Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

C boutons are large, cholinergic, synaptic terminals that arise from local interneurons and specifically contact spinal α-motoneurons (MNs). C boutons characteristically display a postsynaptic specialization consisting of an endoplasmic reticulum-related subsurface cistern (SSC) of unknown function. In the present work, by using confocal microscopy and ultrastructural immunolabeling, we demonstrate that neuregulin-1 (NRG1) accumulates in the SSC of mouse spinal MNs. We also show that the NRG1 receptors erbB2 and erbB4 are presynaptically localized within C boutons, suggesting that NRG1-based retrograde signaling may occur in this type of synapse. In most of the cranial nuclei, MNs display the same pattern of NRG1 distribution as that observed in spinal cord MNs. Conversely, MNs in oculomotor nuclei, which are spared in amyotrophic lateral sclerosis (ALS), lack both C boutons and SSC-associated NRG1. NRG1 in spinal MNs is developmentally regulated and depends on the maintenance of nerve-muscle interactions, as we show after nerve transection experiments. Changes in NRG1 in C boutons were also investigated in mouse models of MN diseases: i.e., spinal muscular atrophy (SMNΔ7) and ALS (SOD1(G93A)). In both models, a transient increase in NRG1 in C boutons occurs during disease progression. These data increase our understanding of the role of C boutons in MN physiology and pathology.

BackgroundExtracellular vesicles (EVs) act as key mediators of intercellular communication and are secreted and taken up by all cell types in the central nervous system (CNS). While detailed study of EV-based signaling is likely to significantly advance our understanding of human neurobiology, the technical challenges of isolating EVs from CNS tissues have limited their characterization using ‘omics’ technologies. We therefore developed a new Protein Organic Solvent Precipitation (PROSPR) method that can efficiently isolate the EV repertoire from human biological samples.ResultsIn the current report, we present a novel experimental workflow that outlines the process of sample extraction and enrichment of CNS-derived EVs using PROSPR. Subsequent LC-MS/MS-based proteomic profiling of EVs enriched from brain homogenates successfully identified 86 of the top 100 exosomal markers. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes, while also expanded the known human EV-associated proteome with 685 novel identifications. Similarly, lipidomic characterization of enriched CNS vesicles not only identified previously reported EV-specific lipid families (PS, SM, lysoPC, lysoPE) but also uncovered novel lipid isoforms not previously detected in human EVs. Finally, dedicated flow cytometry of PROSPR-CNS-EVs revealed that ~80 % of total microparticles observed were exosomes ranging in diameter from ≤100 nm to 300 nm.ConclusionsThese data demonstrate that the optimized use of PROSPR represents an easy-to-perform and inexpensive method of enriching EVs from human CNS tissues for detailed characterization by ‘omics’ technologies. We predict that widespread use of the methodology described herein will greatly accelerate the study of EVs biology in neuroscience.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0108-1) contains supplementary material, which is available to authorized users.

Protein deamidation has been proposed to represent a "molecular clock" that progressively disrupts protein structure and function in human degenerative diseases and natural aging. Importantly, this spontaneous process can also modify therapeutic proteins by altering their purity, stability, bioactivity, and antigenicity during drug synthesis and storage. Deamidation occurs non-enzymatically in vivo, but can also take place spontaneously in vitro, hence artificial deamidation during proteomic sample preparation can hamper efforts to identify and quantify endogenous deamidation of complex proteomes. To overcome this, mass spectrometry (MS) can be used to conduct rigorous site-specific characterization of protein deamidation due to the high sensitivity, speed, and specificity offered by this technique. This article reviews recent progress in MS analysis of protein deamidation and discusses the strengths and limitations of common "top-down" and "bottom-up" approaches. Recent advances in sample preparation methods, chromatographic separation, MS technology, and data processing have for the first time enabled the accurate and reliable characterization of protein modifications in complex biological samples, yielding important new data on how deamidation occurs across the entire proteome of human cells and tissues. These technological advances will lead to a better understanding of how deamidation contributes to the pathology of biological aging and major degenerative diseases. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:677-692, 2017.

Atherosclerosis arises from leukocyte infiltration and thickening of the artery walls and constitutes a major component of vascular disease pathology, but the molecular events underpinning this process are not fully understood. Proteins containing an Asn-Gly-Arg (NGR) motif readily undergo deamidation of asparagine to generate isoDGR structures that bind to integrin αvβ3 on circulating leukocytes. Here we report the identification of isoDGR motifs in human atherosclerotic plaque components including extracellular matrix (ECM) proteins fibronectin and tenascin C, which have been strongly implicated in human atherosclerosis. We further demonstrate that deamidation of NGR motifs in fibronectin and tenascin C leads to increased adhesion of the monocytic cell line U937 and enhanced binding of primary human monocytes, except in the presence of a αvβ3-blocking antibody or the αv-selective inhibitor cilengitide. In contrast, under the same deamidating conditions monocyte-macrophages displayed only weak binding to the alternative ECM component vitronectin which lacks NGR motifs. Together, these findings confirm a critical role for isoDGR motifs in mediating leukocyte adhesion to the ECM via integrin αvβ3 and suggest that protein deamidation may promote the pathological progression of human atherosclerosis by enhancing monocyte recruitment to developing plaques.

BackgroundDementia risk in women is higher than in men, but the molecular neuropathology of this gender difference remains poorly defined. In this study, we used unbiased, discovery-driven quantitative proteomics to assess the molecular basis of gender influences on risk of Alzheimer’s disease with cerebrovascular disease (AD + CVD).ResultsWe detected modulation of several redox proteins in the temporal lobe of AD + CVD subjects, and we observed sex-specific alterations in the white matter (WM) and mitochondria proteomes of female patients. Functional proteomic analysis of AD + CVD brain tissues revealed increased citrullination of arginine and deamidation of glutamine residues of myelin basic protein (MBP) in female which impaired degradation of degenerated MBP and resulted in accumulation of non-functional MBP in WM. Female patients also displayed down-regulation of ATP sub-units and cytochromes, suggesting increased severity of mitochondria impairment in women.ConclusionsOur study demonstrates that gender-linked modulation of white matter and mitochondria proteomes influences neuropathology of the temporal lobe in AD + CVD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0205-7) contains supplementary material, which is available to authorized users.

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Vascular factors that reduce blood flow to the brain are involved in apparition and progression of dementia. We hypothesized that cerebral hypoperfusion (CH) might alter the molecular compositions of brain intercellular communication mechanisms while affecting the neurovascular unit in preclinical and clinical human dementias. To test that hypothesis, mice were subjected to bilateral common carotid stenosis (BCAS) and the molecular compositions of brain-derived and circulating extracellular vesicles (EVs) were assessed. Murine brain vesicle profiles were then analyzed in parallel with brain EVs from post-mortem subjects affected by preclinical Alzheimer's Disease (AD) and mixed dementias. Brain EVs were identified with molecular mediators of hypoxia responses, neuroprotection and neurotoxicity in BCAS mice, patterns also partially resembled by subjects with preclinical AD and mixed dementias. Together these findings indicate that brain EVs represent a promising source of therapeutic targets and circulating markers of neurovascular insult in idiopathic dementias. Furthermore, the results obtained generate novel and compelling hypotheses about the molecular involvement of the vascular component in the etiology of human dementias.