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Identification and proteomic profiling of exosomes in human cerebrospinal fluid.

ABSTRACT: Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: 1) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals.CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR).Flotillin 1 and tumor susceptibility gene 101 (TSG101), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin 1. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-161 ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 81% for ions detected in two or more samples.Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller 'real life' clinical samples; a key challenge in the development of exosomes as translational tools.

SUBMITTER: Street JM

PROVIDER: S-EPMC3275480 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Identification and proteomic profiling of exosomes in human cerebrospinal fluid.

Street Jonathan M JM Barran Perdita E PE Mackay C Logan CL Weidt Stefan S Balmforth Craig C Walsh Tim S TS Chalmers Rod T A RT Webb David J DJ Dear James W JW

Journal of translational medicine 20120105

<h4>Background</h4>Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study ...[more]

PMID: 22221959

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Project description:BackgroundX-linked retinoschisis (XLRS) is a vitreoretinal degenerative disorder causing vision deterioration, due to structural defects in retina. The hallmark of this disease includes radial streaks arising from the fovea and splitting of inner retinal layers (schisis). Although these retinal changes are attributed to mutations in the retinoschisin gene, schisis is also observed in patients who do not carry mutations. In addition, the origin of intraschisis fluid, the triggering point of schisis formation and its progression are largely unknown still. So far, there is no report on the complete proteomic analysis of this fluid. Schisis fluid proteome could reflect biochemical changes in the disease condition, helping in better understanding and management of retinoschisis. Therefore it was of interest to investigate the intraschisis fluid proteome using high-resolution mass spectrometry.MethodsTwo male XLRS patients (aged 4 and 40 years) underwent clinical and genetic evaluation followed by surgical extraction of intraschisis fluids. The two fluid samples were resolved on a SDS-PAGE and the processed peptides were analyzed by Q-Exactive plus hybrid quadrupole-Orbitrap mass spectrometry. Functional annotation of the identified proteins was performed using Ingenuity pathway analysis software.ResultsMass spectrometry analysis detected 770 nonredundant proteins in the intraschisis fluid. Retinol dehydrogenase 14 was found to be abundant in the schisis fluid. Gene ontology based analysis indicated that 19% of the intraschisis fluid proteins were localized to the extracellular matrix and 15% of the proteins were involved in signal transduction. Functional annotation identified three primary canonical pathways to be associated with the schisis fluid proteome viz., LXR/RXR activation, complement system and acute phase response signalling, which are involved in immune and inflammatory responses. Collectively, our results show that intraschisis fluid comprises specific inflammatory proteins which highly reflect the disease environment.ConclusionBased on our study, it is suggested that inflammation might play a key role in the pathogenesis of XLRS. To our knowledge, this is the first report describing the complete proteome of intraschisis fluid, which could serve as a template for future research and facilitate the development of therapeutic modalities for XLRS.

Dataset Information

Identification and proteomic profiling of exosomes in human cerebrospinal fluid.

Publications

Identification and proteomic profiling of exosomes in human cerebrospinal fluid.

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