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 αII-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.

Keywords: Biomarkers; Exosome; Glial injury; Microvesicles; Neuronal injury; Systems biology; Traumatic brain injury.

Figure 1.

Human CSF microvesicles/exosome (MV/E) isolation protocol.

Figure 2.

Microvesicles/exosome sizing analysis with dynamic light scattering (DLS). (A) representative size distribution of a MV/E sample from TBI CSF. (B) Summary of size and concentration results of MV/E from 2 control and 2 TBI CSF samples.

Figure 3.

Electron micrograph (EM) images showing microvesicles/exosomes isolated from either control CSF or from human TBI CSF. PBS buffer only was included as negative control. Scale bar was as indicated (500 nm), while yellow arrows indicate MV/E.

Figure 4.

Presence of neuronal and glial proteolytic biomarkers αII-Spectrin and its breakdown products (SBDPs) and GFAP and its BDP in MV/E samples isolated from control and TBI CSF. (A) Immunoblots images showing the presence of these markers in TBI CSF isolated MV/E. (B) quantification of levels of these protein markers in MV/E preparations isolated from TBI vs. control CSF. **p < 0.01 (statistical significant). For standardization, each lane was loaded with protein from 3 ×10⁸ MV/E particles. Sample size: control n=4, TB, n= 19.

Figure 5.

Presence of neural marker UCH-L1, synaptic marker synaptophysin and exosome maker Alix in MV/E samples isolated from control and TBI CSF. (A) Immunoblots images showing the presence of these markers in TBI and control CSF isolated MV/E. (B) Quantification of levels of these protein markers in MV/E preparations isolated from TBI vs. control CSF. ** p < 0.01 (statistical significant). For standardization, each lane was loaded with protein from 3 ×10⁸ MV/E particles. Sample size: control n=4, TB, n= 19.

Figure 6.

Two-set Venn diagram of the differential unique and combined pathways for protein identified in MV/E from human TB and control CSF.

Figure 7.

Altered targeted molecular functions and biological processes that are unique to the protein present in MV/E from TBI CSF. Shown are the altered pathways involving: neuronal death, oxidative stress, axonal injury, proteolysis and microtubule cytoskeletal assembly implicated in the altered proteins in the TBI CSF-specific proteins. (See Suppl. Table 4 for entity relation, relationship and references involved).

Figure 8.

Global Enriched pathways implicated in the TBI CSF MV/E proteome. Shown are the altered pathways involving: complement activation, cell communication, synaptic endocytosis & exocytosis, cytoskeletal changes, and microtubule cytoskeletal assembly implicated in the altered proteins in the TBI CSF MV/E-specific proteins. (See Suppl. Table 5 for entity relation, relationship and references involved).