The Mechanism Of DMT1 Involved In Amyloid Precursor Protein Processing And Aβ Generation

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.