Transcriptomic Signatures of Neuronally Derived Extracellular Vesicles Reveal the Presence of Olfactory Receptors in Clinical Samples from Traumatic Brain Injury Patients

Abstract

Traumatic brain injury (TBI) is defined as an injury to the brain by external forces which can lead to cellular damage and the disruption of normal central nervous system functions. The recently approved blood-based biomarkers GFAP and UCH-L1 can only detect injuries which are detectable on CT, and are not sensitive enough to diagnose milder injuries or concussion. Exosomes are small microvesicles which are released from the cell as a part of extracellular communication in normal as well as diseased states. The objective of this study was to identify the messenger RNA content of the exosomes released by injured neurons to identify new potential blood-based biomarkers for TBI. Human severe traumatic brain injury samples were used for this study. RNA was isolated from neuronal exosomes and total transcriptomic sequencing was performed. RNA sequencing data from neuronal exosomes isolated from serum showed mRNA transcripts of several neuronal genes. In particular, mRNAs of several olfactory receptor genes were present at elevated concentrations in the neuronal exosomes. Some of these genes were OR10A6, OR14A2, OR6F1, OR1B1, and OR1L1. RNA sequencing data from exosomes isolated from CSF showed a similar elevation of these olfactory receptors. We further validated the expression of these samples in serum samples of mild TBI patients, and a similar up-regulation of these olfactory receptors was observed. The data from these experiments suggest that damage to the neurons in the olfactory neuroepithelium as well as in the brain following a TBI may cause the release of mRNA from these receptors in the exosomes. Hence, olfactory receptors can be further explored as biomarkers for the diagnosis of TBI.

Keywords: biomarkers; exosomes; traumatic brain injury.

Figure 1

Detection of EVs from serum samples (scale bar = 100 nm). (A) Electron microscopy image of samples after total EV isolation (B) Electron microscopy image of samples after neuronal EV isolation.

Figure 2

Differentially expressed mRNA. (A) Fold change against statistical significance. Red spots represent up-regulated genes with significant differential expression; blue spots are down-regulated genes; and gray spots are genes with non-differential expression. Dotted line represents the threshold for level of significance (p < 0.05) (B) Heat map of gene expression data from neuronally derived EVs. Heat plot depicts 99 differentially expressed mRNA genes. Each column represents one sample; each row represents one gene.

Figure 3

Bubble plots of KEGG and GO mRNA gene enrichment analysis. (A) KEGG analysis showing olfactory transduction pathway as highly enriched. (B) GO analysis showing olfactory receptor activity as one of the most significant.

Figure 4

Expression of olfactory receptor genes in sTBI and non-TBI control samples. (A) OR1L1 expression in sTBI and non-TBI controls (p = 0.0359, N = 5). (B) OR4C13 expression in sTBI and non-TBI controls (p = 0.0053, N = 5).

Figure 5

Expression of olfactory receptor genes OR1L1 and OR4C13. (A) OR1L1 expression in mTBI and non-TBI control samples (p < 0.001, N = 5). (B) OR4C13 expression in mTBI and non-TBI control samples (p < 0.001, N = 37 (mTBI), n = 10 (controls)).