Study On The Alterations Of Calmodulin Signal Pathway In Prion Disease

Prion diseases,also known as transmissible spongiform encephalopathy(TSE),are a group of degenerative diseases which affect nerve system of human beings and other mammals,and always have long incubation period and 100% mortality rate.In recent years,the incidence of prion diseases have risen on a global scale,both in the crowd and fauna,while the long latency,high infectivity and 100% lethality of prion disease will be associated with public health causing great potential harm.The pathogen of prion disease is prion,which is a kind of non-nucleic acid,self-replication and infectious protein(Pr PSc).It has the ability of conformational conversion of protease-sensitive cellular prion(Pr PC)to protease-resistance scrapie fibre-like prion protein(Pr PSc).A large amount of Pr PSc deposited in the central nervous system lead to loss of nerve cell death,reactive glial cell proliferation and sponge vacuolar degeneration,which are the primary neuropathological changes of prion diseases.Calmodulin(CaM)is a ubiquitous calcium-binding protein that binds up to four calcium ions and acts as a major transconductor for calcium signaling.The binding of Ca2 + induces a conformational change in CaM that exposes its hydrophobic residues that promote the interaction of the Ca2 + / CaM complex with many targeting proteins and then modulate their function.Changes in CaM and its downstream substrates have been reported in some neurodegenerative diseases,but are rarely described in prion diseases.In this study,CaM and its related proteins were detected in the brains of scrapie agent 139A-infected mice and scrapie agent 263K-infected hamster as well as prion-infected cell line SMB-S15 by Western blot,immunohistochemistry,immunohistofluorescence assay.Analysis of Western blot showed that the levels of CaMKII,phosphorylated CaMKII(p-CaMKII)and p-CaMKIV were increased in the brains of 139 A infected mice.The c AMP response element binding protein(CREB)which is the downstream control protein of CaMKs and its phosphorylated form(p-CREB)were up-regulated,while brain-derived neurotrophic factor(BDNF)expression was significantly down-regulated.Immunohistochemical experiments found that CaM is mainly distributed in some cerebral regions of 139A-infected mice,such as cortex,thalamus and cerebellum.For 263K-infected hamsters,the brain CaM level began to increase at the early stage of prion infection and remained at a high level until the end of the disease.Immunohistochemical results showed increased CaM predominantly accumulated in the 263K-infected hamster's cortex,thalamus and cerebellar regions.Immunofluorescence assay showed well co-localizationl of CaM-and Neu N-positive cells.However,related kinases,such as total and phosphorylated forms of CaMKII and CaMKIV,and their downstream proteins,such as CREB and BDNF were reduced in the brains of 263K-infected hamsters.Further studies revealed a significant increase in the S-nitrosylation(SNO)form of CaM in the brain of 263K-infected hamsters.In the prion-infected cell line SMB-S15,the CaM level was significantly higher than that of the control cell line SMB-PS,while downstream proteins such as CaMKII,p-CaMKII,CREB and BDNF also increased,especially in the nucleus.However,SNO-CaM was not detected in prion-infected SMB-S15 cell lines.In summary,our data from in vivo and in vitro suggest that CaM increases abnormally in prion infection.Increased CaM can trigger activation of its downstream kinase in cultured cells and brains of 139A-infected mice.While in brains of 263K-infected hamsters,the increased CaM appears to no longer activate its downstream kinase,possibly due to its activity being affected by increased SNO-CaM during prion infection.