The Expression Of ZnTs In The Brain Of Human Alzheimer's Disease And Transgenic Mouse And The Inhibitory Effect Of ZnT1 Gene Silencing On Aβ Secretion

PrefaceAlzheimer'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^thamino 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.MethodsAD 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. Results1.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 vesselsImmunohistochemimistry 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 hippocampusWestern 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 AβCo-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 ZnT1RT-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 transportZinquin 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 APPWestern 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 AβsecretionElisa 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 cellsMTT 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 cellsMTT 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.Conclusion1.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.