The Investigation Of Regulatory Role From BACE1 Interaction Proteins In AD Pathogenesis

Alzheimer's disease is a progressive cognitive disorder and the main feature of this disease is the primary central nervous system degeneration.Synaptic dysfunction or the neuronal death takes place in the hippocampus and cerebral cortex,which seriously harms the health especially in elders.The typical pathological features are extensive cortical atrophy,senile plaques formed by extracellular deposits of amyloid beta?A??,and Neurofibrillary Tangles?NFTs?formed due to the aggregates of hyperphosphorylated tau protein.A?consists of the A?40 and A?42,which is derived from the?-amyloid precursor protein?APP?through sequential cleavages by?-and?-secretase enzymatic activity.?-secretase is the key to the pathogenic process of AD.PS1 is the active center of the?-secretase,participating in the APP hydrolysis process.Previous studies show that PS1 can co-localize with the BACE1.The deficiency of the PS1 can not only inhibit the activity of the BACE1 but also decrease the expression of C-terminal products.Based on these results,we propose that PS1can regulate both the expression and activity of BACE1,and then affect the level of APP Proteolytic cleavage.We seek to uncover the mechanism of how exactly PS1 regulates BACE1 to provide a further theoretical basis in understanding the pathogenesis of AD.In this project,we aim at several aspects:the influence that PS1 exerts on expression,activity,and transportation of BACE1;the influence that FAD related PS1 mutations exert on the activity of BACE1 and the process of APP proteolysis cleavage.We are committed to screen proteins which interact with BACE1,selecting specific one that will be interesting to us.Then we can investigate its interaction with BACE1 and effect it has on the APP proteolysis.Through our research,we provide reliable experiment data to support the above hypothesis.1.We used Co-immunoprecipitation,immunocytochemistry?including MEF and primary neurons?to confirm that BACE1 interacted with PS1.When APPswe plasmid was transfected into MEF cells,we found that BACE1 was inactivated because of lack of PS1,compared with control group.When PS1 is transfected into MEF cells,the protein and mRNA expression level increase dramatically.Furthermore,dual luciferase report system reveals that this upregulation of the transcriptional level is the result of PS1 upgrading the promoter activity of BACE1.?-secretase inhibitor L-685,458 can reverse the effect induced by PS1^-/-PS2^-/-cells overexpressing the PS1.The results together indicate that PS1 can regulate the BACE1.2.We used sucrose density gradient centrifugation to separate organelles and test them with Western Blot.When PS1 was transfected,the distribution of the BACE1 decreased distinctly in layer 4 and increased in layer 3.This indicated that BACE1 migrated from layer 4 to layer 3,the composition of which was endoplasmic reticulum?ER?.So PS1 can change the distribution of BACE1 in subcellular structure,through extension of the dwelling time in ER.Through the method of Immunofluorescence and Time-lapes,BACE1distributed evenly in cytoplasm,co-localizing with ER,Golgi apparatus,and early endosome slightly without transfecting PS1.On the contrary,when transfecting PS1,BACE1 migrated to periphery of nucleus distinctly,co-localizing with Calnexin in high amount,indicating that PS1 can force BACE1 to migrate to ER.In the meantime,the transfection of PS1 increases the co-localization of BACE1 with Syn-6 and EEA1,which shows that PS1 improved the maturity of BACE1 in Golgi apparatus.Because BACE1interact with APP in early endosome,the transfection of PS1 promoted BACE1 binding with APP to perform the functions.The results above manifest that PS1 can extend the dwelling time of BACE1 in ER,improve the distribution of the BACE1 in ER and Golgi.In conclusion,PS1 affects the distribution and activity of BACE1 through acting on the synthesis and maturity of BACE1 precursor.3.Weconstructed16plasmidsbythemutationsofPS1relatedwith FAD,transiently transfecting them into 2EB2 cells in order to test protein expression level in the process of APP cleavage using Western Blot and ELISA.We found that with the exception of a few mutations that dominantly had negative effects,most of the mutations dramatically increased the APP and CTFs cleavage,upgrading the amount of A?42,relative to A?40.Among the mutations with positive effects,some of them upregulated the level of BACE1 protein expression,through enhancing its promoter activity,leading to cleavage of more APP protein.The others enhanced the activity of BACE1 protein itself to result in more APP cleavage.Through the LC-MS/MS,the results showed that PS1 mutations,I143V,M146 and S170F,performed the same functions in the cleavage pathways?APP,C99 and C83 cleavage pathway?;M233V,I143T,H163P,I381V and L392V upgraded the ratio of Ab42/40,through enhancing both the A?48-A?38 and A?49-A?40 cleavage pathways in the process of C99 cleavage,while these mutations dominated the A?48-A?38cleavage pathway in the process of C83 cleavage;?E9,G217A,Q223R and G384A show synergy between APP cleavage pathway and C99/C83 cleavage pathway.Therefore,different PS1 mutations show selective cleavage of long A?.4.Using the co-immunoprecipitation and mass spectrometry to the proteins that interacted with BACE1,we found the 891 proteins reported to be functional through PEAKS and SWISSPROT.Then we finally chose the target protein ZNF335 through the biological process and circuit analysis,which was verified by co-immunoprecipitation and Immunocelluler chemistry.When transient transfection of ZNF335 and BACE1,ZNF335enhanced the level of BCAE1 protein expression,APP cleavage,CTFs expression and Ab42 secretion.Through the Real-time PCR,the results show that the transcriptional level of ZNF335 increases significantly in the brain tissues of AD patients and AD model mouse,compared with the normal aged.In the meantime,the ZNF335 is mainly distributed in the neurons.This protein expression level in the cortex and hippocampus decreases in wild type mouse with age,on the contrary,upgrades in AD mouse with age.5.We constructed the model of ZNF335 gene knock-down AD mouse?shZNF335?.The Immunohistochemistry shows that ZNF335 expression level decreases significantly in the shZNF335 group,compared with the scramble group.During the Morris Water Maze task,the results indicate that shZNF335 mouse show a better leaning capacity with shorter escape time and routine than the scramble group.Furthermore,after knocking down the ZNF335,the Ab expression level decreases dramatically in the brain tissues of AD mouse.The result together reveals that ZNF335 can regulate the activity of BACE1,influencing the APP cleavage pathway to affect the expression level of the A?.