Proteomics Analysis Of The Pooled CSF Samples Of Patients With Sporadic Creutzfeldt-Jakob Disease And The Study Of The Alteration Of ADAM10in The Prion Infection

Transmissible spongiform encephalopathy (TSE), also known as prion disease, are a kind of rare neurodegenerative disorders that afflict human beings, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, and fatal familial insomnia (FFI), sheep and goat (scrapie), cattle (bovine spongiform encephalopathy, BSE), and other animals. Among which, CJD is the main type of human TSE, including sporadic, inherited, iatrogenic and varient origin. This study contains two individual parts, including proteomics analysis of the pooled CSF samples of patients with sporadic CJD (sCJD), and the study of the alteration of ADAM10in prion infection.Part I:Proteomics analysis of the pooled CSF samples of patients with Creutzfeldt-Jakob diseaseTo assess the potential expression difference of proteins in cerebrospinal fluids (CSF) between CJD and non-CJD patients, the pooled CSF samples from39Chinese probable sCJD patients and from52non-CJD cases were comparably analyzed with the methodology of TMT-labeling and RP-RP-UPLC-MS/MS. Totally,437possible proteins were identified in the tested CSF specimen, among them,49proteins with95% confidence interval. Differential assays showed among those49CSF proteins,12were up-regulated and13were down-regulated significantly in the sCJD compared to non-CJD. The most affected pathway of the differentially expressed proteins in CSF of sCJD was complement and coagulation cascade. Western blots for six selected changed proteins in the pooled CSF samples revealed the similar altering profiles in the groups of sCJD and non-CJD as proteomics. Furthermore, CSF samples from24CJD patients and24non-CJD patients were randomly selected and subjected individually into the Western blots of an increased protein (phosphoglycerate mutase1) and a decreased one (alpha-1-antichymotrysin), which is also confirmed the altering tendency of these identified proteins.Determinations of biomarkers in CSF have been already widely used in the diagnoses of certain CNS diseases. However, most of the targets of diagnostic methods focus on full-length proteins, whose relative molecular weights are usually larger than10kilodaltons (kDa). The profiles of small proteins or degraded peptides from "biomarker" proteins in CSF of CNS diseases, especially CJD, still remain unknown. To find out the potential difference of protein profiles in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, especially in the fraction ranging from1-10kDa, the CSF samples of40probable sporadic CJD (sCJD) patients,32non-CJD cases with dementia and17non-CJD cases without dementia were separately pooled and enriched by the magnetic beads based weak cation exchange chromatography (MB-WCX). After trypsin digestion, each enriched CSF was separated and identified by RP-HPLC-ESI-QTOF MS/MS. In total,42,53and47signals of proteins were identified in the pooled CSF fraction less than10kDa of probable sCJD, non-CJD with dementia and non-CJD without dementia, respectively. Compared with that of probable sCJD, the similarity of CSF protein profiles of non-CJD with dementia (76.2%) were higher than that of non-CJD without dementia (57.1%). Nine CSF proteins were found to be specially observed in probable sCJD group. Those data indicate that proteomic assay of CSF is a powerful technique not only for selection of the potential biomarkers for the development of diagnostic tool of CJD, but also for supplement of useful scientific clues for understanding the CSF homeostasis during the pathogenesis of prion diseases. Part Ⅱ:The study of the alteration of ADAM10in prion infectionIn this study, we confirmed that ADAM10was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between native PrPc and ADAM10was observed not only in various cultured neuronal cell lines, but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10(after removal of its prodomain) molecules showed the binding activity with the native PrPc. Remarkably more PrP-ADAM10complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion infected SMB-S15cells confirmed no detectable PrP-ADAM10complex in the cellular lysates and PrP-ADAM10colocalization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10in the brain homogenates of scrapie agent263K-infected hamsters and agent ME7-infected mice were also almost diminished at terminal stage, showing time-dependent decreases during incubation period. Our data here provide the solid molecular basis for endoproteolysis of ADAM10on PrP molecules and interaction between ADAM10and PrP. Obvious loss of ADAM10during prion infection in vitro and in vivo highlights that ADAM10may play essential pathophysiological roles in prion replication and accumulation.