Comprehensive Proteomic Profiling of Urinary Exosomes and Identification of Potential Non-invasive Early Biomarkers of Alzheimer's Disease in 5XFAD Mouse Model

Abstract

Background: Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by irreversible progressive cognitive deficits. Identification of candidate biomarkers, before amyloid-β-plaque deposition occurs, is therefore of great importance for early intervention of AD.

Objective: To investigate the potential non-invasive early biomarkers of AD in 5XFAD mouse model, we investigate the proteome of urinary exosomes present in 1-month-old (before amyloid-β accumulation) 5XFAD mouse models and their littermate controls. Another two groups of 2 and 6 months-old urinary samples were collected for monitoring the dynamic change of target proteins during AD progression.

Methods: Proteomic, bioinformatics analysis, multiple reaction monitoring (MRM), western blotting (WB) or ELISA were performed for analyzing these urinary exosomes.

Results: A total of 316 proteins including 44 brain cell markers were identified using liquid chromatography tandem mass spectrometry. Importantly, 18 proteins were unique to the 5XFAD group. Eighty-eight proteins including 11 brain cell markers were differentially expressed. Twenty-two proteins were selected to be verified by WB. Furthermore, based on an independent set of 12 urinary exosomes samples, five in these proteins were further confirmed significant difference. Notably, Annexin 2 and Clusterin displayed significant decreased in AD model during the course detected by ELISA. AOAH, Clusterin, and Ly86 are also brain cell markers that were first reported differential expression in urinary exosomes of AD model.

Conclusion: Our data demonstrated that some urinary exosome proteins, especially Annexin 2 and Clusterin, as nanometer-sized particles, enable detection of differences before amyloid-β-plaque deposition in 5XFAD mouse model, which may present an ideal non-invasive source of biomarkers for prevention of AD.

Keywords: 5XFAD mouse model; Alzheimer’s disease; biomarkers; early diagnosis; urinary exosome proteome.

FIGURE 1

Workflow of isolation, characterization, and proteomics of urinary exosomes in 5XFAD transgenic mouse model of AD and littermate control. The proteins were analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), MRM, WB, and ELISA. Differential proteins were analyzed by bioinformatics analysis. See text for details.

FIGURE 2

Characterization of urinary exosomes. (A) NTA of exosomes purified urinary exosomes. (B) The observation of exosomes by transmission electron microscopy (TEM). Bar = 100 nm. (C) After protein quantification, exosomes were lysed to perform immunoblot with specific monoclonal antibodies against typical exosome proteins were used to validate the quality of exosmes. Identification of Alix, CD10, CD63, Flotillin 1, and TSG101 in urinary exosomes (20 ug total protein per load) by Western blot, Albumin is a negative control protein were used to validate the quality of our isolation technique.

FIGURE 3

Comparison of the proteomics analysis by hierarchical cluster and gene ontology. (A) Heatmap illustrating the classification of control and 5XFAD mice (x axis) based on the deferentially expressed proteins (y axis) and unbiased clustering analysis in urinary exosomes. (B–D) Pie diagrams of Gene ontology (GO) analysis of the differentially expressed proteins was performed to study the specific MFs, BP, and CCs (the top 15 counts are shown) Comparison between control and 5XFAD mice samples in term of number of genes identified for different GO terms. Amounts of the top 15 terms with P-value < 0.05.

FIGURE 4

Comparison of the proteomics analysis by KEGG pathway and PPI network analysis. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (the top 15 proteins with the greatest fold enrichment are shown). Dot plots of enriched KEGG pathways in differential proteins. The x axis shows the fold enrichment of each KEGG pathway, whereas the color denotes the P-value and the area of the dots represents the number of genes assigned to each KEGG pathway. The y axis displays KEGG pathway term. (B) PPI analysis showed the differential proteins are involved in different network. Red indicated upregulated genes, and green indicated downregulated genes in the present study.

FIGURE 5

PPI analysis and multiple reaction monitoring (MRM) assays for the target proteins from control and 5XFAD mice. (A) PPI analysis showed the relationship of the target differential proteins. More lines between two proteins represented more possible interaction. (B) The relative peak area of 22 target proteins were measured by MRM between control and 5XFAD mice. All data are presented as mean ± SD in triplicate experiments; Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 6

Western blots (WB) and quantitative analysis of 11 differential proteins in the urinary exosomes isolated from control and 5XFAD mice. (A) Same aliquot of urinary exosomes isolated from three control samples and three 5XFAD mice samples. AOAH, Clusterin, Ceruloplasmin, Cathepsin B, ENPP2, FUCA2, HEXB, Ly86, Pcolce, Preol, and PrP proteins were detected by WB in sequence. Representative blots of the tested proteins and the molecular weight of standard proteins were presented. (B–L) Quantitative analysis of each of the tested protein’s relative fold change, respectively. All data are presented as mean ± SD in triplicate experiments; Student’s t-test; *P < 0.05; **P < 0.01.

FIGURE 7

Western blots (WB) and quantitative analysis of the other 11 differential proteins in the urinary exosomes isolated from control and 5XFAD mice. (A) Same aliquot of urinary exosomes isolated from three control samples and three 5XFAD mice samples. ALDH3B2, Angiotensin, Annexin 2, Apolipoprotein A I, EEF2, FUCA1, NEU1, QSOX1, RHCG, Smpdl3a, and TGM4 proteins were detected by WB in sequence. Representative blots of the tested proteins and the molecular weight of standard proteins were presented. (B–L) Quantitative analysis of each of the tested protein’s relative fold change, respectively. All data are presented as mean ± SD in triplicate experiments; Student’s t-test; *P < 0.05; **P < 0.01.

FIGURE 8

Western blots (WB) and quantitative analysis of an independent set of the urinary exosomes isolated from control and 5XFAD mice. (A) Same aliquot of urinary exosomes isolated from six control samples and six 5XFAD mice samples. AOAH, NEU1, Annexin 2, Clusterin, and Ly86 proteins were further detected by WB in sequence. Alix and Flotillin 1 were tested as urinary exosomes biomarkers. Representative blots of the tested proteins and the molecular weight of standard proteins were presented. (B–F) Quantitative analysis of each of the tested protein’s relative fold change, respectively. All data are presented as mean ± SD in triplicate experiments; Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 9

(A) The schematic diagram of the pathological feature of 5XFAD mice model during the AD progression. (B,C) Quantification of Annexin 2 and Clusterin in urinary exosome samples using ELISA from 1, 2, and 6-month-old 5XFAD mice and their littermate control. Each point represents value for one sample, and horizontal line in point clusters depicts mean level for that group. All data are presented as mean ± SD in triplicate experiments; Student’s t-test; **P < 0.01; ***P < 0.001.