【作者】 郑玮；

【导师】 王占友；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 前言阿尔茨海默病（Alzheimer’s disease,AD）是一种发生于老年及老年前期的以进行性痴呆为主要特征的神经系统退行性疾病,其主要临床特征是进行性记忆丧失、认知缺陷及严重的精神障碍。AD的一个主要病理学标志是脑部出现淀粉样沉积,即老年斑（senil plaque,SP）。老年斑的核心成分是一种由39-43个氨基酸残基构成的小神经肽—Aβ（β-amyloid peptide）,Aβ由淀粉样前体蛋白（amyloidprecursor protein,APP）经过β-分泌酶及γ-分泌酶水解并分泌至细胞外。在正常生理状态下,人脑内存在纳摩尔级浓度的可溶性Aβ,对神经元具有神经营养作用。但在AD时,APP的异常剪切加工、Aβ的形成及其从可溶状态到不溶状态的转变是AD发病机制中的关键环节,近年来的研究表明铁、铜和锌等金属离子与APP、β-分泌酶、γ-分泌酶和Tau蛋白的基因表达密切相关,从而参与Aβ分泌、沉积。以金属离子及金属离子转运蛋白为切入点,解析AD发病机制并寻找AD治疗靶点已经成为AD治疗学的热点之一。二价金属离子转运体（divalent metal transporter 1,DMT1）属于自然抵抗相关的巨噬细胞蛋白家族。DMT1蛋白有12个跨膜域,根据DMT1 mRNA的3’端非翻译区（3’-untranslational region,3’-UTR）是否带有铁反应元件（iron-responseelement,IRE）,可将DMT1分为DMT1-IRE和DMT1-nonIRE两种异构体,二者均定位于细胞膜和胞内循环的内吞小泡膜上,参与多种二价金属离子尤其是铁离子的转运,是维持细胞内二价金属离子稳态的重要金属转运蛋白之一。近年来有关DMT1与神经退行性疾病发病机理的研究引人关注,通过对帕金森病（Parkinson disease,PD）患者和PD模型鼠的研究表明,DMT1在黑质的表达显著增强;而DMT1功能缺失鼠（mk/mk小鼠）却能拮抗1-甲基-4-苯-1,2,3,6-四氢吡啶（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP）和6-羟多巴胺诱导的PD症状和黑质神经元死亡。令人感兴趣的是老龄大鼠脑内DMT1表达呈年龄性增高趋势;多种炎症因子也能使DMT1在脑内表达上调,这两种情况均是诱发AD的重要危险因素。然而,有关DMT1与AD发病机理的研究尚未见报道。本研究对DMT1-IRE和DMT1-nonlRE在AD病人和APP/PS1转基因小鼠脑内的表达变化、定位分布及与Aβ的相关性进行系统分析,应用RNA干扰技术（RNAinterference,RNAi）探讨DMT1基因沉默阻抑APP表达和Aβ分泌的效果,对深入探讨脑金属代谢紊乱与AD病理生理机制具有重要意义。实验方法采用AD病人尸检脑组织、双转染人APP695swe基因和人早老素（presenilin,PS-1）突变基因的AD模型小鼠（APP/PS1小鼠）及稳定转染APP695swe基因的SH-SY5Y细胞（APPsw细胞）为研究对象,应用免疫荧光双标和激光共聚焦扫描显微技术（Confocal Laser Scanning Microscopy,CLSM）检测DMT1在AD病人和APP/PS1小鼠脑内的定位分布及其与Aβ在老年斑内的位置关系,应用Western Blot技术检测APP/PS1小鼠大脑皮层和海马及APPsw细胞DMT1的表达变化,应用免疫共沉淀技术（co-immunoprecipitation,co-IP）检测APP/PS1转基因小鼠脑内DMT1和Aβ的蛋白相关性,应用RNAi技术阻抑DMT1基因在稳定转染APPsw cDNA和空质粒neo的SH-SY5Y细胞的表达,并应用Western Blot技术检测RNAi对DMT1的基因沉默效果,应用Calcein AM荧光褪色光度法、RT-PCR技术、Western Blot技术、免疫荧光双标技术、ELISA技术、MTT技术检测DMT1-RNAi对细胞铁离子转运、APP表达、Aβ分泌和细胞生存率的影响。实验结果一、DMT1异常分布和表达参与AD发生的在体研究1、DMT1在AD病人及APP/PS1转基因小鼠脑内的分布免疫组织化学结果显示,DMT1-IRE和DMT1-nonIRE免疫阳性反应产物均呈棕黄色,一在大脑皮层及海马内均可见DMT1阳性的老年斑分布,斑块大小不等,形状为圆形或不规则形状,边界较清晰。高倍镜下可见DMT1-IRE和DMT1-nonIRE广泛分布到整个老年斑内。此外,DMT1-IRE还广泛表达于淀粉样变性的血管壁及其周围。免疫荧光双标的共聚焦激光扫描结果显示,在AD病人和APP/PS1转基因小鼠脑内,Aβ免疫阳性的老年斑广泛分布于大脑皮层及海马,几乎所有Aβ阳性的老年斑均有不同程度的DMT1表达,即DMT1和Aβ共存于老年斑内。高倍镜观察可见在APP/PS1转基因小鼠脑内,Aβ主要分布于老年斑的核心,DMT1-IRE与DMT1-nonIRE在整个老年斑内均有分布,中心部位免疫阳性反应最强。而且DMT-IRE免疫荧光除了分布在老年斑中,还可见于淀粉样变性的血管壁及其周围。2、DMT1在APP/PS1转基因小鼠大脑皮层及海马内的表达变化Western Blot结果显示,DMT1在APP/PS1转基因小鼠大脑皮层和海马内的表达均明显高于野生型对照小鼠。其中DMT1-IRE和DMT1-nonIRE在大脑皮层的表达量分别是野生型小鼠的155.4%和158.5%;在海马的表达量分别是野生型小鼠的256.8%和239.9%。3、DMT1与Aβ蛋白相关性分析Co-IP结果显示,应用Aβ抗体对APP/PS1转基因小鼠大脑皮层蛋白进行免疫共沉淀,经过琼脂糖凝胶电泳,未见DMT1-IRE和DMT1-nonIRE条带。二、DMT1异常分布和表达参与AD发生的离体研究1、APPSW细胞APP695表达及Aβ1-42分泌显著增多Western Blot检测转染APPsw基因的SH-SY5Y细胞APP695水平显著高于对照组转空质粒的neo细胞。ELISA法检测APPsw细胞和neo细胞的条件培养基中Aβ1-42的水平,发现APPsw细胞分泌的Aβ1-42为86 pg/ml/protein,明显高于neo细胞分泌的8 pg/ml/protein。2、DMT1在转染人APPsw基因的SH-SY5Y细胞中的表达变化应用转染人APPsw基因的SH-SY5Y细胞株作为AD细胞模型,免疫荧光双标的共聚焦激光扫描结果显示,APPsw细胞呈Aβ免疫阳性,而且DMT1-IRE和DMT1-nonIRE与Aβ共同表达。Western Blot结果显示,DMT1-IRE和DMT1-nonIRE在APPsw细胞的表达均明显高于转染空质粒neo的对照组细胞。3、FeSO₄对APPSW细胞和neo细胞表达DMT1的影响Western Blot结果显示,10和50μM的FeSO₄可使neo细胞表达DMT1-IRE显著增高,100、200和500 gM的FeSO₄处理后DMT1-IRE的表达降低,但是APPsw细胞经不同浓度的FeSO₄处理后,DMT1-IRE表达均显著升高,并具有随着FeSO₄浓度升高表达增高的趋势。不同浓度FeSO₄处理后,neo细胞和APPsw细胞表达的DMT1-nonIRE均有不同程度的增高,但与FeSO₄的浓度不具有相关性,而且APPsw细胞增高的程度更为显著。结果提示DMT1在APPsw细胞中的表达不受铁离子的负反馈调控,始终处于较高的水平。三、DMT1参与APP表达和Aβ分泌的机制研究1、RNAi对DMT1基因的沉默效果检测Western Blot结果显示,RNAi可明显抑制APPsw细胞的DMT1蛋白表达,干扰后48h为抑制效果最佳时间点,DMT1-IRE表达量为对照组的40%,DMT1-nonIRE表达量仅为对照组的20%。Calcein AM荧光褪色光度法结果显示,在APPsw细胞,DMT1-RNAi后48h,APPsw细胞内的荧光强度明显高于未干扰细胞,表明干扰后铁内流减少。2、DMT1-RNAi对APP表达和Aβ分泌的影响RT-PCR结果显示,RNAi可明显抑制APP mRNA的表达,其中RNAi处理48 h后,APPsw细胞和neo细胞的APP mRNA水平分别降低了36.4%和29.2%析因分析显示DMT1-RNAi对APPsw细胞的APP mRNA表达的影响更为显著。Western Blot结果显示,RNAi可明显抑制APPsw细胞的APP695蛋白表达,并且抑制程度与干扰效率呈时间依赖性。neo细胞的APP695表达也有一定程度的降低。析因设计方差分析结果显示,DMT1-RNAi后,ApPsw细胞的APP695蛋白表达量降低的更为显著。ELISA结果显示,干扰48 h后,APPsw分泌的Aβ1-42由干扰前的86pg/ml/protein下降为62 pg/ml/protein。neo细胞分泌的Aβ1-42也有一定程度的降低,但并无统计学意义。3、DMT1-RNAi可拮抗金属离子对细胞生存率的影响MTT结果显示,APPsw细胞生存率随培养液中FeSO₄、FeCl₃、CuSO₄和ZnSO₄浓度的升高而降低,选择可使APPsw细胞的生存率降低20%左右的100μM FeSO₄、100μM FeCl₃、50μM CuSO₄和50μM ZnSO₄作为处理因素。正常培养条件下,DMT1-RNAi对细胞生存率几乎没有影响,但是RNAi可以逆转100μM FeSO₄、100μM FeCl₃和50μM CuSO₄引起的APPsw细胞生存率降低。但对50μM ZnSO₄处理的APPsw细胞生存率没有影响。4、DMT1-RNAi可拮抗金属离子诱导的APP蛋白表达上调Aβ分泌Western Blot结果显示,100μM FeSO₄、100μM FeCl₃和50μM CuSO₄处理可分别使APPsw细胞APP695蛋白表达升高,而DMT1-RNAi可拮抗金属离子诱导的APP蛋白表达上调。但DMT1-RNAi对50μM ZnSO₄处理的APPsw细胞表达的APP695水平没有影响。ELISA结果显示,100μM FeSO₄可使APPsw分泌的Aβ1-42从86 pg/ml/protein升高至98 pg/ml/protein,而DMT1-RNAi后APPsw细胞分泌Aβ1-42的水平降低至90pg/ml/protein。结论1、DMT1-IRE和DMT1-nonIRE与Aβ共表达于AD患者和APP/PS1转基因小鼠脑内老年斑和淀粉样变性的血管壁及其周围脑组织。DMT1-IRE和DMT1-nonIRE在APP/PS1转基因小鼠大脑皮层和海马的表达均高于野生型小鼠。2、DMT1-IRE和DMT1-nonlRE在稳定转染APPsw基因的SH-SY5Y细胞表达均高于转染空质粒的SH-SY5Y细胞,并且不受Fe²⁺的反馈调控。。3、DMT1-RNAi可明显降低稳定转染APPsw基因的SH-SY5Y细胞的APP表达和Aβ分泌,而且APP的表达抑制程度与DMT1-RNAi效率一致。4、金属离子铁、铜和锌能降低APPsw细胞的生存率,而DMT1-RNAi可拮抗金属离子的作用,提高细胞生存率;同时铁、铜和锌能上调APPsw细胞内APP蛋白表达水平,使分泌性Aβ产生增多,DMT1-RNAi可显著降低金属离子诱导的APP蛋白表达水平和Aβ1-42分泌水平。更多还原

【Abstract】 PrefaceAlzheimer’s disease（AD） is a disease clinically characterized by progressive intellectual deterioration.With the gradual ageing of the population,the morbidity of AD increased year by year.It had brought heavy burden to the society and family and become one of the fatal disease that hazard to human health.AD is pathologically characterized by senile plaques（SP） formed by pathological deposition ofβ-amyloid（Aβ）,neurofibrillary tangles（NFT）.Aβis the key factor in the pathologic process of AD and generated from the amyloid precursor protein（APP） by a proteolytic activity ofβ-andγ-secretase.In the past decade,a significant body of evidence has pointed to the "amyloid cascade" event as the major causative factor in AD,with Aβproviding the initial insult.Although a central role Aβplays in the pathogenesis of AD is indisputable,considerable evidence indicates that Aβproduction is not the sole culprit in AD pathogenesis.Aβis a cleavage product ofβ-amyloid precursor protein（APP） and is generated by the combined actions of the proteolytic enzymesβ-andγ-secretases,a process that occurs normally under physiological conditions,and at physiological（nanomolar） concentrations it has even been shown to possess neurotrophic properties in culture cells.Therefore,it is conceivable that there may be an abnormally modified "rogue" form of soluble Aβthat is particularly neurotoxic in AD.The "metal hypothesis of Alzheimer’s disease" proposes that it is the interaction of Aβwith specific metals that drives Aβpathogenicity and downstreams AD pathology.Divalent metal transporter 1（DMT1） also known as Nramp2（natural resistance associated macrophage protein 2） or DCT1（divalent cation transporter 1）,is a newly discovered proton-coupled metal-ion transport protein,and it is the first mammalian transmembrane iron transporter.It is a widely expressed protein with 12 putative transmembrane-spanning domains.DMT1 is responsible for the uptake of a broad range of divalent metal ions including Fe²⁺,Zn²⁺,Mn²⁺,Co²⁺,Cd²⁺,Cu²⁺,Ni²⁺ and Pb²⁺. Four isoforms of DMT1 protein are distinguished arising from their variant mRNA transcripts that vary both at their 5’-UTR and 3’-UTR.Two of these transcripts contain an IRE in their 3’-ends and two do not.Thus,the C-terminal DMT1 protein isoforms are designated DMT1-IRE and DMT1-nonIRE.Interestingly,a recent study on rat brain has shown that the expression of both the-IRE and-nonlRE DMT1 isoforms is increased during aging,the main risk factor in AD.It has also been shown that the expression of DMT1 is modulated by inflammatory factors,which are important in the cascade of events leading to the neuronal loss in AD.Therefore,it is reasonable to speculate that changes in DMT1 expression may contribute to the neuropathogenesis of AD.Although it is well known that DMT1 contributes to neurodegeneration in animal models of Parkinson’s disease,a comprehensive description of DMT1 in the AD pathogenesis has not been established.In the present study,we characterized the distributions and expressions of DMT1 isoforms in the senile plaques of postmortem AD brain and APP/PS1 transgenic mouse brain.Then we developed a SH-SY5Y cell line overexpressing human APP Swedish mutant（APPsw）.Using this system,we demonstrated the vital role DMT1 plays on APP processing and Aβgeneration.Based on the results of the current study,it is reasonable to hypothesize that a DMT1-dependent increase in ions plays a role in the neuropathophysiology of AD.MethodsAD patient brains,APP/PS1 transgenic mice and SH-SYSY cells stable transfected APPsw or APP gene were used for the present study.The distribution patterns of DMT1 in these brains and the positional relation between DMT1 and Aβwere detected by double immunofluorescence and confocal laser scanning microscopy. The changes of DMT1 expression levels in the APP/PS1 transgenic mouse cerebral cortex and hippocampus were studied by western blot analyses. Co-immunoprecipitation（co-IP） was used to detect the molecular correlation between Aβand DMT1 in the APP/PS1 transgenic mice brains.RNA interference（RNAi） technology was used to inhibite the expression of DMT1 gene in SH-SY5Y cells stable transfected APPsw cDNA.The gene silencing effect of DMT1-RNAi was detected by RT-PCR and Western Blot.Calcein AM fluorescence technology,Western Blot,double immunofluorescence techniques,ELISA technology and MTT were used to detect the effects of DMT1 RNAi on metal ions transport,APP expression,Aβsecretion and viability of cells.Results1.The expression and distribution of DMT1 in AD pathogenisis in vitro.（1） Abundant expression of DMT1 in SP of AD human brain and APP/PS transgenic mice.Immunohistochemimistry results revealed that DMT1 immunopositive reaction products were brown.DMT1-positive plaques were round or irregular and distributed throughout the cortex and hippocampus with vary size and clear boundary.At higher magnification,DMT1 were extensively expressed in all parts of the plaques.Our data also showed an abundant expression of DMT1 in the amyloid angiopathic vessels.Double-immunofluorescence staining for Aβand one of the DMT1 showed that the Aβ-positive plaques were widely distributed in the APP/PS1 transgenic mouse cerebral cortex and the hippocampus,DMT1 were expressed in most of the Aβcontaining plaques,i.e.Aβand DMT1 protein were co-expressed in the senile plaques. At higher magnification,the Aβwas dense in the core of plaques,and DMT1-IRE and DMT-nonIRE were present in most Aβ-positive plaques.DMT1-IRE and DMT-nonIRE exhibited similar staining patterns.Both of them were present in all parts of the plaques, but particularly dense in the center.（2） Altered expression of DMT1 in the APP/PS1 transgenic mouse cerebral cortex and hippocampusWestern blot results showed that the expressions of DMT1 were increased in the hippocampus and cortex of APPswe/PS1dE9 transgenic mice.The expressions of DMT1-IRE and DMT1-nonIRE were 155.4%and 158.5%of wild-type control mice in the cerebral cortex;256.8%and 139.9%in the hippocampus.（3） Molecular Correlation Analysis of DMT1 and AβCo-IP results showed that using Aβantibodies to perform the co-immunoprecipitation of DMT1 and Aβin the APP/PS1 transgenic mouse cerebral cortex,after agarose gel electrophoresis,no DMT1 band could be seen. 2.The expression and distribution of DMT1 in AD pathogenisis in vivo.（1） The APP695 and Aβlevel of APPsw cells increased significantly.Western blot results showed that the expressions of APP695 were increased in the SH-SY5Y cells transfected by APPswe gene.ELISA was performed to assess the Aβlevel in the conditioned medium of APPsw cells and neo cells.The APPsw cells exhibited an increased Aβ1-42 level as compared with neo cells as with 86 pg/ml/protein versus 8 pg/ml/protein（normalized to total protein levels）.（2） Protein Levels of DMT1 Increased in SH-SY5Y Cells Expressing Human APPsw.Immunofluorescence labeling and confocal analysis showed that DMT1-IRE and DMT1-nonIRE were mainly distributed in a punctate pattern within the cytoplasm. Both DMT1-IRE and DMT1-nonIRE were co-localized with Aβin APPsw cells. Immunoblotting revealed significant increase in the levels of DMT1-IRE and DMT1-nonIRE by 58.4%and 36.1%respectively following APPsw transfection compared to empty vector transfection.（3） DMT1 was distinctly expressed in APPsw cells or neo cells treated with different concentration ferrous sulfate.In neo clls,10 and 50μM ferrous iron significantly increased,but 100,200 and 500μM of ferrous iron induced reduced protein levels of DMT1-IRE.In APPsw cells, significant increases in cellular protein levels of DMT1-IRE were observed after exposure at the same gradient concentration.DMT1-nonlRE level was not fully dependent on ferrous iron concentration and significant increased both in neo cells and APPsw cells.3.The mechenism of DMT1 involved in the expression of APP and Aβsecretion.（1） The effect of RNAi to silence genes DMT1Western Blot results showed that RNAi could significantly inhibit DMT1-IRE and DMT1-nonIRE expression in APPsw-cells,and the inhibiting rate can reach 60%and 80%,respectively 48 h after RNAi. Calcein AM results showed that ferrous sulfate quenched calcein fluorescence in a time-dependent manner.Importantly,the fluorescence intensity in DMT1 silencing cells was stronger than that in control cells,suggesting that siRNA treatment led to reduction of ferrous uptake in APPsw cells.（2） The effect of DMT1-RNAi on the expression of APP and AβsecretionAfter transfection of DMT1 siRNA,cells were harvested and subjected to RT-PCR and Western blot analyses.APP mRNA levels of neo cells and APPsw cells decreased by 29.2%,36.4%,respectively after treating with DMT1 siRNA for 48 h.Immunoblot analysis revealed that 48 h DMT1 siRNA treatment decreased APP695 levels in neo cells and APPsw cells by 25.9%and 42.7%,respectively.ELISA results showed that RNAi significantly inhibitted Aβsecretion,the amount of Aβin the culture medium was decreased from 86 pg/ml/protein to 62 pg/ml/protein in SH-SY5Y cells stable transfected APPsw gene 48 h after RNAi.（3） DMT1-RNAi incereaed viability of APPsw cells treated with metal ions.Forty-eight hours after transfection of DMT1 RNAi,the cells were treated with metal ions for another 24h.MTT analysis revealed that cells viabilities were all dramatically increased in the APPsw cells treated with DMT1 siRNA as compared with controls.（4） DMT1-RNAi attenuated the increase of APP695 and Aβ1-42 induced by metal ions.APP695 levels of APPsw cells were increased significantly when the cells were incubated with 100μM ferrous,100μM ferric,and 50μM copper respectively. However,DMT1 RNAi treatment significantly decreased APP695 levels by 32.9%, 28.6%,and 27.5%in APPsw cells when the cells were incubated with 100μM ferrous, 100μM ferric,and 50μM copper respectively.After treatment with DMT1 siRNA for 48 h and then incubation with 100μM ferrous sulfate for another 24h,ELISA results showed that DMT1 siRNA treatment significantly decreased Aβ1-42 level as with 90 pg/ml/protein（RNAi+） versus 98 pg/ml/protein（RNAi-）. Conclusion1.DMT1 and Aβare co-expressed in the senile plaques.Different DMT1 has different localization in the senile plaques.The expression of DMT1-IRE and DMT1-nonIRE in the cerebral cortex and hippocampus of APP/PS1 transgenic mice are higher than those in wild-type mice.2.The levels of DMT1-IRE and DMT1-nonlRE in APPsw transfection are higer than empty vector transfection.Furthermore,DMT1-IRE and DMT1-nonIRE level of APPsw cells were not fedback regulated by ferrous iron concentration and maintain high levels.3.DMT1-RNAi could significantly reduce the APP expression and Aβsecretion in SH-SY5Y cells stable transfected APPsw gene.4.DMT1-RNAi can improve the viability of SH-SY5Y cells stable transfected APPsw gene in the environment of high ferrous iron,ferric iron and copper level.Most important DMT1-RNAi can attenuated the increase of APP695 and Aβ1-42 induced by metal ions.更多还原