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. 2020 Jun 15;37(12):1418-1430.
doi: 10.1089/neu.2019.6826. Epub 2020 Mar 11.

Alterations in Plasma microRNA and Protein Levels in War Veterans with Chronic Mild Traumatic Brain Injury

Vikas Ghai  1 , Shannon Fallen  1 , David Baxter  1 , Kelsey Scherler  1 , Taek-Kyun Kim  1 , Yong Zhou  1 , James S Meabon  2   3 , Aric F Logsdon  4   5 , William A Banks  4   5 , Abigail G Schindler  3   4 , David G Cook  4   5   6 , Elaine R Peskind  2   3 , Inyoul Lee  1 , Kai Wang  1
Affiliations

Alterations in Plasma microRNA and Protein Levels in War Veterans with Chronic Mild Traumatic Brain Injury

Vikas Ghai et al. J Neurotrauma. .

Abstract

Blast-related mild traumatic brain injury (mTBI) is considered the "signature" injury of the wars in Iraq and Afghanistan. Identifying biomarkers that could aid in diagnosis and assessment of chronic mTBI are urgently needed, as little progress has been made toward identifying blood-based biomarkers of repetitive mTBI in the chronic state. Addressing this knowledge gap is especially important in the population of military veterans who are receiving assessment and care often years after their last exposure. Circulating microRNAs (miRNAs), especially those encapsulated in extracellular vesicles (EVs), have gained interest as a source of biomarkers for neurological conditions. To identify biomarkers for chronic mTBI, we used next generation sequencing (NGS) to analyze miRNAs in plasma and plasma-derived EVs from 27 Iraq and Afghanistan war veterans with blast-related chronic mTBI, 11 deployed veteran non-TBI controls, and 31 civilian controls. We identified 32 miRNAs in plasma and 45 miRNAs in EVs that significantly changed in the chronic mTBI cohort compared with control groups. These miRNAs were predominantly associated with pathways involved in neuronal function, vascular remodeling, blood-brain barrier integrity, and neuroinflammation. In addition, the plasma proteome was analyzed and showed that the concentrations of C-reactive protein (CRP) and membrane metalloendopeptidase (MME) were elevated in chronic mTBI samples. These plasma miRNAs and proteins could potentially be used as biomarkers and provide insights into the molecular processes associated with the long-term health outcomes associated with blast-related chronic mTBI.

Keywords: EVs; biomarkers; mTBI; miRNAs; veterans.

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
Plasma and extracellular vesicle (EV) microRNAs (miRNAs) that show differential concentration changes between traumatic brain injured (TBI) and control study participants. (A–C) Volcano plots showing plasma miRNA log2 fold change (log2FC) versus p value between (A) chronic mild TBI (mTBI) subjects and community controls (CC), (B) chronic mTBI subjects and deployed veterans with no history of TBI (DC), and (C) DC and CC. (D) Overlap between miRNAs that show concentration differences in A–C. (E–F) Volcano plots showing plasma EV miRNA log2FC versus p value between (E) chronic mTBI subjects and CC, (F) chronic mTBI subjects and DC, and (G) DC and CC. (H) Overlap between miRNAs that show concentration differences in E–F. For volcano plots, gray unfilled circles represent miRNAs with a p value >0.05, and red circles represent miRNAs with a p value <0.05, and a log2FC > ±1 (fold change >2). Color image is available online.
FIG. 2.
FIG. 2.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) verification of extracellular vesicle (EV) microRNAs (miRNAs) that show differential concentration changes between traumatic brain injury (TBI) and control subjects. (A) Concentration levels of eight EV miRNAs selected for follow-up for qRT-PCR verification. (B) qRT-PCR verification of eight EV miRNAs in the same set of samples. (C) qRT-PCR verification of EV miRNAs in a separate cohort of 40 individuals (20 mild TBI [mTBI] and 20 community controls [CC]). Results are shown as log2 fold change of ΔCt values (max cycles – [Ct reference – Ct target]) for qRT-PCR or log2 fold change of reads per million (RPM) for small RNA sequencing (sRNAseq). miR-16-5p was used as a reference standard for qRT-PCR. Statistically significant results are designated by an asterisks (*p < 0.05, **p < 0.01). mTBI, dark gray bars, CC, white bars, DC (deployed controls), light gray bars.
FIG. 3.
FIG. 3.
Enriched biological pathways and processes associated with traumatic brain injury (TBI). Selected pathway analysis showing fold enrichment (based on Database for Annotation, Visualization and Integrated Discovery [DAVID] functional analysis) of Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways/processes associated with plasma (gray bars) and extracellular vesicle (EV) (black bars) microRNA (miRNA) targets. The enrichment factor is calculated as –log10(false discovery rate [FDR] corrected p value), with >1.3 enrichment factor considered statistically significant (FDR corrected p value <0.05 – dashed line).
FIG. 4.
FIG. 4.
Circulating plasma levels of C-reactive protein (CRP) and metalloendopeptidase (MME) are associated with traumatic brain injury (TBI). (A) Relative protein abundance for CRP levels measured by isobaric tag for relative and absolute quantitation (iTRAQ) for community controls (CC), deployed controls (DC), and mild TBI (mTBI) subject pools (average of two pools each). (B) log2 transformed CRP levels measured by enzyme-linked immunosorbent assay (ELISA) for individual CC, DC, and mTBI individuals. (C) log2 transformed MME levels (in normalized protein eXpression [NPX] units) measured by Proximity Extension Assay (PEA) for individual CC, DC, and mTBI.
FIG. 5.
FIG. 5.
Regulation of vascular endothelial growth factor (VEGF) and neurotrophin signaling pathways by microRNAs (miRNAs) associated with mild traumatic brain injury (mTBI). Network diagram showing miRNAs associated with TBI from plasma or extracellular vesicles (EVs) targeting mRNAs of the VEGF and neurotrophin signaling pathway, including downstream signaling effectors that regulate key biological processes associated with these pathways. miRNAs (yellow diamonds) are shown regulating plasma targets (blue edges and nodes), EV targets (red edges and nodes), or targets identified in both plasma and EV (purple edges and nodes). Color image is available online.
See this image and copyright information in PMC

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