老年痴呆实验动物模型研制进展

1、HighMedianLowMillionsYear美国 85岁以上的US Population Demographics 85 Years of Age or Older: 1950-2050Average life span in USAMale: 72.2 years oldFemales: 79.10 years old美国老年痴呆发病率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 s

2、ocioeconomic levels.The US Census Bureau predicts that AD will affect 14 million Americans by 2050.美国老年痴呆病人数量 (百万) AD Patients In USA 1980 2005 2050老年痴呆神经病理Neuropathology of AD神经元纤维缠结(N FT )Neurofibrillary TanglesTau protein(细胞内)老年斑(SP) Senile Plaques淀粉样蛋白-Amyloid (A)(细胞外)理想的老年痴呆实验动物模型标志 Idea AD Ani

3、mal Models老年痴呆病人老年痴呆动物模型_病理表现神经元纤维缠结 细胞内形成tau蛋白纤维老年斑 细胞外形成 淀粉样蛋白纤维生化特点 Tau蛋白超磷酸化 类似AD Ab, Tau蛋白降低溶解性类似AD行为改变 记忆力下降或丧失, 行为测试显示 记忆力下降或丧失_实验动物模型重复性, do not reduce the gene expressing level实验动物模型实用性, drug screen, mechanism study老年痴呆实验动物模型种类Types of AD Animal Models基因种类: 正常和变异的基因 Normal and mutant genes

4、Tau 蛋白, Tau protein b淀粉样前蛋白APP Presenin1, 2, PS1,2 载脂蛋白E, Apolipoprotein E (ApoE)酶 b secratase Cyclin dependent kinase5 (CDK5) glycogen synthase kinase3 (GSK3)动物种类: 非脊椎动物模型 果蝇模型 Drosophila model 线虫模型 Nematode Caenorhabditis elegans model 脊椎动物模型 小鼠模型 mouse model 大鼠模型 rat model Alzheimer-type neuropat

5、hology in transgenic mice overexpressing V717F b-amyloid precursor protein. Dora Games et al., Nature 373, 523-527 Tau transgenic C. elegans.Kraemer et al., PNAS, 100:9982-9985Tau transgenic drosophilaJackson et al., Neuron 34:509-519Gtz et al., Molecular Psychiatry 9, 664老年痴呆实验动物模型种类(续)Types of AD

6、Animal Models基因数量 单基因模型 Single Tg model, Tau, APP, PS1 双基因模型 double Tg model, Tau+APP, APP+PS1 三基因模型 triple Tg model, Tau+APP+PS1基因表达的细胞 神经元细胞 neuron 神经胶质细胞 Neuronal glial cell 少突胶质细胞 Oligodendrocyte 星形胶质细胞 AstrocyteTau and APP double Tg mice.Lowis et al., Science 293:1487-1491Makoto Higuchi, Bin Zh

7、ang, Mark S. Forman, Yasumasa Yoshiyama, John Q. Trojanowski, and Virginia M.-Y. Lee J. Neurosci., 25: 9434, 2005 Mark S. Forman,Devika Lal, Bin Zhang, Deepa V. Dabir, Eric Swanson, Virginia M.-Y. Lee,and John Q. Trojanowski The Journal of Neuroscience,2005, 25:35391. Prepare your DNA + promoter2. T

8、ransform fertilized eggsHarvest freshly fertilized eggs before the sperm head has become a pronucleus. Inject the male pronucleus with your DNA. When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.3. Implant the embryos in a

9、 pseudopregnant foster mother and proceed offsprings. -Over express protein 转基因动物模型 transgenic animal model. -基因敲除动物模型 gene knockout animal model - 基因敲入动物模型 gene knockin animal model -条件基因动物模型 conditional animal model基因引入动物的方法老年痴呆实验动物模型种类(续)Types of AD Animal Models 基因启动子种类 -人Prion 基因启动子human PrP pr

10、omoter (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murine PrP promoter， -鼠CNP基因启动子murine CNP olygodendrocyte promoter -鼠胶质原纤维酸性蛋白启动子 murine GFAP promoter-鼠钙调蛋白激酶IIa 启动子 Mouse CaMKIIa promotor (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.

11、 淀粉样蛋白连锁反应假说 The Amyloid Cascade HypothesisBy Dennis Selkoe, Harvard UniversityII. 载脂蛋白对淀粉样蛋白的作用假说 Effects of ApoE on Amyloid-b Hypothesis By David M. Holtzman, Washington UniversityIII. 蛋白质错误折叠和轴突转运障碍假说 Tau Related Axonal Transport Dysfunction Hypothesis By John Trojanowski, University of Pennsylva

12、nia2005 Update老年痴呆发病机理研究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淀粉样蛋白连锁反应假说 The Amyloid Cascade HypothesisII. 载脂蛋白对 A的作用假说 Effects of ApoE on Amyloid-b Hypothesis

13、II. 载脂蛋白对 A的作用假说 (续) Effects of ApoE on Amyloid-b Hypothesis 蛋白质错误折叠和轴突转运障碍假说 Protein Misfolding and Axonal Transport Dysfunction HypothesisMechanisms Of Protein Misfolding And Brain Amyloidosis Forman M, Trojanowski, JQ, Lee VM-Y. Nat Med, 2004 a-synucleintauvesiclesTau aggregation/hyperphosphoryla

14、tionDecreased MT bound tauMT depolymerization Impaired axonal transportAxonal degeneration & disconnectionMicrotubulesPathologicalaggregatesDeleterious Effects Of Tau Amyloidosis 人类六个 tau 蛋白亚型的形成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

15、14E2E3R1R1R1R1R1R1R2R2R2R3R3R3R3R3R3R4R4R4R4R4R4111111441412383410381352Tau ProteinIsoforms4R/2N4R/1N4R/0N3R/2N3R/1N3R/0NE2E2E2E3Amino terminal repeats氨基末端重复序列Microtubule bindingrepeatsAlternatively splicedGENETranscribedAlternatively splicedTranslated微管结合重复序列Tau基因的变异Mutations in the Tau GeneEXON 1E

16、XON 9EXON 11EXON 12EXON 13K257TI260VG272VE342VV337MR406WG389RR5H/LL266VS320FK369I-AAUAAGAAGCUGGAUCUU-AAT-CCGIntron 9N279KL284LDN296N296HN296NDK280L315RS352LQ336REXON 10P301SP301LS305NS305SINTRON 10SPLICESILENCERINTRON 10SPLICEMODULATORIntron 10+3+11+13+12+14+16EXON 10The R406W mutation, located on e

17、xon 13 of the tau gene, was identified in 3 distinct FTDP-17 kindreds.精氨酸 色氨酸正常 Tau蛋白的功能Normal Tau Protein大量的低分子重量的微管相关旦白 An abundant low molecular weight microtubule-associated protein 分布在轴突 predominantly in axons促进并稳定微管聚合, 维持正常的神经元轴突转运 Promotes microtubule (MT) polymerization, binds to MTs and sta

18、bilizes MTs 磷酸化降低与微管的结合能力 Phosphorylation negatively regulates the binding of tau to MTs正常Tau旦白的功能: 稳定微管并维持正常的神经元轴突转运Normal Tau Stabilizes MTs To Maintain Neuronal Transport Tau病变引起神经元轴突转运障碍 While Tau Pathology Can Disrupt Neuronal Transport With Catastrophic Consequences Equivalent To A Train Wreck

19、 假说 Hypothesis R406W Tau 转基因小鼠的研制Generation of T40hWT and T40R406W Tau Tg mice精氨酸 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 蛋白表达水平溶解性降低微管结合能力下降

20、方法：免疫电泳N-Tg: 正常非转基因小鼠hWT: 正常 Tau 转基因小鼠RW: RW Tau 变异转基因小鼠溶液强度RAB RIPA FA细胞内Tau病变: 类似神经元纤维缠结(NFT)脊髓新皮质小脑海马新皮质RW Tg mice Develop Somatodendritic Tau Pathology Resembling NFTs正常Tau 转基因小鼠RW变异转基因小鼠FTDP-17: Frontal temple dementia Parkinsonsm-17 Chromason额颞叶痴呆巴金氏病-17染色体方法：免疫组化免疫电镜研究:RW Tau 蛋白的纤维形成12m海马小脑新皮

21、质脊髓低倍 高倍RW: RW Tau 变异转基因小鼠RW Tau 蛋白的分布改变: 从轴突到细胞体小脑新皮质Accumulation of Tau in the Neuronal Cell Soma方法：免疫组化正常 Tau 转基因小鼠RW Tau 变异转基因小鼠RW Tau 蛋白的分布改变: 从轴突到细胞体Absence of Tau Staining in the Massy Fibers of Hippocus in RW Mice 海马方法：免疫组化hWT: 正常 Tau 转基因小鼠RW: RW Tau 变异转基因小鼠Tau 蛋白抗体 低分子量神经丝抗体神经轴突转运的机制Molecul

22、ar Machinery For Axonal Transport轴突Axon神经元胞体 Neuronal Cell body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缓慢轴突转运研究Slow Axonal Transport -35S-labeled methionine (甲硫氨酸) was microinjected into the L4-6 ventral horn using a stereotaxic ap

23、paratus. -Groups of animals were sacrificed 7 days after microinjection for the analysis of slow axonal transport. The L4-5 ventral roots were removed and cut into consecutive 5 segments of 2 mm length from proximal to distal. 2 m6m RW line 37RW Tau 蛋白的轴突转运障碍方法：免疫沉淀 (IP), 免疫电泳hWT: 正常 Tau 转基因小鼠RW: RW

24、 Tau 变异转基因小鼠Tau 蛋白抗体 低分子量神经丝抗体12 m6m Slow Axonal Tau Transport is retarded in the early stage of RW Mice RW line 60RW Tau 蛋白的轴突转运障碍RW line 37方法：免疫沉淀 (IP), 免疫电泳hWT: 正常 Tau 转基因小鼠RW: RW Tau 变异转基因小鼠Tau 蛋旦白抗体 低分子量神经丝抗体NFM17026RW Tau 蛋白的轴突转运障碍方法：免疫沉淀 (IP), 免疫电泳N-Tg: 正常非转基因小鼠hWT: 正常 Tau 转基因小鼠RW: RW Tau 变异转

25、基因小鼠中分子量神经丝抗体Tau 旦白抗体脊髓腹根结扎Since abnormal PHF tau proteins could not perform the normal function of binding to and stabilizing microtubules (MTs) in the polymerized state, could further effect MT functions, especially fast axonal transport.The hypothesis: MT stabilizing drugs such as PaxceedTM deliv

26、ering to the CNS could have therapeutic benefit in brains with tauopathies假说 Hypothesis TaxolR (Paclitaxel, mw 854) -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 canc

27、ers; -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 (Cremaphor formulation),

28、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. 微管稳定药物MT Stabilizing DrugsOver express the shortest

29、 tau isoform (T44) with mouse PrP promotor;Tau inclusions begin to accumulate in spinal cord of 9 month old T44 tau Tg mice in association with the onset of motor impairments.Neuron, Ishihara et al., 1999T44 Tau Tg miceNFTs accumulate with age in cortical neurons of T44 tau Tg mice. Pre-embedding im

30、muno-EM shows tau immunoreactive straight filaments in cortical NFTs of a 24 month old T44 tau Tg mouse. AJP, Ishihara et al., 2001T44 Tau Tg mice- Retardation of fast axonal transport has been fund in these mice at 12 months old.Neuron, Ishihara et al., 1999 快速轴突转运障碍低剂量微管稳定药物改善轴突转运Fast Axonal Trans

31、port in the Ventral Root o is Improved in the Low Dose of PaxceedTM Treated T44 Tg MiceB: Ratio of proteins amount In each segment = Treated / ShamC: Percentage of TP Proteins in each segments = (protein in 1 segment / total proteins in 5 segments) x 100/100Bin Zhang, Arpita Maiti, Sharon Shively, F

32、ara Lakhani, Gaye McDonald-Jones, Jennifer Bruce, Edward B. Lee, Sharon X. Xie, Sonali Joyce, Chi Li, Philip M. Toleikis, Virginia M.-Y. Lee, and John Q. Trojanowski PNAS 2005 102: 227-231中等剂量微管稳定药物改善轴突转运 Fast Axonal Transport in the Ventral Root is Improved in the Medium Dose of PaxceedTM Treated T

33、44 Tg MiceMedium大剂量微管稳定药物减缓轴突转运 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%Bin Zhang, Arpita

34、Maiti, Sharon Shively, Fara Lakhani, Gaye McDonald-Jones, Jennifer Bruce, Edward B. Lee, Sharon X. Xie, Sonali Joyce, Chi Li, Philip M. Toleikis, Virginia M.-Y. Lee, and John Q. Trojanowski PNAS 2005 102: 227-231Stable Detyrosinated Tubulin is Increased in the Ventral Root of T44 Tg Mice Treated wit

35、h 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 C