ZnTs在Alzheimer病人和转基因鼠脑内表达及ZnT1基因沉默阻抑Aβ分泌的研究
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摘要
前言
阿尔茨海默病(Alzheimer's disease,AD)是主要发生在老年人的一种以进行性痴呆为临床特征的神经退行性疾病。随着人口的逐渐老龄化,AD的发病率呈逐年上升趋势,给社会和家庭带来了沉重的负担,由于缺乏有效的治疗手段,AD已成为危害人类健康的主要致死性疾病之一。AD的典型病理特征包括:β-淀粉样蛋白(β-amyloid,Aβ)在脑内病理性沉积形成老年斑、神经原纤维缠结(neurofibrillary tangles,NFT)和大脑淀粉样血管病(cerebral amyloid angiopath,CAA)。Aβ是AD发生和发展的中心环节,体内的Aβ由β-淀粉样前体蛋白(amyloidprecursor protein,APP)经β-和γ-分泌酶水解而成。到目前为止,AD的具体发病机制还不是十分清楚,但越来越多的证据表明,锌离子在AD的发病和病理过程中起着关键性的作用。锌离子能够通过链接相邻Aβ分子的第13位上的组氨酸而导致Aβ聚集;与Aβ生成密切相关的γ-分泌酶为锌结合蛋白,细胞内的锌离子具有抑制γ-分泌酶裂解APP向细胞外分泌Aβ的作用;口服金属螯合剂可以明显抑制APP转基因小鼠脑内老年斑的形成。因此,调节细胞内外金属离子稳态,维持Aβ生成与降解的动态平衡,已经成为目前AD治疗学的一个重要策略。
锌离子不能自由通过细胞膜,特定的转运体和膜通道参与锌的转运和代谢。锌转运体(zinc transporter,ZnT)是参与脑锌代谢的重要蛋白家族之一,目前至少拥有7个成员(ZnT1-7),除ZnT2外均在脑内表达,其主要功能是将锌离子转运出细胞或聚集在细胞器内:ZnT1定位在细胞膜上,负责将细胞内的锌离子向细胞外转运;ZnT3定位在突触小泡膜上,其功能是将锌离子转运并聚集在突触小泡内;ZnT4定位在胞质小泡膜上,可将细胞质内的锌离子转运到胞质小泡内;ZnT5-7定位在高尔基复合体,参与高尔基复合体内外的锌离子转运过程。最新的研究表明,ZnT1,4,6在AD病人大脑内的表达明显增强,而敲除ZnT3基因的APP转基因惺?其脑内Aβ老年斑数量、血管淀粉样改变均明显减少,提示ZnT与锌离子共同参与了AD的发病和病理过程。
本研究对锌离子和ZnT在AD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(RNA interference,RNAi)技术探讨ZnT1基因沉默阻抑APP转染细胞Aβ分泌的效果,对深入探讨脑锌代谢紊乱与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、APP/PS1转基因小鼠及稳定转染APPsw、APP基因的SH-SY5Y细胞(APPsw细胞、APP细胞)为研究对象,应用浸入式金属自显影技术(autometallography,AMG)检测AD病人和APP/PS1转基因小鼠脑内锌含量的变化,应用免疫荧光双标和激光共聚焦扫描显微技术检测ZnTs在AD病人和APP/PS1转基因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转基因小鼠大脑皮层及海马内ZnTs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ZnTs和Aβ的蛋白相关性,应用RNAi技术阻抑ZnT1基因在稳定转染APPsw、APP基因的SH-SY5Y细胞的表达,并应用RT-PCR和Western Blot技术检测RNAi对ZnT1的基因沉默效果,应用Zinquin荧光技术、Western Blot技术、免疫荧光双标技术、ELISA技术、MTT技术检测ZnT1-RNAi对细胞锌离子转运、APP表达、Aβ分泌和细胞活力的影响。
实验结果
1、锌离子在AD病人及APP/PS1转基因小鼠脑内的分布
AMG结果显示,AMG阳性的老年斑广泛分布于AD病人和APP/PS1转基因小鼠大脑皮质和海马等脑区,在血管壁及其周围也可见到明显的呈棕黑色的AMG阳性反应产物。AMG阳性的老年斑呈圆形或不规则形,大小不等,在AD病人大脑中多具有明显的致密核心,而在APP/PS1转基因小鼠大脑内多呈中空状。
2、ZnTs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,ZnTs免疫阳性反应产物呈棕黄色,在大脑皮层及海马内均可见ZnTs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ZnT1和ZnT4广泛分布到整个老年斑内,ZnT3、ZnT5及ZnT6主要分布在斑块周围变性的神经元突起内,而ZnT7则主要表达于老年斑的中心部位。此外,ZnTs还广泛表达于淀粉样变性的血管壁及其周围,其中以ZnT3染色最深。
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的齿状回、CA1-CA3区域,几乎所有Aβ阳性的老年斑均有不同程度的ZnTs表达,即ZnTs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,而不同的ZnTs在老年斑内的位置分布存在一定差异(同免疫组织化学染色结果),ZnT3免疫荧光除了分布在大脑皮层和海马的Aβ老年斑中,还可见于海马苔藓纤维和淀粉样变性的血管壁及其周围。
3、ZnTs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,ZnTs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ZnT1,3,4,5,6,7在大脑皮层的表达量分别是野生型小鼠的196.6%,201.9%,150.3%,134.2%,162.0%,122.9%;在海马的表达量分别是野生型小鼠的280.1%,398.6%,277.1%,168.2%,142.2%,282.3%。
4、ZnTs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ZnTs条带。
5、RNAi对ZnT1基因的沉默效果
RT-PCR结果显示,RNAi可在mRNA水平明显抑制APPsw细胞的ZnT1表达,干扰后24h即可出现ZnT1的表达下降,干扰后48h为抑制效果最佳时间点,ZnT1表达量仅为对照组的22%。
Western Blot结果显示,RNAi可明显抑制APPsw细胞的ZnT1蛋白表达,干扰后48h的抑制率达80%。
6、ZnT1-RNAi对锌离子转运的影响
Zinquin荧光染色结果显示,在APPsw细胞,ZnT1-RNAi后48h,可见锌离子在细胞内的聚集明显高于未干扰的对照组细胞。
7、ZnT1-RNAi对APP表达的影响
Western Blot结果显示,RNAi可明显抑制APPsw、APP细胞的APP蛋白表达,干扰后48h,APPsw、APP细胞的APP蛋白表达量分别为对照组的58%和57%。
免疫荧光双标和荧光显微镜观察结果显示,RNAi可明显抑制APPsw细胞的ZnT1和APP表达,与对照组相比,二者的免疫荧光均明显减弱。
8、ZnT1-RNAi对Aβ分泌量的影响
Elisa结果显示,RNAi可明显抑制APPsw、APP细胞的Aβ分泌,干扰48h后,APPsw和APP细胞培养液内的Aβ含量分别降低49%和37%。
9、锌离子对细胞活力的影响
MTT结果显示,正常培养的APPsw、APP和空质粒转染的SH-SY5Y细胞(NEO细胞)活力几乎没有差别;10μM氯化锌处理可分别降低APPsw、APP细胞活力19.7%和13.9%,而对NEO细胞活力没有影响;50μM氯化锌处理可分别降低APPsw、APP、NEO细胞活力48.5%、42.7%、30%;100μM氯化锌处理可分别降低APPsw、APP、NEO细胞活力66.6%、56.5%、46.1%。
10、ZnT1-RNAi对细胞活力的影响
MTT结果显示,正常培养条件下,ZnT1-RNAi对细胞活力几乎没有影响,在50μM氯化锌处理的情况下,ZnT1-RNAi可分别提高APPsw、APP细胞活力14.4%、10.5%。
结论
1、AD病人和APP/PS1转基因小鼠脑内的老年斑和淀粉样变性的血管壁及其周围组织富含锌离子。AMG技术对检测游离锌离子具有高度特异性和敏感性,是研究锌离子在AD病人和APP/PS1转基因小鼠脑内分布的有利手段。
2、ZnTs与Aβ共表达于老年斑和淀粉样变性的血管壁及其周围。不同ZnTs在老年斑内的定位分布具有差异性。ZnTs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。
3、ZnT1-RNAi可明显降低稳定转染APPsw,APP基因的SH-SY5Y细胞的APP表达和Aβ分泌。
4、ZnT1-RNAi可提高稳定转染APPsw,APP基因的SH-SY5Y细胞在高锌环境下的细胞活力。
Preface
Alzheimer's disease(AD)is a disease cinically 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)and cerebral amyloid angiopathy(CAA).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. By now,the pathogenes of AD is not very clear,but more and more evidences suggested that zinc played a key role in the pathogenes and pathologic process of AD. Zinc ions can trigger a deposition of Aβby connecting the 13~(th)amino acides between the adjecent Aβmolecules.γ-secretase is a zinc binding protein,and cytoplasmic zinc has the function to decrease the extracellular secretion of insoluble Aβby inhibitting theγ-secretase cracking APP.Metal chelating agents have been shown to inhibit the formation of amyloid plaques in APP transgenic mouse brains.Therefore,the regulation of metal ion homeostasis and maintain of the formation and degradation of Aβhave become an important therapeutic strategy of AD.Zinc cannot travel across biological membranes by passive diffusion.Specific membrane transporters and channels involved in its transfer and metabolism.Zinc transporter(ZnT)is one of the important protein family involved in zinc metabolism of brain.Until now,seven members of the ZnT family(ZnT1-7)have been characterized and except for ZnT2,all the ZnTs are expressed in the brain.ZnT family members are responsible for the extrusion of zinc outside the cytoplasm to the extracellular space or intracellular organelles.ZnT1,an ubiquitous zinc transporter localized on the plasma membrane, serves an essential function of zinc efflux from the cell.ZnT3 is mainly localized in the membranes of zinc-rich synaptic vesicles and involved in the release of zinc ions in the synaptic vesicles.ZnT4 is localized on the intracellular vesicular membrane and functions to increase vesicular zinc concentration.ZnT5,ZnT6 and ZnT7 are localized on the Golgi apparatus and believed to facilitate the translocation of the cytoplasmic zinc into the Golgi apparatus.Recently,it has been reported that there were significant alterations in the expression of ZnT1,ZnT4 and ZnT6 in AD patient brains,and genetic ablation of ZnT3 in the Tg2576 Alzheimer mouse model inhibited the formation of amyloid plaques and CAA,indicating the roles of ZnT and zinc ions in the pathogenes and pathologic process of AD.
In summary,it has scientific significance to analyse the expression and distribution of zinc ions and ZnT and correlation between ZnT and Aβin the AD brain, as well as the further study about zinc metabolism and its correlation to the pathophysiological mechanism of AD.
Methods
AD patient brains,APP/PS1 transgenic mice and SH-SY5Y cells stable transfected APPsw or APP gene were used for the present study.The levels of free zinc ions in AD patient brains and APP/PS1 transgenic mice brains were evaluated by immersion autometallography(AMG).The distribution patterns of ZnTs in these brains and the positional relation between ZnTs and Aβwere detected by double immunofluorescence and confocal laser scanning microscopy.The changes of ZnTs 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 ZnTs in the APP/PS1 transgenic mice brains.RNA interference(RNAi)technology was used to inhibite the expression of ZnT1 gene in SH-SY5Y cells stably transfected APPsw or APP gene.The gene silencing effect of ZnT1-RNAi was detected by RT-PCR and Western Blot.Zinquin fluorescence technology,Western Blot,double immunofluorescence techniques, ELISA technology and MTT were used to detect the effects of ZnT1 RNAi on zinc ions transport,APP expression,Aβsecretion and activity of cells.
Results
1.Distribution of zinc ions in the human AD and APP/PS1 transgenic mice brains.
AMG results showed that AMG-positive plaques were widely distributed in the cerebral cortex and hippocampus of APP/PS1 transgenic mouse.Brown black AMG positive reaction products could be seen clearly in the vascular wall and its surrounding. The AMG-positive plaques were round or irregular in shape and different in size and morphology.In the AD patients,a majority of the AMG-stained plaques had a dence core,while in the APP/PS1 transgenic mice,most plaques were rosette-shaped with a non-zinc stained interior.
2.Abundant expression of ZnTs in SP and amyloid angiopathic vessels
Immunohistochemimistry results revealed that ZnTs immunopositive reaction products were brown.ZnTs-positive plaques were round or irregular and distributed throughout the cortex and hippocampus with vary size and clear boundary.At higher magnification,ZnT1 and ZnT4 were extensively expressed in all parts of the plaques. ZnT3,ZnT5 and ZnT6 were expressed prominently in the degenerating neurites in the peripheral part of the plaques,while ZnT7 was present in the core of the plaques.Our data also showed an abundant expression of ZnTs in the amyloid angiopathic vessels. ZnT3 immunoreactivity was the most intense.
Double-immunofluorescence staining for Aβand one of the ZnTs showed that the Aβ-positive plaques were widely distributed in the APP/PS1 transgenic mouse cerebral cortex and the DG and CA1-CA3 region of the hippocampus,ZnTs were expressed in most of the Aβcontaining plaques,i.e.Aβand ZnTs protein were co-expressed in the senile plaques.At higher magnification,the Aβwas located in the core of plaques,and ZnTs,however,exhibited different staining patterns(similar to immunohistochemical results).Apart from senile plaques,an intense ZnT3 fluorescence was also seen in the hippocampal mossy fibers and the wall of amyloid angiopathic vessels.
3.Altered expression of ZnTs in the APP/PS1 transgenic mouse cerebral cortex and hippocampus
Western blot results showed that the expressions of ZnTs were increased in the hippocampus and cortex of APPswe/PS1dE9 transgenic mice.The expressions of ZnT1, 3,4,5,6 and 7 were 196.6%,201.9%,150.3%,134.2%,162.0%,122.9%of wild-type control mice in the cerebral cortex;280.1%,398.6%,277.1%,168.2%,142.2%, 282.3%in the hippocampus.
4.Molecular Correlation Analysis of ZnTs and Aβ
Co-IP results showed that using Aβantibodies to perform the co-immunoprecipitation of ZnTs and Aβin the APP/PS1 transgenic mouse cerebral cortex,after agarose gel electrophoresis,no ZnTs band could be seen.
5.The effect of RNAi to silence genes ZnT1
RT-PCR results show that RNAi significantly inhibited the expression of ZnT1 at mRNA level in SH-SY5Y cells stable transfected APPsw gene.The inhibitory effect beginning to show 24 h after RNAi and 48 h after RNAi was the most effective time, the expression of ZnT1 account for only 22%of control group.
Western Blot results showed that RNAi could significantly inhibit ZnT1 expression in APPsw-cells,and the inhibiting rate can reach 80%48 h after RNAi.
6.The effect of ZnT1-RNAi on zinc ion transport
Zinquin staining results showed that the aggregation of zinc ions in the SH-SY5Y cells stable transfected APPsw gene(APPsw cells)was significantly higher than that of the control group 48 h after RNAi.
7.The effect of ZnT1-RNAi on the expression of APP
Western Blot results showed that RNAi could significantly inhibit the APP expression in SH-SY5Y cells stable transfected APPsw or APP gene,the expression of APP account for only 58%or 57%of control group in SH-SY5Y cells stable transfected APPsw or APP gene 48 h after RNAi.
Double immunofluorescence and fluorescence microscopy showed that RNAi could significantly inhibit the APP expression in SH-SY5Y cells stable transfected APPsw gene,the immunofluorescence of them was significantly weakened compared with control group.
8.The effect of ZnT1-RNAi on Aβsecretion
Elisa results showed that RNAi significantly inhibitted Aβsecretion,the amount of Aβin the culture medium was decreased by 49%and 37%respectively in SH-SY5Y cells stable transfected APPsw and APP gene 48 h after RNAi.
9.Effect of zinc ions on the activity of cells
MTT results show that there are almost no activity difference among normal cultured SH-SY5Y cells stable transfected APPsw gene,APP gene or empty vector (NEO).The cell activity were reduced by 19.7%or 13.9%in SH-SY5Y cells stable transfected APPsw or APP gene after 10μM zinc chloride treatment.The cell activity can be reduced by 48.5%,42.7%,30%in APPsw,APP or NEO cells after 50μM zinc chloride treatment.The cell activity can be reduced by 66.6%,56.5%,46.1%in APPsw, APP or NEO cells after 100μM zinc chloride treatment.
10.Effect of ZnT1-RNAi on the activity of cells
MTT results show that ZnT1-RNAi did not affect cell viability at normal cultured state,and can improve cell viability of APPsw,APP cell cultured with 50μM zinc chloride by 14.4%and 10.5%respectively.
Conclusion
1.Zinc ion is enriched in the senile plaques and the wall and the vicinity of CAA changed vessels of AD patients and APP/PS1 transgenic mice brain.AMG,a high specificity and sensitivity technology to detect free zinc ions,is a favorable means to study the distribution of zinc ions in the AD patients and APP/PS1 transgenic mice brain.
2.ZnTs and Aβare co-expressed in the senile plaques and the wall and the vicinity of CAA changed vessels.Different ZnTs has different localization in the senile plaques. The expression of ZnTs in the cerebral cortex and hippocampus of APP/PS1 transgenic mice is higher than that in wild-type mice.
3.ZnT1-RNAi could significantly reduce the APP expression and Aβsecretion in SH-SY5Y cells stable transfected APPsw or APP gene.
4.ZnT1-RNAi can improve the viability of SH-SY5Y cells stable transfected APPsw or APP gene in the environment of high zinc level.
阿尔茨海默病(Alzheimer's disease,AD)是主要发生在老年人的一种以进行性痴呆为临床特征的神经退行性疾病。随着人口的逐渐老龄化,AD的发病率呈逐年上升趋势,给社会和家庭带来了沉重的负担,由于缺乏有效的治疗手段,AD已成为危害人类健康的主要致死性疾病之一。AD的典型病理特征包括:β-淀粉样蛋白(β-amyloid,Aβ)在脑内病理性沉积形成老年斑、神经原纤维缠结(neurofibrillary tangles,NFT)和大脑淀粉样血管病(cerebral amyloid angiopath,CAA)。Aβ是AD发生和发展的中心环节,体内的Aβ由β-淀粉样前体蛋白(amyloidprecursor protein,APP)经β-和γ-分泌酶水解而成。到目前为止,AD的具体发病机制还不是十分清楚,但越来越多的证据表明,锌离子在AD的发病和病理过程中起着关键性的作用。锌离子能够通过链接相邻Aβ分子的第13位上的组氨酸而导致Aβ聚集;与Aβ生成密切相关的γ-分泌酶为锌结合蛋白,细胞内的锌离子具有抑制γ-分泌酶裂解APP向细胞外分泌Aβ的作用;口服金属螯合剂可以明显抑制APP转基因小鼠脑内老年斑的形成。因此,调节细胞内外金属离子稳态,维持Aβ生成与降解的动态平衡,已经成为目前AD治疗学的一个重要策略。
锌离子不能自由通过细胞膜,特定的转运体和膜通道参与锌的转运和代谢。锌转运体(zinc transporter,ZnT)是参与脑锌代谢的重要蛋白家族之一,目前至少拥有7个成员(ZnT1-7),除ZnT2外均在脑内表达,其主要功能是将锌离子转运出细胞或聚集在细胞器内:ZnT1定位在细胞膜上,负责将细胞内的锌离子向细胞外转运;ZnT3定位在突触小泡膜上,其功能是将锌离子转运并聚集在突触小泡内;ZnT4定位在胞质小泡膜上,可将细胞质内的锌离子转运到胞质小泡内;ZnT5-7定位在高尔基复合体,参与高尔基复合体内外的锌离子转运过程。最新的研究表明,ZnT1,4,6在AD病人大脑内的表达明显增强,而敲除ZnT3基因的APP转基因惺?其脑内Aβ老年斑数量、血管淀粉样改变均明显减少,提示ZnT与锌离子共同参与了AD的发病和病理过程。
本研究对锌离子和ZnT在AD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(RNA interference,RNAi)技术探讨ZnT1基因沉默阻抑APP转染细胞Aβ分泌的效果,对深入探讨脑锌代谢紊乱与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、APP/PS1转基因小鼠及稳定转染APPsw、APP基因的SH-SY5Y细胞(APPsw细胞、APP细胞)为研究对象,应用浸入式金属自显影技术(autometallography,AMG)检测AD病人和APP/PS1转基因小鼠脑内锌含量的变化,应用免疫荧光双标和激光共聚焦扫描显微技术检测ZnTs在AD病人和APP/PS1转基因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转基因小鼠大脑皮层及海马内ZnTs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ZnTs和Aβ的蛋白相关性,应用RNAi技术阻抑ZnT1基因在稳定转染APPsw、APP基因的SH-SY5Y细胞的表达,并应用RT-PCR和Western Blot技术检测RNAi对ZnT1的基因沉默效果,应用Zinquin荧光技术、Western Blot技术、免疫荧光双标技术、ELISA技术、MTT技术检测ZnT1-RNAi对细胞锌离子转运、APP表达、Aβ分泌和细胞活力的影响。
实验结果
1、锌离子在AD病人及APP/PS1转基因小鼠脑内的分布
AMG结果显示,AMG阳性的老年斑广泛分布于AD病人和APP/PS1转基因小鼠大脑皮质和海马等脑区,在血管壁及其周围也可见到明显的呈棕黑色的AMG阳性反应产物。AMG阳性的老年斑呈圆形或不规则形,大小不等,在AD病人大脑中多具有明显的致密核心,而在APP/PS1转基因小鼠大脑内多呈中空状。
2、ZnTs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,ZnTs免疫阳性反应产物呈棕黄色,在大脑皮层及海马内均可见ZnTs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ZnT1和ZnT4广泛分布到整个老年斑内,ZnT3、ZnT5及ZnT6主要分布在斑块周围变性的神经元突起内,而ZnT7则主要表达于老年斑的中心部位。此外,ZnTs还广泛表达于淀粉样变性的血管壁及其周围,其中以ZnT3染色最深。
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的齿状回、CA1-CA3区域,几乎所有Aβ阳性的老年斑均有不同程度的ZnTs表达,即ZnTs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,而不同的ZnTs在老年斑内的位置分布存在一定差异(同免疫组织化学染色结果),ZnT3免疫荧光除了分布在大脑皮层和海马的Aβ老年斑中,还可见于海马苔藓纤维和淀粉样变性的血管壁及其周围。
3、ZnTs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,ZnTs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ZnT1,3,4,5,6,7在大脑皮层的表达量分别是野生型小鼠的196.6%,201.9%,150.3%,134.2%,162.0%,122.9%;在海马的表达量分别是野生型小鼠的280.1%,398.6%,277.1%,168.2%,142.2%,282.3%。
4、ZnTs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ZnTs条带。
5、RNAi对ZnT1基因的沉默效果
RT-PCR结果显示,RNAi可在mRNA水平明显抑制APPsw细胞的ZnT1表达,干扰后24h即可出现ZnT1的表达下降,干扰后48h为抑制效果最佳时间点,ZnT1表达量仅为对照组的22%。
Western Blot结果显示,RNAi可明显抑制APPsw细胞的ZnT1蛋白表达,干扰后48h的抑制率达80%。
6、ZnT1-RNAi对锌离子转运的影响
Zinquin荧光染色结果显示,在APPsw细胞,ZnT1-RNAi后48h,可见锌离子在细胞内的聚集明显高于未干扰的对照组细胞。
7、ZnT1-RNAi对APP表达的影响
Western Blot结果显示,RNAi可明显抑制APPsw、APP细胞的APP蛋白表达,干扰后48h,APPsw、APP细胞的APP蛋白表达量分别为对照组的58%和57%。
免疫荧光双标和荧光显微镜观察结果显示,RNAi可明显抑制APPsw细胞的ZnT1和APP表达,与对照组相比,二者的免疫荧光均明显减弱。
8、ZnT1-RNAi对Aβ分泌量的影响
Elisa结果显示,RNAi可明显抑制APPsw、APP细胞的Aβ分泌,干扰48h后,APPsw和APP细胞培养液内的Aβ含量分别降低49%和37%。
9、锌离子对细胞活力的影响
MTT结果显示,正常培养的APPsw、APP和空质粒转染的SH-SY5Y细胞(NEO细胞)活力几乎没有差别;10μM氯化锌处理可分别降低APPsw、APP细胞活力19.7%和13.9%,而对NEO细胞活力没有影响;50μM氯化锌处理可分别降低APPsw、APP、NEO细胞活力48.5%、42.7%、30%;100μM氯化锌处理可分别降低APPsw、APP、NEO细胞活力66.6%、56.5%、46.1%。
10、ZnT1-RNAi对细胞活力的影响
MTT结果显示,正常培养条件下,ZnT1-RNAi对细胞活力几乎没有影响,在50μM氯化锌处理的情况下,ZnT1-RNAi可分别提高APPsw、APP细胞活力14.4%、10.5%。
结论
1、AD病人和APP/PS1转基因小鼠脑内的老年斑和淀粉样变性的血管壁及其周围组织富含锌离子。AMG技术对检测游离锌离子具有高度特异性和敏感性,是研究锌离子在AD病人和APP/PS1转基因小鼠脑内分布的有利手段。
2、ZnTs与Aβ共表达于老年斑和淀粉样变性的血管壁及其周围。不同ZnTs在老年斑内的定位分布具有差异性。ZnTs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。
3、ZnT1-RNAi可明显降低稳定转染APPsw,APP基因的SH-SY5Y细胞的APP表达和Aβ分泌。
4、ZnT1-RNAi可提高稳定转染APPsw,APP基因的SH-SY5Y细胞在高锌环境下的细胞活力。
Preface
Alzheimer's disease(AD)is a disease cinically 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)and cerebral amyloid angiopathy(CAA).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. By now,the pathogenes of AD is not very clear,but more and more evidences suggested that zinc played a key role in the pathogenes and pathologic process of AD. Zinc ions can trigger a deposition of Aβby connecting the 13~(th)amino acides between the adjecent Aβmolecules.γ-secretase is a zinc binding protein,and cytoplasmic zinc has the function to decrease the extracellular secretion of insoluble Aβby inhibitting theγ-secretase cracking APP.Metal chelating agents have been shown to inhibit the formation of amyloid plaques in APP transgenic mouse brains.Therefore,the regulation of metal ion homeostasis and maintain of the formation and degradation of Aβhave become an important therapeutic strategy of AD.Zinc cannot travel across biological membranes by passive diffusion.Specific membrane transporters and channels involved in its transfer and metabolism.Zinc transporter(ZnT)is one of the important protein family involved in zinc metabolism of brain.Until now,seven members of the ZnT family(ZnT1-7)have been characterized and except for ZnT2,all the ZnTs are expressed in the brain.ZnT family members are responsible for the extrusion of zinc outside the cytoplasm to the extracellular space or intracellular organelles.ZnT1,an ubiquitous zinc transporter localized on the plasma membrane, serves an essential function of zinc efflux from the cell.ZnT3 is mainly localized in the membranes of zinc-rich synaptic vesicles and involved in the release of zinc ions in the synaptic vesicles.ZnT4 is localized on the intracellular vesicular membrane and functions to increase vesicular zinc concentration.ZnT5,ZnT6 and ZnT7 are localized on the Golgi apparatus and believed to facilitate the translocation of the cytoplasmic zinc into the Golgi apparatus.Recently,it has been reported that there were significant alterations in the expression of ZnT1,ZnT4 and ZnT6 in AD patient brains,and genetic ablation of ZnT3 in the Tg2576 Alzheimer mouse model inhibited the formation of amyloid plaques and CAA,indicating the roles of ZnT and zinc ions in the pathogenes and pathologic process of AD.
In summary,it has scientific significance to analyse the expression and distribution of zinc ions and ZnT and correlation between ZnT and Aβin the AD brain, as well as the further study about zinc metabolism and its correlation to the pathophysiological mechanism of AD.
Methods
AD patient brains,APP/PS1 transgenic mice and SH-SY5Y cells stable transfected APPsw or APP gene were used for the present study.The levels of free zinc ions in AD patient brains and APP/PS1 transgenic mice brains were evaluated by immersion autometallography(AMG).The distribution patterns of ZnTs in these brains and the positional relation between ZnTs and Aβwere detected by double immunofluorescence and confocal laser scanning microscopy.The changes of ZnTs 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 ZnTs in the APP/PS1 transgenic mice brains.RNA interference(RNAi)technology was used to inhibite the expression of ZnT1 gene in SH-SY5Y cells stably transfected APPsw or APP gene.The gene silencing effect of ZnT1-RNAi was detected by RT-PCR and Western Blot.Zinquin fluorescence technology,Western Blot,double immunofluorescence techniques, ELISA technology and MTT were used to detect the effects of ZnT1 RNAi on zinc ions transport,APP expression,Aβsecretion and activity of cells.
Results
1.Distribution of zinc ions in the human AD and APP/PS1 transgenic mice brains.
AMG results showed that AMG-positive plaques were widely distributed in the cerebral cortex and hippocampus of APP/PS1 transgenic mouse.Brown black AMG positive reaction products could be seen clearly in the vascular wall and its surrounding. The AMG-positive plaques were round or irregular in shape and different in size and morphology.In the AD patients,a majority of the AMG-stained plaques had a dence core,while in the APP/PS1 transgenic mice,most plaques were rosette-shaped with a non-zinc stained interior.
2.Abundant expression of ZnTs in SP and amyloid angiopathic vessels
Immunohistochemimistry results revealed that ZnTs immunopositive reaction products were brown.ZnTs-positive plaques were round or irregular and distributed throughout the cortex and hippocampus with vary size and clear boundary.At higher magnification,ZnT1 and ZnT4 were extensively expressed in all parts of the plaques. ZnT3,ZnT5 and ZnT6 were expressed prominently in the degenerating neurites in the peripheral part of the plaques,while ZnT7 was present in the core of the plaques.Our data also showed an abundant expression of ZnTs in the amyloid angiopathic vessels. ZnT3 immunoreactivity was the most intense.
Double-immunofluorescence staining for Aβand one of the ZnTs showed that the Aβ-positive plaques were widely distributed in the APP/PS1 transgenic mouse cerebral cortex and the DG and CA1-CA3 region of the hippocampus,ZnTs were expressed in most of the Aβcontaining plaques,i.e.Aβand ZnTs protein were co-expressed in the senile plaques.At higher magnification,the Aβwas located in the core of plaques,and ZnTs,however,exhibited different staining patterns(similar to immunohistochemical results).Apart from senile plaques,an intense ZnT3 fluorescence was also seen in the hippocampal mossy fibers and the wall of amyloid angiopathic vessels.
3.Altered expression of ZnTs in the APP/PS1 transgenic mouse cerebral cortex and hippocampus
Western blot results showed that the expressions of ZnTs were increased in the hippocampus and cortex of APPswe/PS1dE9 transgenic mice.The expressions of ZnT1, 3,4,5,6 and 7 were 196.6%,201.9%,150.3%,134.2%,162.0%,122.9%of wild-type control mice in the cerebral cortex;280.1%,398.6%,277.1%,168.2%,142.2%, 282.3%in the hippocampus.
4.Molecular Correlation Analysis of ZnTs and Aβ
Co-IP results showed that using Aβantibodies to perform the co-immunoprecipitation of ZnTs and Aβin the APP/PS1 transgenic mouse cerebral cortex,after agarose gel electrophoresis,no ZnTs band could be seen.
5.The effect of RNAi to silence genes ZnT1
RT-PCR results show that RNAi significantly inhibited the expression of ZnT1 at mRNA level in SH-SY5Y cells stable transfected APPsw gene.The inhibitory effect beginning to show 24 h after RNAi and 48 h after RNAi was the most effective time, the expression of ZnT1 account for only 22%of control group.
Western Blot results showed that RNAi could significantly inhibit ZnT1 expression in APPsw-cells,and the inhibiting rate can reach 80%48 h after RNAi.
6.The effect of ZnT1-RNAi on zinc ion transport
Zinquin staining results showed that the aggregation of zinc ions in the SH-SY5Y cells stable transfected APPsw gene(APPsw cells)was significantly higher than that of the control group 48 h after RNAi.
7.The effect of ZnT1-RNAi on the expression of APP
Western Blot results showed that RNAi could significantly inhibit the APP expression in SH-SY5Y cells stable transfected APPsw or APP gene,the expression of APP account for only 58%or 57%of control group in SH-SY5Y cells stable transfected APPsw or APP gene 48 h after RNAi.
Double immunofluorescence and fluorescence microscopy showed that RNAi could significantly inhibit the APP expression in SH-SY5Y cells stable transfected APPsw gene,the immunofluorescence of them was significantly weakened compared with control group.
8.The effect of ZnT1-RNAi on Aβsecretion
Elisa results showed that RNAi significantly inhibitted Aβsecretion,the amount of Aβin the culture medium was decreased by 49%and 37%respectively in SH-SY5Y cells stable transfected APPsw and APP gene 48 h after RNAi.
9.Effect of zinc ions on the activity of cells
MTT results show that there are almost no activity difference among normal cultured SH-SY5Y cells stable transfected APPsw gene,APP gene or empty vector (NEO).The cell activity were reduced by 19.7%or 13.9%in SH-SY5Y cells stable transfected APPsw or APP gene after 10μM zinc chloride treatment.The cell activity can be reduced by 48.5%,42.7%,30%in APPsw,APP or NEO cells after 50μM zinc chloride treatment.The cell activity can be reduced by 66.6%,56.5%,46.1%in APPsw, APP or NEO cells after 100μM zinc chloride treatment.
10.Effect of ZnT1-RNAi on the activity of cells
MTT results show that ZnT1-RNAi did not affect cell viability at normal cultured state,and can improve cell viability of APPsw,APP cell cultured with 50μM zinc chloride by 14.4%and 10.5%respectively.
Conclusion
1.Zinc ion is enriched in the senile plaques and the wall and the vicinity of CAA changed vessels of AD patients and APP/PS1 transgenic mice brain.AMG,a high specificity and sensitivity technology to detect free zinc ions,is a favorable means to study the distribution of zinc ions in the AD patients and APP/PS1 transgenic mice brain.
2.ZnTs and Aβare co-expressed in the senile plaques and the wall and the vicinity of CAA changed vessels.Different ZnTs has different localization in the senile plaques. The expression of ZnTs in the cerebral cortex and hippocampus of APP/PS1 transgenic mice is higher than that in wild-type mice.
3.ZnT1-RNAi could significantly reduce the APP expression and Aβsecretion in SH-SY5Y cells stable transfected APPsw or APP gene.
4.ZnT1-RNAi can improve the viability of SH-SY5Y cells stable transfected APPsw or APP gene in the environment of high zinc level.
引文
1 Kar S, Slowikowski SP, Westaway D, Mount HT. Interactions between beta-amyloid and central cholinergic neurons: implications for Alzheimer's disease. J Psychiatry Neurosci 2004; 29:427-441.
2 Stoltenberg M, Bruhn M, Sondergaard C, Doering P, West MJ, Larsen A et al. Immersion autometallographic tracing of zinc ions in Alzheimer beta-amyloid plaques. Histochem Cell Biol 2005; 123: 605-611.
3 Samudralwar DL, Diprete CC, Ni BF, Ehmann WD, Markesbery WR. Elemental imbalances in the olfactory pathway in Alzheimer's disease. JNeurol Sci 1995; 130: 139-145.
4 Deibel MA, Ehmann WD, Markesbery WR. Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to oxidative stress. J Neurol Sci 1996; 143:137-142.
5 Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158: 47-52.
6 Miller LM, Wang Q, Telivala TP, Smith RJ, Lanzirotti A, Miklossy J. Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease. J Struct Biol 2006; 155: 30-37.
7 Lee JY, Mook-Jung I, Koh JY. Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice. J Neurosci 1999; 19: RC10.
8 Vallee BL, Auld DS. New perspective on zinc biochemistry: cocatalytic sites in multi-zinc enzymes. Biochemistry 1993; 32: 6493-6500.
9 Perez-Clausell J. Distribution of terminal fields stained for zinc in the neocortex of the rat. J Chem Neuroanat 1996; 11: 99-111.
10 Takeda A. Movement of zinc and its functional significance in the brain. Brain Res Brain Res Rev 2000; 34: 137-148.
11 Takeda A. Zinc homeostasis and functions of zinc in the brain. Biometals 2001; 14: 343-351.
12 Valente T, Auladell C, Perez-Clausell J. Postnatal development of zinc-rich terminal fields in the brain of the rat. Exp Neurol 2002; 174: 215-229.
13 Wang ZY, Stoltenberg M, Jo SM, Huang L, Larsen A, Dahlstrom A et al. Dynamic zinc pools in mouse choroid plexus. Neuroreport 2004; 15: 1801-1804.
14 Christianson DW. Structural biology of zinc. Adv Protein Chem 1991; 42: 281-355.
15 Coleman JE. Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem 1992; 61: 897-946.
16 Frederickson CJ, Kasarskis EJ, Ringo D, Frederickson RE. A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J Neurosci Methods 1987; 20: 91-103.
17 Mancini M, Ricci A, Amenta F. Age-related changes in sulfide-silver staining in the rat neostriatum: a quantitative histochemical study. Neurobiol Aging 1992; 13: 501-504.
18 Takeda A, Suzuki M, Okada S, Oku N. 65Zn localization in rat brain after intracerebroventricular injection of 65Zn-histidine. Brain Res 2000; 863: 241-244.
19 Miro-Bernie N, Sancho-Bielsa FJ, Lopez-Garcia C, Perez-Clausell J. Retrograde transport of sodium selenite and intracellular injection of micro-ruby: a combined method to describe the morphology of zinc-rich neurones. J Neurosci Methods 2003; 127: 199-209.
20 Danscher G, Stoltenberg M, Bruhn M, Sondergaard C, Jensen D. Immersion autometallography: histochemical in situ capturing of zinc ions in catalytic zinc-sulfur nanocrystals. JHistochem Cytochem 2004; 52: 1619-1625.
21 Danscher G, Jensen KB, Frederickson CJ, Kemp K, Andreasen A, Juhl S et al. Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: a proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material. J Neurosci Methods 1997; 76: 53-59.
22 Bush AI, Pettingell WH, Multhaup G, d Paradis M, Vonsattel JP, Gusella JF et al. Rapid induction of Alzheimer A beta amyloid formation by zinc. Science 1994; 265: 1464-1467.
23 Liu ST, Howlett G, Barrow CJ. Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the A beta peptide of Alzheimer's disease. Biochemistry 1999; 38: 9373-9378.
24 Bush AI. Metal complexing agents as therapies for Alzheimer's disease. Neurobiol Aging 2002; 23: 1031-1038.
25 Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001; 30: 665-676.
26 Lee JY, Friedman JE, Angel I, Kozak A, Koh JY. The lipophilic metal chelator DP-109 reduces amyloid pathology in brains of human beta-amyloid precursor protein transgenic mice. Neurobiol Aging 2004; 25: 1315-1321.
27 Zirah S, Kozin SA, Mazur AK, Blond A, Cheminant M, Segalas-Milazzo I et al Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging. J Biol Chem 2006; 281: 2151-2161.
28 Regland B, Lehmann W, Abedini I, Blennow K, Jonsson M, Karlsson I et al. Treatment of Alzheimer's disease with clioquinol. Dement Geriatr Cogn Disord2001; 12: 408-414.
29 Filiz G, Price KA, Caragounis A, Du T, Crouch PJ, White AR. The role of metals in modulating metalloprotease activity in the AD brain. Eur Biophys J 2008; 37: 315-321.
30 Suh SW, Jensen KB, Jensen MS, Silva DS, Kesslak PJ, Danscher G et al. Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains. Brain Res 2000; 852: 274-278.
31 Palmiter RD, Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch 2004; 447: 744-751.
32 Eide DJ. The SLC39 family of metal ion transporters. Pflugers Arch 2004; 447: 796-800.
33 Palmiter RD, Cole TB, Quaife CJ, Findley SD. ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc Natl Acad Sci USA 1996; 93:14934-14939.
34 Colvin RA, Fontaine CP, Laskowski M, Thomas D. Zn2+ transporters and Zn2+ homeostasis in neurons. Eur J Pharmacol 2003; 479: 171-185.
35 Kambe T, Yamaguchi-Iwai Y, Sasaki R, Nagao M. Overview of mammalian zinc transporters. Cell Mol Life Sci 2004; 61: 49-68.
36 Palmiter RD, Cole TB, Findley SD. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 1996; 15:1784-1791.
37 Huang X, Atwood CS, Moir RD, Hartshorn MA, Vonsattel JP, Tanzi RE et al. Zinc-induced Alzheimer's A beta1-40 aggregation is mediated by conformational factors. J Biol Chem 1997; 272: 26464-26470.
38 Huang L, Kirschke CP, Gitschier J. Functional characterization of a novel mammalian zinc transporter, ZnT6. J Biol Chem 2002; 277: 26389-26395.
39 Lovell MA, Smith JL, Xiong S, Markesbery WR. Alterations in zinc transporter protein-1 (ZnT-1) in the brain of subjects with mild cognitive impairment, early, and late-stage Alzheimer's disease. Neurotox Res 2005; 7: 265-271.
40 Smith JL, Xiong S, Markesbery WR, Lovell MA. Altered expression of zinc transporters-4 and -6 in mild cognitive impairment, early and late Alzheimer's disease brain. Neuroscience 2006; 140: 879-888.
41 Lovell MA, Smith JL, Markesbery WR. Elevated zinc transporter-6 in mild cognitive impairment, Alzheimer disease, and pick disease. J Neuropathol Exp Neurol 2006; 65: 489-498.
42 Cousins RJ, McMahon RJ. Integrative aspects of zinc transporters. J Nutr 2000; 130: 1384S-1387S.
43 Wang Z, Danscher G, Kim YK, Dahlstrom A, Mook Jo S. Inhibitory zinc-enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase. Neurosci Lett 2002; 321: 37-40.
44 Wang ZY, Danscher G, Dahlstrom A, Li JY. Zinc transporter 3 and zinc ions in the rodent superior cervical ganglion neurons. Neuroscience 2003; 120: 605-616.
45 Wang ZY, Stoltenberg M, Huang L, Danscher G, Dahlstrom A, Shi Y et al. Abundant expression of zinc transporters in Bergman glia of mouse cerebellum. Brain Res Bull 2005; 64:441-448.
46 Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 1995; 14: 639-649.
47 Nitzan YB, Sekler I, Hershfinkel M, Moran A, Silverman WF. Postnatal regulation of ZnT-1 expression in the mouse brain. Brain Res Dev Brain Res 2002; 137: 149-157.
48 Sekler I, Moran A, Hershfinkel M, Dori A, Margulis A, Birenzweig N et al. Distribution of the zinc transporter ZnT-1 in comparison with chelatable zinc in the mouse brain. J Comp Neurol 2002; 447: 201-209.
49 Andrews GK, Wang H, Dey SK, Palmiter RD. Mouse zinc transporter 1 gene provides an essential function during early embryonic development. Genesis 2004; 40: 74-81.
50 Langmade SJ, Ravindra R, Daniels PJ, Andrews GK. The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J Biol Chem 2000; 275: 34803-34809.
51 Tsuda M, Imaizumi K, Katayama T, Kitagawa K, Wanaka A, Tohyama M et al. Expression of zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil. J Neurosci 1997; 17: 6678-6684.
52 Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD. Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey. Proc Natl Acad Sci USA 1997; 94: 12676-12681.
53 Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl Acad Sci USA 2002; 99: 7705-7710.
54 Cole TB, Wenzel HJ, Kafer KE, Schwartzkroin PA, Palmiter RD. Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene. Proc Natl Acad SciUSA 1999; 96: 1716-1721.
55 Friedlich AL, Lee JY, van Groen T, Cherny RA, Volitakis I, Cole TB et al. Neuronal zinc exchange with the blood vessel wall promotes cerebral amyloid angiopathy in an animal model of Alzheimer's disease. J Neurosci 2004; 24: 3453-3459.
56 Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H et al. Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol 2002; 161: 1869-1879.
57 Wirths O, Multhaup G, Czech C, Blanchard V, Moussaoui S, Tremp G et al. Intraneuronal Abeta accumulation precedes plaque formation in beta-amyloid precursor protein and presenilin-1 double-transgenic mice. Neurosci Lett 2001; 306: 116-120.
58 Shie FS, LeBoeuf RC, Jin LW. Early intraneuronal Abeta deposition in the hippocampus of APP transgenic mice. Neuroreport 2003; 14: 123-129.
59 Kirschke CP, Huang L. ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J Biol Chem 2003; 278: 4096-4102.
60 Baulac S, LaVoie MJ, Kimberly WT, Strahle J, Wolfe MS, Selkoe DJ et al. Functional gamma-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aphl, Pen-2, and gamma-secretase substrates. Neurobiol Dis 2003; 14: 194-204.
61 Hannon GJ. RNA interference. Nature 2002; 418: 244-251.
62 Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A 2002; 99:5515-5520.
63 McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature 2002; 418: 38-39.
64 Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 2001; 15: 2654-2659.
65 Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 2001; 107: 297-307.
66 Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 2001; 107: 465-476.
67 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806-811.
68 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25-33.
69 Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003; 9: 1539-1544.
70 Kao SC, Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem 2004; 279: 1942-1949.
71 Silva JM, Hammond SM, Hannon GJ. RNA interference: a promising approach to antiviral therapy? Trends Mol Med 2002; 8: 505-508.
72 McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002; 3: 737-747.
73 Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 6047-6052.
74 Krichevsky AM, Kosik KS. RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci U S A 2002; 99: 11926-11929.
75 Omi K, Tokunaga K, Hohjoh H. Long-lasting RNAi activity in mammalian neurons. FEBS Lett 2004; 558: 89-95.
76 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63.
1 Liu ST, Howlett G, Barrow CJ. Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the A beta peptide of Alzheimer's disease. Biochemistry 1999; 38: 9373-9378.
2 Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev 1993; 73: 79-118.
3 Vallee BL, Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990; 29: 5647-5659.
4 Koh JY, Choi DW. Zinc toxicity on cultured cortical neurons: involvement of N-methyl-D-aspartate receptors. Neuroscience 1994; 60: 1049-1057.
5 Jia Y, Jeng JM, Sensi SL, Weiss JH. Zn2+ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones. J Physiol 2002; 543: 35-48.
6 Rogers EE, Eide DJ, Guerinot ML. Altered selectivity in an Arabidopsis metal transporter. Proc Natl Acad Sci U S A 2000; 97: 12356-12360.
7 Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 1995; 14: 639-649.
8 Tsuda M, Imaizumi K, Katayama T, Kitagawa K, Wanaka A, Tohyama M et al. Expression of zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil. J Neurosci 1997; 17: 6678-6684.
9 Kim AH, Sheline CT, Tian M, Higashi T, McMahon RJ, Cousins RJ et al. L-type Ca(2+) channel-mediated Zn(2+) toxicity and modulation by ZnT-1 in PC12 cells. Brain Res 2000; 886: 99-107.
10 Cousins RJ, McMahon RJ. Integrative aspects of zinc transporters. J Nutr 2000; 130: 1384S-1387S.
11 Langmade SJ, Ravindra R, Daniels PJ, Andrews GK. The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J Biol Chem 2000; 275: 34803-34809.
12 Palmiter RD, Cole TB, Findley SD. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 1996; 15: 1784-1791.
13 Liuzzi JP, Blanchard RK, Cousins RJ. Differential regulation of zinc transporter 1, 2, and 4 mRNA expression by dietary zinc in rats. J Nutr 2001; 131: 46-52.
14 Kelleher SL, Lonnerdal B. Zinc transporters in the rat mammary gland respond to marginal zinc and vitamin A intakes during lactation. J Nutr 2002; 132: 3280-3285.
15 Liuzzi JP, Bobo JA, Cui L, McMahon RJ, Cousins RJ. Zinc transporters 1, 2 and 4 are differentially expressed and localized in rats during pregnancy and lactation. J Nutr 2003; 133: 342-351.
16 Iguchi K, Usui S, Inoue T, Sugimura Y, Tatematsu M, Hirano K. High-level expression of zinc transporter-2 in the rat lateral and dorsal prostate. J Androl 2002; 23: 819-824.
17 Frederickson RE, Frederickson CJ, Danscher G. In situ binding of bouton zinc reversibly disrupts performance on a spatial memory task. Behav Brain Res 1990; 38: 25-33.
18 Jo SM, Danscher G, Daa Schroder H, Won MH, Cole TB. Zinc-enriched (ZEN) terminals in mouse spinal cord: immunohistochemistry and autometallography. Brain Res 2000; 870: 163-169.
19 Jo SM, Won MH, Cole TB, Jensen MS, Palmiter RD, Danscher G. Zinc-enriched (ZEN) terminals in mouse olfactory bulb. Brain Res 2000; 865: 227-236.
20 Danscher G, Jo SM, Varea E, Wang Z, Cole TB, Schroder HD. Inhibitory zinc-enriched terminals in mouse spinal cord. Neuroscience 2001; 105: 941-947.
21 Wang Z, Li JY, Dahlstrom A, Danscher G. Zinc-enriched GABAergic terminals in mouse spinal cord. Brain Res 2001; 921: 165-172.
22 Wang Z, Danscher G, Kim YK, Dahlstrom A, Mook Jo S. Inhibitory zinc-enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase. Neurosci Lett 2002; 321: 37-40.
23 Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD. Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey. Proc Natl Acad Sci U S A 1997; 94: 12676-12681.
24 Li Y, Hough CJ, Frederickson CJ, Sarvey JM. Induction of mossy fiber --> Ca3 long-term potentiation requires translocation of synaptically released Zn2+. J Neurosci 2001; 21: 8015-8025.
25 Cole TB, Wenzel HJ, Kafer KE, Schwartzkroin PA, Palmiter RD. Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene. Proc Natl Acad Sci U S A 1999; 96: 1716-1721.
26 Gaither LA, Eide DJ. Eukaryotic zinc transporters and their regulation. Biometals 2001; 14: 251-270.
27 Huang L, Gitschier J. A novel gene involved in zinc transport is deficient in the lethal milk mouse. Nat Genet 1997; 17: 292-297.
28 Kambe T, Narita H, Yamaguchi-Iwai Y, Hirose J, Amano T, Sugiura N et al. Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells. J Biol Chem 2002; 277: 19049-19055.
29 Naureckiene S, Ma L, Sreekumar K, Purandare U, Lo CF, Huang Y et al. Functional characterization of Narc 1, a novel proteinase related to proteinase K. Arch Biochem Biophys 2003; 420: 55-67.
30 Kirschke CP, Huang L. ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J Biol Chem 2003; 278: 4096-4102.
31 Palumaa P, Njunkova O, Pokras L, Eriste E, Jornvall H, Sillard R. Evidence for non-isostructural replacement of Zn(2+) with Cd(2+) in the beta-domain of brain-specific metallothionein-3. FEBS Lett 2002; 527: 76-80.
32 Masliah E, Sisk A, Mallory M, Games D. Neurofibrillary pathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. J Neuropathol Exp Neurol 2001; 60: 357-368.
33 Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S. Skeletal and CNS defects in Presenilin-1-deficient mice. Cell 1997; 89: 629-639.
34 Huynh DP, Ho VV, Pulst SM. Characterization and expression of presenilin 1 in mouse brain. Neuroreport 1996; 7: 2423-2428.
35 Borchelt DR, Thinakaran G, Eckman CB, Lee MK, Davenport F, Ratovitsky T et al. Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo. Neuron 1996; 17: 1005-1013.
36 Vito P, Wolozin B, Ganjei JK, Iwasaki K, Lacana E, D'Adamio L. Requirement of the familial Alzheimer's disease gene PS2 for apoptosis. Opposing effect of ALG-3. J Biol Chem 1996; 271:31025-31028.
37 Di Luca M, Pastorino L, Bianchetti A, Perez J, Vignolo LA, Lenzi GL et al. Differential level of platelet amyloid beta precursor protein isoforms: an early marker for Alzheimer disease. Arch Neurol 1998; 55: 1195-1200.
38 Klafki H, Abramowski D, Swoboda R, Paganetti PA, Staufenbiel M. The carboxyl termini of beta-amyloid peptides 1 -40 and 1 -42 are generated by distinct gamma-secretase activities. J Biol Chem 1996; 271: 28655-28659.
39 Bozner P, Grishko V, LeDoux SP, Wilson GL, Chyan YC, Pappolla MA. The amyloid beta protein induces oxidative damage of mitochondrial DNA. J Neuropathol Exp Neurol 1997; 56: 1356-1362.
40 Garcia-Alloza M, Dodwell SA, Meyer-Luehmann M, Hyman BT, Bacskai BJ. Plaque-derived oxidative stress mediates distorted neurite trajectories in the Alzheimer mouse model. J Neuropathol Exp Neurol 2006; 65: 1082-1089.
41 Atwood CS, Huang X, Moir RD, Tanzi RE, Bush AI. Role of free radicals and metal ions in the pathogenesis of Alzheimer's disease. Met Ions Biol Syst 1999; 36: 309-364.
42 Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158: 47-52.
43 Miller LM, Wang Q, Telivala TP, Smith RJ, Lanzirotti A, Miklossy J. Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease. J Struct Biol 2006; 155: 30-37.
44 Barghorn S, Zheng-Fischhofer Q, Ackmann M, Biernat J, von Bergen M, Mandelkow EM et al. Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 2000; 39: 11714-11721.
45 Bush AI, Pettingell WH, Multhaup G, d Paradis M, Vonsattel JP, Gusella JF et al. Rapid induction of Alzheimer A beta amyloid formation by zinc. Science 1994; 265:1464-1467.
46 Yang DS, McLaurin J, Qin K, Westaway D, Fraser PE. Examining the zinc binding site of the amyloid-beta peptide. Eur J Biochem 2000; 267: 6692-6698.
47 Opazo C, Luza S, Villemagne VL, Volitakis I, Rowe C, Barnham KJ et al. Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease. Aging Cell 2006; 5: 69-79.
48 Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001; 30: 665-676.
49 Regland B, Lehmann W, Abedini I, Blennow K, Jonsson M, Karlsson I et al. Treatment of Alzheimer's disease with clioquinol. Dement Geriatr Cogn Disord 2001; 12: 408-414.
50 Lovell MA, Smith JL, Xiong S, Markesbery WR. Alterations in zinc transporter protein-1 (ZnT-1) in the brain of subjects with mild cognitive impairment, early, and late-stage Alzheimer's disease. Neurotox Res 2005; 7: 265-271.
51 Saito T, Takahashi K, Nakagawa N, Hosokawa T, Kurasaki M, Yamanoshita O et al. Deficiencies of hippocampal Zn and ZnT3 accelerate brain aging of Rat. Biochem Biophys Res Commun 2000; 279: 505-511.
52 Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 1996; 274: 99-102.
53 Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl AcadSci USA 2002; 99: 7705-7710.
54 Smith JL, Xiong S, Markesbery WR, Lovell MA. Altered expression of zinc transporters-4 and -6 in mild cognitive impairment, early and late Alzheimer's disease brain. Neuroscience 2006; 140: 879-888.
55 Lovell MA, Smith JL, Markesbery WR. Elevated zinc transporter-6 in mild cognitive impairment, Alzheimer disease, and pick disease. J Neuropathol Exp Neurol 2006; 65: 489-498.
2 Stoltenberg M, Bruhn M, Sondergaard C, Doering P, West MJ, Larsen A et al. Immersion autometallographic tracing of zinc ions in Alzheimer beta-amyloid plaques. Histochem Cell Biol 2005; 123: 605-611.
3 Samudralwar DL, Diprete CC, Ni BF, Ehmann WD, Markesbery WR. Elemental imbalances in the olfactory pathway in Alzheimer's disease. JNeurol Sci 1995; 130: 139-145.
4 Deibel MA, Ehmann WD, Markesbery WR. Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: possible relation to oxidative stress. J Neurol Sci 1996; 143:137-142.
5 Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158: 47-52.
6 Miller LM, Wang Q, Telivala TP, Smith RJ, Lanzirotti A, Miklossy J. Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease. J Struct Biol 2006; 155: 30-37.
7 Lee JY, Mook-Jung I, Koh JY. Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice. J Neurosci 1999; 19: RC10.
8 Vallee BL, Auld DS. New perspective on zinc biochemistry: cocatalytic sites in multi-zinc enzymes. Biochemistry 1993; 32: 6493-6500.
9 Perez-Clausell J. Distribution of terminal fields stained for zinc in the neocortex of the rat. J Chem Neuroanat 1996; 11: 99-111.
10 Takeda A. Movement of zinc and its functional significance in the brain. Brain Res Brain Res Rev 2000; 34: 137-148.
11 Takeda A. Zinc homeostasis and functions of zinc in the brain. Biometals 2001; 14: 343-351.
12 Valente T, Auladell C, Perez-Clausell J. Postnatal development of zinc-rich terminal fields in the brain of the rat. Exp Neurol 2002; 174: 215-229.
13 Wang ZY, Stoltenberg M, Jo SM, Huang L, Larsen A, Dahlstrom A et al. Dynamic zinc pools in mouse choroid plexus. Neuroreport 2004; 15: 1801-1804.
14 Christianson DW. Structural biology of zinc. Adv Protein Chem 1991; 42: 281-355.
15 Coleman JE. Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem 1992; 61: 897-946.
16 Frederickson CJ, Kasarskis EJ, Ringo D, Frederickson RE. A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J Neurosci Methods 1987; 20: 91-103.
17 Mancini M, Ricci A, Amenta F. Age-related changes in sulfide-silver staining in the rat neostriatum: a quantitative histochemical study. Neurobiol Aging 1992; 13: 501-504.
18 Takeda A, Suzuki M, Okada S, Oku N. 65Zn localization in rat brain after intracerebroventricular injection of 65Zn-histidine. Brain Res 2000; 863: 241-244.
19 Miro-Bernie N, Sancho-Bielsa FJ, Lopez-Garcia C, Perez-Clausell J. Retrograde transport of sodium selenite and intracellular injection of micro-ruby: a combined method to describe the morphology of zinc-rich neurones. J Neurosci Methods 2003; 127: 199-209.
20 Danscher G, Stoltenberg M, Bruhn M, Sondergaard C, Jensen D. Immersion autometallography: histochemical in situ capturing of zinc ions in catalytic zinc-sulfur nanocrystals. JHistochem Cytochem 2004; 52: 1619-1625.
21 Danscher G, Jensen KB, Frederickson CJ, Kemp K, Andreasen A, Juhl S et al. Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: a proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material. J Neurosci Methods 1997; 76: 53-59.
22 Bush AI, Pettingell WH, Multhaup G, d Paradis M, Vonsattel JP, Gusella JF et al. Rapid induction of Alzheimer A beta amyloid formation by zinc. Science 1994; 265: 1464-1467.
23 Liu ST, Howlett G, Barrow CJ. Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the A beta peptide of Alzheimer's disease. Biochemistry 1999; 38: 9373-9378.
24 Bush AI. Metal complexing agents as therapies for Alzheimer's disease. Neurobiol Aging 2002; 23: 1031-1038.
25 Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001; 30: 665-676.
26 Lee JY, Friedman JE, Angel I, Kozak A, Koh JY. The lipophilic metal chelator DP-109 reduces amyloid pathology in brains of human beta-amyloid precursor protein transgenic mice. Neurobiol Aging 2004; 25: 1315-1321.
27 Zirah S, Kozin SA, Mazur AK, Blond A, Cheminant M, Segalas-Milazzo I et al Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging. J Biol Chem 2006; 281: 2151-2161.
28 Regland B, Lehmann W, Abedini I, Blennow K, Jonsson M, Karlsson I et al. Treatment of Alzheimer's disease with clioquinol. Dement Geriatr Cogn Disord2001; 12: 408-414.
29 Filiz G, Price KA, Caragounis A, Du T, Crouch PJ, White AR. The role of metals in modulating metalloprotease activity in the AD brain. Eur Biophys J 2008; 37: 315-321.
30 Suh SW, Jensen KB, Jensen MS, Silva DS, Kesslak PJ, Danscher G et al. Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains. Brain Res 2000; 852: 274-278.
31 Palmiter RD, Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch 2004; 447: 744-751.
32 Eide DJ. The SLC39 family of metal ion transporters. Pflugers Arch 2004; 447: 796-800.
33 Palmiter RD, Cole TB, Quaife CJ, Findley SD. ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc Natl Acad Sci USA 1996; 93:14934-14939.
34 Colvin RA, Fontaine CP, Laskowski M, Thomas D. Zn2+ transporters and Zn2+ homeostasis in neurons. Eur J Pharmacol 2003; 479: 171-185.
35 Kambe T, Yamaguchi-Iwai Y, Sasaki R, Nagao M. Overview of mammalian zinc transporters. Cell Mol Life Sci 2004; 61: 49-68.
36 Palmiter RD, Cole TB, Findley SD. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 1996; 15:1784-1791.
37 Huang X, Atwood CS, Moir RD, Hartshorn MA, Vonsattel JP, Tanzi RE et al. Zinc-induced Alzheimer's A beta1-40 aggregation is mediated by conformational factors. J Biol Chem 1997; 272: 26464-26470.
38 Huang L, Kirschke CP, Gitschier J. Functional characterization of a novel mammalian zinc transporter, ZnT6. J Biol Chem 2002; 277: 26389-26395.
39 Lovell MA, Smith JL, Xiong S, Markesbery WR. Alterations in zinc transporter protein-1 (ZnT-1) in the brain of subjects with mild cognitive impairment, early, and late-stage Alzheimer's disease. Neurotox Res 2005; 7: 265-271.
40 Smith JL, Xiong S, Markesbery WR, Lovell MA. Altered expression of zinc transporters-4 and -6 in mild cognitive impairment, early and late Alzheimer's disease brain. Neuroscience 2006; 140: 879-888.
41 Lovell MA, Smith JL, Markesbery WR. Elevated zinc transporter-6 in mild cognitive impairment, Alzheimer disease, and pick disease. J Neuropathol Exp Neurol 2006; 65: 489-498.
42 Cousins RJ, McMahon RJ. Integrative aspects of zinc transporters. J Nutr 2000; 130: 1384S-1387S.
43 Wang Z, Danscher G, Kim YK, Dahlstrom A, Mook Jo S. Inhibitory zinc-enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase. Neurosci Lett 2002; 321: 37-40.
44 Wang ZY, Danscher G, Dahlstrom A, Li JY. Zinc transporter 3 and zinc ions in the rodent superior cervical ganglion neurons. Neuroscience 2003; 120: 605-616.
45 Wang ZY, Stoltenberg M, Huang L, Danscher G, Dahlstrom A, Shi Y et al. Abundant expression of zinc transporters in Bergman glia of mouse cerebellum. Brain Res Bull 2005; 64:441-448.
46 Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 1995; 14: 639-649.
47 Nitzan YB, Sekler I, Hershfinkel M, Moran A, Silverman WF. Postnatal regulation of ZnT-1 expression in the mouse brain. Brain Res Dev Brain Res 2002; 137: 149-157.
48 Sekler I, Moran A, Hershfinkel M, Dori A, Margulis A, Birenzweig N et al. Distribution of the zinc transporter ZnT-1 in comparison with chelatable zinc in the mouse brain. J Comp Neurol 2002; 447: 201-209.
49 Andrews GK, Wang H, Dey SK, Palmiter RD. Mouse zinc transporter 1 gene provides an essential function during early embryonic development. Genesis 2004; 40: 74-81.
50 Langmade SJ, Ravindra R, Daniels PJ, Andrews GK. The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J Biol Chem 2000; 275: 34803-34809.
51 Tsuda M, Imaizumi K, Katayama T, Kitagawa K, Wanaka A, Tohyama M et al. Expression of zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil. J Neurosci 1997; 17: 6678-6684.
52 Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD. Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey. Proc Natl Acad Sci USA 1997; 94: 12676-12681.
53 Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl Acad Sci USA 2002; 99: 7705-7710.
54 Cole TB, Wenzel HJ, Kafer KE, Schwartzkroin PA, Palmiter RD. Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene. Proc Natl Acad SciUSA 1999; 96: 1716-1721.
55 Friedlich AL, Lee JY, van Groen T, Cherny RA, Volitakis I, Cole TB et al. Neuronal zinc exchange with the blood vessel wall promotes cerebral amyloid angiopathy in an animal model of Alzheimer's disease. J Neurosci 2004; 24: 3453-3459.
56 Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H et al. Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol 2002; 161: 1869-1879.
57 Wirths O, Multhaup G, Czech C, Blanchard V, Moussaoui S, Tremp G et al. Intraneuronal Abeta accumulation precedes plaque formation in beta-amyloid precursor protein and presenilin-1 double-transgenic mice. Neurosci Lett 2001; 306: 116-120.
58 Shie FS, LeBoeuf RC, Jin LW. Early intraneuronal Abeta deposition in the hippocampus of APP transgenic mice. Neuroreport 2003; 14: 123-129.
59 Kirschke CP, Huang L. ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J Biol Chem 2003; 278: 4096-4102.
60 Baulac S, LaVoie MJ, Kimberly WT, Strahle J, Wolfe MS, Selkoe DJ et al. Functional gamma-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aphl, Pen-2, and gamma-secretase substrates. Neurobiol Dis 2003; 14: 194-204.
61 Hannon GJ. RNA interference. Nature 2002; 418: 244-251.
62 Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A 2002; 99:5515-5520.
63 McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature 2002; 418: 38-39.
64 Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 2001; 15: 2654-2659.
65 Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 2001; 107: 297-307.
66 Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 2001; 107: 465-476.
67 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806-811.
68 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25-33.
69 Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 2003; 9: 1539-1544.
70 Kao SC, Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem 2004; 279: 1942-1949.
71 Silva JM, Hammond SM, Hannon GJ. RNA interference: a promising approach to antiviral therapy? Trends Mol Med 2002; 8: 505-508.
72 McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002; 3: 737-747.
73 Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 6047-6052.
74 Krichevsky AM, Kosik KS. RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci U S A 2002; 99: 11926-11929.
75 Omi K, Tokunaga K, Hohjoh H. Long-lasting RNAi activity in mammalian neurons. FEBS Lett 2004; 558: 89-95.
76 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63.
1 Liu ST, Howlett G, Barrow CJ. Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the A beta peptide of Alzheimer's disease. Biochemistry 1999; 38: 9373-9378.
2 Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev 1993; 73: 79-118.
3 Vallee BL, Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990; 29: 5647-5659.
4 Koh JY, Choi DW. Zinc toxicity on cultured cortical neurons: involvement of N-methyl-D-aspartate receptors. Neuroscience 1994; 60: 1049-1057.
5 Jia Y, Jeng JM, Sensi SL, Weiss JH. Zn2+ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones. J Physiol 2002; 543: 35-48.
6 Rogers EE, Eide DJ, Guerinot ML. Altered selectivity in an Arabidopsis metal transporter. Proc Natl Acad Sci U S A 2000; 97: 12356-12360.
7 Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 1995; 14: 639-649.
8 Tsuda M, Imaizumi K, Katayama T, Kitagawa K, Wanaka A, Tohyama M et al. Expression of zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil. J Neurosci 1997; 17: 6678-6684.
9 Kim AH, Sheline CT, Tian M, Higashi T, McMahon RJ, Cousins RJ et al. L-type Ca(2+) channel-mediated Zn(2+) toxicity and modulation by ZnT-1 in PC12 cells. Brain Res 2000; 886: 99-107.
10 Cousins RJ, McMahon RJ. Integrative aspects of zinc transporters. J Nutr 2000; 130: 1384S-1387S.
11 Langmade SJ, Ravindra R, Daniels PJ, Andrews GK. The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J Biol Chem 2000; 275: 34803-34809.
12 Palmiter RD, Cole TB, Findley SD. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 1996; 15: 1784-1791.
13 Liuzzi JP, Blanchard RK, Cousins RJ. Differential regulation of zinc transporter 1, 2, and 4 mRNA expression by dietary zinc in rats. J Nutr 2001; 131: 46-52.
14 Kelleher SL, Lonnerdal B. Zinc transporters in the rat mammary gland respond to marginal zinc and vitamin A intakes during lactation. J Nutr 2002; 132: 3280-3285.
15 Liuzzi JP, Bobo JA, Cui L, McMahon RJ, Cousins RJ. Zinc transporters 1, 2 and 4 are differentially expressed and localized in rats during pregnancy and lactation. J Nutr 2003; 133: 342-351.
16 Iguchi K, Usui S, Inoue T, Sugimura Y, Tatematsu M, Hirano K. High-level expression of zinc transporter-2 in the rat lateral and dorsal prostate. J Androl 2002; 23: 819-824.
17 Frederickson RE, Frederickson CJ, Danscher G. In situ binding of bouton zinc reversibly disrupts performance on a spatial memory task. Behav Brain Res 1990; 38: 25-33.
18 Jo SM, Danscher G, Daa Schroder H, Won MH, Cole TB. Zinc-enriched (ZEN) terminals in mouse spinal cord: immunohistochemistry and autometallography. Brain Res 2000; 870: 163-169.
19 Jo SM, Won MH, Cole TB, Jensen MS, Palmiter RD, Danscher G. Zinc-enriched (ZEN) terminals in mouse olfactory bulb. Brain Res 2000; 865: 227-236.
20 Danscher G, Jo SM, Varea E, Wang Z, Cole TB, Schroder HD. Inhibitory zinc-enriched terminals in mouse spinal cord. Neuroscience 2001; 105: 941-947.
21 Wang Z, Li JY, Dahlstrom A, Danscher G. Zinc-enriched GABAergic terminals in mouse spinal cord. Brain Res 2001; 921: 165-172.
22 Wang Z, Danscher G, Kim YK, Dahlstrom A, Mook Jo S. Inhibitory zinc-enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase. Neurosci Lett 2002; 321: 37-40.
23 Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD. Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey. Proc Natl Acad Sci U S A 1997; 94: 12676-12681.
24 Li Y, Hough CJ, Frederickson CJ, Sarvey JM. Induction of mossy fiber --> Ca3 long-term potentiation requires translocation of synaptically released Zn2+. J Neurosci 2001; 21: 8015-8025.
25 Cole TB, Wenzel HJ, Kafer KE, Schwartzkroin PA, Palmiter RD. Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene. Proc Natl Acad Sci U S A 1999; 96: 1716-1721.
26 Gaither LA, Eide DJ. Eukaryotic zinc transporters and their regulation. Biometals 2001; 14: 251-270.
27 Huang L, Gitschier J. A novel gene involved in zinc transport is deficient in the lethal milk mouse. Nat Genet 1997; 17: 292-297.
28 Kambe T, Narita H, Yamaguchi-Iwai Y, Hirose J, Amano T, Sugiura N et al. Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells. J Biol Chem 2002; 277: 19049-19055.
29 Naureckiene S, Ma L, Sreekumar K, Purandare U, Lo CF, Huang Y et al. Functional characterization of Narc 1, a novel proteinase related to proteinase K. Arch Biochem Biophys 2003; 420: 55-67.
30 Kirschke CP, Huang L. ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J Biol Chem 2003; 278: 4096-4102.
31 Palumaa P, Njunkova O, Pokras L, Eriste E, Jornvall H, Sillard R. Evidence for non-isostructural replacement of Zn(2+) with Cd(2+) in the beta-domain of brain-specific metallothionein-3. FEBS Lett 2002; 527: 76-80.
32 Masliah E, Sisk A, Mallory M, Games D. Neurofibrillary pathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. J Neuropathol Exp Neurol 2001; 60: 357-368.
33 Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S. Skeletal and CNS defects in Presenilin-1-deficient mice. Cell 1997; 89: 629-639.
34 Huynh DP, Ho VV, Pulst SM. Characterization and expression of presenilin 1 in mouse brain. Neuroreport 1996; 7: 2423-2428.
35 Borchelt DR, Thinakaran G, Eckman CB, Lee MK, Davenport F, Ratovitsky T et al. Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo. Neuron 1996; 17: 1005-1013.
36 Vito P, Wolozin B, Ganjei JK, Iwasaki K, Lacana E, D'Adamio L. Requirement of the familial Alzheimer's disease gene PS2 for apoptosis. Opposing effect of ALG-3. J Biol Chem 1996; 271:31025-31028.
37 Di Luca M, Pastorino L, Bianchetti A, Perez J, Vignolo LA, Lenzi GL et al. Differential level of platelet amyloid beta precursor protein isoforms: an early marker for Alzheimer disease. Arch Neurol 1998; 55: 1195-1200.
38 Klafki H, Abramowski D, Swoboda R, Paganetti PA, Staufenbiel M. The carboxyl termini of beta-amyloid peptides 1 -40 and 1 -42 are generated by distinct gamma-secretase activities. J Biol Chem 1996; 271: 28655-28659.
39 Bozner P, Grishko V, LeDoux SP, Wilson GL, Chyan YC, Pappolla MA. The amyloid beta protein induces oxidative damage of mitochondrial DNA. J Neuropathol Exp Neurol 1997; 56: 1356-1362.
40 Garcia-Alloza M, Dodwell SA, Meyer-Luehmann M, Hyman BT, Bacskai BJ. Plaque-derived oxidative stress mediates distorted neurite trajectories in the Alzheimer mouse model. J Neuropathol Exp Neurol 2006; 65: 1082-1089.
41 Atwood CS, Huang X, Moir RD, Tanzi RE, Bush AI. Role of free radicals and metal ions in the pathogenesis of Alzheimer's disease. Met Ions Biol Syst 1999; 36: 309-364.
42 Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158: 47-52.
43 Miller LM, Wang Q, Telivala TP, Smith RJ, Lanzirotti A, Miklossy J. Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease. J Struct Biol 2006; 155: 30-37.
44 Barghorn S, Zheng-Fischhofer Q, Ackmann M, Biernat J, von Bergen M, Mandelkow EM et al. Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 2000; 39: 11714-11721.
45 Bush AI, Pettingell WH, Multhaup G, d Paradis M, Vonsattel JP, Gusella JF et al. Rapid induction of Alzheimer A beta amyloid formation by zinc. Science 1994; 265:1464-1467.
46 Yang DS, McLaurin J, Qin K, Westaway D, Fraser PE. Examining the zinc binding site of the amyloid-beta peptide. Eur J Biochem 2000; 267: 6692-6698.
47 Opazo C, Luza S, Villemagne VL, Volitakis I, Rowe C, Barnham KJ et al. Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease. Aging Cell 2006; 5: 69-79.
48 Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA et al. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001; 30: 665-676.
49 Regland B, Lehmann W, Abedini I, Blennow K, Jonsson M, Karlsson I et al. Treatment of Alzheimer's disease with clioquinol. Dement Geriatr Cogn Disord 2001; 12: 408-414.
50 Lovell MA, Smith JL, Xiong S, Markesbery WR. Alterations in zinc transporter protein-1 (ZnT-1) in the brain of subjects with mild cognitive impairment, early, and late-stage Alzheimer's disease. Neurotox Res 2005; 7: 265-271.
51 Saito T, Takahashi K, Nakagawa N, Hosokawa T, Kurasaki M, Yamanoshita O et al. Deficiencies of hippocampal Zn and ZnT3 accelerate brain aging of Rat. Biochem Biophys Res Commun 2000; 279: 505-511.
52 Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 1996; 274: 99-102.
53 Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl AcadSci USA 2002; 99: 7705-7710.
54 Smith JL, Xiong S, Markesbery WR, Lovell MA. Altered expression of zinc transporters-4 and -6 in mild cognitive impairment, early and late Alzheimer's disease brain. Neuroscience 2006; 140: 879-888.
55 Lovell MA, Smith JL, Markesbery WR. Elevated zinc transporter-6 in mild cognitive impairment, Alzheimer disease, and pick disease. J Neuropathol Exp Neurol 2006; 65: 489-498.