Proteomic Analysis Of Plasma Neurogenic Exosomes In Patients With Alzheimer's Disease

Objective:(1)Exploring the influencing factor of Alzheimer's Disease(AD)patients from epidemiological factors to promote the prevention of AD;(2)Using quantitative proteomics techniques to explore the changes of plasma neurogenic exosome proteins in AD patients,to find relevant biomarkers to promote early diagnosis of AD.Methods:(1)Establish a 1:1 matched case-control study,a total of 137 elderly people with cognitive dysfunction who were assessed by a Mini-Cog and a Mini-mental State Examination(MMSE)in a hospital in a certain district of Shenzhen were selected as case groups,while 137 healthy elderly people of the same gender with age differences less than 3 years were selected as the control group,each subject comes from the same area.Self-made questionnaires were used to investigate the dietary status of the subjects.Epidata 3.1 was used to enter the physical examination and questionnaire data,and SPSS19.0 software was used for statistical analysis such as univariate analysis,logistic regression and correlation analysis.(2)A total of 5 groups of AD,MCI patients and 80,60,40-year-old normal people were selected.The ratio of male to female was 1:1 in each group,and there were no significant difference in age between AD,MCI and 80 years old groups(P>0.05).(3)Human plasma-derived exosomes were extracted using ExoQuick,and neuron-derived exosomes were isolated using L1 cell adhesion molecule(L1CAM)b iotinylated antibody.The exosomes were identified by transmission electron microscopy,Nanosight NS500 and Nanoparticle Tracking A nalysis(NTA)software.The exosome proteins were degraded to peptides by alkylation,desalting and enzymatic hydrolysis,and Tandem Mass Tag(TMT)was used,and then the labeled peptides were identified and quantified by LC-ESI-MS/MS.(4)Perseus software was used to analyze which exogenous proteins have statistically significant difference between groups.The above different proteins were an alyzed for bioinformatics using GO(Gene Ontology)analysis,Venn,Volcano Plot,Heatmap,Kyoto Encyclopedia of Genes and Genome s(KEGG),Wikipathway and STRING protein interaction networks.Results:(1)The annual income,education level,monthly intake of vegetables,freshwater prawn/shellfish or eggs,and the frequency of drinking green tea were statistically significant compared with the control group.Conditional logistic regression analysis showed that education(OR=0.651,95%CI: 0.476,0.890)is a protective factor,and more vegetables(OR=2.405,95%CI: 1.568,3.688)and freshw ater shellfish(OR=3.796,95%CI: 1.258,11.453)are risk factors.(2)Correlation and statistical analysis show that the blood glucose level of the case group was higher than that of the control group,and the prevalence of diabetes is higher too;The smaller the patient's Mini-cog score,the smaller the MMSE score,the smaller the egg int ake,the more intake of freshwater prawn/shellfish and vegetables,the larger the BMI value,the higher the blood sugar level;And the higher the patient's MMSE score,the more intake of marine fish and dairy products,the lower the systolic blood pressure and blood sugar level.(3)Electron microscopy and NTA analysis showed that t he exosomes with elliptical and bilayer membrane structure were successfully isolated.A total of 360 proteins were identified by LC-E SI-MS/MS,Variance analysis showed that there were 62 differential proteins between the 5 groups(P<0.05);Further t-test analysis bet ween the two groups found that AD and MCI,AD and 80,MCI and 80,80 and 60,80 and 40,60 and 40,AD and 60,AD and 40,MCI and 60,MCI and 40 group has 8,15,18,41,42,57,41,40,30,36 differential proteins respectively(P<0.05),The proteins up-regulated between groups were 4,6,9,19,31,37,41,40,30,36,respectively,and the down-regulated proteins were 4,9,9,22,11,19,28,17,21 14,14 according to the criteria of expression differ ence Ratio>1.5 or Ratio<0.67.Further bioinformatics analysis results showed that the 62 proteins were involved in 42 biological proces ses(BP),18 molecular functions(MF)and 16 cell Components(CC).The top 7 of the BP were mainly endocytosis,complement acti vation,regulation of endopeptidase activity,and Fc-? receptor signal ing pathway involved in phagocytosis;the top 7 of the MF are mainly serine endopeptidase activity,endopeptidase inhibitor activity,antigen Binding,calcium ion binding,MHC class II protein complex b inding,etc.;CC results indicate that 43 differential proteins belong to exosomal proteins.Signal pathway analysis showed that 62 proteins were involved in 10 signaling pathways including complement and agglutination systems,statin pathway,lipid particle composition,oxidative damage,selenium micronutrient network and PI3K-Akt signaling pathway.The String results indicate that 13 exosomal proteins: A2 M,LCAT,FERMT3,HSP90AB1,APOC1,FCN2,ADIPOQ,A TPB,SPP2,HSP90AA1,FBLN5,PIGR and COMP have interaction with other proteins.Conclusion:(1)High education is protective factor for cognitive dysfunction,and more vegetables and freshwater shellfish are risk f actors.It is recommended to increase the intake of seafood,dairy products and eggs,drink green tea properly,and maintain proper BM I.(2)Neurogenic exosomes can be obtained from peripheral blood by immunoprecipitation.The protein composition of exosomes in AD and MCI patients has changed,and it is expected that the differen tial protein will promote early diagnosis of patients.