Identification and proteomic profiling of exosomes in human cerebrospinal fluid - PubMed (original) (raw)

Identification and proteomic profiling of exosomes in human cerebrospinal fluid

Jonathan M Street et al. J Transl Med. 2012.

Abstract

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

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Figures

Figure 1

Figure 1

A) The pellet resulting from ultracentrifugation of human CSF contained protein. The amount of protein is expressed as per ml of CSF. Samples 1 - 5 are from 5 different study participants. B) The size distribution of protein was different in the pellet resulting from ultracentrifugation of human CSF (CSF pellet) compared with uncentrifuged CSF (whole CSF). C) Western blots for flotillin-1 and TSG-101 on the ultracentrifugation pellet (P) and supernatant (S) from CSF collected from 3 study participants (1-3). Both exosomal markers were enriched in the pellets in comparison to the supernatants. D) Western blot for flotillin-1 and TSG101 on fractions obtained following isopycnic centrifugation. The exosomal markers were present in fractions corresponding to a density of 1.10-1.14 g.cm-3 .

Figure 2

Figure 2

A) Transmission electron micrographs of embedded and negatively stained human CSF. Several structures of the characteristic size and appearance for exosomes are highlighted by arrows. B) Transmission electron micrographs of human CSF exosomes incubated with an anti-flotillin-1 antibody and then a gold nanoparticle tagged secondary antibody. A structure with the appearance of an exosome labelled by 2 gold nanoparticles is demonstrated in the figure.

Figure 3

Figure 3

Ions detected following FT-ICR MS on the pellet that resulted from ultracentrifugation of human CSF. A) The number of ions detected in each sample. Sample 1 - 5 are from 5 different study participants B) The number of ions detected in multiple samples. C) Histogram for the coefficient of variation of ions detected in 2 or more of the 5 CSF ultracentrifugation pellets.

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