Characteristics Of Prion Disease With E200K Mutation And The Study Of Mitophagy In The Process Of Prion Infection

Prion diseases,also known as the transmissible spongiformencephalopathies(TSE),is a rare degenerative disease that causes spongiform pathological changes in the brain tissue of human and animals.The neuropathologic features of prion disease are spongiform vacuol-like degeneration,amyloid plaque deposition,neuron loss,and glial cell proliferation in brain tissue.The pathogenesis is believed to be due to the misfolding of the normal ?-spiral-rich prion protein,PrPC,into the ?-fold-rich PrPSc Since sheep scrapie was first reported in 1730,prion has infected human and more than 20 kinds of animals.Human prion diseases include CJD,FFI,Kuru,GSS and the animal prion diseases includes sheep scrapie,BSE,etc.According to the different pathogenesis,human CJD can be divided into sporadic CJD,familial CJD,itrogenic CJD and variant CJD.This study is divided into two parts and the first part mainly aimed at 30 genetic CJD patients with E200K mutation in our country.The genetic type of prion disease heredity,family,clinical and laboratory features were analyzed in this part.The second part is the study of the mitophagy mediated by the Pink 1-Parkin pathway in the process of prion infection.We took prion infected cells and brain tissue samples of scrapie infected animals as research objects,and demonstrated that prion infection can activate the mitophagy system in vitro and in vivo.Part ?:Characteristics analysis of Chinese patients with E200K mutation of prion diseaseGenetic Creutzfeldt-Jakob disease(gCJD)with E200K mutation is one of the common subtypes of human genetic prion diseases worldwide.In this study,we systematically analyzed 30 Chinese E200K gCJD cases for their epidemiological,clinical,laboratory and genetic features.The patients came from 12 diferent provinces,majority in northern part of China.The onset age varied from 42 to 71 years old(y),with the median of was 57 y.The CYP4X1 gene rs9793471 SNP was tested.Only one patient's rs9793471 genotype was GA and the others'were AA.The gender ratio(M:F)was 1:1.73(11:19).The foremost symptoms and clinical progression of Chinese E200K gCJD patients were quite similar as sporadic CJD cases.Only a few cases(4/30)recalled clearly disease related family history.74.1%(20/27),86.7%(26/30)and 50.0%(13/26)of the cases were CSF 14-3-3 positive,sCJD associated abnormalitie on MRI and special PSWC on EEG,respectively.The median clinical duration was 9 months(varying from 2 to 26 months).All 30 Chinese E200K gCJD patients were M129M and E219E homozygous.21 members from 3 families conducted PRNP sequencing and 16 asymptomatic carriers of E200K mutation with M129M and E219E homozygous were identifed.This is the largest study on E200K gCJD patients in China,which would beneft to the knowledge of E200K gCJD.Part ?:Study on the mitophagy mediated by Pink1-Parkin pathway in prion infectionMitophagy is an important process of removing damaged mitochondria in cells.Mitochondrial dysfunction is directly related to different kinds of neurodegenerative diseases.However,mitophagy in prion diseases still needs further exploration.In this study,we found that in prion-infected cell lines SMB-S15,there were more autophagosomes,swollen and ribbed mitochondria,and damaged mitochondrial structures wrapped in autophagosomes,and we also found molecular evidence of activated autophagosomes,namely,decreased p62 expression and increased LC3? ?expression.Western blots showed decreased levels of mitochondrial membrane proteins TIMM44,TOMM20 and TIMM23,and co-localization of LC3? proteins with mitochondria,suggesting activation of mitophagy in prion-infected SMB-S15 cells.In addition,the expression levels of Pink1 and Parkin were increased,especially in the mitochondrial component.In SMB-S15 cells,the expression level of polyubiquitinated protein decreased,while the expression level of phosphorylated polyubiquitinated protein increased,indicating that Pink1 was activated.The expression level of MFN2 in SMB-S15 cells decreased,while the expression of its ubiquitination form was up-regulated,indicating that Parkin was activated.These results indicate that the mitophagy mediated by the Pink1-Parkin pathway plays an important role in prion infection.SiRNA interference experiments were used to knock down the expression of Pink1 and Parkin in SMB-S15 cells,which could reduce the autophagy activity,but did not seem to affect the expression of TOMM20 and TIMM23.In addition,Western blot and IHC assay were used to detect that the levels of Pink 1 and Parkin in the brain tissues of mice infected with 139A and ME7 were significantly increased at the end of the disease.Immunofluorescence analysis showed that Pink1 signals co-located with GFAP,Ibal and NeuN positive cells in brain tissues of mice infected with scrapie.IHC examined successive sections of brain tissue infected with 139A,ME7 and found that more Pink1 and Parkin positive cells were located in areas where more PrPSc deposited.These results suggest that in both prion-infected cells and prion-infected experimental mouse models,mitophagy is activated through the enhanced Pink1-Parkin pathway,which may also be a cause of neuronal cell necrosis.