CHINA LifeSciences THEMATICISSUE:Frombrainfunctiontotherapy•REVIEW• April2014Vol.57No.4:378–383doi:10.1007/s11427-014-4636-z Bulkendocytosisatneuronalsynapses NGUYENTamH1,QIUXuFeng2,SUNJianYuan2&MEUNIERFredericA1* 1QueenslandBrainInstitute,TheUniversityofQueensland,Brisbane,Queensland4072,Australia;2InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,China ReceivedJanuary17,2014;eptedFebruary24,2014;publishedonlineMarch14,2014 Neurotransmitter-containingsynapticvesicle(SV)fusionwiththenerveterminalplasmamembraneinitiatesneurotransmissioninresponsetoneuronalexcitation.Undermildstimulation,thefusedvesicularmembraneisretrievedviakiss-and-runand/orclathrin-mediatedendocytosis,whichissufficienttomaintainrecyclingofSVs.Whenneuronsarechallengedwithveryhighstimulation,thenumberoffusedSVscanbeextremelyhigh,resultinginsignificantplasmamembraneaddition.Undersuchconditions,ahighercapacityretrievalpathway,bulkendocytosis,isactivatedtoredressthislargemembraneimbalance.Despitefirstbeingdescribedmorethan40yearsago,themolecularmechanismsunderpinningthisimportantprocesshaveyettobeclearlydefined.Inthisreview,wehighlightthecurrentevidenceforbulkendocytosisanditsprevalenceinvariousneuronalmodels,aswellasdiscusstheunderlyingponents. bulkendocytosis,synapticvesiclerecycling,synapse,neuron,neurotransmission,stimulation Citation: NguyenTH,QiuXF,SunJY,MeunierFA.Bulkendocytosisatneuronalsynapses.SciChinaLifeSci,2014,57:378–383,doi:10.1007/s11427-014-4636-z Neuronstransmitinformationbythereleaseofneurotransmittersfromalimitedpoolofsynapticvesicles(SVs)duringstimulation-inducedexocytosis.Duringperiodsofintensestimulation,therateofvesiclereleasecanreachmorethan100Hz,leadingtodepletionofSVs.Itisthereforeessentialthatnerveterminalshaveretrievalmechanism(s)tomaintainthesupplyofSVsandsustainneurotransmission.However,whichmechanismsareactiveatsynapsestomediateSVmembraneretrievaliscurrentlyunclear.Studiesinvarioussynapsemodelshavedemonstratedtheinvolvementofthreebasicmodesofendocytosis:clathrin-mediatedendocytosis(CME)[1],kiss-and-run[2,3]andactivity-dependentbulkendocytosis(ABDE)[48]. CMEisthebestcharacterizedofthethreeSVretrievalpathways,duetoextensiveparallelmolecularstudiesinnon-neuronalcells.ThispathwayretrievessingleSVsdenovofromtheplasmamembraneusingasetofmolecules *Correspondingauthor(email:f.meunier@uq.edu.au) includingthescaffoldingproteinclathrin,togetherwithadaptorandessoryproteins[911].Thekiss-and-runmodeofendocytosisstipulatesthatSVsdonotfullycollapseintotheplasmamembraneandaredirectlyretrievedafterthereleaseofneurotransmitter.ThispathwayinvolvingatransientfusionporeispatiblewiththefastdynamicsofasynapseandwouldalsoaffordenergyefficiencyduetothetransientnatureofSVfusion.However,theexistenceofkiss-and-runintypicallysmallnerveterminalsisdifficulttoassess,withonlyafewstudiespublishedusingvariousfluorescencemethods[1215]orbinationofamperometricandcapacitancemeasurementsinneurosecretorycells.Onerecentstudyusingfluorescentquantaldotstodemonstratedifferentialuptakeandreleaseprovidespromisingevidencetosupporttheexistenceandcontributionofkiss-and-run[16].ThethirdmodeofSVretrieval,ADBE,involvestheformationoflargemembraneinvaginationsorcisternaethatundergofissionfromtheplasmamembranetoforminternal‘bulk’endo- ©TheAuthor(s)2014.Thisarticleispublishedwithopenessat NguyenTH,etal.SciChinaLifeSciApril(2014)Vol.57No.4 379 somes.SVscanthenbudfromthesebulkendosomestoreplenishthevesiclepools.Underconditionsofintenseactivity,bothCMEandkiss-and-runareactiveatcentralsynapses,however,theirlimitedmembraneretrievalcapacitiesmeanthattheyareunabletodealwiththefastervesicledepletionandlargeincreaseinnerveterminalmembranearea.ADBEhasthecapacitytofulfilthisrequirementforhigh-capacitymembraneretrieval.Inthisreview,wediscussthecurrentevidenceforADBEatdifferenttypesofneuronalsynapses,thetechniquesusedforitsdetection,andhighlightsomeofthemolecularmechanismsdrivingitsfunction. 1ADBEatneuronalsynapses ThefirstdescriptionofADBEwasinastudybyHeuserandReeseattheamphibianneuromuscularjunctioninwhichtheyobservedtheappearanceofnumerouslargeintraterminalpartmentsthattheytermed‘cisternae’[1],inresponseto10Hzelectricalstimulationfor10min.Asaresultofstimulation,thenerveterminalsinthesepreparationscontainedsignificantlylesssynapticparedtorestingpreparationsandweremorphologicallydominatedbythepresenceofthelargemembranecisternae.Interestingly,whenpreparationswererestedafter15minstimulation,thecisternaedisappearedandSVsreappeared,suggestingtheSVscouldhavederivedfromthecisternae[1].Sincethisinitialseminalstudy,aconsiderablenumberofstudieshavedemonstratedthatADBEalsoursinrathippocampal[6,17]andcerebellargranule[18]neurons,lampreyreticulospinalsynapses[5],mousecalyxofHeld[19]andauditoryinnerhaircells[20],snake[21]andCaenorhabditiselegansmotorterminals[22],andgoldfishbipolarneurons[8]. Asthenamesuggests,ADBEursinresponsetoelevatedsynapticactivity[8,18,23].Anumberofstudieshaveusedeitherpharmacologicalstimuliattheneuromuscularjunction[24]orpotassiumdepolarizationincentralsynapses[2528]toelicitbulkendocytosis.Theuseofthesenon-physiologicalstimulationmethodsinitiallycasteddoubtsonwhetherbulkendocytosiscouldbeinducedunderphysiologicalconditions.Subsequentstudies,however,showedthatbulkendocytosiscouldbeinducedinvariousneuronalpreparationsincludingamphibianneuromuscularjunction[4],retinalbipolarneurons[8],thecalyxofHeld[19]andprimaryculturedneurons[18]usingelectricalstimulationparadigmswithinthephysiologicalrangeencounteredinvivo(Figure1).ThesedemonstrationsofADBEactivationbystimulationparadigmsmimickingphysiologicalconditionssuggestthatADBEisanimportantSVretrievalpathwayduringperiodsofintensesynapticactivity.Interestingly,otherstudieshavealsoshownthatADBEcanbetriggeredusingprolongedtetanicsimulation[23,31]. 2DetectionofADBEbymembranecapacitancemeasurement ADBEhasbeenclassicallyobservedusingelectronmicroscopicanalysisofphotoconvertedstyryldyes,revealingpreferentialstainingofcisternaebyFM1-43parisontoFM2-10.ThissuggeststhatthesestructurestrapFM1-43andthatthevesiclesthatslowlybudfromthemselectivelyfillthereservevesiclepool[4,5,32].Likewise,opticalimagingofFMdyeinretinalbipolarnerveterminalshasdemonstratedthatmembranecanbeendocytosedintoSVsorlargecisternaestructures[29].Whileopticaltechniquescanprovideexquisitelyhighspatialresolution,particularlyinthecaseofelectronmicroscopy,littleicinformationcanbeobtained.Membranecapacitancemeasurement,asanassayofmembranesurfacearea,hasprovedtobeapowerfulmethodtomonitorSVfusionandretrievalwithahighsignalandtemporal(millisecondtimescale)resolution[33].Thisapproachcanfurtherestimatethefusion/fissionporeicsbymonitoringtheporeconductance[34].Capacitancemeasurementswereinitiallyusedtomonitorvesicleexocytosisandendocytosisinsecretorycellsandhavesincebeenappliedtonerveterminalswithgoodess[35,36].Recently,WuandWuwerethefirsttodemonstratetheinstantofbulkmembranescissionfromtheplasmamembraneinacentralsynapticnerveterminalusingmembranecapacitancemeasurements[19].UsingcalyxofHeldsynapsesinthecentralnervoussystem,briefdownwardcapacitanceshiftsintherangeof20500fFwereobservedfromthecapacitancetracesafterstrongstimulation(Figure1D).Thiscorrelatestoamembraneretrievalthatisapproximately2000-foldlargerthanthecapacitanceofasinglevesicleandconsistentwithamagnitudethatonewouldexpectforADBE.Moreover,thestudyfoundthatADBEurswithin10safterstimulation,muchfasterthanthetimescaleofminutesasestimatedbyelectronmicroscopy[4]andopticalimaging[29].ThesefindingsdemonstratetheexistenceofADBEeventsthatarenotdetectablebyelectronmicroscopyoropticalimagingduetotheirlowtemporalresolution.Importantly,thesefindingsprovideevidencethatADBEisafastmechanismfortheclearanceofponentsfromreleasesitesandmaintenanceofsynaptictransmissionduringsustainedstrongstimulation[19]. 3ThemolecularmachineryofADBE AlthoughADBEwasfirstdescribedmorethan40yearsago,littleisknownabouttheponentsandmechanismsunderpinningthisprocess.Thusfar,onlyahandfulofmoleculeshavebeenidentifiedasplayingrolesinvariousstepsofthismembraneretrievalpathway(Figure2).AsADBEisactivatedinresponsetointensesynapticactivityorstimulation,themolecularsensor(s)thatrespondstothisincreasedactivityisexpectedtobeaponent, 380 NguyenTH,etal.SciChinaLifeSciApril(2014)Vol.57No.4 Figure1Bulkendocytosisisactivatedinresponsetoelevatedneuronalactivityinavarietyofneuronalsynapses.A,ThefirstdescriptionofADBEinthefrogneuromuscularjunctionelicitedby10Hzstimulationfor15min.Notethenumerouslargemembranecisternae(C)andcoatedvesicles(arrow).Schwancellprocessesarealsopresent(s).Magnification,30000×[1].B,ADBEinfrogneuromuscularjunctionprobedusingphotoconversionofthestyryldyeFM1-43andelectronmicroscopy.30Hzstimulationfor1minresultedintheappearanceofFMdyeinc-shapedbulkcisternae[4].C(ad),Bulkendosomesingoldfishretinalbipolarneuronslabelledwithhighmolecularweightfluorescentdextran.C(b)isasingleconfocalsectionthroughthemiddleoftheterminal,C(c)isaprojectionofaz-stackthroughtheentireterminalandC(d)isaprojectionthroughthesameterminalfromtheside.FigurereproducedfromHoltetal.[29]withpermissionofTheSocietyforNeuroscience.D,DetectionofADBEusingmembranecapacitancemeasurementsinmouseCalyxofHeldsynapses.Fissionofbulkendosomesisrepresentedasalargedownwardcapacitanceshift(greyarrowandinset)thaturredpensatorymembraneretrievalinresponsetostimulusof500actionpotentialequivalent(AP-e)at100Hz[19].E,ElectronmicrographsshowingbulkendosomeslabelledwithHRP(blackarrow)inresponseto400actionpotentials(at40Hz)stimulationinnerveterminalsofculturedratcerebellargranuleneurons.FigurereproducedfromClaytonetal.[18]withpermissionofTheSocietyforNeuroscience.F,Bulkendosomesrevealedaslargedonut-shapedstructuresbyfluorescenceimagingofFM1-43dye(a)andultrastructuralanalysisfollowingphotoconversionofFM1-43(b).Notethelargenumberofvesiclescontainingphotoconvertedmaterialincloseproximitytothedelimitingmembraneofthebulkcisternae(inset)[30]. NguyenTH,etal.SciChinaLifeSciApril(2014)Vol.57No.4 381 Figure2SchematicrepresentationoftheinteractionsbetweenkeymoleculesdrivingvariousstagesofADBE.
(1)HighintensitystimulationresultsinCa2+influxintothenerveterminalandactivatingADBEsensormolecule,calcineurin.
(2)Activatedcalcineurindephosphorylatesdynaminwhichthenbindssyndapin(3)toinducemembranecurvatureandtabulationtoformbulkmembraneinvagination,astepthatmayalsoinvolveactin.
(4)Dynaminmediatesthescissionofnascentbulkendosomesfromtheplasmamembrane.
(5)Afterscissionofbulkendosome,dynaminisrephosphorylatedbycyclindependentkinase5(CDK5)andglycogensynthasekinase3(GSK3). withcalcineurinemergingasacandidatetofulfilthisrole.Calcineurinisacalcium-dependentproteinphosphatasethatdephosphorylatesatleasteightnerveterminallylocatedsubstrates,termedthedesphophins,allofwhichareessentialforSVendocytosis[37,38].TwoimportantfactorssupportcalcineurinbeingthesensormoleculeforADBE.Firstly,calcineurinislocalizedtothelasmandhasalowmicromolaraffinityforcalcium[39],meaningthatitisnotsensitivetothelevelofcalciuminfluxassociatedwithmildactivityandwouldonlyencountersufficientlevelsofcalciumduringintenseactivity.Second,ithasbeendemonstratedthatdephosphorylationofoneofitskeysubstrates,thelargeGTPasedynaminI,onlyursatthesamestimulationthresholdthatactivatesADBE[40].Thus,thedephosphorylationfunctionofcalcineurinislinkedtotheactivitydependencyofADBE.Indeed,pharmacologicaldisruptionofcalcineurinfunctionresultsininhibitionofbulkendocytosisinculturedneurons[28]. DynaminisalargeGTPaseproteinresponsibleforthefissionofendocyticvesiclesfromtheplasmamembrane[41].AlthoughtheroleofdynamininCMEiswellestablished,itsinvolvementinbulkendocytosisislesswellunderstood.EvidenceimplicatingdynamininbulkendocytosiswasfirstobtainedinastudybyKeonigandIkedainnerveterminalsofthetemperature-sensitiveDrosophilashibiremutant[42,43].Shibirefliesharbouratemperature-sensitivemutationinthedynaminproteinthatinactivatestheproteinattherestrictivetemperature(>29°C)[43].KeonigandIkeda’sstudydemonstratedthatvesiclerecy- clingaftersynapticdepletionattherestrictivetemperatureursviatwopathways,afastactivezone-associatedpathwayandaslowernon-activezone-associatedpathwaythatinvolvesendosomalintermediates.Importantly,tubularinvaginationsremainedconnectedtotheplasmamembraneinshibiremutantsattherestrictivetemperature,indicatingthatdynaminisrequiredforscissionofthelargepartments.WhiledynaminGTPaseactivityisrequiredforallformsofSVendocytosis,thedephosphorylationofdynaminisofparticularimportanceinADBE.Asmentionedabove,dynaminIisonlydephosphorylatedbycalcineurinduringneuronalactivitythattriggersADBE[40].Furthermore,inhibitionoftheproteinkinasesresponsiblefortherephosphorylationofdynaminIafterstimulation,cyclin-dependentkinase5[28]andglycogensynthasekinase3[44],specificallyblocksADBEbutnotCME.Anotherimportantconsequenceofdynamindephosphorylationisitsinteractionwiththesynapticprotein,syndapinI[45,46].Syndapin,aF-BARdomain-containingproteinthatiscapableofsensingmembranecurvatureandinducingtabulation[47],mayplayanactiveroleindrivingmembranecurvatureuponinitiationofADBE.TheessentialnatureofdynamininADBEisfurtherdemonstratedbythefactthatapplicationofthepotentinhibitorsofdynamin,dynasore[48]andDyngo4aTM[49],resultsinpleteblockofADBEbothinprimaryculturedneurons[40]andattheneuromuscularjunction[30]. Althoughtheactincytoskeletonhaslongbeenimplicatedinvariousstepsoftheendocyticpathway(reviewedin[50]), 382 NguyenTH,etal.SciChinaLifeSciApril(2014)Vol.57No.4 itsroleinADBEisnotwellunderstood.Theactincytoskeletonisimportantintheprocessofmacropinocytosis,theuptakeofextracellularfluidinnon-neuronalcells[51].Inneuronalcells,actindynamicsareessentialforADBEinboththeamphibianneuromuscularjunction[30,31]andgoldfishretinalbipolarneurons[29].TheinvolvementofsyndapinIinADBEprovidesanotherlineofevidenceimplicatingactininthisprocess.TheSH3domainofsyndapincannotonlybinddynaminI,butalsotheneuronalWiskott-Aldrichsyndromeprotein(N-WASP)[52],aproteinthatrecruitsthearp2/plextonucleateactinpolymerization[53].Thus,ithasbeensuggestedthatdephosphorylation-dependentdynaminIrecruitmentofsyndapinIcouldalsorecruitN-WASP,andsubsequentlyarp2/3,topromoteactinpolymerizationatsitesofADBE[54]. Recently,Nguyenetal.demonstratedthatbothdynaminandactinaredifferentiallyrequiredforthedevelopmentandmaturationofADBEattheamphibianneuromuscularjunction[30].Thismaturationprocesswascharacterizedbyatransientbulgingoftheplasmamembranepriortoformationofbulkendosomes.Thedegreeofbulgingincreasedwithstimulationfrequencyandtheplasmalemmasurfaceretrievedfollowingthetransientbulgingcorrelatedwiththesurfacemembraneinternalizedinbulkcisternaeandrecyclingvesicles.Thesefindingssupportatantalisinghypothesisthatthematurationprocessmaycontributetoamechanismbywhichnerveterminalscoupleexocytosisandbulkendocytosis,bysensinghowmuchpresynapticmembranetoretrieveinresponsetosustainedstimulation.However,sofaronlyacorrelationbetweenthedegreeofplasmamembranebulgingandthefrequencyofstimulationhasbeendemonstratedandmoreworkisneededtodissectthefullmechanismsunderpinningthisprocess. 4Concludingremarks ThemolecularmechanismsdrivingADBEarestillnotclearlydefined.However,alargebodyofworkduringthelastdecadehasshedlightonsomekeyaspectsofthisimportantSVretrievalpathway.Thedefiningcharacteristicsofbulkendocytosis,highcapacityformembraneretrievalandactivationinresponsetosynapticactivity,establishthisendocyticpathwayasanimportantphysiologicalprocessinneuronalsynapses.Firstly,ADBEprovidesamechanismbywhichnerveterminalscansustainneurotransmissionunderconditionsofhighstimulationthatcanoverwhelmtherecyclingcapacityoftheCMEand/orkiss-and-runvesiclerecyclingpathways.Second,itisanticipatedthatADBEshouldbeanponentofprocessesthaturinresponsetoelevatedneuronalactivity,suchaslong-termpotentiationandsynapticplasticity.Furthermore,ADBEcouldalsoplayacriticalroleinneuronalpathologiessuchasepilepsy,whichischaracterizedbyburstsofintenseneuronalfiring.Thisisofparticularinterestasarecent studybySmillieandcolleaguesshowedthatADBEcanbeblockedbyBDNF[55],aneurotrophicfactorthathasbeenshowntobereleasedduringepilepticseizures[56].ThuspotentialtherapiesdesignedtotargetADBEareexpectedtodampentheincreasedbrainactivityassociatedwithepilepsy.SuchpossibilitiesprovideexcitingchallengesforfutureresearchintoADBE,andinparticular,themolecularmechanismsthatdrivethisprocess. 1HeuserJE,ReeseTS.Evidenceforrecyclingofsynapticvesiclemembraneduringtransmitterreleaseatthefrogneuromuscularjunction.JCellBiol,1973,57:315–344 2arelliB,HurlbutWP,MauroA.Turnoveroftransmitterandsynapticvesiclesatthefrogneuromuscularjunction.JCellBiol,1973,57:499–524 3FesceR,GrohovazF,ValtortaF,MeldolesiJ.Neurotransmitterrelease:fusionor‘kiss-and-run’?
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