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An optimized method for enrichment of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer's disease.
Journal of Neuroscience Methods 2018 September 2
BACKGROUND: Alzheimer's disease (AD) is the major cause of dementia that has increased dramatically in prevalence over the past several decades. Yet many questions still surround the etiology of AD. Recently, extracellular vesicles (EVs) that transport protein, lipid, and nucleic acids from cell to cell have been implicated in the clearance and propagation of misfolded proteins. Investigation of EVs in AD progression, and their potential diagnostic utility may contribute to understanding and treating AD. However, the challenges of isolating brain-derived EVs have in part hindered these studies.
NEW METHOD: Here, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis.
RESULTS: We demonstrate the isolation of these vesicles and the enrichment of EV proteins compared to sedimentation gradient isolation of vesicles. Moreover, comparative proteomic analysis of brain-derived EVs from healthy and AD mouse brains revealed differences in vesicular content including proteins involved in aging, immune response, and oxidation-reduction maintenance. These changes provide insight into AD-associated neurodegeneration and potential biomarkers of AD. Comparison with existing methods: Recent techniques have used sedimentation sucrose gradients to isolate EVs from brain tissue. However, here we demonstrate the advantages of floatation iodixanol density gradient isolation of small EVs, and provide evidence of EV enrichment by electron microscopy, immunoblot analysis, and quantitative mass spectrometry.
CONCLUSIONS: Together these findings offer a rigorous technique for enriching whole tissue-derived EVs for downstream analyses, and application of this approach to uncovering molecular changes in AD progression and other neurological conditions.
NEW METHOD: Here, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis.
RESULTS: We demonstrate the isolation of these vesicles and the enrichment of EV proteins compared to sedimentation gradient isolation of vesicles. Moreover, comparative proteomic analysis of brain-derived EVs from healthy and AD mouse brains revealed differences in vesicular content including proteins involved in aging, immune response, and oxidation-reduction maintenance. These changes provide insight into AD-associated neurodegeneration and potential biomarkers of AD. Comparison with existing methods: Recent techniques have used sedimentation sucrose gradients to isolate EVs from brain tissue. However, here we demonstrate the advantages of floatation iodixanol density gradient isolation of small EVs, and provide evidence of EV enrichment by electron microscopy, immunoblot analysis, and quantitative mass spectrometry.
CONCLUSIONS: Together these findings offer a rigorous technique for enriching whole tissue-derived EVs for downstream analyses, and application of this approach to uncovering molecular changes in AD progression and other neurological conditions.
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