The Role of Transporters ABCG1/4 And

生物化学与生物物理进展 ProgressinBiochemistryandBiophysics 2014,41(8):765~776 www.pibb.ac.cn TheRoleofTransportersABCG1/4andABCA1 inBrainCholesterolMetabolism*

SUNHai-Mei1),CHENLi2,3),MIAOJin-Wei4)** (1) DepartmentofHistologyandEmbryology,SchoolofBasicMedicalSciences,CapitalMedicalUniversity,Beijing 100069,China; 2) CenterforClinicalandTranslationalResearch,TheResearchInstituteatNationwideChildren sHospital,Columbus,OH 43205,USA; 忆 3) BeijingZhongguancunHospital,Beijing 100190,China; 4) DepartmentofGynecologicOncology,BeijingObstetricsandGynecologyHospital,CapitalMedicalUniversity,Beijing 100006,China)

Abstract Thebrainisthemostcholesterol-richorganinthebodyandconsistsof25%oftotalcholesterol.ATP-bindingcassette (ABC)transportersplayessentialrolesincellularcholesteroleffluxandhomeostasisinthebrain.Recently,ABCG1,ABCG4and ABCA1expressionintheadultbrainhasbeendescribed.Theabsenceofoneormoreofthesetransportershasbeenimplicatedinthe developmentofneurodegenerativediseases.Inthisstudy,wecharacterizedthemRNAandproteinexpressionlevelsofABCG1, ABCG4andABCA1inthedevelopingpostnatalbrainofnormalC57BL/6Jmicefedachowdiet.Westudiedthecorrelationbetween ABCtransportersexpressionandcholesterollevels(freecholesterol,esterifiedcholesterol)inthebrainandserum,toelucidatea potentialroleofthesetransportersincholesterolmetabolisminthebrainandbodyduringpostnatalstages.Wefurtherinvestigatedthe changesofexpressionlevelsofABCG1,ABCG4,ABCA1,cholesterolrelatedgenesandbraincholesterollevelsinABCG1-/-,ABCG4-/- andABCG1-/- ABCG4-/- doubleknockoutmice.ABCA1mRNAexpressionweredetectableinmultipletissues,andABCG1,ABCG4 werehighlyexpressedinadultbrain.ABCG1andABCG4mRNAlevelspeakedat42daysofage,whileABCA1mRNAlevelswere nearbaseline.ABCG1proteinlevelspeakedatday28thendecreased,whileABCG4levelspeakedatday42.ABCA1levelsremained nearbaseline.Interestingly,circulatingplasmaandbrainesterifiedcholesterollevelsexhibitedabiphasicdistribution,whichpeakedat day42.LossofABCG1iscompensatedbyincreasedABCG4andviceversa.LossofbothABCG1andABCG4resultsinaltered expressionofcholesterolsynthesisrelatedgenesandcholesterolaccumulationinthebrain.ThedatasuggestthatABCG1andABCG4, butnotABCA1areimportantfortransporterfunctioninthedevelopingbrain.ABCG1andABCG4playcomplementaryrolesin maintainingbraincholesterolhomeostasis.

Keywords ATP-bindingcassette(ABC)transporters,braindevelopment,cholesterol DOI:10.3724/SP.J.1206.2014.00012

Thebrainisthemostcholesterol-richorganinthe homeostasisandcouldcontributetothe bodyandconsistsof ～ 25%oftotalcholesterol [1]. neurodegenerativediseaseincludingAlzheimer's Almostallcholesterolacquiredinthebrainisderived disease [7].However,theroleofABCG1,ABCG4and from denovo biosynthesisbecauseoftheeffective ABCA1inthebraincholesterolequilibriumremains preventionofblood-brainbarrier [2].Thus,theisolated elusive.Inaddition,brainneuronsswitchfrom cholesterolpoolinthebraindemandsaprecise cholesterolbiosynthesisduringembryonicperiodto mechanismforhomeostasisofthecholesterolcontent. cholesteroluptakefromastrocytesafterbirth [8].Itis TheATP-bindingcassette(ABC)transporter thereforepostulatedthatpostnatalneuronsmayobtain superfamilyhasbeenreportedtoplaycrucialrolesin cellularcholesterolefflux,whichisessentialforthe *ThisworkwassupportedbyagrantfromBeijingNaturalScience cholesterolhomeostasisinthebrain [3].ABCG1, FoundationProjects(7102056). ABCG4andABCA1havebeenrecentlydemonstrated **Correspondingauthor. [4-6] tobeexpressedinthebrain .TheabsenceofABCG1 Tel:86-10-52277296,E-mail:[email protected] isimplicatedinthedisturbanceofcholesterol Received:January10,2014Accepted:June23,2014 ··766 生物化学与生物物理进展 Prog.Biochem.Biophys. 2014;41(8) astrocytes-secretedcholesterol via apolipoproteinE chowdietfoodandwateradlibitum.Animalswere (ApoE)containingHDLparticles[9].TheATP-binding fasted6hbeforedissection.Theprotocolwas cassette(ABC)transportersmediatethetransportof approvedbytheInstitutionalAnimalCareandUse intracellularcholesteroltoApoEandApoA1,the CommitteeinNationwideChildren'sHospital.Brain crucialprocessforHDLsynthesis [10].Therefore,we andserumwereharvestedfromneonatal(1-,and hypothesizethatABCG1,ABCG4andABCA1may 7-day-old),pubertal(14-,21-,28-,35-,42-day-old) expressinspecificcelltypesofthebrainandmediate andadult(12-week-old)mice.Sixmiceperagegroup cholesterolhomeostasisintime-controlledmanner. wereusedexceptfortheneonatalperiod,where10 Inourstudy,wehavecharacterizedthemRNA animalswereutilized.Thetissueswerenotpooledbut andproteinexpressionofABCG1,ABCG4and treatedindividually.ABCG1-/-,ABCG4-/- andABCG1-/- ABCA1inpostnataldevelopingandadultmouse ABCG4-/- micewerekindlyprovidedbyDr.Matthew brains.WestudiedthecorrelationbetweenABC Kennedy'slaboratoryinNationwideChildren'sHospital. transportersexpressionandcholesterollevels(free 1.3 Real鄄time quantitative PCR analysis of cholesterol,esterifiedcholesterol)inthebrainand ABCG1,ABCG4andABCA1mRNAexpression serum,toelucidateapotentialroleofthese TotalRNAwasisolatedfromthetissuesbyusing transportersincholesterolmetabolisminthebrainand Trizolpermanufacturer'sprotocol.Afterisolation, body.WefurtherinvestigatedtheeffectsofABCG1 2 goftotalRNAwasreversetranscribedintocDNA andABCG4onthecholesterollevelsandcholesterol at滋42℃ for1hbyusingOligo(dT)and1 lofiScript synthesisrelatedgenesinthebrainofABCG1-/-, Reversetranscriptase(iScriptTM cDNASyn滋thesiskit)in ABCG4-/- andABCG1 -/- ABCG4-/- doubleknockout a20 lfinalvolume.ThemRNAexpressionof mice.Thesestudieswillleadtoabetterunderstanding ABCG滋1,ABCG4andABCA1weremeasuredusing inneurologicaldiseasewhereaberrantcholesterol primersdesignedformousebyPrimer3software metabolismhasbeenimplicated. (PremierBiosoftInternational,PaloAlto,CA,USA) (Table1).Othergenesexpression(Hmgr,Fpps,Cyp51 1 Materialsandmethods andAcat1)wereanalyzedbyusingprimersshownin 1.1 Reagents Table3.AlltheseprimerswerepurchasedfromIDT. TrizolwaspurchasedfromInvitrogen(Carlsbad, Corp.TheReal-timequantitativePCR(Real-time TM TM CA,USA).iScript cDNASynthesiskit,iQ SYBR PCR)reactionswereperformedusingiQTM SYBR TM GreenSupermixandImmun-blot PVDFmembrane GreenSupermixaccordingtothemanufacturer's werepurchasedfromBio-Rad(Hercules,CA,USA). instructions(Invitrogen,Carlsbad,CA,USA)and AllprimerswereobtainedfromIDT(Coralville,IA, 0.5 mol/Lofeachprimer.InallReal-timePCR TM USA).BCA ProteinAssayKitwasobtainedfrom reacti滋ons,anegativecontrolcorrespondingtoreverse Pierce(Rockford,IL,USA).Rabbitpolyclonal transcriptionalreactionwithoutthereverse anti-ABCA1antibodywaspurchasedfromNovus transcriptaseenzymeandablanksamplewerecarried Biologicals(Littleton,CO,USA).Rabbitpolyclonal out(datanotshown).Amplificationofthe anti-ABCG1andanti-ABCG4antibodywasobtained housekeepergenepeptidylprolylisomeraseA(PpiA) fromOpenBiosystems(Huntsville,AL,USA). cDNAwasusedasinternalcontroltoquantifythe Monoclonalanti- -tubulinantibody,peroxidase- expressionofagivengeneinReal-timePCRs [11]. 茁 conjugatedanti-rabbitIgG,peroxidase-conjugated QuantificationwasperformedwiththeOpticalSystem anti-mouseIgGandKodakBiomaxMSfilmwere software,version1.0(Bio-Rad).Productsofreaction TM purchasedfromSigma(St.Louis,MO,USA).ECL werevisualizedbyelectrophoresisina1.5%agarose Westernblottingdetectionreagentswaspurchased gel,stainedwithethidiumbromideandphotographed fromGEHealthcare(Piscataway,NJ,USA).Totaland underaUVtransilluminator. freecholesterolkitswerepurchasedfromWako 1.4 ImmunoblotsanalysisofABCG1,ABCG4 ChemicalsUSA,Inc(Richmond,UA,USA). andABCA1proteinexpression 1.2 Mice Proteinsweremeasuredaccordingtothe C57BL/6Jwildtype(WT)micewereobtained manufacturer'sinstructions(BCATM ProteinAssaykit) fromJAX,USA.Animalswerekeptundernatural andBSAasastandard.Sampleswerepreparedfor lightingconditions,andtheywereallowedstandard electrophoresisbyboilingtheminthecorresponding 孙海梅, 等：转运蛋白 ABCG1/4 和 ABCA1 对脑胆固醇的调节作用 ··767 2014;41(8)

volumeofsamplebuffer2 (8mol/Lurea,70mmol/L determinedbysubtractingthefreefractionfromthe Tris-HClpH6.8,3%SD伊S,0.005%Bromopheonol totalcholesterolfraction. blue,and5% -mercaptoethanol)for5min.Proteins 1.6 Analysisofdataandstatisticalanalysis (50 g)werere茁solvedona10%sodiumdodecylsulfate- TheimmunoreactivebandsinWesternblots poly滋acrylamidegelelectrophoresis(SDS-PAGE),and obtainedforeachtimeintervalstudiedwerescanned electro-transferredontonitrocellulosemembranes. withalaserscanner.Theintensityofthe Membraneswereblockedwith3%blottinggrade immunoreactivebandwasquantifiedbyScionimage blockerinTris-bufferedsaline(TBS:140mmol/L program(ScionCorporation,Frederick,MD,USA). NaCl,50mmol/LTris-HCl,pH7.4)for60minat Thevalueswerenormalizedtothe1dayvaluesfor 37 ℃ andthenincubatedwithanti-ABCG1(1 ∶ 50 developmentalstudies.Thedataareexpressedasthe dilution),anti-ABCG4(1∶ 50dilution),anti-ABCA1 x 依 s.Student's t-testwasusedforcomparisons (1 ∶ 300dilution)ormonoclonalanti- tubulin betweentwogroups.Differenceswereconsidered 茁 (1∶ 5000dilution)overnightat4℃.Membraneswere statisticallysignificantas P <0.05.DataforReal-time thoroughlyrinsedandincubatedwithperoxidase- PCRwerealsoexpressedasthe x 依 s.Allanalyses conjugatedanti-rabbitIgG(1 ∶ 3000dilution)or werecarriedoutwithSigmaplot9.0software(SPSS peroxidase-conjugatedanti-mouseIgG(1 ∶ 5000 Inc.,Chicago,IL,USA). dilution).Antigen-antibodycomplexeswererevealed 2 Results bychemiluminescenceandthemembraneswere exposedtoKodakbiomaxMSfilm. 2.1 ABCG1 and ABCG4, but not ABCA1, 1.5 Free cholesterol (FC) and esterified mainlyexpressedinadultbrain cholesterol(EC)levelsassaysinmousebrainand ThemRNAexpressionprofileofABCG1, serum ABCG4andABCA1wereevaluatedbyReal-time Freeandesterifiedcholesterollevelsofmouse PCRusingtotalRNAisolatedfrommousedifferent brainandserumweredetermined.Sampleswere tissuesinadulthood.PeptidylprolylisomeraseA preparedbyamodifiedFolchmethod[11-12].Inbrief,the (PpiA),ahousekeepergene,wasusedasinternal brainwashomogenizedwitha2∶1Chloroform- controlsinceitisexpressedconstitutivelyand [11] methanolmixture.Extractswerewashedbyaddition constantlyinallsamples .ABCG1,ABCG4and of0.9mol/LNaClsolution.Thelowerchloroform ABCA1primers(Table1)amplifiedasingleproduct layercontainingthelipidswastransferredtoawheaten withanexpectedsizeof120bp,120bpand125bpby vialanddrieddownunderNitrogen.Thesampleswere Real-timePCR(datanotshown).Anegativecontrol resolubilizedin250ml95%ethanolandquantified correspondingtoRTreactionwithoutthereverse usingakitfortotalandfreecholesterolper transcriptaseenzymewascarriedoutandshowedno manufacturesprotocol(WakoChemicals,USA,Inc. PCRproductamplification(datanotshown). Richmond,VA,USA).Esterifiedcholesterolwas

Table1 SpecificprimersdesignedformouseABCG1,ABCG4andABCA1 GenBankaccession Primers Gene Productsize/bp numbers Sense Anti-sense ABCG1 NM_009593 5 GGGGAAAGGTCTCCAATCTC3 5 TGTTCTGATCCCCGTACTCC3 120 忆忆忆忆 ABCG4 NM_138955 5 GCGTGGTTACCAACCTGATT3 5 TGGGGTTCAGGTCTCCATAC3 120 忆忆忆忆 ABCA1 NM_013454 5 AACAGTTTGTGGCCCTTTTG3 5 CACAATCAGGCTGAAGACCA3 125 忆忆忆忆 PpiA NM_008907 5 GCATACAGGTCCTGGCATCT3 5 CTTCCCAAAGACCACATGCT3 121 忆忆忆忆

ABCG1,ABCG4andABCA1expressionwere brownadiposetissue(BAT),adrenalgland,muscle detectableinmultipletissues,includingtestis,brain, andlung(Figure1).ABCG1washighlyexpressedin spleen,kidney,liver,whiteadiposetissue(WAT), brainandnextinlung.Spleen,kidney,WAT,BAT, ··768 生物化学与生物物理进展 Prog.Biochem.Biophys. 2014;41(8) andadrenalglandcontainedlowbutappreciablelevels moderateABCG4mRNAexpressioninWAT,BAT, ofABCG1mRNAexpression(Figure1a).The adrenalgland,andmuscle,whichhasnotreported ABCG1mRNAexpressioninthetestis,liverand before.Aspreviousdescribed,ABCA1mRNAbroadly muscleweredetectable.Ontheotherhand,ABCG4 expressedofinmanytissueswithprominentmRNA wasstronglyexpressedinthebrain(Figure1b).The expressioninliver,BAT,adrenalgland,andlung [13] ABCG4mRNAexpressedintestis,kidney,liver,and (Figure1c). lungwereatverylowlevels.Surprisingly,wedetected

(a) 0.004 (b) 0.006 0.005 0.003 0.004 0.002 0.003 0.002 0.001 0.001 0 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

Fig.1 mRNAexpressionprofileofABCG1,ABCG4andABCA1inadultmousetissues TotalRNAwasextractedfromadultC57BL/6Jmousetissuesonchowdiet.Afterthereversetranscription,amplificationwascarriedoutwith0.5 mol/L 滋 ofmouseABCG1,ABCG4orABCA1primers(Table1).Thehistogramsshow:(a)ABCG1,(b)ABCG4and(c)ABCA1mRNAexpressioninseveral mousedifferenttissues.AlldatawerenormalizedtotheinternalreferencePpiAandexpressedasfoldincreaserelativetonormalizedreferencevalue. Therelativevaluesarecomparableingroups. 1:Testis; 2:Brain; 3:Spleen; 4: Kidney; 5:Liver; 6:WAT; 7:BAT; 8:Adrenalgland; 9:Muscle; 10:Lung.

2.2 ABCG1andABCG4mRNAlevelspeakedat (Figure2, n=6).ABCG1andABCG4mRNAlevels 42daysofage,whileABCA1mRNAlevelswere tendedtoincreasewithdevelopmentandreachedthe nearbaseline highestlevelat42dayswiththefoldvalueof26.7 TodeterminethepatternofABCG1,ABCG4and (+P <0.05,42versus1day)or2.75(*P <0.01,42 ABCA1expressioninthebrainduringdevelopment, versus1day)(Figure2a, n=6).IncontrasttoABCG1, wemeasuredtheirmRNAlevelsinmousebrainat ABCG4mRNAlevelstendedtoelevatefrom1to14 varioustimepointsduringpostnataldevelopment.The daysthensignificantlydecreasedfrom14to21days mRNAexpressionofABCG1,ABCG4andABCA1 (+P <0.04,21versus14day)beforerisingagainfrom obtainedfromneonatal(1-,and7-day-old),pubertal 21daystoadulthood(Figure2b, n=6).Interestingly,the (14-,21-,35-,and42-day-old),andadult(>55days) levelsofABCA1mRNAexpressionwerelowinthe micebrainwerestudiedbyRealtimeQ-PCRanalyses. developmentalbrainandremainedatthislevel ABCG1orABCG4mRNAexpressiondifferedgreatly throughoutallthedevelopmentalattimepointstested fromABCA1inmicebrainduringdevelopment (Figure2c, n=6). 孙海梅, 等：转运蛋白 ABCG1/4 和 ABCA1 对脑胆固醇的调节作用 ··769 2014;41(8)

versus1day)beforedroppingfrom28daysto adulthood.Incontrast,ABCG4proteinlevels (a) 0.020 significantlyincreasedfrom21to28days(+P<0.03) 0.015 andreachedthemaximallevelsfrom42daysto + adulthood(*P <0.0142versus1day).Surprisingly, 0.010 荫 ABCA1proteinlevelswerelowandrelatively 荫 0.005 unchangedthroughoutdevelopment. 荫荫荫荫 0 荫荫 (a) 1d 7d 14d 21d 28d 35d 42d Ad 1d 7d 14d 21d 28d 35d 42d Ad

(b) ABCG1 0.008 ABCG4 0.006 * ▲ ABCA1 0.004 ▲ + ▲ ▲ ▲ ▲ + -Tubulin 0.002 ▲ ▲ 茁

0 (b) 5 1d 7d 14d 21d 28d 35d 42d Ad

0 1d 7d 14d 21d 28d 35d 42d Ad Fig.2 mRNAexpressionprofileofABCG1,ABCG4and ABCA1inmousebrainduringpostnataldevelopment RealtimeQ-PCRwasperformedforABCG1(a),ABCG4(b)and Fig.3 ProteinexpressionprofileofABCG1,ABCG4and ABCA1(c)genesinmousebrainduringpostnataldevelopment.Alldata ABCA1inmousebrainduringpostnataldevelopment werenormalizedtotheinternalreferencePpiAandexpressedasfold 50 gofproteinfrommicedevelopmentalbrainweresubjectedto12% 滋 increaserelativetonormalizedreferencevalue.(a)Theincreasebetween ofSDS-PAGEandWesternblotanalyseswereperformedbyusing

42and1day(+P<0.05, n=6)wassignificant.(b)Thedecreasefrom14 anti-ABCG1,ABCG4andABCA1antibodies.RepresentativeWestern

to21days(+P<0.04, n=6)andtheincreasefrom21to28days(+P< blotsresultsshowingthat74kuABCG1,74kuABCG4and220ku 0.05, n=6)andtheelevationbetween42and1day(*P <0.01, n=6)were ABCA1werepresentinmousebrainweeklythroughoutdevelopment significant.d:Daysold.Ad:Adult.Thedatawereobtainedfromsix (a).Theimmunoreactivebandswerescannedandtheirintensitieswere independentexperimentsforeachagegroup.Neonatal:1d,7dand14d; quantifiedusingtheScionImageprogram(b).Alldatawerenormalized Pubertal:21d,28d,35dand42d;Adult:12-week-oldmice. totheinternalcontrol -tubulinamounts.Theintensitiesofthebands 茁 obtainedinsixindependentexperimentswererelativetoABCG1, ABCG4andABCA1proteinlevelsat1day(1fold).Thevaluesshown ofABCG1(● ● ),ABCG4(),andABCA1(▼ ▼)are x 依 s.

TheincreaseofABCG1proteinlevelsbetween28and1day(+P<0.05, 2.3 ABCG1proteinlevelspeakedatday28then n=6)wassignificant.AndtheelevationofABCG4proteinlevelsfrom decreased,whileABCG4levelspeakedatday42, 21to28days(+P<0.03, n=6),andbetween42and1day(*P <0.01, n= andABCA1levelsremainednearbaseline 6)weresignificant.d:Days;Ad:Adult. RepresentativeWesternblotsofABCG1,ABCG4 andABCA1areshowninFigure3a.Protein expressionofABCG1(74ku),ABCG4(74ku)and 2.4 Circulating plasma and brain esterified ABCA1(220ku)variedduringpostnataldevelopment cholesterollevelsexhibitedabiphasicdistribution, inC57BC/Jmice(Figure3b, n=6).ABCG1protein whichpeakedatday42 levelsrosefrom1to28daysafterbirth(+P<0.05,28 InordertoevaluatethepotentialrolesofABCG1, ··770 生物化学与生物物理进展 Prog.Biochem.Biophys. 2014;41(8)

ABCG4andABCA1inthebraincholesterol Inthebrain,FCvalueswerenear19 g metabolismduringdevelopment,wemeasuredthe cholesterol/mgtotalproteinatday1ofpostna滋tal levelsoffreecholesterol(FC)andesterified (Figure4n=4)androseto37 gcholesterol/mgtotal 滋 cholesterol(EC)inthebrainandserum.The proteinby7days(+++P <0.002,1versus7day)before developmentalFCandEClevelvariationsinthebrain droppingtobaselinelevelsthroughadulthood(14 g andserumareshowninFigure4. cholesterol/mgtotalprotein).Asexpected,ECvalu滋es weremorethanthreetimestheFCvaluesinthebrain. Surprisingly,theincreaseinEClevelsfrom1to14 (a) 160 days(***P <0.0001)wasfollowedbyasignificant 140 decrease(***P <0.005)from14to21daysandthena 120 荫 markedincreasefrom28daystoadulthoodandpeaking 100 *** ***荫 byday42withthevalueof119 gcholesterol/mg 80 荫*** 滋 60 totalprotein(***P <0.00001,42versus35day) +++ *荫** 40 荫荫 (Figure4a, n=4).TheEClevelswasdroppedin 荫 +++ *** 20 *** adulthood(***P <0.00005). 0 荫 SerumFCandEClevelswassimilartothatinthe 20 - 1d 7d 14d 21d 28d 35d 42d Ad brain(Figure4b, n=4).However,serumFClevels werelowandconstantthroughoutalldevelopmentat (b) timepoints.SerumECvalueswere24mg 160 cholesterol/dlserumatday1,peakingat118mg 140 cholesterol/dlserumbyday42.SerumECvalues 120 *荫** 100 showedabiphasicprofilesimilartothatofEClevels *荫** inbrainexceptthatthechangefrom7-to-14dayswas 80 *荫** 60 *荫** 荫 notstatisticallysignificant(P>1). 40 *荫** 荫 2.5 ABCG1andABCG4expressedreciprocally -/- 20 荫 inAbcg1orAbcg4knockoutmice (Abcg1 or 0 -/- 1d 7d 14d 21d 28d 35d 42d Ad Abcg4 ) SincedistincttemporalexpressionofABCG1and ABCG4,butnotABCA1,isseeninbrainduring postnataldevelopmentalstage,wefurtherinvestigated Fig.4 Freecholesterol (FC)andesterifiedcholesterol thechangesofABCG1andABCG4expressioninthe -/- -/- (EC) levelsinmousebrainandserum brainofABCG1 andABCG4 mice.Asassessedby duringpostnataldevelopment quantitativeReal-timePCR,diminishedlevelof WTC57BC/6Jmicebrainandserumwereisolated.Freeandesterified ABCG1mRNA,butelevatedlevelofABCG4mRNA cholesterolweredeterminedafterisolationasdescribedinmaterialsand wasfoundinABCG1 -/- (Figure5a).Similarreciprocal methods.(a)EC(● ● )andFC()areexpressedin g 滋 -/- cholesterol/mgtotalproteinformousebrain.Thevaluesshownare x 依 s relationshipwasalsoobservedinABCG4 , offourindependentexperiments.TheincreaseofFClevelsfrom1to7 representedbydecreasedABCG4expressionbut days(***P <0.002, n=4)wassignificant.TheincreaseofEClevels increasedABCG1expressioninthebrain(Figure5b). from1to14days(***P <0.0001, n=4)andfrom28to42days(***P < Westernblotanalysesusinganti-ABCG1and 0.00001, 4),andthedecreaseofEClevelsfrom14to21days(*** < n= P ABCG4antibodiesrevealedincreasedexpressionlevel 0.005, n=4)andfrom42toadulthood(***P <0.00005, n=4)were significant.(b)EC(● ● )andFC()areexpressedinmg ofABCG4proteinandconcomitantlydiminishedlevel -/- cholesterol/dlserum.Thevaluesshownare x 依 s offourindependent ofABCG1proteininABCG1 mice(Figure5c).In experiments.TheincreaseofEClevelsfrom1to7days(***P < contrast,ABCG4-/- micecontainedhighlevelsof 0.00001, n=4)andfrom28to42days(***P <0.002, n=4)were ABCG1proteinandverylowlevelsofABCG4protein significant.ThedecreaseofEClevelsfrom14to21days(***P <0.003, (Figure5d). n=4)andfrom42daystoadulthood(***P <0.0004, n=4)were significant.d:Days;Ad:Adult. 孙海梅, 等：转运蛋白 ABCG1/4 和 ABCA1 对脑胆固醇的调节作用 ··771 2014;41(8)

(b) (a) ABCG1 ABCG4 ABCG4 ABCG1 2.0 mRNA 4 mRNA 2.0 mRNA 4 mRNA * 1.5 3 1.5 3 *

1.0 2 1.0 2

0.5 1 0.5 1 * * 0 0 0 0 WTABCG1-/- WTABCG1-/- WTABCG4-/- WTABCG4-/-

(d) ABCG1 (c) ABCG4 6 5 5 * 4 * 4 3 3 2 2 1 1 0 0 -/- WTABCG1-/- WTABCG4

Fig.5 ComplementaryexpressionofABCG1andABCG4inABCG1-/- andABCG4-/- mice (a,b)RealtimeRT-PCRwasperformedforABCG1andABCG4genesinABCG1-/- andABCG4-/- mice.Alldatawerenormalizedtotheinternal referencePpiAandareshownrelativetotheexpressionofeachgeneproductincontrolwild-type(WT)mice(n=10,*P<0.001 vs.WT).(c,d)Western blotanalysisconfirmedreciprocalexpressionofABCG1andABCG4.50 gofproteinfromthebrainofABCG1-/-,ABCG4-/- miceandWTmicewere 滋 loadedto12%SDS-PAGEandWesternblotanalyseswereperformedusinganti-ABCG1andABCG4antibodies.Semi-quantificationof immunoreactivebandintensitywereperformedandarepresentativeblotwasshown.Dataareshownas x 依 s ofthreeindependentexperiments(*P < 0.001 vs.WT).

2.6 Brainfreeandesterifiedcholesterollevels ABCG1andABCG4genes(Table2).Thesefindings increasedinA月悦郧1-/- A月悦郧4-/- mice suggestthatABCG1andABCG4compensateforeach ToelucidatetheeffectsofABCG1andABCG4 otherintheregulationofcholesterolhomeostasis,and intheregulationofcholesterolhomeostasis,we onlylossofbothtransportersleadstocholesterol measuredthelevelsoffreeandesterifiedcholesterolin accumulationinthebrain. thebrainofABCG1-/-,ABCG4-/- andABCG1ABCG4 BraintissuesweredissectedoutfromABCG1-/-, doubleknockoutmice(ABCG1-/- ABCG4-/-).Wefound ABCG4 -/- andABCG1-/- ABCG4-/- miceandbrainfree thatfreeandesterifiedcholesterollevelsremain andesterifiedcholesterolweremeasured.Free normalinthebrainofthemicewitheitherABCG1 cholesterolandesterifiedcholesterolareexpressedin geneorABCG4genewasdeleted(ABCG1 -/- or gcholesterol/mgtotalproteinformousebrain.The 滋 ABCG4-/-).However,significantlyincreasedlevelsof valuesshownare x 依 s.Significantincreaseoffree freeandesterifiedcholesterolswerefoundinthe cholesterolandesterifiedcholesterollevelswas brainfromABCG1-/- ABCG4-/- mice,whichlackboth noticedinthebrainsofABCG1 -/- ABCG4-/- compared

Table2 FreecholesterolandesterifiedcholesterollevelsinABCG1-/-,ABCG4-/- andABCG1-/- ABCG4-/- brains WildType ABCG1-/- ABCG4-/- WildType ABCG1-/- ABCG4-/- Cholesterol (n=5) (n=10) (n=10) (n=5) (n=10) Freecholesterol 14.9 0.6 15.4 0.8 15.2 0.3 14.2 0.6 25.9 0.8* 依依依依依 Esterifiedcholesterol 85.7 1.2 89.4 1.0 91.2 2.3 86.1 0.9 109.2 2.7* 依依依依依 ··772 生物化学与生物物理进展 Prog.Biochem.Biophys. 2014;41(8) toABCG1 -/- orABCG4-/- (*P <0.001, n=10ineach decreasedinABCG1-/- ABCG4-/- mice,wefurther group).Thereisnosignificantchangeofcholesterol investigatedthechangesofcholesterolsynthesis levelsbetweenABCG1-/- andWTorABCG4-/- andWT relatedgenesinthebrain.RealtimeRT-PCRwas (P >0.05; n=5inWTgroup). appliedtoamplifythegeneexpressionusingthe 2.7 ChangesofgenesexpressioninABCG1 -/- specificprimerslistedinTable3andquantification ABCG4-/- micebrains analysiswasperformed. Braincholesterollevelsweresignificantly

Table3 SpecificprimersdesignedformouseHMGR,FPPS,CYP51,ACAT1andABCA1 GenBank Primers Gene accession Productsize/bp numbers Sense Anti-sense HMGR NM_008255 5 CACAAGCTGGAAACTGGTGA3 5 GAAGAAGTAGGCCCCCAATC3 197 忆忆忆忆 FPPS AF_309508 5 TGTACATGGCAGGCATTGAT3 5 GAGGAGAGGCTCGTAGCAGA3 201 忆忆忆忆 CYP51 NM_013454 5 CCTTCACTCTCAGCCTCGTC3 5 GCCCACCATGGTAAAGCTAA3 203 忆忆忆忆 ACAT1 NM_144784.3 5 CCAGATGTGGTGGTGAAAGA3 5 ATTCGTGCCAATGGCTTAAC3 200 忆忆忆忆 ABCA1 NM_020010 5 AACAGTTTGTGGCCCTTTTG3 5 CACAATCAGGCTGAAGACCA3 125 忆忆忆忆

OurdatashowthatmRNAlevelofHMG-CoA esterifiedcholesterolenzyme,wassignificantly reductase(HMGR)andfarnesyldiphosphatesynthase decreasedinthebrainsofABCG1-/- ABCG4-/- mice (FPPS),whichinvolvesinthebiosynthesisoffree (Figure6).Consistentwithdefectiveeffluxand cholesterol,wereremarkablydecreasedinABCG1-/- cholesterolaccumulation,otherATP-bindingcassette ABCG4-/- mice.ThemRNAsencodinglanosterol transporter,ABCA1expressionwassignificantly demethylase(CYP51)wasalsodramaticallyreduced increasedinABCG1-/- ABCG4-/- brain(Figure6). inthebrainsofABCG1-/- ABCG4-/- mice.Striking Thesechangesingenesexpressionsuggestthat changesofgeneexpressionofAcyl-coenzymeA: cholesterolhomeostasiswasalteredinthebrain cholesterolacyltransferase1(ACAT1),aspecific followinglackofABCG1andABCG4.

2.0 3.0 HMGR FPPS CYP51 ACAT1 ABCA1 2.5 * 1.5 2.0 1.0 1.5 + * + 1.0 0.5 * 0.5 0 0 1 2 1 2 1 2 1 2 1 2

Fig.6 mRNAexpressionlevelsofHMGR,FPPS,CYP51,ACAT1andABCA1inthebrainsofABCG1-/- ABCG4-/- mice Real-timePCRwasperformedforHMGR,FPPS,CYP51,ACAT1andABCA1genesinthebrainsofABCG1-/- ABCG4-/- andWTmice.Alldatawere normalizedtotheinternalreferencePpiAandexpressedasfoldincreaserelativetonormalizedinternalcontrol.Thedatawereobtainedfromfour -/- -/- independentexperiments(*P <0.01; +P <0.05; n=4ineachgroup). 1:WT; 2:ABCG1 ABCG4 .

transportersABCG1,ABCG4andABCA1mRNAs 3 Discussion areexpressedinavarietyoftissuesofadultmale Inthisstudy,wehavedemonstratedthattheABC C57BL/6Jmiceonachowdiet.Whenadjustedto 孙海梅, 等：转运蛋白 ABCG1/4 和 ABCA1 对脑胆固醇的调节作用 ··773 2014;41(8) comparablelevels,wewereparticularlystruckbythe increasedremarkablyduringthefirstthreeweeksof data,whichshowsthatABCG1andABCG4mRNA postnatallife [14].Contributingtothedramatic levelswereseveralfoldhigherthanABCA1mRNA developmentalgrowthofthemousebrainisan levelsintheadultbrain(seeFigure1).Wewere elevationinlipids,whichisrequiredintheprocessof interestedinunderstandingthefunctionofthese neuronsdifferentiationormyelinogenesis.SinceABC transportersinthedevelopingbrain;thereforewe transportersplaycrucialrolesinbrainlipidtransport determinedthemRNAandproteinlevelsduring whichisnecessaryforefficientneuronsdifferentiation postnataldevelopment.Weweresurprisedtoseethat oroligodendrocytesmyelination,itispredictedthatthe thethreeABCtransportermRNAexpressionlevels uniqueexpressionofABCtransportersmayvary varydistinctlyduringpostnatalbraindevelopment(see dramaticallyduringmousebraindevelopment [2].Our Figure2).ABCG1mRNAlevelsrosesteadilywith studiessupportthishypothesisandarethefirstto expressionatP42andstayedelevatedthroughout indicatethatABCG1andABCG4proteinlevelsare adulthood.ABCG4mRNAlevelsexhibitedabiphasic expressedwidelyinthebrainthroughoutdevelopment; expressionpatternwithpeaksintheprepubescent andtheexpressionpatternvariedwiththe phase(P7 ～ P14),andagainatP42intoadulthood, developmentaltimeperiod.ElevatedFCandEC similartoABCG1.ABCA1mRNAlevelswereat levelsinthebrainby7daysafterbirth,combinedwith baselinefromP1throughadulthood.Interestingly, increasingmRNA/proteinlevelsofABCG1and ABCtransportersproteinlevelsdidnotcorrelateto ABCG4inbrainduringneonataldevelopment, theirmRNAexpressionlevels(seeFigure3).In suggeststhatABCG1/ABCG4areassociatedwith particular,ABCG1proteinlevelspeakedatP28then cholesterolrelease.Moreover,highlevelsofABCG1 decreased,eventhoughABCG1mRNAlevelswere andABCG4,butnotABCA1,mRNAlevelsinadult elevated.ABCG4proteinlevelswerebaselinethrough brainsuggestthatABCG1andABCG4haverolesin P21andthenclimbedrapidly,peakingatP42.The theregulationoflipidhomeostasisduringthelate proteinlevelsdidnotshowthesamebiphasic developmentalperiod. expressionlevelsasthemRNA.Additionally,when Aspreviouslydemonstrated,astrocytesproduce serumandbrainsterollevelsweremeasured,esterified cholesterolinthebrainandsynthesizeapoEcontaining cholesterollevelsinboththebrainandserumhada nascentHDLtoremovescholesterolfrombrain [15]. biphasicdistribution(seeFigure4).Interestingly, ABCG1hasbeenimplicatedinremovingcholesterol fromP1～ P7inthebrain,freecholesterollevelswere fromastrocytesandneurons via theinteractionofthe higherthanesterifiedcholesterollevels. transporterswithHDLparticles [14-17].Thus,we IncontrasttoapreviousstudythatABCA1 hypothesizedthatABCG1andABCG4mayhavean mRNAwasonlydetectedintheearlypostnatalbrain[4], importantroleinapolipoprotein-dependentcholesterol wefoundthatitisexpressedinthemousebrain effluxfromastrocytesandneuronsandourdatainthe throughoutthepostnataldevelopmentstagesaswellas developingbrainsupportthistheory. adultperiod.BecauseABCtransportersareassumedto Inthepresentstudy,wealsofoundthe mediatecholesteroleffluxprocess,ABCA1expressed significantlydecreasedABCG4mRNAlevelsinbrain inthedevelopmentalbrainislikelyresponsiblefor by21days,whichisaccompaniedwiththediminished cholesterolefflux.However,therelativelylow esterifiedcholesterollevel.Consideringtheprevious expressionofABCA1mRNAandproteinthroughout observationthattherateofaccumulationofsterolpool postnatalbraindevelopmentimpliesthatABCA1may markedlydecreasedafterthreeweeksofbirth [1],the notplayamajorroleincholesterolefflux,resultingin neuronaldifferentiationandsynapsedevelopment thepreventionofcholesterollossbeforethe tendedtoreduceastheanimalmatures,weassumed blood-brainbarrierisfullydeveloped.Incontrast, thatsuchaneffectoftheeliminationinABCG4 ABCG1andABCG4appeartobethemajorly transcriptionmayreflectthedecreasedneedof expressedtransportersinthebrainduringpostnatal cholesterol,whichwouldcompromisethebrain developmentandarelikelytobeimportantfor development. cholesterolefflux. Furthermore,ABCG4mRNAandproteinlevels Asalreadynoted,themousebrainvolume areremarkablyelevatedafter28daysduring ··774 生物化学与生物物理进展 Prog.Biochem.Biophys. 2014;41(8) development,whichisaccompaniedwiththeslight accumulationofcholesterolandalteredexpressionof decreaseofABCG1proteinlevels.Theevidenceof genesinvolvedinthebiosynthesisoffreecholesterol. complementaryexpressionofABCG1andABCG4 Althoughbrainmitochondrialcholesteroloverloading proteinsduringlatedevelopmentsuggestthatABCG1 hasbeenreportedtocauseneuronalapoptosisina andABCG4couldfunctionallycompensateforone varietiesofthecentralnervoussystem(CNS)diseases anotherinredistributionofcholesterollevelsinthe withgenemutationsorgeneticanimalsmodelswhich brain.However,ABCG1andABCG4protein affectcholesterolhomeostasis [18-21],nosignificant expressionarenotcoincidentwiththemRNA pathologicalormorphologicalchangeswerefoundin expressionduringdevelopment.Onecouldexpectthat theofthebrainofABCG1-/- ABCG4-/- (datanot anotherregulatorymechanismofthetransporters shown).UpregulationofABCA1secondarytothe existsatthetranslationallevels.However,this deficiencyofABCG1andABCG4indoubleknockout remainstobedetermined. micemayincreasecholesteroleffluxfromthebrain Serumfreecholesterollevelswerelowthrough usingalternativecholesteroltransportpathwaysand alldevelopmentaltimepoints,whichisconsistentwith ameliorateneuronalstresscausedbycholesterol whathavepreviouslybeendemonstratedinadult accumulation.Morepronouncedelevationin mice [11].Inaddition,esterifiedcholesterollevels cholesterollevelsanddefectsintheCNSareexpected increasedsteadilythroughoutdevelopmentreaching tobeseeninthemicewithcombineddeficiencyof previouslydescribedlevelsbyadulthood. ABCG1,ABCG4andABCA1. Unexpectedlyhowever,therewasabiphasic Inconclusion,distincttemporalexpressionof distributionwithaminorpeakfrom7～14days. 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转运蛋白 ABCG1/4 和 ABCA1 对脑胆固醇的调节作用 *

孙海梅 1) 陈莉2,3) 苗劲蔚 4)** (1) 首都医科大学基础医学院，组织学与胚胎学教研室，北京 100069； 2) CenterforClinicalandTranslationalResearch,TheResearchInstituteatNationwideChildren sHospital,Columbus,OH 43205,USA；忆 3) 北京市中关村医院，北京 100190； 4)首都医科大学附属北京妇产医院妇瘤科，北京 100006)

摘要脑是富含胆固醇的器官，机体大约有 25%的胆固醇集中在脑组织中． ATP 结合盒超家族转运蛋白对脑组织中胆固醇的膜外转运和动态平衡起着重要的调节作用．研究发现，ATP 结合盒超家族转运蛋白亚体 ABCG1、ABCG4 和 ABCA1 在成体脑组织中存在不同程度的表达，一种或多种亚体的缺失可以导致神经退行性病变．然而，ATP 结合盒超家族转运蛋白亚体对脑发育过程中脑胆固醇动态变化的调节缺乏相关性的报道．在本研究中，从低胆固醇饮食喂养的 C57BL/6J 小鼠中获取出生后不同发育时期的脑组织，对 ABCG1、ABCG4 和 ABCA1 的 mRNA 与蛋白质表达水平进行测定，并对脑组织和血清中 ATP 结合盒超家族转运蛋白的表达水平与胆固醇水平的相关性进行研究．同时，使用 ABCG1、ABCG4单一基因敲除鼠和 ABCG1、ABCG4双基因敲除鼠，研究 ATP 结合盒超家族转运蛋白对与胆固醇合成的相关基因表达的影响以及对脑组织胆固醇代谢的调节作用．结果发现，ABCG1、ABCG4 和 ABCA1 在机体多个器官中均有表达，但 ABCG1 和 ABCG4 在小鼠脑组织中表达量最高．在脑组织发育过程中，ABCG1 和 ABCG4mRNA 水平呈现明显的表达时效性，小鼠于出生后 42 天达到峰值，而 ABCA1mRNA 的表达水平无明显变化．血清和脑组织中中酯化型胆固醇水平呈双高峰分布，也于出生后 42 天达到最高．基因敲除鼠模型显示，单一敲除 ABCG1 或者 ABCG4基因对脑组织胆固醇水平无明显影响，而 ABCG1 和 ABCG4基因的同时缺失导致脑胆固醇水平显著升高，并明显降低胆固醇合成相关基因的表达水平．本研究表明，在脑发育成熟过程中，ATP 结合盒超家族转运蛋白亚体 ABCG1 和 ABCG4，而非 ABCA1，以调节脑胆固醇的膜外转运；ABCG1 和 ABCG4 互补调控脑胆固醇的动态平衡．

关键词 ATP 结合盒超家族转运蛋白，脑发育，胆固醇学科分类号 Q7 DOI:10.3724/SP.J.1206.2014.00012

* 北京市自然科学基金资助项目(7102056). ** 通讯联系人. Tel:010-52277296,E-mail:[email protected] 收稿日期：2014-01-10，接受日期：2014-06-23