【作者】 谷峰；

【导师】 胡应和； 赵政；

【作者基本信息】 华东师范大学 ， 生理学， 2008， 博士

【摘要】 随着人口老龄化的发展,阿尔茨海默病（Alzheimer’s disease,AD）患者数量逐年增多,但目前尚缺乏有效的治疗手段,给社会和家庭带来沉重的负担。AD是一种中枢神经系统退行性疾病,细胞外β淀粉样蛋白（β-amyloid,Aβ）沉积形成的老年斑（senile plaques,SP）,和细胞内高度磷酸化的tau蛋白形成的神经纤维缠结（neurofibrillary tangles,NFT）是两个显著的神经病理特征。Aβ由β淀粉样蛋白前体（amyloid precursor protein,APP）经β分泌酶和γ分泌酶裂解生成,早老素（presenilin,PS）包括PS1和PS2,是γ分泌酶的主要活性成分,对Aβ的生成至关重要。此外,PS蛋白在胚胎发育、神经母细胞分化、神经元凋亡、tau蛋白过度磷酸化和神经细胞黏附中也具有重要作用。PS cDKO（conditional double knockout,cDKO）小鼠前脑（皮层和海马）PS基因被特异性敲除,出生3周后,PS1和PS2基因才开始特异性失活,并逐渐出现年龄依赖性的神经退行性病变和脑组织病理学异常,如突触丢失、神经元死亡、大脑皮层萎缩、海马神经元分子层和胼胝体萎缩,侧脑室和第三脑室扩大,tau蛋白过度磷酸化。2个月即出现记忆力和突触可塑性降低,并且随着年龄的增长逐渐加重。因此PS cDKO小鼠是目前较好的研究PS功能的转基因动物模型,也是较好的AD转基因模型,通过基因操作的方式模拟了AD的神经退行性病变和痴呆的症状。由于PS1和PS2在前脑特异性失活,Aβ生成减少,未见SP,也没有Aβ介导的氧化应激,因而,PS cDKO小鼠的发病机制和主流的“Aβ毒性学说”不符。有研究表明,PS cDKO小鼠学习记忆能力下降并非由于大量的Aβ聚集,而是由于PS功能丧失,炎症相关基因表达增加所致。蛋白质是主要功能的执行者,其表达和翻译后修饰（post translational modification,PTM）对AD的病理过程有至关重要的影响,目前在PS cDKO小鼠中知之甚少。因此,我们利用二维液相色谱（two-dimensional liquid chromatography,2-DL）分离的蛋白质组学研究技术和其它分子生物学方法,观察PS cDKO小鼠与野生型（wild-type,WT）小鼠脑组织的蛋白表达差异;同时,分析PS cDKO小鼠脑组织的氧化应激情况和蛋白质的氧化修饰,在蛋白水平上探讨PS cDKO小鼠AD样病理变化的分子机制。另一方面,与PS cDKO相反,NR2B转基因小鼠是一种学习记忆增强的模式动物,关于NR2B小鼠脑组织的蛋白表达情况,目前的了解也不多。因此,采用蛋白质组学方法,对这两种模式动物的脑组织蛋白同时进行研究,更加有利于分析蛋白在AD中的功能。本论文分为4章。第一章分析了目前常用的几种蛋白质分离方法,重点讨论了蛋白样品的纯化方法,以及我们所采用的二维液相色谱分离系统PF2D方法的稳定性、可靠性,为后面的实验奠定基础。第二章分别分离并鉴定了不同月龄的PS cDKO小鼠与WT小鼠、NR2B小鼠与WT小鼠脑组织的蛋白质差异表达,并对部分差异蛋白采用Western blot进行了确认,同时,分析了这些差异蛋白与神经退行性疾病（包括AD）的关系。第三章研究了不同月龄的PS cDKO小鼠脑组织和肝组织的氧化产物和抗氧化物水平,并对其氧化应激情况进行了分析。第四章综述了目前人们对PS、NR2B和AD的研究,AD的蛋白质组学研究,以及AD与氧化应激等方面的认识。具体如下:首先,对PF2D中的蛋白样品处理方法和系统的重复性进行摸索。由于脑组织中含有较多的脂质和脂蛋白等杂质,对于等电聚焦色谱的分离效果有一定的影响,因此我们对不同的蛋白除脂方法进行了比较,如超速离心、丙酮沉淀、异丙醇/正己烷、Freon和Cleanasite法。最终我们选择了蛋白损失少、易操作且重复性高的Freon和Cleanasite法除脂。重复上样后分离结果显示,系统一维和二维的重复性都较好。峰面积和蛋白浓度成正比,从而保证了用于蛋白差异表达分析时的可靠性。其次,我们利用上述实验系统,分别分离鉴定了不同月龄的PS cDKO、NR2B小鼠脑组织的蛋白表达谱。PS cDKO的结果显示,与WT小鼠相比,2月龄显著差异的峰有26个（6个下调,20个上调）,4月龄有51个（33个下调,18个上调）,6月龄有108个（44个下调,64个上调）。对6月龄小鼠部分差异表达的峰进行基质辅助激光解吸离子化飞行时间质谱（matrix assisted laser desorotion/ionizationtime-of-flight,MALDI-TOF）鉴定,有二氢嘧啶相关蛋白2（dihydropyrimidinase-like2,DRP-2）、异柠檬酸脱氢酶、丙酮酸脱氢酶、微管蛋白、星型胶质细胞丝状酸性蛋白（glial fibrillary acidic protein,GFAP）、肌酸激酶BB和液泡H⁺-ATP酶B2等蛋白,它们与神经元生长和可塑性、神经元损伤以及ATP合成等功能密切相关。用Western blot方法检测其中的GFAP表达水平,结果显示6月龄PS cDKO小鼠海马与WT相比表达水平升高,和蛋白组学结果趋势一致。NR2B的结果显示,相对于匹配的WT小鼠,NR2B转基因小鼠有显著性差异的峰共有了2个,其中有34个上调、38个下调。经质谱鉴定出的26个蛋白中有14个上调如谷氨酰胺合成酶（glutamine synthetase,GS）、网格蛋白轻链B（Clathrinlight chain B,Lcb）、NADH2脱氢酶、ATP合成酶等,12个下调如鸟核苷酸释放因子1、Pin2/TRF1交互作用蛋白等,它们和神经递质、突触可塑性、神经元发育、细胞循环、能量代谢、基因转录等生理功能相关。Western blot方法检测结果表明,GS和Lcb蛋白在NR2B转基因小鼠中表达均升高,与蛋白质组学方法结果的趋势一致,进一步证实了二维液相色谱分离、MALDI-TOF/TOF质谱鉴定方法的可靠。对鉴定出的蛋白进行功能分析,发现PS cDKO小鼠和NR2B转基因小鼠实验中鉴定出的蛋白,有许多和能量代谢密切相关,表明能量代谢对神经元的功能非常重要。ATP合成和神经元的可塑性与学习记忆能力有密切关系。这为进一步探究PS cDKO转基因小鼠的病理机制、学习记忆能力下降的分子机制,寻找治疗AD可能的药物靶点奠定了基础。第三,之前有关氧化应激在AD中的作用的实验都是建立在Aβ生成增加的基础上,而PS cDKO小鼠脑内Aβ生成是减少的。本论文中我们通过检测氧化产物包括丙二醛（malondialdehyde,MDA）、羧基化蛋白（carbonylated protein）、iPF_2α-Ⅵ（F₂-isoprostanes-Ⅵ）和抗氧化酶如超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（Catalase,CAT）、谷胱甘肽过氧化酶（Glutathione peroxidase,GSH-px）水平分析PS cDKO小鼠不同年龄阶段的大脑皮层和肝组织的氧化应激情况。实验结果表明,PS cDKO小鼠大脑皮层MDA水平与对照组相比,2、4、7月龄都维持在较高水平,有统计学差异;蛋白被氧化后产生的羧基化蛋白的含量与对照组相比也明显升高,在2、4和7月龄组分别升高51%、61%和41%。应用Oxyblot免疫标记羧基蛋白也进一步证实了这个趋势。PS cDKO小鼠大脑皮层的抗氧化能力也相应有所改变:CAT活性在4月龄和7月龄时明显下降;SOD在2、4和7月龄均有升高,其中2月龄和7月龄组和对照组相比,分别升高了12%和45%,差异具有统计学意义。各组PS cDKO小鼠皮层GSH-px活性与对照组比较差别无统计学意义,但2月龄雄性小鼠皮层GSH-px活性与同月龄同性别的对照组相比,则明显升高。GFAP表达也呈年龄相关性的增加,在2个月时表达就有所升高,与MDA和蛋白氧化水平相一致,进一步证实了PS cDKO小鼠的氧化应激过程和免疫炎症等病理过程密切相关。我们还利用气相色谱-质谱（gas chromatograph-mass spectrometer,GC-MS）检测小鼠大脑皮层脂质过氧化物isoprostanes的活性异构体之一iPF_2α-Ⅵ。结果表明,PS cDKO小鼠大脑皮层的iPF_2α-Ⅵ含量随着年龄增长迅速增高,在4和7月龄时和WT相比有显著性差异;血浆中的iPF_2×-Ⅵ在7月龄时显著增高,但雌性PScDKO小鼠在4和7月龄都有显著性差异。最后,我们对蛋白的氧化修饰进行了初步探讨。利用肼化生物胞素标记氧化修饰的羧基化蛋白,再用链霉亲和素纯化后,酶解、LCQ-MS进行质谱鉴定。实验结果表明,酮戊二酸脱氢酶、肌动蛋白α亚型、血红蛋白β1亚单位和丝/苏氨酸蛋白激酶BUB1在PS cDKO小鼠大脑皮层中发生氧化修饰。综上所述,我们利用二维液相色谱分离和质谱的方法验证了PS cDKO小鼠和NR2B转基因小鼠脑组织中多类蛋白水平发生改变。对这些蛋白的功能进行分析,发现尤其与能量代谢和神经可塑性功能密切相关;大脑皮层的氧化应激在病理过程的早期阶段已经开始出现,是对PS丧失功能的一个早期反应,这一反应和脑Aβ沉积无关;同时鉴定出部分氧化修饰的蛋白;在PS cDKO AD样模型小鼠中,氧化应激和神经元损伤之间的因果关系尚需进一步研究。更多还原

【Abstract】 Alzheimer’s disease（AD） is a neurodegeneration disease of central nervous system characterized by senile plaques（SP） which is formed by extracellularβ-amyloid（Aβ） deposits and neurofibrillary tangles（NFT） which is formed by tau hyper-phosphoralation.Presenilin（PS） is an essential component ofγ-secretase, which cleaves the C-terminal ofβ-amyloid precursor protein（APP） to produce Aβthat accumulates into SP.In addition to the critical role of PS in Aβformation in the brain,PS also has many other functions such as embryo development,neuroblast differentiation,neuron apoptosis,tau hyper-phosphoralation and neural cell adhesion.The forebrain-specific（cerebral cortex and hippocampus） and conditional PSI and PS2 double knock-out（PS cDKO） mice appear inactivation of PS at 3 weeks of age postnatally.They exhibit age-dependent AD-like neurodegenerative phenotypes although the brain Aβwas decreased,such as synaptic loss,neuronal cell death, astrogliosis,cerebral cortex,hippocampus and corpus callosum atrophy,lateral ventricle and third ventricle enlargement and tau hyperphosphorylation.Memory loss and synaptic plasticity impairments start from 2 months of age and become more serious progressively.Thus,derived from genetic manipulation of AD genes that reproduce the central features of AD,namely neurodegeneration and dementia,PS cDKO mice is an ideal model for the study of PS functions in adult mice and AD pathology.In PS cDKO mice,Aβproduction is decreased and the oxidative stress induced by Aβis not found.It is not coincidence with the conventional "Aβhypothesis" of AD pathogenesis.Several important studies have indicated that memory impairment and neurodegeneration in PS cDKO mice are not caused by Aβaccumulation but related to the loss of PS function with the evidence of differential up-regulation of inflammatory markers in the cerebral cortex.As a function executor, protein,especially protein expression and post-translational modification（PTM）,is more important to the AD pathogenesis.Unfortunately,up to now,the proteomics of PS cDKO mice is still unknown.In this study,a two-dimensional liquid chromatography-based（2-DL） proteomic approach and other molecular biology methods were used to observe the differentially expressed proteins between PS cDKO and wild-type（WT） mice,and the identification of oxidative modified proteins and oxidative stress status in the brain of PS cDKO mice were also performed.The molecular mechanism of AD-like pathology of PS cDKO mice on the protein level was further investigated.On the other hand,the NR2B transgenic（Tg） mice are the model mice with enhanced learning and memory.However,information about the proteomics of NR2B Tg mice is also scant.It would be thus more helpful to the analysis of protein functions in AD when we compare the expression patterns of brain proteins between these two transgenic models via the proteomics approach.This thesis is composed of four chapters.Chapter 1 will mainly describe the establishment of the methods for purification of brain tissue proteins and the reproducibility and reliability of the 2-DL system PF2D.Chapter 2 describes the proteomic investigation of proteins in brain tissues and the identification of the differentially expressed proteins between PS cDKO and the age-,gender-matched WT mice over several age points,and those between NR2B and age-,gender-matched WT mice,respectively.The expression levels of some proteins were further verified by using the Western blot method.The relationship of these differentially expressed proteins with AD pathogenesis will further be discussed.In chapter 3,we present the results from the studies of oxidative stress of PS cDKO mice at different age point by detecting oxidative productions and anti-oxidative enzymes.Finally,we will give a comprehensive literature review in terms of the most recent advances in physiological and pathological relevance of PS and NR2B in neurodegeneration as well as learning and memory functions,and in proteomic studies of AD and the oxidative stress as a major function in AD pathogenesis.Firstly we optimized the method of brain tissue protein purification to make sure that protein could be well separated by PF2D.The brain tissue is aboundant of lipid as much as about 63%of the dry matter of the brain.High contents of lipid and lipid-associated protein in the brain sample may cause the charge loss on the surface of protein and dramatically reduce the binding efficiency of proteins to the stationary phase of the CF column during the first dimensional fractionation.We initially tried several approaches for removing lipids from the rude protein extracts,such as ultra-centrifugation,acetone precipitation,isopropanol/hexane,Freon and Cleanascite. Freon and Cleanascite methods were proved to be superior over the others in terms of its simplicity and a higher protein recovery rate.Thus,it was used to purify the brain proteins in our protein profiling experiments.The good reproducibility of 1st dimension and 2nd dimension is demonstrated after we loaded two repeat samples. Peak area is in direct ratio with protein concentration.Both of them assure the reliability of the system for analysis the differentially expressed protein.Secondly,we separated proteins of brain tissue by using the PF2D system and searched differentially expressed peaks by the Delt vue software.By comparing with the age- and gender-matched WT mice,26 peaks（6 up-regulation and 20 down-regulation） in 2-month-old PS cDKO mice,51 peaks（18 up-regulation and 33 down-regulation） in 4-month-old PS cDKO mice and 108 peaks（64 up-regulation and 44 down-regulation） in 6-month-old PS cDKO mice were observed.Differentially expressed peaks of 6-month-old PS cDKO mice from the second dimensional chromatography were selected for protein identification using the PMF and MS/MS data from matrix assisted laser desorption/ionization time-of-flight（MALDI-TOF） mass spectrometric analysis.Dihydropyrimidinase-like 2（DRP-2）,isocitrate dehydrogenase,pyruvate dehydrogenase,creatine kinase,glial fibrillary acidic protein （GFAP）,tubulin were identified and they have relations with neuron development and plasticity,neuron injury and ATP synthesis.To further corroborate the expression profiling data from the present proteomic experiment,GFAP was selected for Western blotting analysis by using specific antibody.The result showed that the mean level of the protein in the 6-month-old PS cDKO mice were elevated compared to WT mice’s, similar to that observed in the proteomic data.Totally,72 differentially expressed proteins were mapped with 34 proteins up-regulation and 38 down-regulation in 6-month-old NR2B Tg mice.Twenty six cortical proteins with altered expression level were identified by MALDI-TOF/TOF in the NR2B Tg mice.Among them,14 proteins were up-regulated,while 12 proteins were down-regulated,including glutamine synthetase（GS）,guanine nucleotide-releasing factor 1,carbonic anhydrase,clathrin light chain B（Lcb）, enolase 1,ATP synthase,cytochrome c,THO complex 4,and M-phase phosphoprotein 1.They play important roles in cell cycle control,neuronal development,RNA transcription as well as energy and metabolic homeostasis.These findings were further corroborated in an independent group of and WT mice by Western blot analysis of two selected proteins:Lcb and GS. From analysis of functions of these identified protein in PS cDKO and NR2B Tg mice,we found many proteins have relations with energy metabolic homeostasis, suggesting an important role of proteins associated energy metabolism in the neuron normal functions,and ATP synthesis has close relations with neuron plasticity and learning and memory.These results provide important information in understanding the AD pathological mechanism of PS cDKO mice.Thirdly,previous investigations into oxidative imbalance implicated in underlying AD pathogenic mechanism have been largely based on the AD-like amyloidosis in transgenic animal models.However,data derived from PS cDKO that presents the loss-of-function of pathogenic mechanism of AD with reduced Aβproduction are currently scant.The present study aimed primarily to investigate oxidative stress status in the PS cDKO mice with different ages by measuring levels of peroxidation products including malondialdehyde（MDA）,carbonylated proteins and F₂-isoprostanes-Ⅵ(iPF_2α-Ⅵ) and anti-oxidative enzymes such as superoxide dismutase（SOD）,catalase（CAT） and glutathione peroxidase（GSH-px） in response to the presenilin inactivation in the brain.Our results show that levels of lipid peroxidation product MDA were significantly elevated in the cortex of PS cDKO mice at the age as early as 2-month, and the oxidative stress were even severe and maintained at a higher level during the ages from 4- to 7-month,as compared with those of age- and gender-matched. Accordantly,protein oxidative modification levels,as measured as protein carbonyl content by DNPH assay,in the cerebral cortex samples were also significantly higher by 51%,61%and 41%in 2-,4- and 7-month age groups of PS cDKO mice, respectively,as compared with those of WT controls.The finding was further confirmed in the mice,especially in those at ages of 4 and 7-month,using Oxyblot immunolabeling of carbonylated proteins.Levels of anti-oxidative enzymes also have changes.Cortical CAT activities were significantly decreased by 58%and 46%in the 4- and 7-month mice,respectively,compared to that of matched controls. Contrastingly,cortical SOD activities were markedly higher in 2-month and 7-month PS cDKO mice by 12%and 45%,respectively,compared to WT controls.There was no significant difference in cortical GSH-px activities between WT and PS cDKO mice over all age points.However,in 2-month male PS cDKO mice the activities of this enzyme appeared to be increased significantly compared to age- and gender-matched WT.GFAP expression was increased in the cortices of PS cDKO mice in an age-related manner,similar to the changes shown in the MDA and protein oxidation results above.It revealed that the interaction relationship between oxidative stress and inflammatory may be closely associated with the underlying loss-of-function pathogenesis of AD.We also measured the levels of iPF_2α-Ⅵby gas chromatograph-mass spectrometer（GC-MS）.iPF_2α-Ⅵis one of active isoforms of isoprostanes which were produced by lipid peroxidation of cell memberane.Results show that cerebral cortex iPF_2α-Ⅵcontents increased in an age-dependent manner,and have significantly difference in 4-month and 7-month PS cDKO mice compared with WT’s.Contents of plasma iPF_2α-Ⅵsignificantly increased in 7-month PS cDKO mice,and increased in 4- and 7-month male PS cDKO mice.In the present study,we also conducted a preliminary investigation in identification of oxidative modified proteins in the brain of the AD Tg model by using biocytin hydrazide-streptavidin affinity methodology coupled to microTOFQ proteomics approach.2-oxoglutarate dehydrogenase E1 component,alpha-actin-2, hemoglobin subunit beta-land mitotic checkpoint serine/threonine-protein kinase BUB1 were identified only in PS cDKO mice cortex.In conclusion,we identified many proteins by 2-DL coupled to MALDI-TOF/TOF approach and found changes of protein levels to be mainly associated with energy metabolism and neuron plasticity in both PS cDKO and NR2B Tg mice compared to WT mice.Enhanced oxidative stress in the cortex of PS cDKO mice is an early event responsible for the loss-of-function of PS towards the AD-like pathologies unrelated with the brain amyloidosis.The cause-effect relationship between oxidative stress and inflammatory response in this PS-deficient AD-like model needs to be further elucidated.更多还原