Study On The Distribution And Mechanism Of DMT1 In APP/PS1 Transgenic Mice

Objectives: Alzheimer's disease(AD)is a common dementia worldwide.The main features of AD are senile plaques formed by the deposition of ?-amyloid peptides(A?)and neurofibrillary tangle(NFT)formed by the core of hyperphosphorylated Tau protein.In recent years,several studies have shown that brain metal metabolism disorders are related to neurological diseases.Increased brain iron and imbalance of iron metabolism control molecules may play an important role in the pathogenesis of AD.DMT1(divalent metal transporter,DMT1)is a metal transporter that maintains the steady state of divalent metal ions in cells.It is the main importer of iron ions in intestinal cells and red blood cells.The abnormality of iron will cause neurotoxicity,and it will form active oxygen in the hippocampus and other areas related to neurodegenerative diseases,and eventually cause brain death.Studies have shown that abnormal distribution and expression of DMT1 in AD brain is closely related to the formation of A? and senile plaques and neuronal death.This study aimed to explore the expression and distribution of DMT1 in the brain of APP/PS1 transgenic mice.To explore the effect of DMT1 gene on the expression of APP and its related hydrolases.Using DMT1 as an entry point,the role of DMT1 in the induction of cytotoxicity by A?1-42 oligomer was studied using the S-DMT1 cell model.It provides new ideas for finding potential and effective drug target therapy for AD.Methods: 1.Immunofluorescence double labeling staining method was used to detect the location and distribution of DMT1 and A? in the senile plaques of APP/PS1 transgenic mice.Western blot was used to detect the protein expression level of DMT1 in APP/PS1 transgenic mouse brain.2.Immunofluorescence double labeling staining method was used to detect the distribution of DMT1 in APPsw cells and neo cells.Western blot detection of DMT1 expression in APPsw cells and neo cells 3.Taking SH-SY5 Y cells stably expressing DMT1 gene and Vector cells transfected with empty vector as the research object,Western blot method was used to detect the protein expression of APP and its hydrolase Level.4.A?1-42 monomer is prepared into A?1-42 oligomer.Different concentrations of A?1-42 oligomer were used to induce Vector cells and S-DMT1 cells.5.Use CCK8 to screen out the A? oligomer concentration corresponding to the appropriate ratio of cell activity,and use this concentration for 24 hours of cell culture.6.Heochst33258 staining was used to observe the apoptosis of Vector cells and S-DMT1 cells before and after administration.7.Use Calcein-AM staining to observe whether there is any difference in the number of viable cells and cell morphology.8.Using Western blot method to detect the expression levels of autophagy-related proteins LC3,P62 and apoptosis-related proteins Caspase-3,Bax,BCL2.Compare the difference between Vector cells and S-DMT1 cells before and after administration.Results: 1.Immunofluorescence double-labeling staining revealed that in the cortex and hippocampus of APP/PS1 mice,A? and DMT1 were co-localized in senile plaques.Western blot results showed that DMT1 expression increased in cerebral cortex and hippocampus of APP/PS1 transgenic mice.2.Double-labeled immunofluorescence staining revealed that APPsw cells and neo cells had a positive distribution of DMT1,and DMT1 and A? co-localized in APPsw cells.Western blot results showed that the expression of DMT1 in APPsw cells was significantly higher than that in neo cells.3.Western blot results show that DMT1 affects the expression of APP and its hydrolase.4.CCK8 cell viability experiments showed that the cell viability gradually decreased with the increase of A? oligomer concentration,and the cell viability of S-DMT1 cells decreased more significantly than that of Vector cells.5.Calcein-AM results showed that the number of viable cells decreased significantly after administration of A? oligomers and the number of viable cells in the S-DMT1 group was significantly lower than that in the Vector group.6.Western blot method was used to detect the expression level of autophagyrelated protein.The results showed that the autophagy level of the cells in the administration group increased significantly,and the DMT1 overexpression autophagy protein increased more significantly.7.Heochst33258 staining results showed that after the administration of A? oligomers,the nuclei of the cells contracted and collapsed,the cells apoptotic,and the nuclear damage of DMT1 overexpressing cells was more obvious.8.Western blotting showed that the levels of apoptosis-related proteins were significantly increased after administration of A? oligomers,and the increase was more significant in the DMT1 overexpression group.Conclusion: 1.DMT1 participates in the formation of A? senile plaques by promoting the expression of APP and its hydrolase.2.DMT1 aggravates the cytotoxicity of A?1-42 oligomer by regulating the expression of autophagy and apoptosis-related proteins.