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1、老年痴呆实验动物模型 研制进展 Research Progress of Animal Models for Alzheimers Disease 张 斌 (Bin Zhang) M.D., Ph.D. Center for Neurodegenerative Disease Research, School of Medicine University of Pennsylvania Philadelphia, PA USA High Median Low Millions Year 美国 85岁岁以上的人口统计统计 US Population Demographics 85 Years o
2、f Age or Older: 1950-2050 Average life span in USA Male: 72.2 years old Females: 79.10 years old 美国老年痴呆发发病率 AD Incidence In US AD 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
3、 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 老年痴呆神经经病理 Neuropathology of AD 神经元纤维缠结(N FT ) Neurofibrillary Tangles Tau protein (细细胞内) 老年斑(SP) Senile Plaques 淀粉样蛋白 -Amyloid (A) (细细胞外) 理想的老年痴呆实验动物模型标标志 Idea AD Animal Models 老年痴呆病人老年痴呆动物模型 _ 病理表现现神经元纤维缠结 细细胞内形
4、成tau蛋白纤维 老年斑 细细胞外形成 淀粉样蛋白纤维 生化特点 Tau蛋白超磷酸化 类类似AD Ab, Tau蛋白降低溶解性类类似AD 行为为改变变 记忆记忆 力下降或丧丧失, 行为测试显为测试显 示 记忆记忆 力下降或丧丧失 _ 实验动物模型重复性, do not reduce the gene expressing level 实验动物模型实实用性, drug screen, mechanism study 老年痴呆实验动物模型种类类 Types of AD Animal Models 基因种类类: 正常和变异的基因 Normal and mutant genes Tau 蛋白, Tau
5、 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) 动动物种类类: 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 neuropathology
6、 in transgenic mice overexpressing V717F b-amyloid precursor protein. Dora Games et al., Nature 373, 523- 527 Tau transgeni c C. elegans. Kraemer et al., PNAS, 100:9982- 9985 Tau transgenic drosophila Jackson et al., Neuron 34:509-519 Gtz et al., Molecular Psychiatry 9, 664 老年痴呆实验动物模型种类类(续续) Types o
7、f AD Animal Models 基因数量 单单基因模型 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 基因表达的细细胞 神经经元细细胞 neuron 神经经胶质细质细胞 Neuronal glial cell 少突胶质细质细胞 Oligodendrocyte 星形胶质细质细胞 Astrocyte Tau and APP double Tg mice. Lowis et al., Science 293:1487- 1491
8、Makoto Higuchi, Bin Zhang, 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, 25:3539 1. Prepar
9、e your DNA + promoter 2. Transform fertilized eggs Harvest 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.
10、 3. Implant the embryos in a 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类(续续) Types of AD Animal Models
11、基因启动动子种类类 -人Prion 基因启动子 human PrP promoter (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. Sa
12、ntaCruz et al., Science, 309: 476-481, 2005 I. 淀粉样蛋白连锁反应假说 The Amyloid Cascade Hypothesis By Dennis Selkoe, Harvard University II. 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 University III. 蛋白质错误 折叠和轴突转运障碍假说 Tau Related Axonal Transport Dysfunction Hypot
13、hesis By John Trojanowski, University of Pennsylvania 2005 Update 老年痴呆发发病机理研究 AD Pathogenesis Studies Dennis 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 淀粉样蛋白连锁连锁 反应应假说说 The Amyloid Cascade Hypo
14、thesis II. 载载脂蛋白对对 A的作用假说说 Effects of ApoE on Amyloid-b Hypothesis II. 载载脂蛋白对对 A的作用假说说 (续续) Effects of ApoE on Amyloid-b Hypothesis III. 蛋白质错误质错误 折叠和轴轴突转转运障碍假说说 IV.Protein Misfolding and Axonal Transport Dysfunction Hypothesis Mechanisms Of Protein Misfolding And Brain Amyloidosis Forman M, Trojanow
15、ski, JQ, Lee VM-Y. Nat Med, 2004 a-synuclein tau vesicles Tau aggregation/hyperphosphorylation Decreased MT bound tau MT depolymerization Impaired axonal transport Axonal degeneration -appears to arrest cells in mitosis by stabilizing spindle MTs. -induce MT polymer mass in cell and “MT bundling”; -
16、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), PaxceedTM is a Micellar formulation that does not cause the inflammatory reactions; -Micellar paclitaxel is soluble in sa
17、line and once in the bloodstream, the micelles break down so that the paclitaxel can bind to serum albumin for bioavailability. 微管稳稳定药药物 MT Stabilizing Drugs -Over express the shortest tau isoform (T44) with mouse PrP promotor; Tau inclusions begin to accumulate in spinal cord of 9 month old T44 tau
18、 Tg mice in association with the onset of motor impairments. Neuron, Ishihara et al., 1999 T44 Tau Tg mice NFTs accumulate with age in cortical neurons of T44 tau Tg mice. Pre-embedding immuno-EM shows tau immunoreactive straight filaments in cortical NFTs of a 24 month old T44 tau Tg mouse. AJP, Is
19、hihara et al., 2001 T44 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 轴突转转运 障碍 低剂剂量微管稳稳定药药物改善轴轴突转转运 Fast Axonal Transport in the Ventral Root o is Improved in the Low Dose of PaxceedTM Treated T44 Tg Mice B: Ratio o
20、f proteins amount In each segment = Treated / Sham C: Percentage of TP Proteins in each segments = (protein in 1 segment / total proteins in 5 segments) x 100/100 Bin Zhang, Arpita Maiti, Sharon Shively, Fara Lakhani, Gaye McDonald-Jones, Jennifer Bruce, Edward B. Lee, Sharon X. Xie, Sonali Joyce, C
21、hi Li, Philip M. Toleikis, Virginia M.-Y. Lee, and John Q. Trojanowski PNAS 2005 102: 227-231 中等剂剂量微管稳稳定药药物改善轴轴突转转运 Fast Axonal Transport in the Ventral Root is Improved in the Medium Dose of PaxceedTM Treated T44 Tg Mice Medium 大剂剂量微管稳稳定药药物减缓缓轴轴突转转运 Fast Axonal Transport in the Ventral Root is Reta
22、rded in the High Dose of PaxceedTM Treated T44 Tg Mice MTs in the VR Axons of the Tau Tg Mice Treated with PaxceedTM The Numbers of MTs in the VR Axons Increase in the tau Tg Mice Treated with PaxceedTM % % % % % Bin Zhang, Arpita Maiti, Sharon Shively, Fara Lakhani, Gaye McDonald-Jones, Jennifer Br
23、uce, 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 Stable Detyrosinated Tubulin is Increased in the Ventral Root of T44 Tg Mice Treated with PaxceedTM Summary Dose dependant improvement of fast axonal transp
24、ort 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 ta
25、uopathies. Acknowledgement Angiotech Pharmaceuticals Inc. Canada Arpita Maiti, Philip M. Toleikis, The University of Pennsylvania The Center for Neurodegenerative Disease Research Makoto Higuchi, MD. PhD. Yasumasa Yoshiyama, MD. PhD. Sona Sundarraj Mark S. Forman, MD. PhD. Lewis Z. Leng, Christine K
26、aminski, Susan Leight, Virginia M.-Y. Lee MS. PhD John Q. Trojanowski MD. PhD Acknowledgements The Center for Neurodegenerative Disease Research Makoto Higuchi, MD. PhD. Yasumasa Yoshiyama, MD. PhD. Sona Sundarraj Mark S. Forman, MD. PhD. Lewis Z. Leng, Christine Kaminski, Susan Leight, Virginia M.-
27、Y. Lee MS. PhD John Q. Trojanowski MD. PhD The University of Pennsylvania USA AD Animal Mmodel Comparison Privacy Policy 2004 Nature Publishing Group II. 载载脂蛋白对对 A的作用假说说 (续续) Effects of ApoE on Amyloid-b Hypothesis AD is a clinicopathological syndrome in which different gene defects can leaddirectly
28、 or indirectlyto altered APP expression or proteolytic processing or to changes in A stability or aggregation. These result in a chronic imbalance between A production and clearance. Gradual accumulation of aggregated A initiates a complex, multistep cascade that includes gliosis, inflammatory changes, neuritic/synaptic change, tangles and transmitter loss. By Dennis Selkoe. Harvard University I. 淀粉样蛋白连锁反应假说 The Amyloid Cascade Hypothesis