Abstract
BackgroundAlzheimer’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.ObjectiveTo 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.MethodsProteomic, bioinformatics analysis, multiple reaction monitoring (MRM), western blotting (WB) or ELISA were performed for analyzing these urinary exosomes.ResultsA 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.ConclusionOur 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.
Highlights
Alzheimer’s disease is a chronic age-associated neurodegenerative disease related to irreversible cognitive impairment and progressive dementia (Dos et al, 2018)
Mice expressing 5 human mutations (5XFAD mouse model of Alzheimer’s disease (AD)) in amyloid precursor protein (APP) and PS1(B6SJLTg[APP∗K670N∗M671L∗I716V∗V717I, PSEN1∗M146∗L286V] 6799Vas/J) under neuron-specific elements of the Thy1 promoter were purchased from The Jackson Laboratory and crossed to wild type SJL mice to generate offspring hemizygous for the APP and PS1 transgenes (Oakley et al, 2006)
The positive mice tested by Polymerase Chain Reaction (PCR) (Transgene forward: AGG ACT GAC CAC TCG ACC AG; transgene reverse: CGG GGG TCT AGT TCT GCA T) were named as 5XFAD mice, littermate mice tested by PCR as negative are named as control group
Summary
Alzheimer’s disease is a chronic age-associated neurodegenerative disease related to irreversible cognitive impairment and progressive dementia (Dos et al, 2018). Exosomes are small extracellular nano-sized vesicles between 40 and 160 nm in diameter (Kalluri and LeBleu, 2020) that were first described in the 1980s (Johnstone et al, 1987) They can be isolated from a variety of body fluids, making them amenable to proteomic and transcriptomic analyses; this supports the hypothesis that their analysis may provide valuable information for disease diagnosis and monitoring (Mitchell et al, 2009). Urinary exosomes contain approximately 3% of total urine protein based on a previous report (Zhou et al, 2006b) and provide a promising source for biomarker discovery because of the ability to detect proteins with relatively low abundance and the reduced complexity of the urinary proteome (Pisitkun et al, 2004; Wang et al, 2012). Identification of candidate biomarkers, before amyloid-β-plaque deposition occurs, is of great importance for early intervention of AD
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