【作者】 张明；

【导师】 王玉炯； 贺林；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 维生素A是一种多效的微量营养元素,维生素A对于早期神经系统发育和成年神经再生有重要的调节作用。成年维生素A缺乏的啮齿类动物表现出运动能力和学习记忆功能的异常。一些研究表明维生素A通路与精神分裂症有潜在的联系:维生素A通路调控许多精神分裂症候选基因的表达;精神分裂症病人血浆和脑脊液中维生素A转运蛋白TTR和APOE含量异常;精神分裂症脑组织中维生素A转运与合成相关分子表达异常。此外维生素A通路还与阿尔茨海默症(AD)有关。维生素A可以调控淀粉样前体蛋白(APP)基因,以及APP水解酶的表达;维生素A缺乏动物表现出一些AD相关的分子变化:淀粉样蛋白聚集,胆碱乙酰转移酶和视黄酸受体RARα的下调。在本研究的第一部分工作中,我们检测了早期维生素A缺乏(VAD)小鼠的运动能力、长期记忆能力和热痛刺激的敏感性,并检测了早期VAD和谷氨酸受体拮抗剂MK-801(5-甲基二氢丙环庚烯亚胺马来酸)的双重作用对小鼠行为的影响。我们发现早期VAD小鼠展现出更高的旷场移动和旷场直立能力,以及热痛敏感性的增加;早期VAD小鼠对MK-801的注射更敏感:增加了MK-801诱导的刻板行为和共济失调行为,增加了MK-801对旷场直立的抑制作用,MK-801和维生素A缺乏的交互作用导致热痛不敏感。这些行为学的异常反映了某些类似精神分裂症的行为,如旷场行为增加和MK-801诱导的刻板行为增加。然而我们并没有发现早期VAD对长期恐惧记忆(步下法实验)的影响。此后我们利用蛋白质组学技术研究了早期VAD雄性小鼠海马、皮层和肝脏组织的蛋白质水平差异。我们还研究了神经胶质瘤细胞U251中维生素A活性形式视黄酸的合成抑制对蛋白质组的影响。我们鉴定了海马中23个VAD相关蛋白点,皮层中46个差异蛋白点,肝脏中38个差异蛋白点和U251细胞39个差异蛋白点。通过生物信息学分析(Ingenuity pathway analysis),我们发现VAD皮层和神经胶质细胞中的差异蛋白汇聚在氧化压力反应,神经递质代谢(丝氨酸和谷氨酸),线粒体功能和能量代谢异常(ATP合成); VAD在皮层中可能通过影响谷氨酸NMDA受体2B、细胞骨架和调控转录因子,来改变神经细胞的迁移、生长、发育和神经突触的可塑性。其中3磷酸甘油酸脱氢酶(PHGDH)在视黄酸合成抑制的U251细胞中显著上调得到了Western blot的验证,而PHGDH同样在VAD皮层中显著上调。在VAD雄性小鼠海马中差异蛋白汇聚在脂代谢、神经轴突生长和突触塑形性等功能。VAD可能通过调节细胞骨架和细胞膜相关蛋白来影响神经递质的释放,多巴胺信号通路,谷氨酸NMDA受体2B介导的通路,以及神经细胞的再生和迁移。在肝脏组织中,维生素A缺乏可以造成糖代谢和脂代谢的异常,这同我们在皮层中的发现是一致的。因此我们认为早期VAD可能影响皮层和海马能量代谢和细胞骨架,影响谷氨酸通路和多巴胺通路,从而造成小鼠的类精神分裂症行为(旷场行为增加,MK-801诱导的刻板行为增加)。这些结果进一步支持维生素A缺乏与神经精神疾病有关的假说。SynapsinⅡ是一种神经元磷酸化蛋白,位于神经突触囊泡,与细胞骨架相连,参与调节神经递质的释放。有一些报道提示SynapsinⅡ与精神分裂症有关:精神分裂症病人海马与皮层中Synapsin II mRNA与蛋白含量降低;关联分析表明Synapsin II与精神分裂症显著相关(汉族人群,韩国人群,北欧人);此外Synapsin II基因敲除小鼠表现出类精神分裂症的行为异常:感觉门控,社交退缩。SynapsinⅡ由2种剪切异形体:SynapsinⅡa和SynapsinⅡb。以往的研究表明在双向凝胶上每种SynapsinⅡ剪切异形体均表现出多个翻译后修饰亚型。然而目前对于SynapsinⅡ翻译后修饰的信息了解甚少。在本研究的第二部分工作中,我们利用多种蛋白酶水解和串联质谱技术(LC-ESI-QTOF, LC-ESI-TRAP),研究了大鼠和小鼠海马组织中SynapsinIIa和SynapsinIIb的蛋白序列以及翻译后修饰信息。我们总共鉴定了12个小鼠海马SynapsinII蛋白点(6个IIa蛋白点,6个IIb蛋白点)和13个大鼠海马SynapsinII蛋白点(6个IIa蛋白点,7个IIb蛋白点),其中18个蛋白点得到90%以上的串联质谱序列覆盖率。我们提供了每个SynapsinII剪切体翻译后修饰蛋白点的全面翻译后修饰信息(包括磷酸化,甲基化,乙酰化,脱氨基化等)。我们还发现了7个新的磷酸化位点,其中3个经过磷酸酶处理得到验证(大鼠SynapsinIIa的T422,SynapsinIIb的S426和T338),其他4个为大鼠SynapsinIIa的S546、SynapsinIIb的T422和S446,以及小鼠SynapsinIIa的S446。我们在SynapsinIIb中发现了N339的糖基化修饰。这些翻译后修饰信息对于今后系统研究SynapsinII在调节神经递质释放中的机制有重要意义。更多还原

【Abstract】 Vitamin A is a micronutrient having multiple functions, including regulating early nervous system development and adult neurogenesis. Vitamin A deficiency (VAD) in adult rodents led to impairments of motor function and learning and memory abilities. And many clues suggest that Vitamin A cascade is associated with schizophrenia: Vitamin A pathway regulates the expression of some schizophrenia risk genes; the protein levels of Vitamin A transporters (TTR and APOE) are dysregulated in schizophrenia patients; the expression of Vitamin A transport and synthesis related molecules were found dysregulated. Furthermore, Vitamin A cascade is linked to Alzheimer disease. Vitamin A could regulate the gene expression of amyloid precursor protein (APP) and APP hydrolases. VAD animals displayed AD like molecular changes: APP accumulation, down-regulation of choline acetyltransferase and RARα.In the first part, we studied the motor ability, long term memory and thermal nociception in early VAD mouse, and detected the effect of combination of VAD and MK-801 (a NMDA receptor antagonist) on those behaviors. We found that early VAD mouse displayed higher locomotion and rearing abilities, and VAD induced pain sensitivity. Moreover, early VAD mouse showed hypersensitivity to MK-801 injection: VAD increased MK-801 induced stereotypy and ataxia, and amplified the suppression effect of MK-801 on rearing; the combination of VAD and MK-801 also lead to pain insensitivity. These abnormal behaviors reflect some faces of schizophrenia like symptoms: such as hyperactivity of locomotion and increased MK-801 induced stereotypy. However we didn’t find the influence of VAD on long term horrible memory (step down test).After that, we took the proteomis technology to study the proteome changes in hippocampus, cortex and liver tissues of early VAD male mouse. We also studied the influences of retinoic acid (the active form of Vitamin A) synthesis inhibition upon proteome changes in U251 astrocytoma cells. We identified 23 VAD related protein spots in hippocampus, 46 altered protein spots in cortex, 38 altered protein spots in liver and 39 altered protein spots in U251 cells. Through bioinformatics analysis (Ingenuity pathway analysis, IPA), we found that VAD related proteins in cortex and U251 cells clustered to biological functions: oxidative stress response, neurotransmitters metabolism (serine and glutamate), mitochondrial function and energy metabolism (ATP synthesis); in cortex, VAD could influence NMDA 2B receptor signaling, cytoskeleton and transcript regulation factors, therefore altered the neuronal migration、growth、development and neuroplasticity. With western blot, up-regulation of PHGDH was also confirmed in U251 cells after inhibiting retinoic acid synthesis, and this up-regulation of PHGDH was also observed in cortex of VAD male mouse. The VAD altered proteins in hippocampus clustered to lipid metabolism, axonal guidance signaling and neuroplasticity. VAD could regulate the cytoskeleton and cell membrane related proteins to influence the neurotransmitters releasing, dopamine signaling, NMDA 2B receptor mediated signaling,neurogenesis and neuronal migration. In liver, VAD could induce abnormal carbohydrate metabolism and lipid metabolism, which were corresponding to our findings in cortex. Therefore all results suggest that early VAD could influence energy metabolism and cytoskeleton in cortex and hippocampus, influence the glutamate signaling and dopamine signaling, and finally lead to schizophrenia like behaviors in mouse: increased locomotion and increased MK-801 induced stereotypy. These findings further support the hypothesis that vitamin A pathway is associated with neuropsychiatric diseases.SynapsinⅡis a neuronal phosphoprotein, located in nervous synaptic vesicles. SynapinⅡis connected to cytoskeleton and participates in regulating the realease of neurotransmitters. Previous reports suggested that synapinⅡis associated to schizophrenia. The mRNA and protein level of synapinⅡwere found down-regulated in hippocampus of schizophrenia patients. There are significant association between some SNPs of synapinⅡand schizophrenia in Han, Korean and north European population. Furthermore, synapinⅡgene knockout mouse displayed some schizophrenia like behaviors: impairment of sensorimotor gating and social interaction. There are 2 kinds of splicing isoforms of synapinⅡ: synapinⅡa and synapinⅡb. There are several post-translational modification (PTM) variants of synapinⅡsplicing isoforms in 2-dimensonal gels. However, until now the knowledge of PTMs in synapinⅡisoforms is still limited.In the second part, we used multiple proteolytic enzymes and tandem mass spectrometric technologies (LC-ESI-QTOF, LC-ESI-TRAP) to study the protein sequence and PTMs of synapsin IIa and synapsin IIb in rat and mouse hippocampus. We identified 12 synapsin II protein spots in mouse hippocampus (6 IIa and 6 IIb) and 13 synapsin II proteins in rat hippocampus (6 IIa and 7 IIb).Among those, we got more than 90% sequence coverage (MS/MS) of 18 spots. We provided multiple PTM information of each PTM variants of synapsinII splicing isoforms, including phosphorylation, methylation, acetylation and deamidation. We found 7 new phosphorylation sites, 3 of them have been confirmed using phosphatase (T422 of rat synapsin IIa,S426 and T338 of rat synapsin IIb). Other 4 new phosphorylation sites were: S546 of rat synapsin IIa, T422 and S446 of rat synapsin IIb, S446 of mouse synapsin IIa. Furthermore, we found a new glycosylation site: N339 of rat synapsin IIb by MS/MS. These findings of PTMs provide important basis for annotating the molecular mechanisms of synapsin II in regulating neurotransmitters releasing in near future.更多还原