Alzheimer's Disease (AD) is a progressive neurodegenerative disorder characterized by the gradual deterioration of cognitive function and memory loss. It is the most common cause of dementia, affecting millions of individuals worldwide. Despite extensive research efforts, there is still no cure for AD, and available treatments only offer symptomatic relief, highlighting the urgent need for effective diagnostic tools and therapeutic strategies.
Early diagnosis of AD is crucial as it enables timely intervention and treatment, potentially slowing down disease progression and improving patient outcomes. However, current diagnostic methods, which often involve invasive procedures or expensive imaging techniques, are not suitable for routine screening, particularly in the preclinical stages of the disease. Therefore, there is a pressing need to identify non-invasive and reliable biomarkers for early detection of AD.
Exosomes have emerged as promising candidates for biomarker discovery due to their role in intercellular communication and their ability to carry a cargo of proteins, nucleic acids, and lipids derived from their parent cells. Urinary exosomes, in particular, have gained attention as potential sources of biomarkers for neurological disorders like AD. Urine is readily accessible and can be collected non-invasively, making it an attractive biofluid for biomarker research.
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Case. Identification of Potential Urinary Exosomal Biomarkers for Early Detection of Alzheimer's Disease in 5XFAD Mouse Model (1)
Research Background
The 5XFAD mouse model used in this study is a widely used transgenic model that overexpresses mutant forms of amyloid precursor protein (APP) and presenilin 1 (PS1), leading to the accelerated production and aggregation of amyloid-beta (Aβ) peptides, a hallmark of AD pathology. This model recapitulates key features of AD, including amyloid plaque deposition and cognitive deficits, making it a valuable tool for studying the disease mechanisms and testing potential therapeutic interventions.
By leveraging the 5XFAD mouse model and utilizing advanced analytical techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting, liquid chromatography-mass spectrometry (LC-MS/MS), multiple reaction monitoring (MRM), and enzyme-linked immunosorbent assay (ELISA), the researchers aimed to identify specific proteins present in urinary exosomes that could serve as early biomarkers for AD. This comprehensive approach allowed for the identification, validation, and further characterization of potential biomarkers associated with AD pathology, with the ultimate goal of improving early diagnosis and intervention strategies for this devastating disease.
Sample Source
The study utilized the 5XFAD mouse model as a mimic of Alzheimer's disease, alongside corresponding control group samples. Urine served as the sample source, and purified urinary exosome samples were obtained through thorough sample processing and exosome isolation techniques.
Technical Methods
Various technical methods were employed, including nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) observation, Western blot analysis, liquid chromatography-mass spectrometry (LC-MS/MS), multiple reaction monitoring (MRM), and enzyme-linked immunosorbent assay (ELISA). Through these techniques, qualitative and quantitative analyses of proteins in urinary exosomes were conducted, and potential biomarkers were validated and further investigated.
Research Objective
The aim of this study was to identify potential urinary exosomal biomarkers associated with the 5XFAD mouse model as candidate markers for early detection of Alzheimer's disease. By comprehensively utilizing various technical approaches, the protein composition of urinary exosomes was analyzed in depth to explore their relationship with AD pathology, providing theoretical basis and experimental support for the early diagnosis and treatment of AD.
Technical Approach
Research Content:
STEP 1 Sample Collection
Urine samples were collected from both 5XFAD mice and age-matched control mice, followed by isolation and extraction of exosomes.
STEP 2 Exosome Characterization
Nanoparticle tracking analysis (NTA): Urinary exosomes from both the control and 5XFAD groups were subjected to NTA for quantitative analysis. The mean diameters of exosomes were 120.8 ± 43.0 nm and 103.2 ± 34.1 nm for the control and 5XFAD groups, respectively. The exosomes derived from the urine of 5XFAD mice exhibited a smaller mean diameter compared to those from the control group, yet their concentration was higher.
Transmission electron microscopy (TEM) validation of exosome morphology: Exosomes exhibited flattened spherical or rounded shapes, with a bilayer membrane structure.
WB validation of exosomal marker expression: Expression of exosomal transmembrane proteins CD10, CD63, and Flotillin 1 was detected, along with cytoplasmic proteins Alix and TSG101. Albumin was used to assess the influence of urine on exosomes, indicating the purity of exosomes isolated from urine and absence of contamination, as well as the presence of exosomal-specific markers.
STEP 3: Proteomic Analysis
Differential Protein Screening
The exosomal proteins were subjected to LC-MS/MS for qualitative and quantitative analysis. Significant differences were observed in 88 urinary exosomal proteins between the 5XFAD and control groups. In comparison to the control group, 53 proteins were up-regulated (ratio ≥ 1.2, P-value < 0.05) and 27 proteins were down-regulated (ratio ≤ 0.83, P-value < 0.05) in the 5XFAD group.
Cluster analysis revealed hierarchical protein clusters with similar expression profiles. Eighteen proteins were unique to the 5XFAD group, whereas only one unique protein was found in the control group. Among these differentially expressed proteins, 11 were also cell markers in the brain and were selected for further investigation. Specifically, at least 30 proteins have been reported to be associated with the pathological mechanisms or development of AD, while six were identified as direct AD biomarkers and have great potential to be considered as candidate urinary exosomal biomarkers of AD.
Proteomic Cluster Analysis and Comparison with GO Analysis
Using differential protein expression data and unbiased clustering analysis of urinary exosomal proteins, GO pie chart analysis was conducted to study specific molecular functions (MFs), biological processes (BPs), and cellular components (CCs) between samples from the control and 5XFAD mice groups. Comparison was made based on the number of genes identified for different GO terms determined by the various GO terms.
Validation of Differential Proteins by WB, ELISA, and MRM Targeted Protein Analysis
Based on qualitative and quantitative analysis, there are 88 urinary exosomal proteins showing significant differences between the 5XFAD and control groups. Among these, 22 proteins were selected for further validation based on proteomic analysis. To elucidate the relationships among the target proteins more clearly and intuitively, protein-protein interaction (PPI) network analysis and multiple reaction monitoring (MRM) analysis were performed for the 22 target proteins.
PPI analysis and MRM analysis of target proteins in the control and 5XFAD mice revealed the relationships among the differential target proteins. The more lines between two proteins, the greater the likelihood of interaction between them. MRM measured the relative peak areas of 22 target proteins between the control and 5XFAD mice.
The results showed:
FUCA2, Ly86, PrP, Annexin 2, and Fuca1 exhibited significant differences between the two groups. WB, ELISA, and MRM targeted protein analyses validated the 22 target proteins. Compared to the control group, HEXB (lysosomal and/or autophagic pathway protein) significantly increased in urinary exosomal proteins, consistent with the differential expression of HEXB in the brains of 5XFAD mice. Additionally, four proteins, AOAH, Clusterin, Ly86, and Preol, exhibited the same trend, with significant differences observed in proteomic and WB analyses. Compared to the control group, the levels of AOAH were significantly elevated in the 5XFAD group; conversely, the levels of Clusterin, Ly86, and Preol were significantly decreased.
The 11 differential proteins isolated from urinary exosomes of the control and 5XFAD mice were subjected to WB and quantitative analysis for validation.
A total of 316 proteins were identified, including 44 brain cell markers. Importantly, 18 proteins were uniquely present in the 5XFAD group. A total of 88 proteins (including 11 brain cell markers) showed differential expression. WB validation further confirmed significant differences in five of these proteins.
This study followed a clear methodology, involving the collection of urinary exosome samples from both control and model mice, followed by proteomic analysis to identify differentially expressed proteins. Subsequently, the selected differential proteins were validated, and the results indicated that some urinary exosome proteins, particularly Annexin 2 and Clusterin, could detect differences before the deposition of amyloid-beta plaques, making them potentially ideal non-invasive biomarkers for the prevention of AD.
Reference
- Song, Zhiqi, et al. "Comprehensive proteomic profiling of urinary exosomes and identification of potential non-invasive early biomarkers of Alzheimer's disease in 5XFAD mouse model." Frontiers in Genetics 11 (2020): 565479.