentageofthetotalnumberofanimalspergroup)issignificantlyreducedbyMEblockadeofTACEactivity.D,The
ceptors(SemyanovandKullmann,2001).
meanageatfirstovulationisalsosignificantlydelayedinTAPI-2-implantedrats.Errorbarsrepresentthemean(cid:4)SEMageatfirst
Infact,theMEisnotonlyrichinglialcells
ovulation.***p(cid:7)0.001versuscontrolgroup;n(cid:8)6ratspergroup).AHA,Anteriorhypothalamicarea;DMH,dorsomedial
but also in axons from both neuroendo-
hypothalamus;OC,opticchiasm;POA,preopticarea;PVH,paraventricularhypothalamicnucleus;SC,suprachiasmaticnucleus;
crine neurons that release their secretory
VMH,ventromedialnucleus.
products into the portal vasculature and
non-neuroendocrineneuronscontrolling
Our in vivo studies show that TACE-like activity, but not
neurosecretion(Kobayashietal.,1972;Page,1994).
TACEmRNAabundance,increasesselectivelyintheMEonthe
The need for simultaneous activation of AMPARs and
dayoffirstproestrus.Thissuggeststhat,asobservedinhuman
mGluRstoenhanceTACEactivityandinduceTGF(cid:1)releaseis
cellsinvitro(Blacketal.,1997),theavailabilityofTACEwithin
consistentwithpreviousobservationsshowingthatsuchacoac-
theMEdependsoninputsregulatingitsproteaseactivityrather
tivationisrequiredtobothinduceastrocyticglutamateandPGE
2 thanonchangesingeneexpression.ThepubertalincreaseinME
release(Bezzietal.,1998)andtransactivateglialerbBreceptors
TACE activity may be caused, at least in part, by the elevated
(Dziedzicetal.,2003).Becausehypothalamicastrocytescontain
estrogenlevelsthatcharacterizethisphaseofsexualdevelopment
Ca2(cid:1)-impermeable (GluR2), in addition to Ca2(cid:1)-permeable
(OjedaandTerasawa,2002).Asshownhere,thiseffectisdirectly
(GluR3),AMPARs(Dziedzicetal.,2003),itisplausiblethatthe
exerted on astrocytes, but it may also result from an action of
underlyingmechanismofthiscodependencyinvolvesthefunc-
estrogen on glutamatergic neurons signaling to astrocytes. By
tionalassociationofCa2(cid:1)-impermeableAMPARswithmGluRs
showingthatglialcellsoftheMEarerichinTACEandthatthein
tocausealonger-lastingCa2(cid:1)mobilization,asshownrecentlyin
vivopharmacologicalblockadeofTACEactivitytargetedtothe
hippocampal interneurons (Topolnik et al., 2005). In fact, our
MEdelaysthefirstovulation,ourresultssuggestthatactivation
resultsshowthatboththemagnitudeanddurationofCa2(cid:1)mo-
ofmetalloproteaseactivityinthisregionoftheneuroendocrine
bilizationaregreaterwhenastrocytesaresubjectedtoconcomi-
brainisarequiredstepintheprocessbywhichglialcellsfacilitate
tantAMPA–mGluRstimulationthanwheneachreceptorisstim-
theinitiationoffemalepuberty.Theresultsalsoprovideevidence
ulatedseparately.Suchanenhancedresponsemightbenecessary
foraninvolvementofTACEintheprocessbywhichglutamater-
toensureadequateactivationofPKC,themostimportantregu-
gic neurons communicate with glial cells to activate erbB
latory mechanism controlling protein ectodomain shedding in
receptor-mediatedsignalingandraisethepossibilitythatsucha
general(KiesslingandGordon,1998)andTACEactivityinpar-
mechanism might be broadly used throughout the brain for
ticular(Monteroetal.,2002).Inkeepingwiththisconcept,our
neuron-to-gliacommunication.
results show that PKC is required for glutamate receptor-
dependentTACEactivation.BecauseCa2(cid:1)enhancestheactivity References
ofTGF(cid:1)convertasesotherthanTACE(C.Blobel,personalcom- AndrewsWW,MizejewskiGJ,OjedaSR (1981) Developmentofestradiol-
munication),itispossiblethatglialAMPARs/mGluRsmightuse positive feedback on luteinizing hormone release in the female rat: a
another metalloprotease, in addition to TACE, to elicit TGF(cid:1) quantitativestudy.Endocrinology109:1404–1413.
release.Ofnote,ourresultsshowthatglutamate-inducedTGF(cid:1) ApostolakisEM,GaraiJ,LohmannJE,ClarkJH,O’MalleyBW (2000) Epi-
release from hypothalamic astrocytes, but not basal TGF(cid:1)re- dermal growth factor activates reproductive behavior independent of
ovariansteroidsinfemalerodents.MolEndocrinol14:1086–1098.
lease, was blocked by TAPI-2, suggesting that, as previously
Arribas J, Coodly L, Vollmer P, Kishimoto TK, Rose-John S, Massague J
showninimmortalizedmousemonocytes(Doedensetal.,2003), (1996) Diversecellsurfaceproteinectodomainsareshedbyasystem
basalTGF(cid:1)sheddingisaTACE-independentprocess. sensitivetometalloproteaseinhibitors.JBiolChem271:11376–11382.