Study On Changes Of Tau Protein Secretion By Nerve Cells After Prion Infection

Prion disease is a kind of degenerative encephalopathy which infects the central nervous system of human and many mammals.As an intracellular microtubule-associated protein,tau protein is significantly increased in cerebrospinal fluid of patients with various neurodegenerative diseases.For the past few years studies have reaveled that nerve cells and non-nerve cells can secrete tau protein specifically.These studies suggest that tau protein can be secreted abnormally when suffering from neurodegenerative diseases.However,the secretion of tau protein in prion diseases has not been thoroughly documented.In order to investigate the secretion of total tau protein after prion infection,the levels of total tau protein in SMB-S15 cell and its control cell line SMB-PS was determinated by ELISA and Western blot.ELISA results displayed which the levels of total tau protein in medium of SMB-S15 cells was significantly higher than that in SMB-PS cells.Besides,Western blot datas manifested that the levels of total tau protein in SMB-S15 cell lysate were markedly increased compared to those of SMB-PS cells.Moreover,positive signals of total tau protein were found both in medium of SMB-PS and SMB-S15 cells.After uniformization with the intracelluar total tau protein in lysate,the levels of total tau protein in medium of SMB-S15 cells were dramatically higher than those in SMB-PS cells,which was in line with the datas of ELISA.In this study,four monoclonal antibodies against exon 2 and exon 10 of tau protein were prepared and were evaluated in terms of affinity in the immune response,species reactivity and adaptability to prion models.Then,an indirect ELISA technique was conducted with prepared monoclonal antibodies to detect.The levels of tau isoforms with exon 2 and 10(E2/E10-tau)in medium of SMB-PS and SMB-S15 cells.The results showed that both SMB-PS and SMB-S15cells were able to secrete E2/E10-tau,but there was no remarkable variation between two types cell lines.To probe into the role of phosphorylation on the ability of prion-infected neurons to secret tau protein,the levels of tau protein phosphorylated at Thr181,Thr205,Ser396 and Ser404(pT181/pT205/pS396/pS404-tau)in medium of SMB-PS and SMB-S15 cells were detected by Western blot.The results showed that pS396/pT205-tau could be secreted by SMB-PS and SMB-S15 cells,but the secretion level of SMB-S15 cells was significantly higher than that of SMB-PS cells.Meanwhile,pS404/pT181-tau can not be secreted by those two cell lines.To further inquiry the relation of the increased secretion of pS396/pT205-tau in the culture medium with prion replication in SMB-S15 cells,total tau and pT205/pS396-tau levels in culture medium of SMB-RES cells obtained from 7 days of resveratrol treatment to completely remove PrP~Scc from SMB-S15 cells were determined by ELISA and Western blot.The results showed that the ability to secret total tau protein and pS396/pT205-tau by SMB-RES cell was restored compared to SMB-S15 cell after prion was removed.This study demonstrates that the ability of nerve cells to secrete tau protein was increased after prion infection,which can be reversed by prion removal.In addition,it was found that the presence of exons 2 and 10 may not be the main factor that affects the secretion of tau protein in nerve cells after prion infection,while the phosphorylation of different sites of tau protein may be the key factor affecting this process.These data help to explain the possible causes of the increased tau protein in the cerebrospinal fluid of patients with prion disease,and provide fundamental basis for understanding the pathogenesis of prion disease.The monoclonal antibodies against exon 2 and exon 10 of tau protein prepared in this study have high specificity,strong antigen binding ability and wide application range,which will become a powerful tool to understand the role of different tau protein isoforms in neurodegenerative diseases.