凋亡相关基因caspase_3和NF_B在_省略_Tg2576转基因小鼠海马内的

37NeurosciBullFebruary1,2010,26(1):37-46.·OriginalArticle·*Theseauthorscontributedequallytothiswork.Correspondingauthor:Jin-BoDENGTel:0378-3880292;Fax:[phone]Email:[e-mail]:1673-7067(2010)01-0037-10Receiveddate:2009-06-12;Accepteddate:2009-09-18Expressionoftheapoptosis-relatedproteinscaspase-3andNF-κBinthehippocampusofTg2576miceYan-LiNIU*,Wei-JuanZHANG*,PingWU,BinLIU,Guo-TaoSUN,Dong-MingYU,Jin-BoDENGInstituteofNeurobiologyandLaboratoryofNeurobiology,NursingCollegeofHenanUniversity,Kaifeng475004,China©ShanghaiInstitutesforBiologicalSciences,CASandSpringer-VerlagBerlinHeidelberg2010Abstract:ObjectiveToinvestigatetherelationsbetweenneuroapoptosisandtheonsetanddevelopmentofAlzheimer’sdisease(AD),especiallytheroleofNF-κBintheregulationofneuroapoptosis.MethodsCaspase-3andNF-κB(p50)expressionsintheCA3regionofthehippocampusinAPPsweTg2576transgenicmicewerestudiedfrompostnatalday0-180,usingNisslstaining,immunohistochemistryandRT-PCRmethods.ResultsBothneuronalapoptosisandNF-κBactivitydecreasedgraduallywiththeincreaseofageinwildtypeandTg2576mice.However,thenumberofcaspase-3-positiveorNF-κB-positivepyramidalcellsinTg2576micewasgreaterthanthatinage-matchedwildtypemice,withsignificantdifferencesafterpostnatalday14(P0.01orP0.05).Linearregressionanalysesofcaspase-3andNF-κBexpressiondemonstratedacorrelationbetweenneuroapoptosisandactivityofNF-κB.ConclusionTheprocessofneuroapoptosisisconsistentwiththeonsetanddevelopmentofAD.Furthermore,theobservedcorrelationbetweenneuroapoptosisandNF-κBactivitysug-gestsaroleofNF-κBinhippocampalneuroapoptosis.Keywords:Alzheimer’sdisease;Tg2576transgenicmice;caspase-3;hippocampus;apoptosis;NF-κB1IntroductionAlzheimer’sdisease(AD)isadevastatingdiseaseaf-fectingthehealthofmanypeopleagedover65years.Symp-tomsofADincludememoryloss,languagedeterioration,poorjudgment,confusionandmoodswings.Theneuropa-thologyofADischaracterizedbyamyloidplaques,neurofibril-larytanglesanddegenerationofneuronsandsynapses[1].Inthepresentstudy,neuroapoptosiswasinvestigatedinAPPsweTg2576transgenicmiceatdifferentdevelopmentalstagesusingcaspase-3immunocytochemistryinanefforttofurtherunderstandthedevelopmentofneuroapoptosisinAD.Inaddition,activationofNF-κBinthehippocampuswasanalyzed.UnderstandingtheroleofNF-κBinneuroapoptosiswillbebeneficialtodevelopmentofnovelstrategiesforphar-macologicalinterventioninmanyneurodegenerativediseases.2Materialsandmethods2.1AnimalsAPPsweTg2576transgenicmicewereusedinthepresentstudy.Hemizygousmales(transgenicmice,TM)encodingtheSwedishFADdoublemutation,HuAPP695SWEunderthecontrolofahamsterprionproteinpromoter[2],werecrossedwithwildtype(WT)femalemiceonahybridC57BL/6-SJLbackground.AllthemicewerebornandbredintheanimalhousingfacilitiesattheInstituteofNeurobiologyatHenanUniversity,undera12-hlight/darkcycle.TailDNAwasextractedfromP10animals(P=postnataldays;P0=thefirst24hafterbirth)andgenotypingwasperformedbyPCRtodetecthAPPswepositiveanimalsusingthefollowingprimers:5'GTGGATAACCCCTCCCCCAGCCTAGACCA38NeurosciBullFebruary1,2010,26(1):37-463'and5'CTGACCACTCGACCAGGTTCTGGGT3'.Off-springgenotypeswereidentifiedasWT(-/-,normalmicelackingaPCRband)andheterozygote(+/-,witha466bpPCRband).Forthebehaviour,Tg2576miceappearedslowerinreactionthanWTmicedidafterpostnatal3months.Inaddition,Tg2576miceusuallycouldnotliveforlongerthan8months.Thesurvivalrateattheageof6monthsforTg2576micewas82%vs95%forWT.Attheageofoneyear,thesurvivalrateofTg2576micewaslessthan10%comparedto91%ofWTmice.BehaviourandsurvivalratesinTg2576micewereconsistentwithADonsetandprogression.Inor-dertofindtheprogressivecourseofAD,severalagepointswereselectedtoinvestigateneuroapoptosisandbrainpa-thologyofTg2576mice,includingP0,P7,P14,P30,P90andP180.Age-matchedWTlittermatesservedascontrol.Foreachagegroup,atleast8micewereincluded,amongwhich5wereforhistologicalanalysisand3wereforreversetran-scription-polymerasechainreaction(RT-PCR).Totally96micewereusedforpathologicalandRT-PCRanalyses.2.2HEstainingandNisslstainingmethodsPriortosacrifice,animalsweregivenalethaloverdoseofsodiumpentobar-bital(80mg/kg,i.p.)andthenperfusedtranscardiallywith4%paraformaldehydein0.1mol/Lphosphatebuffer(PB;pH7.2).Thewholebrainwasremovedusingafinespatula.Afterimmersioninfreshfixativefor1-2dat4ºC,brainsweredehy-dratedingradientsofethanolandembeddedinparaffin.Coronalsectionsofhippocampuswerecutata5-μmthickness,andHEstainingandNisslmethodwerecarriedout.2.3Caspase-3/NF-κBimmunohistochemistryandhema-toxylincounterstainingHippocampalsectionswerepreparedasdescribedabove(2.2).Toeliminatetheactivityofendog-enousperoxidases,sectionswereincubatedwith3%H2O2preparedin0.1mol/LPBfor15min.Afterbeingwashed,sectionswereincubatedfor30minin0.5%normalgoatse-rumtoblocknon-specificbinding.Thenrabbitanti-activatedcaspase-3(1:500;R&D)orrabbitanti-NF-κB,p50antibody(1:200,SantaCruz)wasaddedandsectionswereincubatedovernightat4ºC.Afterthat,sectionswereincubatedwithHRP-conjugatedgoatanti-rabbitIgG(1:500,Upstate)for3hatroomtemperature.Finally,sect