Source:Journal of Neuroscience Methods
Author(s): Stephanie N. Hurwitz, Li Sun, Kalonji Y. Cole, Charles R. Ford, James M. Olcese, David G. Meckes
BackgroundAlzheimer'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 methodHere, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis.ResultsWe 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.ConclusionsTogether 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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