Salivary microRNAs: Diagnostic markers of mild traumatic brain injury in contact-sport

Concussion is difficult to diagnose, particularly when symptoms are atypical or late in presenting. An accurate and timely initial assessment is crucial for clinical management. Cerebral spinal fluid (CSF) and blood markers of traumatic brain injury show promising results but their clinical applicability in concussion has significant limitations. In the study, we explored saliva as a new source of biomarkers of concussion. Saliva samples of concussed players were collected after 48–72 h from concussion and analyzed by high-throughput technologies. A discovery group of 10 concussed rugby professional and semiprofessional athletes and 10 non-concussed matched controls was used for the analysis of 92 inflammatory proteins by the Proseek-Multiplex-Inflammation technology. In addition, saliva samples of 6 concussed and 6 non-concussed athletes were used to screen 800 human microRNAs (miRNAs) by the Nanostring Technology. The results were then validated by RT-qPCR in an enlarged cohort (validation group) comprising 22 concussed athletes. Results showed, no significant variations of the 65 inflammatory proteins detected in saliva between groups but 5 microRNAs, miR-27b-3p (p = 0.016), let-7i-5p (p = 0.001), miR-142-3p (p = 0.008), miR-107 (p = 0.028), miR-135b-5p (p = 0.017) significantly upregulated in concussed athletes. Univariate ROC curve analysis showed that the differentially expressed miRNAs could be considered good classifiers of concussion. Further analyses showed significant correlation between these microRNAs and Reaction Time component of the ImPACT concussion assessment tool. In addition, biocomputation analysis predicted the involvement of these microRNAs in important biological processes that might be related to trauma, such as response to hypoxia, cell death, neurogenesis, axon repair and myelination. Ease of access and non-invasiveness of saliva samples make these biomarkers particularly suitable for concussion assessment

Seasonal Variation of Myostatin Gene Expression in Pectoralis Muscle of House Sparrows (Passer domesticus) Is Consistent with a Role in Regulating Thermogenic Capacity and Cold Tolerance

ABSTRACT Winter acclimatization in small birds overwintering in cold climates, including house sparrows (Passer domesticus), is associated with improved cold tolerance, elevated summit metabolic rates (M sum p maximum cold-induced metabolic rate), and increased pectoralis muscle mass compared to summer birds. Myostatin is a potent autocrine/paracrine inhibitor of skeletal muscle growth in mammals and birds and is a potential candidate for regulation of seasonal phenotypic flexibility in birds. As a first step toward examining such a role for myostatin in small birds, we measured summer and winter gene expression of myostatin and its potential metalloproteinase activators TLL-1 and TLL-2 in house sparrows from southeastern South Dakota. Gene expression of myostatin decreased significantly in winter, with summer values exceeding winter values by 1.52-fold. Moreover, gene expression of TLL-1 was also significantly reduced in winter, with summer values exceeding winter values by 1.55-fold. These data are consistent with the hypothesis that the winter increases in pectoralis muscle mass, M sum , and cold tolerance in house sparrows are mediated by reduced levels of myostatin and its activator TLL-1, and they suggest the possibility that myostatin may be a common mediator of phenotypic flexibility of muscle mass in birds

Mitochondria-Associated MicroRNAs in Rat Hippocampus Following Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI; however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI; however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12h following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI

Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy

Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data

Transcriptional profiling predicts behavioral performance in experimental mouse model of traumatic brain injury (TBI)

Abstract Traumatic brain injury (TBI) is a prevailing cause of disability and death. There are no curative therapies or robust predictors of long-term neurodegenerative disease. Treatment is challenging due to poor understanding of the cellular responses and mechanisms underlying TBI. We linked transcriptional profiling, cytokine profiling, and behavioral scoring in experimental TBI to discover new insights into immune-mediated mechanisms of TBI. Mice received ipsilateral moderate controlled cortical impaction. Luminex analyses showed upregulation of inflammatory cytokines in injured cortex, hippocampus, and spinal cord. Neurobehavioral evaluations were performed, then hippocampi isolated at 28d post-TBI for RNAseq. Pathway analyses of upregulated genes were functionally enriched for glial activation, proliferation, and neuronal development. Genes involved in synaptic transmission, glutamate receptor signaling, and neuronal migration were downregulated. Partial least squares (PLS) regression successfully modeled the relationship between differentially expressed genes and behavioral scores (R2=0.94). A core 10-gene signature predicted TBI severity and behavioral score. PLS discriminant analysis (PLSDA) classified TBI severity. VIP scores were used to define a 20-gene signature in TBI based on high VIP score, high fold change, and contribution to biological function. A PLSDA model using only these 20 genes was cross-validated by bootstrap method (AUROC=0.93), indicating that these genes may identify the critical TBI-altered processes. Altogether, our analyses revealed TBI-related biological pathways, potential therapeutic targets, and gene signatures as biomarkers for TBI severity and behavioral outcomes.</jats:p

Down-Regulation of miR-23a-3p Mediates Irradiation-Induced Neuronal Apoptosis

Radiation-induced central nervous system toxicity is a significant risk factor for patients receiving cancer radiotherapy. Surprisingly, the mechanisms responsible for the DNA damage-triggered neuronal cell death following irradiation have yet to be deciphered. Using primary cortical neuronal cultures in vitro, we demonstrated that X-ray exposure induces the mitochondrial pathway of intrinsic apoptosis and that miR-23a-3p plays a significant role in the regulation of this process. Primary cortical neurons exposed to irradiation show the activation of DNA-damage response pathways, including the sequential phosphorylation of ATM kinase, histone H2AX, and p53. This is followed by the p53-dependent up-regulation of the pro-apoptotic Bcl2 family molecules, including the BH3-only molecules PUMA, Noxa, and Bim, leading to mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c, which activates caspase-dependent apoptosis. miR-23a-3p, a negative regulator of specific pro-apoptotic Bcl-2 family molecules, is rapidly decreased after neuronal irradiation. By increasing the degradation of PUMA and Noxa mRNAs in the RNA-induced silencing complex (RISC), the administration of the miR-23a-3p mimic inhibits the irradiation-induced up-regulation of Noxa and Puma. These changes result in an attenuation of apoptotic processes such as MOMP, the release of cytochrome c and caspases activation, and a reduction in neuronal cell death. The neuroprotective effects of miR-23a-3p administration may not only involve the direct inhibition of pro-apoptotic Bcl-2 molecules downstream of p53 but also include the attenuation of secondary DNA damage upstream of p53. Importantly, we demonstrated that brain irradiation in vivo results in the down-regulation of miR-23a-3p and the elevation of pro-apoptotic Bcl2-family molecules PUMA, Noxa, and Bax, not only broadly in the cortex and hippocampus, except for Bax, which was up-regulated only in the hippocampus but also selectively in isolated neuronal populations from the irradiated brain. Overall, our data suggest that miR-23a-3p down-regulation contributes to irradiation-induced intrinsic pathways of neuronal apoptosis. These regulated pathways of neurodegeneration may be the target of effective neuroprotective strategies using miR-23a-3p mimics to block their development and increase neuronal survival after irradiation