Decreased level of olfactory receptors in blood cells following traumatic brain injury and potential association with tauopathy

Abstract

Traumatic brain injury (TBI) is a leading cause of death and disability among children and young adults in the United States. In this study, we explored whether changes in the gene expression profile of peripheral blood mononuclear cells (PBMC) may provide a clinically assessable "window" into the brain, reflecting molecular alterations following TBI that might contribute to the onset and progression of TBI clinical complications. We identified three olfactory receptor (OR) TBI biomarkers that are aberrantly down-regulated in PBMC specimens from TBI subjects. Down-regulation of these OR biomarkers in PBMC was correlated with the severity of brain injury and TBI-specific symptoms. A two- biomarker panel comprised of OR11H1 and OR4M1 provided the best criterion for segregating the TBI and control cases with 90% accuracy, 83.3% sensitivity, and 100% specificity. We found that the OR biomarkers are ectopically expressed in multiple brain regions, including the entorhinal-hippocampus system known to play an important role in memory formation and consolidation. Activation of OR4M1 led to attenuation of abnormal tau phosphorylation, possibly through JNK signaling pathway. Our results suggested that addition of the two-OR biomarker model to current diagnostic criteria may lead to improved TBI detection for clinical trials, and decreased expression of OR TBI biomarkers might be associated with TBI-induced tauopathy. Future studies exploring the physiological relevance of OR TBI biomarkers in the normal brain and in the brain following TBI will provide a better understanding of the biological mechanisms underlying TBI and insights into novel therapeutic targets for TBI.

Fig. 1

Development of blood biological indices (biomarkers) capable of correctly segregating TBI and control cases. Gene expression profile analysis of PBMC specimens from TBI and 5 age-, gender-, and education-matched control cases using a microarray platform (Affymetrix) led to the identification of a panel of 102 candidate biomarker genes. A) The 108 differentially-regulated genes identified were subjected to unsupervised hierarchical clustering analysis using the UPGMA algorithm with cosine correlation as the similarity metric. Results are presented as a heat map (left panel) demonstrating that the content of the 108 biomarker panel is able to correctly segregate TBI from control cases. B) 7 ORs and one OR pseudogene that are down-regulated in PBMC of TBI cases.

Fig. 2

Down-regulation of OR genes in PBMC of TBI cases provide a sensitive and specific criterion for distinguishing TBI from control cases. mRNA contents for each of the 7 candidate OR biomarker genes in TBI and control cases were analyzed by microarray (A) or independent qPCR (B). Bar graphs represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by student t-test, TBI versus control. The efficacy of using biomarker contents from PBMC as a criterion to correctly segregate TBI and control cases was tested by unsupervised clustering analysis using the UPGMA algorithm with cosine correlation as the similarity metric. C) The accuracy, sensitivity, and specificity of OR11H1, OR4M1, OR52N5, or panels of ORs to distinguish TBI from control cases. D) A heat map graphically depicting the efficacy of using the two biomarker panel to distinguish TBI cases and control cases by unsupervised clustering analysis.

Fig. 3

Validation of OR TBI biomarkers in a veteran study cohort. The mRNA contents for (A) OR4M1, (B) OR11H1, and (C) OR52N5 in TBI and control cases from a veteran study cohort were analyzed by qPCR. Bar graphs represent mean + SEM. *p < 0.05 by student t-test, TBI versus control. D) The accuracy, sensitivity, and specificity of OR11H1, OR4M1, OR52N5, or panels of ORs to distinguish veteran TBI from control veteran cases. E) A heat map graphically depicting the efficacy of using the two biomarker panel to distinguish veteran TBI cases and control veteran cases by unsupervised clustering analysis.

Fig. 4

Olfactory receptor TBI biomarker contents in PBMCs are inversely correlated with TBI severity and long-term neuropsychological complications. Correlation analysis was used to test the potential association between PBMC OR11H1, OR4M1, and OR52N5 mRNA content and the severity of TBI injury or self-reported measures of TBI complications. OR11H1 (A), OR4M1 (B), and OR52N5 (C) content in PBMCs are inversely correlated with TBI severity. D) Correlation coefficients and p-values of associations between individual OR biomarker content in PBMCs and TBI severity, TBI-specific symptoms (a summation of 25 cognitive symptoms that are sensitive and specific to TBI) and self-assessment of mood.

Fig 5

Olfactory receptor TBI biomarkers expression in the brain. OR11H1, OR4M1, and OR52N5 mRNA expression by (A) RT-PCR and (B) relative expression level in postmortem superior temporal gyrus (BM22), hippocampal formation (HF), occipital cortex (BM17), and entorhinal cortex (BM36) specimens from neurologically normal cases. Expression pattern of (C) OR4M1, (D) OR11H1, and (E) OR52N5 in the brain by genome-wide microarray from the Allen Human Brain Atlas [30].

Fig. 6

Activation of OR4M1 resulted in reduced tau phosphorylation via JNK signaling pathway. Primary cortico-hippocampal neuron culture was transduced with lentiviral particles overexpressin OR4M1 or control lentiviral particles by spin infection (250 g × 90 min at 30°C). A) Transduced cells were treated by (1) hedione, (2) (+) citronellal, (3) acetophenone, (4) pyrazine, and (5) 2-isobutyl-3-methopyrazine (all at 10 μM, Sigma-Aldrich) for 10 min and cAMP assay was performed. Transduced neurons were also treated with acetophenone for 1 h, and multiplex luminex assay was performed using the Milliplex xMAP 8-plex multipathway signaling-phosphoprotein kit (Millipore) according to manufacturer's protocol: (B) Change of JNK phosphorylation on Thr183/Tyr185 and (C) ERK1/2 phosphorylation on Thr185/Tyr187. JNK and tau phosphorylation were also measured by (D) western blot analysis using antibodies recognizing phospho-JNK, phospho-ERK, or phospho-tau (PHF-1 epitope) and quantified (E, F) using GAPDH as a loading control. *p<0.05, **p < 0.01, ***p < 0.001 by two-tailed student t-test.

Fig. 7

Scheme of working hypothesis.