The Study Of Pathogenic Proteins Of Neurally Derived Serum Exosomes As The Early Diagnostic Biomarker Of Alzheimer's Disease

Objective:1.To extract and identify the neurally derived plasma exosomes from the patients with dementia.2.To compare pathogenic proteins of neurally derived plasma exosomes,including A?1-42?P-T181-tau and P-S396-tau in patients with either Alzheimer's disease(AD)or non-AD.3.To analysis the sensitivity and specificity of pathogenic proteins of neuraly derived plasma exosomes from AD by comparing with the ? amyloid protein PET which had been verified as a biomarker of AD.Methods:1.38 patients with mild cognitive impairment(MCI)and dementias were recruited for this study between June 2015 and December 2016 at cognitive impairment outpatient department of Tianjin Medical University General Hospital.The patients were divided into two groups AD group including AD patients(n=19)and MCI due to AD patients(n=5)according to the NIA-AA creteria,and non-AD group including MCI(n=7)and frontotemporal dementia(FTD)(n=7).All patients extracted blood sample 5ml since morning on an empty stomach.After centrifugation,total exsomes were extracted by ExoQuick Exosome Precipitation Solution.Neurally derived exosomes were isolated by using L1 cell adhesion molecule.The exosomes were analyzed by transmission electron microscopy for its size and appearance and by an NS500 nanoparticle tracking system for counting.Exosome proteins were quantified using Enzyme linked immunosorbent assay(ELISA)for the exosomal marker CD81,human A?1-42,human P-T181-tau and human P-S396-tau.The level of A?1-42?P-T181-tau and P-S396-tau were analyzed in order to explore the sensitivity and specificity for the diagnosis of AD.The levels of pathogenic proteins have been corrected by normalization with the exosomal marker CD81 in order to compare the level of A?1-42?P-T181-tau and P-S396-tau.2.11C-labeled Pittsburgh compound B(PiB)PET imaging was assessed with visual analyze.According to the result of PiB PET,levels of A?1-42?P-T181-tau and P-S396-tau in extracts of neutrally derived blood exosomes were used to analyse the sensitivity and specificity as the early diagnostic biomarker of AD.Results:1.Exosomes are small membranous vesicles with a size ranging from 63 to 100 nm and appear with a characteristic round or cup-shaped morphology in patients with plasma,which were identified by transmission electron microscopy.Exosomes from AD patients(n=3)and non-AD patients(n=3),respectively,contained 1.09±0.024×109/ml and 0.98±0.067×109/ml with the Nanosight NS500 system and Nanoparticle Tracking Analysis(NTA),while there were no statistically significant differences among groups(P > 0.05).After normalized by the exosomal marker CD81,exosomal concentrations of A?1-42?P-T181-tau and P-S396-tau in AD group(10.52±3.87pg/ml?105.18±28.96pg/ml?7.91±2.67pg/ml)were higher than non-AD group(6.76±1.67pg/ml,58.93±16.45pg/ml?4.57±1.01pg/ml),P < 0.05.2.On the basis of PiB PET for the diagnosis of AD,receiver operating characteristics(ROC)analyses were conducted.The area under the curve(AUC)for A?1-42?P-T181-tau and P-S396-tau from the ROC analysis were 0.801?0.917 and 0.839.Cut-off for every protein were 8.03pg/ml?80.10pg/ml and 6.59pg/ml.The sensitivities of A?1-42?P-T181-tau and P-S396-tau were 85%?100% and 100%,respectively,distinguishing AD from non-AD.The specificities of A?1-42 ?P-T181-tau and P-S396-tau were 67.89%?77.78% and 66.67% respectively for the diagnosis of AD and MCI due to AD.Conclusion:1.The neurally derived exosomes were obtained from peripheral blood using the method of immunoprecipitation.2.Levels of A?1-42?P-T181-tau and P-S396-tau in extracts of neurally derived plasma exosomes in AD group were higher than non-AD.3.The sensitivity and specificity of pathogenic proteins of neuraly derived plasma exosomes for the diagnosis of AD was a higher,as considered a class good biomarker for the diagnosis of AD.