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

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.

Association of Peripheral Serum MicroRNAs With Persistent Phantom Limb Pain in Individuals With Amputation.

OBJECTIVE: Individuals with major limb amputation(s) frequently experience phantom limb sensations, which are described as vivid impressions of either parts or entire missing limb(s). Despite the high incidence and prevalence of phantom limb pain, the underlying pathophysiology of phantom limb pain remains poorly understood. The objective of this study was to evaluate a possible role of microRNAs in the pathophysiology of phantom limb pain. DESIGN: Adults with acquired limb amputation and varying degrees of phantom limb pain consented to provide clinical data and blood samples. One hundred forty participants with single or multiple limb amputation(s) were enrolled. The Visual analog scale and neuropathic pain symptom inventory were administered to evaluate the pain. Serum samples were analyzed for microRNA expression and bioinformatic analysis was performed. RESULTS: Sixty-seven participants did not experience phantom limb pain, whereas 73 participants experienced varying severities of phantom limb pain measured on a pain scale. Linear regression analysis suggested that the time since amputation is inversely related to severity of the pain. A significantly increased expression of 16 microRNAs was observed in participants experiencing phantom limb pain. Bioinformatic analysis shows a possible role of these microRNAs in regulating genes expressed in peripheral neuropathy. CONCLUSIONS: This study provides the first evidence of association of microRNA in phantom limb pain.

Decreases in Dorsal Cervical Spinal Cord White Matter Tract Integrity Are Associated with Elevated Levels of Serum MicroRNA Biomarkers in NCAA Division I Collegiate Football Players.

This prospective, controlled, observational cohort study assessed the performance of a novel panel of serum microRNA (miRNA) biomarkers relative to findings on cervical spinal cord magnetic resonance imaging (MRI) in collegiate football players. There were 44 participants included in the study: 30 non-athlete control subjects and 14 male collegiate football athletes participating in a Division I Football Bowl Subdivision of the National Collegiate Athletic Association. Diffuse tensor MRI and blood samples were acquired within the week before the athletic season began and within the week after the last game of the season. All miRNAs were significantly higher in athletes regardless of their fractional anisotropy (FA) values (p < 0.001), even those considered to be in the "normal" range of FA for white and gray matter integrity in the cervical spinal cord. miRNA biomarkers were most significantly correlated with FA of the white matter (WM) tracts of the dorsal (posterior) spinal cord; particularly, the fasciculus gracilis, fasciculus cuneatus, lateral corticospinal tract, rubrospinal tract, lateral reticulospinal tract, spinal lemniscus, and spinothalamic and -reticular tracts. Areas under the curve for miRNA biomarkers predicting lower FA of WM dorsal (posterior) cervical spinal tracts, therefore lower white matter integrity (connectivity), were miR-505* = 0.75 (0.54-0.96), miR-30d = 0.74 (0.52-0.95), and miR-92a = 0.75 (0.53-0.98). Should these findings be replicated in a larger cohort of athletes, these markers could potentially serve as measures of neuroimaging abnormalities in athletes at risk for concussion and subconcussive injuries to the cervical spinal cord.

MicroRNAs in Basolateral Amygdala Associated with Stress and Fear Memories Regulate Rapid Eye Movement Sleep in Rats.

Stress-related sleep disturbances are distressing clinical symptoms in posttraumatic stress disorder patients. Intensely stressful events and their memories change rapid eye movement (REM) sleep in animal models. REM sleep varies with individual differences of stress resilience or vulnerability. The basolateral amygdala (BLA) is a primary mediator of the effects of stress and fear memories on sleep. However, the molecular mechanisms in BLA regulating the effects of fear conditioning, shock training (ST) and context re-exposure (CTX) on REM sleep are not well known. MicroRNAs (miRNAs) are small, non-coding RNAs and posttranscriptional gene regulators of diverse biological processes. The aim of this study is to investigate ST- and CTX-altered miRNAs in the BLA of resilience and vulnerable animals and on REM sleep regulation. MiRNAs expression profiles in BLA were generated following ST and CTX using the Taqman Low Density rodent microRNA array. The altered BLA miRNAs expression and REM sleep reduction observed in ST and CTX vulnerable animals. AntagomiR-221 microinjection into BLA for one of the upregulated miRNAs, miR-221 in BLA, attenuated the REM sleep reduction. This study suggests that miRNAs in the BLA may play a significant role in mediating the effects of stress and fear memories on REM sleep.

Exosomal MicroRNAs Released by Activated Astrocytes as Potential Neuroinflammatory Biomarkers.

Neuroinflammation is a hallmark of several neurodegenerative diseases and disorders, including traumatic brain injury (TBI). Neuroinflammation results in the activation of glial cells which exacerbates the neuroinflammatory process by secretion of pro-inflammatory cytokines and results in disruption of glial transmission networks. The glial cells, including astrocytes, play a critical role in the maintenance of homeostasis in the brain. Activated astrocytes release several factors as part of the inflammatory process including cytokines, proteins, and microRNAs (miRNAs). MiRNAs are noncoding RNA molecules involved in normal physiological processes and disease pathogenesis. MiRNAs have been implicated as important cell signaling molecules, and they are potential diagnostic biomarkers and therapeutic targets for various diseases, including neurological disorders. Exosomal miRNAs released by astrocytic response to neuroinflammation is not yet studied. In this study, primary human astrocytes were activated by IL-1ß stimulation and we examined astrocytic exosomal miRNA cargo released in a neuroinflammatory stress model. Results indicate that acute neuroinflammation and oxidative stress induced by IL-1ß generates the release of a specific subset of miRNAs via exosomes, which may have a potential role in regulating the inflammatory response. Additionally, these miRNAs may serve as potential biomarkers of neuroinflammation associated with neurological disorders and injuries.

Brain Perfusion Mediates the Relationship Between miRNA Levels and Postural Control.

Transcriptomics, regional cerebral blood flow (rCBF), and a virtual reality-based spatial motor task were integrated using mediation analysis in a novel demonstration of "imaging omics." Data collected in National Collegiate Athletic Association (NCAA) Division I football athletes cleared for play before in-season training showed significant relationships in 1) elevated levels of miR-30d and miR-92a to elevated putamen rCBF, 2) elevated putamen rCBF to compromised Balance scores, and 3) compromised Balance scores to elevated microRNA (miRNA) levels. rCBF acted as a consistent mediator variable (Sobel's test P < 0.05) between abnormal miRNA levels and compromised Balance scores. Given the involvement of these miRNAs in inflammation and immune function and that vascular perfusion is a component of the inflammatory response, these findings support a chronic inflammatory model in these athletes with 11 years of average football exposure. rCBF, a systems biology measure, was necessary for miRNA to affect behavior.

Elevations in MicroRNA Biomarkers in Serum Are Associated with Measures of Concussion, Neurocognitive Function, and Subconcussive Trauma over a Single National Collegiate Athletic Association Division I Season in Collegiate Football Players.

This prospective controlled observational cohort study assessed the performance of a novel panel of serum microRNA (miRNA) biomarkers on indicators of concussion, subconcussive impacts, and neurocognitive function in collegiate football players over the playing season. Male collegiate student football athletes participating in a Division I Football Bowl Subdivision of the National Collegiate Athletic Association (NCAA) were enrolled. There were a total of 53 participants included in the study, 30 non-athlete control subjects and 23 male collegiate student football athletes. Neurocognitive assessments and blood samples were taken within the week before the athletic season began and within the week after the last game of the season and measured for a panel of pre-selected miRNA biomarkers. All the athletes had elevated levels of circulating miRNAs at the beginning of the season compared with control subjects (p < 0.001). Athletes with the lowest standard assessment of concussion (SAC) scores at the beginning of the season had the highest levels of miRNAs. The area under the curve (AUC) for predicting pre-season SAC scores were miR-195 (0.90), miR-20a (0.89), miR-151-5p (0.86), miR-505* (0.85), miR-9-3p (0.77), and miR-362-3p (0.76). In athletes with declining neurocognitive function over the season, concentrations of miRNAs increased over same period. There were significant negative correlations with miR-505* (p = 0.011), miR-30d (p = 0.007), miR-92 (p = 0.033), and (p = 0.008). The miRNAs correlating with balance problems were miR-505* (p = 0.007), miR-30d (p = 0.028), and miR-151-5p (p = 0.023). Those correlating with poor reaction times were miR-20a (0.043), miR-505* (p = 0.049), miR-30d (p = 0.031), miR-92 (p = 0.015), and miR-151-5p (p = 0.044). Select miRNAs were associated with baseline concussion assessments at the beginning of the season and with neurocognitive changes from pre to post-season in collegiate football players. Should these findings be replicated in a larger cohort of athletes, these markers could potentially serve as measures of neurocognitive status in athletes at risk for concussion and subconcussive injuries.

Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts.

Background: Chikungunya virus (CHIKV) is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF) on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.

Hypothesis: Exosomal microRNAs as potential biomarkers for schizophrenia.

Schizophrenia is a serious mental disorder with lifelong morbidity and increased mortality. Currently, the diagnosis of the disorder is based on patient history and clinical examination, but it has a low inter-rater reliability and validity. Various biological variables, such as event related potentials, hormonal levels, brain ventricular volume and hippocampal size, have been put forth as objective markers to diagnose schizophrenia, but none with the desired sensitivity and specificity. It has been shown that microRNAs play a vital role in gene regulation in schizophrenia and have been proposed as possible biomarkers for the disease. When compared to the free microRNAs in the body fluids, exosomal microRNAs are more resistant to degradation and are easier to isolate. There are no studies reporting exosomal microRNAs as biomarkers for schizophrenia, but we hypothesize that exosomal microRNAs will be found to be potential biomarkers for diagnosis, prognosis assessment and medication response to patients with this disease.

Differential host gene responses from infection with neurovirulent and partially-neurovirulent strains of Venezuelan equine encephalitis virus.

BACKGROUND: Venezuelan equine encephalitis virus (VEEV) is an alphavirus in the family Togaviridae. VEEV causes a bi-phasic illness in mice where primary replication in lymphoid organs is followed by entry into the central nervous system (CNS). The CNS phase of infection is marked by encephalitis and large scale neuronal death ultimately resulting in death. Molecular determinants of VEEV neurovirulence are not well understood. In this study, host gene expression response to highly neurovirulent VEEV (V3000 strain) infection was compared with that of a partially neurovirulent VEEV (V3034 strain) to identify host factors associated with VEEV neurovirulence. METHODS: Whole genome microarrays were performed to identify the significantly modulated genes. Microarray observations were classified into three categories i.e., genes that were similarly modulated against both V3000 and V3034 infections, and genes that were uniquely modulated in infection with V3034 or V3000. Histologic sections of spleen and brain were evaluated by hematoxylin and eosin stains from all the mice. RESULTS: V3000 infection induced a greater degree of pathology in both the spleen and brain tissue of infected mice compared to V3034 infection. Genes commonly modulated in the spleens after V3000 or V3034 infection were associated with innate immune responses, inflammation and antigen presentation, however, V3000 induced a gene response profile that suggests a stronger inflammatory and apoptotic response compared to V3034. In the brain, both the strains of VEEV induced an innate immune response reflected by an upregulation of the genes involved in antigen presentation, interferon response, and inflammation. Similar to the spleen, V3000 was found to induce a stronger inflammatory response than V3034 in terms of induction of pro-inflammatory genes and associated pathways. Ccl2, Ccl5, Ccl6, and Ly6 were uniquely upregulated in V3000 infected mouse brains and correlated with the extensive inflammation observed in the brain. CONCLUSION: The common gene profile identified from V3000 and V3034 exposure can help in understanding a generalized host response to VEEV infection. Inflammatory genes that were uniquely identified in mouse brains with V3000 infection will help in better understanding the lethal neurovirulence of VEEV. Future studies are needed to explore the roles played by the genes identified in VEEV induced encephalitis.

Differential expression of microRNAs in the brains of mice subjected to increasing grade of mild traumatic brain injury.

OBJECTIVE: To investigate the effect of heterogeneity in mTBI on miRNA expression in mouse brain and to identify molecular pathways targeted by the modulated miRNAs. METHODS: A weight drop device was used to induce four increasing grades of mTBI. MiRNA expression was evaluated using TaqMan rodent miRNA arrays. Bioinformatics analysis was done using the DIANA miRPath tool and Ingenuity Pathway Analysis software. Histology of brain sections was evaluated using H&E staining. RESULTS: No histologic lesions were observed in the brains of injured mice; however, significant modulation in miRNA expression profile was observed. Global miRNA profiling indicated a trend of decrease in the number of modulated miRNAs from 24 hours to day 7 post-injury, except for the most severe grade of mTBI. Canonical pathways like calcium signalling, synaptic pathways and axon guidance pathway were the major targets of the modulated miRNAs. Network correlation analyses indicated an interaction between the modulated miRNAs and putative protein biomarkers of TBI. CONCLUSIONS: The data demonstrated that varying intensities of mTBI induced a differential miRNA expression profile in the brain post-injury. Pathways such as calcium and synaptic signalling were major targets of modulated miRNAs and may play a role in the pathophysiology of mTBI.

A Panel of Serum MiRNA Biomarkers for the Diagnosis of Severe to Mild Traumatic Brain Injury in Humans.

MicroRNAs (MiRNAs) are small endogenous RNA molecules and have emerged as novel serum diagnostic biomarkers for several diseases due to their stability and detection at minute quantities. In this study, we have identified a serum miRNA signature in human serum samples of mild to severe TBI, which can be used for diagnosis of mild and moderate TBI (MMTBI). Human serum samples of MMTBI, severe TBI (STBI), orthopedic injury and healthy controls were used and miRNA profiling was done using taqman real time PCR. The real time PCR data for the MMTBI, STBI and orthopedic injury was normalized to the control samples which showed upregulation of 39, 37 and 33 miRNAs in MMTBI, STBI and orthopedic injury groups respectively. TBI groups were compared to orthopedic injury group and an up-regulation of 18 and 20 miRNAs in MMTBI and STBI groups was observed. Among these, a signature of 10 miRNAs was found to be present in both MMTBI and STBI groups. These 10 miRNAs were validated in cerebrospinal fluid (CSF) from STBI and four miRNAs were found to be upregulated in CSF. In conclusion, we identified a subset of 10 unique miRNAs which can be used for diagnosis of MMTBI and STBI.

Identification of serum microRNA signatures for diagnosis of mild traumatic brain injury in a closed head injury model.

Wars in Iraq and Afghanistan have highlighted the problems of diagnosis and treatment of mild traumatic brain injury (mTBI). MTBI is a heterogeneous injury that may lead to the development of neurological and behavioral disorders. In the absence of specific diagnostic markers, mTBI is often unnoticed or misdiagnosed. In this study, mice were induced with increasing levels of mTBI and microRNA (miRNA) changes in the serum were determined. MTBI was induced by varying weight and fall height of the impactor rod resulting in four different severity grades of the mTBI. Injuries were characterized as mild by assessing with the neurobehavioral severity scale-revised (NSS-R) at day 1 post injury. Open field locomotion and acoustic startle response showed behavioral and sensory motor deficits in 3 of the 4 injury groups at day 1 post injury. All of the animals recovered after day 1 with no significant neurobehavioral alteration by day 30 post injury. Serum microRNA (miRNA) profiles clearly differentiated injured from uninjured animals. Overall, the number of miRNAs that were significantly modulated in injured animals over the sham controls increased with the severity of the injury. Thirteen miRNAs were found to identify mTBI regardless of its severity within the mild spectrum of injury. Bioinformatics analyses revealed that the more severe brain injuries were associated with a greater number of miRNAs involved in brain related functions. The evaluation of serum miRNA may help to identify the severity of brain injury and the risk of developing adverse effects after TBI.

Serum and amygdala microRNA signatures of posttraumatic stress: fear correlation and biomarker potential.

Exposure to acute traumatic stress can cause permanent changes in neurological circuitry and may lead to the development of an anxiety disorder known as posttraumatic stress disorder (PTSD). Current diagnosis of PTSD is based on clinical or behavioral symptom assessment, however, these are not definitive due to overlapping symptoms with other psychiatric disorders or mild traumatic brain injury (mTBI). No FDA approved diagnostic tests or biomarkers are currently available for diagnosis of PTSD. Recently, circulating miRNAs have emerged as novel biomarkers of many diseases. In this study, we have examined the altered expression of serum and amygdala miRNAs in an animal model of PTSD. Differentially expressed and statistically significant miRNAs in serum were validated for their presence in amygdala of corresponding animals. A panel of nine stress-responsive miRNAs viz., miR-142-5p, miR-19b, miR-1928, miR-223-3p, miR-322∗, miR-324, miR-421-3p and miR-463∗ and miR-674∗ were identified, and may have potential as biomarker(s) for PTSD. Further validations by bioinformatics and system biology approaches indicate that five miRNAs such as miR-142-5p, miR-19b, miR-1928, miR-223 and miR-421-3p may play a potential role in the regulation of genes associated with delayed and exaggerated fear. To the best of our knowledge, this is the first report demonstrating the plausibility of using circulating miRNAs as biomarkers of PTSD.

Artificial microRNAs can effectively inhibit replication of Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus is a member of the alphavirus family and genus togaviridae. VEEV is highly infectious in aerosol form and has been weaponized in the past making it a potential biothreat agent. At present, there are no FDA approved antiviral treatments or vaccines for VEEV. Artificial microRNAs are small molecules which are expressed through endogenous microRNA machinery by RNA polymerase II. These artificial microRNAs effectively inhibit gene expression and are non-toxic to the host cell. VEEV RNA dependent RNA polymerase (RdRp) is central to VEEV replication. Therefore, we hypothesize that targeted inhibition of VEEV RdRp using artificial microRNAs may efficiently inhibit VEEV replication. Five artificial microRNAs were tested in vitro in BHK cells. Three of these artificial miRNAs showed significant inhibition of VEEV replication. Further, these microRNAs were cloned into the expression vector in combination to see the synergistic effect on VEEV replication. Combination of more than one miRNA did not result in significant inhibition of virus replication. In conclusion, we have shown that RNAi through artificial microRNAs effectively inhibits VEEV replication and is significantly less toxic in comparison to siRNAs.

MicroRNA let-7i is a promising serum biomarker for blast-induced traumatic brain injury.

Blast-induced traumatic brain injury (TBI) is of significant concern in soldiers returning from the current conflicts in Iraq and Afghanistan. Incidents of TBI have increased significantly in the current conflicts compared to previous wars, and a majority of these injuries are caused by improvised explosive devices. Currently, no specific technique or biomarker is available for diagnosing TBI when no obvious clinical symptoms are present. Micro-RNAs are small RNA (~ 22nts) molecules that are expressed endogenously and play an important role in regulating gene expression. MicroRNAs have emerged as novel serum diagnostic biomarkers for various diseases. In this study, we studied the effect of blast overpressure injury on the microRNA signatures in the serum of rats. Rats were exposed to three serial 120-kPa blast overpressure exposures through a shockwave tube. Blood and cerebrospinal fluid were collected at various time points after injury, and microRNA modulation was analyzed using real-time PCR. Five microRNAs were significantly modulated in the serum samples of these animals at three time points post-injury. Further, we also found that the levels of microRNA let-7i are also elevated in cerebrospinal fluid post-blast wave exposure. The presence of microRNA in both serum and cerebrospinal fluid immediately after injury makes microRNA let-7i an ideal candidate for further studies of biomarkers in TBI.

Role of adhesion molecules and inflammation in Venezuelan equine encephalitis virus infected mouse brain.

BACKGROUND: Neuroinvasion of Venezuelan equine encephalitis virus (VEEV) and subsequent initiation of inflammation in the brain plays a crucial role in the outcome of VEEV infection in mice. Adhesion molecules expressed on microvascular endothelial cells in the brain have been implicated in the modulation of the blood brain barrier (BBB) and inflammation in brain but their role in VEEV pathogenesis is not very well understood. In this study, we evaluated the expression of extracellular matrix and adhesion molecules genes in the brain of VEEV infected mice. FINDINGS: Several cell to cell adhesion molecules and extracellular matrix protein genes such as ICAM-1, VCAM-1, CD44, Cadherins, integrins, MMPs and Timp1 were differentially regulated post-VEEV infection. ICAM-1 knock-out (IKO) mice infected with VEEV had markedly reduced inflammation in the brain and demonstrated a delay in the onset of clinical symptoms of disease. A differential regulation of inflammatory genes was observed in the IKO mice brain compared to their WT counterparts. CONCLUSIONS: These results improve our present understanding of VEEV induced inflammation in mouse brain.

Analysis of microRNAs induced by Venezuelan equine encephalitis virus infection in mouse brain.

MicroRNAs (miRNA) are small RNA (approximately 22nts) molecules that are expressed endogenously in cells and play an important role in regulating gene expression. Recent studies have shown that cellular miRNA plays a very important role in the pathogenesis of viral infection. Venezuelan equine encephalitis virus (VEEV) is an RNA virus and is a member of the genus Alphavirus in the family Togaviridae. VEEV is infectious in aerosol form and is a potential biothreat agent. In this study, we report for the first time that VEEV infection in mice brain causes modulation of miRNA expression. Pathway analyses showed that majority of these miRNAs are involved in the neuronal development and function. Target gene prediction of the modulated miRNAs correlates with our recently reported mRNA expression in VEEV infected mice brain.