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Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury.

ABSTRACT: Recently, there have been emerging interests in the area of microvesicles and exosome (MV/E) released from brain cells in relation to neurodegenerative diseases. However, only limited studies focused on MV/E released post-traumatic brain injury (TBI) as they highlight on the mechanistic roles of released proteins. This study sought to examine if CSF samples from severe TBI patients contain MV/E with unique protein contents. First, nanoparticle tracking analysis determined MV/E from TBI have a mode of 74-98 nm in diameter, while control CSF MV/E have a mode of 99-104 nm. Also, there are more MV/E were isolated from TBI CSF (27.8-33.6?×?108/mL) than from control CSF (13.1-18.5?×?108/mL). Transmission electron microscopy (TEM) visualization also confirmed characteristic MV/E morphology. Using targeted immunoblotting approach, we observed the presence of several known TBI biomarkers such as ?II-spectrin breakdown products (BDPs), GFAP, and its BDPs and UCH-L1 in higher concentrations in MV/E from TBI CSF than their counterparts from control CSF. Furthermore, we found presynaptic terminal protein synaptophysin and known exosome marker Alix enriched in MV/E from human TBI CSF. In parallel, we conducted nRPLC-tandem mass spectrometry-based proteomic analysis of two control and two TBI CSF samples. Ninety-one proteins were identified with high confidence in MV/E from control CSF, whereas 466 proteins were identified in the counterpart from TBI CSF. MV/E isolated from human CSF contain cytoskeletal proteins, neurite-outgrowth related proteins, and synaptic proteins, extracellular matrix proteins, and complement protein C1q subcomponent subunit B. Taken together, following severe TBI, the injured human brain released increased number of extracellular microvesicles/exosomes (MV/E) into CSF. These TBI MV/E contain several known TBI biomarkers and previously undescribed brain protein markers. It is also possible that such TBI-specific MV/E might contain cell to cell communication factors related to both cell death signaling a well as neurodegeneration pathways.

SUBMITTER: Manek R

PROVIDER: S-EPMC6359938 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury.

Manek Rachna R Moghieb Ahmed A Yang Zhihui Z Kumar Dhwani D Kobessiy Firas F Sarkis George Anis GA Raghavan Vijaya V Wang Kevin K W KKW

Molecular neurobiology 20171129 7

Recently, there have been emerging interests in the area of microvesicles and exosome (MV/E) released from brain cells in relation to neurodegenerative diseases. However, only limited studies focused on MV/E released post-traumatic brain injury (TBI) as they highlight on the mechanistic roles of released proteins. This study sought to examine if CSF samples from severe TBI patients contain MV/E with unique protein contents. First, nanoparticle tracking analysis determined MV/E from TBI have a mo ...[more]

PMID: 29188495

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Project description:BACKGROUND:Proteomic analysis of cerebrospinal fluid (CSF) has shown great promise in identifying potential markers of injury in neurodegenerative diseases [1-13]. Here we compared CSF proteomes in healthy individuals, with patients diagnosed with traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) in order to characterize molecular biomarkers which might identify these different clinical states and describe different molecular mechanisms active in each disease state. METHODS:Patients presenting to the Neurosurgery service at the Louisiana State University Hospital-Shreveport with an admitting diagnosis of TBI or SAH were prospectively enrolled. Patients undergoing CSF sampling for diagnostic procedures were also enrolled as controls. CSF aliquots were subjected to 2-dimensional gel electrophoresis (2D GE) and spot percentage densities analyzed. Increased or decreased spot expression (compared to controls) was defined in terms of in spot percentages, with spots showing consistent expression change across TBI or SAH specimens being followed up by Matrix-Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). Polypeptide masses generated were matched to known standards using a search of the NCBI and/or GenPept databases for protein matches. Eight hundred fifteen separately identifiable polypeptide migration spots were identified on 2D GE gels. MALDI-MS successfully identified 13 of 22 selected 2D GE spots as recognizable polypeptides. RESULTS:Statistically significant changes were noted in the expression of fibrinogen, carbonic anhydrase-I (CA-I), peroxiredoxin-2 (Prx-2), both ? and ? chains of hemoglobin, serotransferrin (Tf) and N-terminal haptoglobin (Hp) in TBI and SAH specimens, as compared to controls. The greatest mean fold change among all specimens was seen in CA-I and Hp at 30.7 and -25.7, respectively. TBI specimens trended toward greater mean increases in CA-I and Prx-2 and greater mean decreases in Hp and Tf. CONCLUSIONS:Consistent CSF elevation of CA-I and Prx-2 with concurrent depletion of Hp and Tf may represent a useful combination of biomarkers for the prediction of severity and prognosis following brain injury.

Project description:BackgroundTraumatic brain injury (TBI) can result in an acute coagulopathy including platelet dysfunction that can contribute to ongoing intracranial hemorrhage. Previous studies have shown adenosine diphosphate (ADP)-induced platelet aggregation to be reduced after TBI. In addition, circulating microvesicles (MVs) are increased following TBI and have been shown to play a role in post-TBI coagulopathy and platelet function. We hypothesized that post-TBI MVs would affect platelet aggregation in a murine head injury model.MethodsModerate TBI was performed using a weight-drop method in male C57BL6 mice. Whole blood, plasma, MVs, and MV-poor plasma were isolated from blood collected 10 minutes following TBI and were mixed separately with whole blood from uninjured mice. Platelet aggregation was measured with Multiplate impedance platelet aggregometry in response to ADP. The ADP P2Y12 receptor inhibitor, R-138727, was incubated with plasma and MVs from TBI mice, and platelet inhibition was again measured.ResultsWhole blood taken from 10-minute post-TBI mice demonstrated diminished ADP-induced platelet aggregation compared with sham mice. When mixed with normal donor blood, post-TBI plasma and MVs induced diminished ADP-induced platelet aggregation compared with sham plasma and sham MVs. By contrast, the addition of post-TBI MV-poor plasma to normal blood did not change ADP-induced platelet aggregation. The observed dysfunction in post-TBI ADP platelet aggregation was prevented by the pretreatment of post-TBI plasma with R-138727. Treatment of post-TBI MVs with R-138727 resulted in similar findings of improved ADP-induced platelet aggregation compared with nontreated post-TBI MVs.ConclusionAdenosine diphosphate-induced platelet aggregation is inhibited acutely following TBI in a murine model. This platelet inhibition is reproduced in normal blood by the introduction of post-TBI plasma and MVs. Furthermore, observed platelet dysfunction is prevented when post-TBI plasma and MVs are treated with an inhibitor of the P2Y12 ADP receptor. Clinically observed post-TBI platelet dysfunction may therefore be partially explained by the presence of the ADP P2Y12 receptor within post-TBI MVs.Level of evidenceLevel III.

Dataset Information

Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury.

Publications

Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury.

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