老年痴呆实验动物模型老年痴呆实验动物模型研制进展研制进展Research Progress of Animal Models for Alzheimers Disease张张 斌斌(Bin Zhang) M.D., Ph.D.Center for Neurodegenerative Disease Research, School of Medicine University of PennsylvaniaPhiladelphia, PA USAHighMedianLowMillionsYear美国美国 85岁岁以上的以上的人口人口统计统计US Population Demographics 85 Years of Age or Older: 1950-2050Average life span in USAMale: 72.2 years oldFemales: 79.10 years old美国美国老年痴呆老年痴呆发发病率病率AD Incidence In USAD afflicts 4 million Americans 100,000 die/year.AD occurs in men and women of all ethnic groups and at all socioeconomic levels.The US Census Bureau predicts that AD will affect 14 million Americans by 2050.美国美国老年痴呆老年痴呆病人数量病人数量 (百万百万)AD Patients In USA 1980 2005 2050老年痴呆老年痴呆神神经经病理病理Neuropathology of AD神经元纤维缠结神经元纤维缠结(N FT )Neurofibrillary TanglesTau protein(细细胞内胞内)老年斑老年斑(SP) Senile Plaques淀粉样蛋白淀粉样蛋白-Amyloid (A)(细细胞外胞外)理想的理想的老年痴呆实验老年痴呆实验动物动物模型模型标标志志 Idea AD Animal Models老年痴呆老年痴呆病人病人老年痴呆老年痴呆动物动物模型模型_病理表病理表现现神经元纤维缠结神经元纤维缠结 细细胞内形成胞内形成tautau蛋白蛋白纤维纤维老年老年斑斑 细细胞外形成胞外形成 淀粉样蛋白纤维淀粉样蛋白纤维生化特点生化特点 Tau蛋白超磷酸化蛋白超磷酸化 类类似似AD A Ab b, , TauTau蛋白降低溶解性蛋白降低溶解性类类似似AD行行为为改改变变 记忆记忆力下降或力下降或丧丧失失, , 行行为测试显为测试显示示 记忆记忆力下降或力下降或丧丧失失_实验实验动物动物模型模型重复性重复性, do not reduce the gene expressing level, do not reduce the gene expressing level实验实验动物动物模型模型实实用性用性, drug screen, mechanism study, drug screen, mechanism study老年痴呆实验动物模型老年痴呆实验动物模型种种类类Types of AD Animal Models基因种基因种类类: 正常和变异的基因NormalandmutantgenesTau蛋白,Tauproteinb淀粉样前蛋白APPPresenin1,2,PS1,2载脂蛋白E,ApolipoproteinE(ApoE)酶bsecrataseCyclindependentkinase5(CDK5)glycogensynthasekinase3(GSK3)动动物种物种类类:非脊椎动物模型果蝇模型Drosophilamodel线虫模型NematodeCaenorhabditiselegansmodel脊椎动物模型小鼠模型mousemodel大鼠模型ratmodelAlzheimer-typeneuropathologyintransgenicmiceoverexpressingV717Fb-amyloidprecursorprotein.DoraGamesetal.,Nature373,523-527Tau transgenic C. elegans.Kraemer et al., PNAS, 100:9982-9985Tau transgenic drosophilaJackson et al., Neuron 34:509-519Gtzetal.,MolecularPsychiatry9,664老年痴呆实验动物模型老年痴呆实验动物模型种种类类(续续)Types of AD Animal Models基因数量基因数量 单单基因模型基因模型 Single Tg model, Tau, APP, PS1 双基因模型双基因模型 double Tg model, Tau+APP, APP+PS1 三基因模型三基因模型 triple Tg model, Tau+APP+PS1基因表达的基因表达的细细胞胞 神神经经元元细细胞胞 neuron 神神经经胶胶质细质细胞胞 Neuronal glial cell 少突胶少突胶质细质细胞胞 Oligodendrocyte 星形胶星形胶质细质细胞胞 AstrocyteTauandAPPdoubleTgmice.Lowisetal.,Science293:1487-1491Makoto Higuchi, Bin Zhang, Mark S. Forman, Yasumasa Yoshiyama, John Q. Trojanowski, and Virginia M.-Y. Lee J. Neurosci., 25: 9434, 2005 MarkS.Forman,DevikaLal,BinZhang,DeepaV.Dabir,EricSwanson,VirginiaM.-Y.Lee,andJohnQ.TrojanowskiTheJournalofNeuroscience,2005,25:35391.PrepareyourDNA+promoter2.TransformfertilizedeggsHarvestfreshlyfertilizedeggsbeforethespermheadhasbecomeapronucleus.InjectthemalepronucleuswithyourDNA.Whenthepronucleihavefusedtoformthediploidzygotenucleus,allowthezygotetodividebymitosistoforma2-cellembryo.3.Implanttheembryosinapseudopregnantfostermotherandproceedoffsprings. -Overexpressprotein转基因动物模型transgenicanimalmodel.-基因敲除动物模型geneknockoutanimalmodel-基因敲入动物模型geneknockinanimalmodel-条件基因动物模型conditionalanimalmodel基因引入基因引入动物动物的方法的方法老年痴呆实验动物模型老年痴呆实验动物模型种种类类(续续)Types of AD Animal Models 基因启基因启动动子种子种类类 -人Prion基因启动子humanPrPpromoter(极微小的蛋白质颗粒，类似于病毒，但不含核酸，被认为是绵羊痒病和某些神经系统变性疾病的传染介质)-鼠Prion基因启动子murinePrPpromoter，-鼠CNP基因启动子murineCNPolygodendrocytepromoter-鼠胶质原纤维酸性蛋白启动子murineGFAPpromoter-鼠钙调蛋白激酶IIa启动子MouseCaMKIIapromotor(鼠产生的启动子，钙调蛋白激酶IIa驱动基因表达在产后二周的前脑和海马部位) K. SantaCruz et al., Science, 309: 476-481, 2005I. 淀粉样蛋白连锁反应假说TheAmyloidCascadeHypothesisByDennisSelkoe,HarvardUniversityII.载脂蛋白对淀粉样蛋白的作用假说EffectsofApoEonAmyloid-bHypothesisByDavidM.Holtzman,WashingtonUniversityIII.蛋白质错误折叠和轴突转运障碍假说TauRelatedAxonalTransportDysfunctionHypothesisByJohnTrojanowski,UniversityofPennsylvania2005Update老年痴呆老年痴呆发发病机理研究病机理研究AD Pathogenesis StudiesDennis Selkoe, Nature. 360(6405):672-4 , Proc Natl Acad Sci U S A 91(25):11993-7.Cai XD et al., Science, 259 :514-6. Suzuki N et. al., Science, 264:1336-40淀粉样蛋白淀粉样蛋白连锁连锁反反应应假假说说 The Amyloid Cascade HypothesisII. 载载脂蛋白脂蛋白对对 A的作用假的作用假说说 Effects of ApoE on Amyloid-b b HypothesisII. 载载脂蛋白脂蛋白对对 A的作用假的作用假说说 (续续) Effects of ApoE on Amyloid-b b HypothesisIII. 蛋白蛋白质错误质错误折叠和折叠和轴轴突突转转运障碍假运障碍假说说 Protein Misfolding and Axonal Transport Dysfunction HypothesisMechanisms Of Protein Misfolding And Brain Amyloidosis FormanM,Trojanowski,JQ,LeeVM-Y.NatMed,2004 a a-synucleintauvesiclesTau aggregation/hyperphosphorylationDecreased MT bound tauMT depolymerization Impaired axonal transportAxonal degeneration & disconnectionMicrotubulesPathologicalaggregatesDeleterious Effects Of Tau Amyloidosis 人人类类六个六个 tau 蛋白蛋白亚亚型型的形成的形成Human tau gene is alternatively spliced to produce six isoformsExons: 0 1 2 3 4 4a 5 6 7 8 9 10 11 12 13 14E2E3R1R1R1R1R1R1R2R2R2R3R3R3R3R3R3R4R4R4R4R4R4111111441412383410381352Tau ProteinIsoforms4R/2N4R/1N4R/0N3R/2N3R/1N3R/0NE2E2E2E3Aminoterminalrepeats氨基末端重复序列MicrotubulebindingrepeatsAlternativelysplicedGENETranscribedAlternativelysplicedTranslated微管结合重复序列Tau基因的基因的变变异异Mutations in the Tau GeneEXON 1EXON 9EXON 11EXON 12EXON 13K257TI260VG272VE342VV337MR406WG389RR5H/LL266VS320FK369I-AAUAAGAAGCUGGAUCUU-AAT-CCGIntron 9N279KL284LD DN296N296HN296ND DK280L315RS352LQ336REXON 10P301SP301LS305NS305SINTRON 10SPLICESILENCERINTRON 10SPLICEMODULATORIntron 10+3+11+13+12+14+16EXON 10TheR406Wmutation,locatedonexon13ofthetaugene,wasidentifiedin3distinctFTDP-17kindreds.精氨酸精氨酸 色氨酸色氨酸正常正常 Tau蛋白蛋白的功能的功能Normal Tau Protein大量的低分子重量的微管相关旦白大量的低分子重量的微管相关旦白 Anabundantlowmolecularweightmicrotubule-associatedprotein分布在分布在轴轴突突 predominantlyinaxons促促进进并并稳稳定微管聚合定微管聚合, 维维持正常的神持正常的神经经元元轴轴突突转转运运 Promotesmicrotubule(MT)polymerization,bindstoMTsandstabilizesMTs磷酸化磷酸化降低与微管的降低与微管的结结合能力合能力 PhosphorylationnegativelyregulatesthebindingoftautoMTs正常正常Tau旦白的功能旦白的功能: 稳定微管并维持正常的神经元元轴突转运Normal Tau Stabilizes MTs To Maintain Neuronal Transport Tau病病变变引起神引起神经经元元轴轴突突转转运障碍运障碍 While Tau Pathology Can Disrupt Neuronal Transport With Catastrophic Consequences Equivalent To A Train Wreck 假假说说 Hypothesis R406W Tau 转转基因小鼠的研制基因小鼠的研制Generation of T40hWT and T40R406W Tau Tg mice精氨酸精氨酸 色氨酸色氨酸Bin Zhang, Makoto Higuchi, Yasumasa Yoshiyama, Takeshi Ishihara, Mark S. Forman, Dan Martinez, Sonali Joyce, John Q. Trojanowski, and Virginia M.-Y. Lee , J Neurosci J. Neurosci., 24: 4657 RW Tau 蛋蛋白表达水平白表达水平溶解性溶解性降低降低微管微管结结合能力下降合能力下降方法：免疫方法：免疫电电泳泳N-Tg: 正常非正常非转转基因小鼠基因小鼠hWT: 正常正常 Tau 转转基因小鼠基因小鼠RW: RW Tau 变变异异转转基因小鼠基因小鼠溶液溶液强强度度RAB RIPA FA细细胞内胞内Tau病病变变: 类类似似神经元纤维缠结神经元纤维缠结(NFT)脊髓脊髓新皮新皮质质小小脑脑海海马马新皮新皮质质RWTgmiceDevelopSomatodendriticTauPathologyResemblingNFTs正常正常Tau 转转基因小鼠基因小鼠RW变变异异转转基因小鼠基因小鼠FTDP-17: FrontaltempledementiaParkinsonsm-17Chromason额颞叶额颞叶痴呆痴呆巴金氏巴金氏病病-17染色体染色体方法：免疫方法：免疫组组化化免疫免疫电镜电镜研究研究:RW Tau 蛋白白的的纤维纤维形成形成12m12m海海马马小小脑脑新皮新皮质质脊髓脊髓低倍低倍 高倍高倍RW: RW Tau 变变异异转转基因小鼠基因小鼠RW Tau 蛋蛋白白的分布的分布改改变变: : 从从轴轴突突到到细细胞体胞体小小脑脑新皮新皮质质Accumulation of Tau in the Neuronal Cell Soma方法：免疫方法：免疫组组化化正常正常 Tau 转转基因小鼠基因小鼠RW Tau 变变异异转转基因小鼠基因小鼠RW Tau 蛋蛋白白的分布的分布改改变变: : 从从轴轴突突到到细细胞体胞体Absence of Tau Staining in the Massy Fibers of Hippocampus in RW Mice 海海马马方法：方法：免疫免疫组组化化hWT: 正常正常 Tau 转转基因小鼠基因小鼠RW: RW Tau 变变异异转转基因小鼠基因小鼠Tau 蛋蛋白白抗体抗体 低分子量神低分子量神经丝经丝抗体抗体神神经轴经轴突突转转运的机制运的机制Molecular Machinery For Axonal Transport轴突Axon神经元胞体NeuronalCellbody顺行转运,逆行转运Fast transport: 1-4 mm/hour, Vesicles, TransmittersSlow Transport: 0.2-0.4 mm/day, NFs, Tub, Actin缓缓慢慢轴轴突突转转运研究运研究Slow Axonal Transport -35S-labeledmethionine(甲硫氨酸)wasmicroinjectedintotheL4-6ventralhornusingastereotaxicapparatus.-Groupsofanimalsweresacrificed7daysaftermicroinjectionfortheanalysisofslowaxonaltransport.TheL4-5ventralrootswereremovedandcutintoconsecutive5segmentsof2mmlengthfromproximaltodistal.2 m6m RW line 37RW Tau 蛋蛋白白的的轴轴突突转转运运障碍障碍方法：方法：免疫沉淀免疫沉淀 (IP), (IP), 免疫免疫电电泳泳hWT: 正常正常 Tau 转转基因小鼠基因小鼠RW: RW Tau 变变异异转转基因小鼠基因小鼠Tau 蛋蛋白抗体白抗体 低分子量神低分子量神经丝经丝抗体抗体12 m6m SlowAxonalTauTransportisretardedintheearlystageofRWMiceRW line 60RW Tau 蛋蛋白白的的轴轴突突转转运运障碍障碍RW line 37方法：方法：免疫沉淀免疫沉淀 (IP), (IP), 免疫免疫电电泳泳hWT: 正常正常 Tau 转转基因小鼠基因小鼠RW: RW Tau 变变异异转转基因小鼠基因小鼠Tau 蛋蛋旦白抗体旦白抗体 低分子量神低分子量神经丝经丝抗体抗体NFM17026RW Tau 蛋蛋白白的的轴轴突突转转运运障碍障碍方法：免疫沉淀方法：免疫沉淀 (IP), (IP), 免疫免疫电电泳泳N-Tg: 正常非正常非转转基因小鼠基因小鼠hWT: 正常正常 Tau 转转基因小鼠基因小鼠RW: RW Tau 变变异异转转基因小鼠基因小鼠中中分子量神分子量神经丝经丝抗体抗体Tau 旦白旦白抗体抗体脊髓腹根脊髓腹根结结扎扎SinceabnormalPHFtauproteinscouldnotperformthenormalfunctionofbindingtoandstabilizingmicrotubules(MTs)inthepolymerizedstate,couldfurthereffectMTfunctions,especiallyfastaxonaltransport.Thehypothesis:MT stabilizing drugs such as PaxceedTM delivering to the CNS could have therapeutic benefit in brains with tauopathies假假说说 Hypothesis TaxolR (Paclitaxel,mw854)(Paclitaxel,mw854)-isolated in 1971 from plant, the pacific yew Taxus brevifonia -the first of a class of MT stabilizing drug which was approved for use in the treatment of breast, ovarian and lung cancers; -appears to arrest cells in mitosis by stabilizing spindle MTs. -induce MT polymer mass in cell and “MT bundling”; -readily soluble in methanol, but limited soluble in water; PaxceedTM -A new MT Stabilizing anti-neoplastic agent by Angiotech Inc.; -In contrast to Taxol (CremaphorCremaphorformulation)formulation), PaxceedTM is a Micellar formulation that does not cause the inflammatory reactions; -Micellar paclitaxel is soluble in saline and once in the bloodstream, the micelles break down so that the paclitaxel can bind to serum albumin for bioavailability. 微管微管稳稳定定药药物物MT Stabilizing Drugs-Overexpresstheshortesttauisoform(T44)withmousePrPpromotor;Tauinclusionsbegintoaccumulateinspinalcordof9montholdT44tauTgmiceinassociationwiththeonsetofmotorimpairments.Neuron,Ishiharaetal.,1999T44 Tau Tg miceNFTsaccumulatewithageincorticalneuronsofT44tauTgmice.Pre-embeddingimmuno-EMshowstauimmunoreactivestraightfilamentsincorticalNFTsofa24montholdT44tauTgmouse.AJP,Ishiharaetal.,2001T44 Tau Tg mice- Retardation of fast axonal transport has been fund in these mice at 12 months old.Neuron,Ishiharaetal.,1999 快速快速轴轴突突转转运运障碍障碍低低剂剂量量微管微管稳稳定定药药物物改善改善轴轴突突转转运运Fast Axonal Transport in the Ventral Root o is Improved in the Low Dose of PaxceedTM Treated T44 Tg MiceB:RatioofproteinsamountIneachsegment=Treated/ShamC:PercentageofTPProteinsineachsegments=(proteinin1segment/totalproteinsin5segments)x100/100BinZhang,ArpitaMaiti,SharonShively,FaraLakhani,GayeMcDonald-Jones,JenniferBruce,EdwardB.Lee,SharonX.Xie,SonaliJoyce,ChiLi,PhilipM.Toleikis,VirginiaM.-Y.Lee,andJohnQ.TrojanowskiPNAS2005102:227-231中等中等剂剂量微管量微管稳稳定定药药物物改善改善轴轴突突转转运运 Fast Axonal Transport in the Ventral Root is Improved in the Medium Dose of PaxceedTM Treated T44 Tg MiceMedium大大剂剂量微管量微管稳稳定定药药物物减减缓缓轴轴突突转转运运 Fast Axonal Transport in the Ventral Root is Retarded in the High Dose of PaxceedTM Treated T44 Tg MiceMTs in the VR Axons of the Tau Tg Mice Treated with PaxceedTMThe Numbers of MTs in the VR Axons Increase in the tau Tg Mice Treated with PaxceedTM%BinZhang,ArpitaMaiti,SharonShively,FaraLakhani,GayeMcDonald-Jones,JenniferBruce,EdwardB.Lee,SharonX.Xie,SonaliJoyce,ChiLi,PhilipM.Toleikis,VirginiaM.-Y.Lee,andJohnQ.TrojanowskiPNAS2005102:227-231Stable Detyrosinated Tubulin is Increased in the Ventral Root of T44 Tg Mice Treated with PaxceedTMSummaryDose dependant improvement of fast axonal transport in PrpT44 Tg mice by PaxceedTM treatment indicates that PaxceedTM treatment in the tau transgenic mice with existed retarded axonal transport is effective. MT stabilizing drugs, PaxceedTM could be a candidate for the further studies in treatment of AD and other neurodegenerative diseases with tauopathies.AcknowledgementAngiotech Pharmaceuticals Inc. Canada Arpita Maiti, Philip M. Toleikis,The University of PennsylvaniaThe Center for Neurodegenerative Disease ResearchMakotoHiguchi,MD.PhD.YasumasaYoshiyama,MD.PhD.SonaSundarrajMarkS.Forman,MD.PhD.LewisZ.Leng,ChristineKaminski,SusanLeight,VirginiaM.-Y.LeeMS.PhDJohnQ.TrojanowskiMD.PhDAcknowledgementsThe Center for Neurodegenerative Disease ResearchMakoto Higuchi, MD. PhD.Yasumasa Yoshiyama, MD. PhD.Sona SundarrajMark S. Forman, MD. PhD. Lewis Z. Leng, Christine Kaminski, Susan Leight, Virginia M.-Y. Lee MS. PhD John Q. Trojanowski MD. PhDThe University of Pennsylvania USAAD Animal Mmodel ComparisonPrivacy Policy 2004 Nature Publishing GroupII. 载载脂蛋白脂蛋白对对 A的作用假的作用假说说 (续续) Effects of ApoE on Amyloid-b b HypothesisADisaclinicopathologicalsyndromeinwhichdifferentgenedefectscanleaddirectlyorindirectlytoalteredAPPexpressionorproteolyticprocessingortochangesinAstabilityoraggregation.TheseresultinachronicimbalancebetweenAproductionandclearance.GradualaccumulationofaggregatedAinitiatesacomplex,multistepcascadethatincludesgliosis,inflammatorychanges,neuritic/synapticchange,tanglesandtransmitterloss.By Dennis Selkoe. Harvard UniversityI. 淀粉样蛋白淀粉样蛋白连锁反应假说 The Amyloid Cascade Hypothesis