miRNAs as Potential Biomarkers for Traumatic Brain Injury: Pathway From Diagnosis to Neurorehabilitation.

BACKGROUND: Biomarkers that can advance precision neurorehabilitation of the traumatic brain injury (TBI) are needed. MicroRNAs (miRNAs) have biological properties that could make them well suited for playing key roles in differential diagnoses and prognoses and informing likelihood of responsiveness to specific treatments. OBJECTIVE: To review the evidence of miRNA alterations after TBI and evaluate the state of science relative to potential neurorehabilitation applications of TBI-specific miRNAs. METHODS: This scoping review includes 57 animal and human studies evaluating miRNAs after TBI. PubMed, Scopus, and Google Scholar search engines were used. RESULTS: Gold standard analytic steps for miRNA biomarker assessment are presented. Published studies evaluating the evidence for miRNAs as potential biomarkers for TBI diagnosis, severity, natural recovery, and treatment-induced outcomes were reviewed including statistical evaluation. Growing evidence for specific miRNAs, including miR21, as TBI biomarkers is presented. CONCLUSIONS: There is evidence of differential miRNA expression in TBI in both human and animal models; however, gaps need to be filled in terms of replication using rigorous, standardized methods to isolate a consistent set of miRNA changes. Longitudinal studies in TBI are needed to understand how miRNAs could be implemented as biomarkers in clinical practice.

Age-dependent Effects of 17ß-estradiol on the dynamics of estrogen receptor ß (ERß) protein-protein interactions in the ventral hippocampus.

Recent clinical evidence suggests that the neuroprotective and beneficial effects of hormone therapy may be limited by factors related to age and reproductive status. The patient's age and length of time without circulating ovarian hormones are likely to be key factors in the specific neurological outcomes of hormone therapy. However, the mechanisms underlying age-related changes in hormone efficacy have not been determined. We hypothesized that there are intrinsic changes in estrogen receptor ß (ERß) function that determine its ability to mediate the actions of 17ß-estradiol (E2) in brain regions such as the ventral hippocampus. In this study, we identified and quantified a subset of ERß protein interactions in the ventral hippocampus that were significantly altered by E2 replacement in young and aged animals, using two-dimensional differential gel electrophoresis coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. This study demonstrates quantitative changes in ERß protein-protein interactions with E2 replacement that are dependent upon age in the ventral hippocampus and how these changes could alter processes such as transcriptional regulation. Thus, our data provide evidence that changes in ERß protein interactions are a potential mechanism for age-related changes in E2 responsiveness in the brain after menopause.

Biomarker Discovery in Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating problem. Overall, the mortality rate associated with aSAH is 32% to 67%, which makes it the most lethal type of hemorrhagic stroke. Once the aneurysm has been treated, cerebral vasospasm is the leading cause of morbidity and mortality associated with aSAH. Thus, ability to effectively prevent or treat cerebral vasospasm could result in significantly improved survival and quality of life for aSAH patients. Unfortunately, partly because of poor understanding of the mechanisms of vasospasm, current diagnosis and treatment can be inconsistent and/or ineffective. Current treatment methods include primarily medical therapy and endovascular methods. Alone, or in combination, these measures can be of benefit in some patients. However, they are not uniformly efficacious and, on an individual basis, they can present significant risks. These risks include stroke, cardiovascular compromise, and death. More effective diagnosis and treatment strategies could significantly improve patient outcomes after aSAH. Unfortunately, clinically reliable biomarker for cerebral vasospasm has yet to be identified. Biomarker discovery may facilitate earlier diagnosis of vasospasm and improved monitoring of the response to treatment. It may help in stratifying patients into categories of risk to develop vasospasm, which could subsequently guide therapy. Indeed, biomarker research may suggest "vasospasm phenotypes" that can be used to guide the most effective type of therapy for that particular patient. The purpose of this manuscript is to review the current cerebral vasospasm biomarker literature. METHODS: An extensive PubMed literature search was performed. We identified over 100 English language articles with key words cerebral vasospasm and biomarkers. Some of these articles and related references were used as the basis of this review. We focused on related human studies performed within the past 10 years. RESULTS: In this review, we focus on recent work identifying molecular markers of cerebral vasospasm following aSAH and the current understanding of the utility of these markers. We highlight novel approaches such as the use of cellular microparticles for the evaluation of cerebral vasospasm. CONCLUSIONS: Although multiple molecules have been proposed, no single molecule has been shown to be a clinically reliable biomarker for cerebral vasospasm. This is not surprising based on the complex pathogenesis of cerebral vasospasm. Indeed, it is unlikely that a single biomarker will be clinically effective and reliable for predicting cerebral vasospasm. Instead, cerebral vasospasm may be best predicted by a panel of markers and the temporal progression of their relative levels after aSAH. Many such candidate molecules are reviewed herein and can be categorized as markers of cell damage, inflammation, changes in metabolism and vascular tone as well as microparticle-derived biomarkers. Among these, microparticle-derived biomarkers seem to be promising and lend themselves to further study. Biomarker discovery may facilitate earlier diagnosis of vasospasm and improved monitoring of the response to treatment. Ultimately, it may guide in the development of safer and more effective therapies for the most dreaded of aSAH complications.

Binge-pattern alcohol exposure during puberty induces sexually dimorphic changes in genes regulating the HPA axis.

Maternal alcohol consumption during critical periods of fetal brain development leads to devastating long-term consequences on adult reproductive physiology, cognitive function, and social behaviors. However, very little is known about the long-term consequences of alcohol consumption during puberty, which is perhaps an equally dynamic and critical period of brain development. Alcohol abuse during adulthood has been linked with an increase in clinically diagnosed anxiety disorders, yet the etiology and neurochemical mechanisms of alcohol-induced anxiety behavior is unknown. In this study, we determined the effects of binge ethanol exposure during puberty on two critical central regulators of stress and anxiety behavior: corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP). Our results showed that ethanol increased plasma corticosterone (CORT) levels in both sexes, yet binge-treated animals had significantly lower CORT levels than animals exposed to a single dose, suggesting that the hypothalamo-pituitary-adrenal (HPA) axis habituated to the repeated stressful stimuli of ethanol. Binge ethanol exposure also significantly increased CRH and AVP gene expression in the paraventricular nucleus of males, but not females. Overall, our results demonstrate that binge ethanol exposure during puberty changes the central expression of stress-related genes in a sex-specific manner, potentially leading to permanent dysregulation of the HPA axis and long-term behavioral consequences.

Microparticle derived proteins as potential biomarkers for cerebral vasospasm post subarachnoid hemorrhage. A preliminary study.

OBJECTIVE: Cerebral vasospasm (CV) and associated secondary brain injury are major contributors to death and disability after aneurysmal subarachnoid hemorrhage (aSAH). Microparticles (MP) are small vesicular micro-molecules released by red and white blood cells, platelets and endothelial cells that can change rapidly and specifically depending on the type of cellular insult. They may serve as useful tools to target a specific pool of proteins associated with the development of CV post aSAH. In these studies, our goal was to use targeted MP-derived protein isolation to find reliable biomarkers indicating increased risk for the development of CV. We hypothesize that there are specific early changes in MP-derived protein expression in CV patients. These proteins may be useful as biomarkers for CV and may help us to further understand the mechanism for the development of CV. Patients Adult male and female patients with angiographically confirmed aSAH and an external ventricular drain (EVD) placed for medical or surgical needs were included in this study. Patients were closely monitored for CV development. Cerebrospinal fluid (CSF) was collected daily until EVD was removed. METHODS: Microparticles were isolated using serial ultra centrifugation. Differential protein expression in CSF microparticles was analyzed by a mass spectroscopy based system using isotopically-tagged peptides to profile proteins and determine their relative concentrations in individual patient samples. These proteins were correlated with the patient's clinical data and used to identify candidates for biomarkers predictive of CV. RESULTS: Over 140 proteins were isolated from CSF microparticles. Proteomic and molecular pathways analysis revealed marked differential expression of proteins in patients with CV. We identified specific candidate proteins that could potentially serve as early biomarkers for CV. ApoE, ApoD, synaptic nuclear envelope protein 1, clusterin, α-1-acid glycoprotein, plasma protease C1 inhibitor, and prostaglandin H2 D isomerase were downregulated in patients who developed CV post aSAH. Haptoglobin, fibrinogen α and γ chain, synaptic nuclear envelope protein 2, and hemoglobin subunits α and ß were upregulated. Some of these proteins are associated with immune and metabolic processes and some have been specifically associated with cerebrovascular disease states. CONCLUSIONS: This is the first preliminary demonstration that there is differential protein expression in CSF microparticles from CV patients. Alone or in combination, these and other proteins may be useful as reliable biomarkers to guide in stratifying patients into categories of risk to develop CV post aSAH. These results will deepen our understanding of the mechanisms of cerebral vasospasm and potentially facilitate the development of safer and more effective therapies therapies for cerebral vasospasm.

Drug-Paired Contextual Stimuli Increase Dendritic Spine Dynamics in Select Nucleus Accumbens Neurons.

Repeated exposure to amphetamine leads to both associative conditioning and nonassociative sensitization. Here we assessed the contribution of neuronal ensembles in the nucleus accumbens (NAcc) to these behaviors. Animals exposed to amphetamine IP or in the ventral tegmental area (VTA) showed a sensitized locomotor response when challenged with amphetamine weeks later. Both exposure routes also increased ΔFosB levels in the NAcc. Further characterization of these ΔFosB+ neurons, however, revealed that amphetamine had no effect on dendritic spine density or size, indicating that these neurons do not undergo changes in dendritic spine morphology that accompany the expression of nonassociative sensitization. Additional experiments determined how neurons in the NAcc contribute to the expression of associative conditioning. A discrimination learning procedure was used to expose rats to IP or VTA amphetamine either Paired or Unpaired with an open field. As expected, compared with Controls, Paired rats administered IP amphetamine subsequently showed a conditioned locomotor response when challenged with saline in the open field, an effect accompanied by an increase in c-Fos+ neurons in the medial NAcc. Further characterization of these c-Fos+ cells revealed that Paired rats showed an increase in the density of dendritic spines and the frequency of medium-sized spines in the NAcc. In contrast, Paired rats previously exposed to VTA amphetamine showed neither conditioned locomotion nor conditioned c-Fos+ expression. Together, these results suggest a role for c-Fos+ neurons in the medial NAcc and rapid changes in the morphology of their dendritic spines in the expression of conditioning evoked by amphetamine-paired contextual stimuli.

Stimuli associated with the presence or absence of amphetamine regulate cytoskeletal signaling and behavior.

Drug-paired stimuli rapidly enlarge dendritic spines in the nucleus accumbens (NAcc). While increases in spine size and shape are supported by rearrangement of the actin cytoskeleton and facilitate the synaptic expression of AMPA-type glutamate receptors, it remains unclear whether drug-related stimuli can influence signaling pathways known to regulate these changes in spine morphology. These pathways were studied in rats trained on a discrimination learning paradigm using subcellular fractionation and protein immunoblotting to isolate proteins within dendritic spine compartments in the NAcc shell. An open field chamber was repeatedly associated with amphetamine in one group (Paired) and explicitly unpaired with amphetamine in another (Unpaired). Rats in a third group were exposed to the open field but never administered amphetamine (Control). When administered saline and returned to the open field one week later, Paired rats as expected displayed a conditioned locomotor response relative to rats in the other two groups. NAcc shell tissues were harvested immediately after this 30-minute test. Re-exposing Paired rats to the drug-paired excitatory context significantly decreased p-GluA2(S880), an effect consistent with reduced internalization of this subunit and increased spine proliferation in these rats. In contrast, re-exposing Unpaired rats to the drug-unpaired context, capable of inhibiting conditioned responding in these animals, significantly decreased levels of both actin binding protein Arp2/3 and p-cofilin, consistent with spine volatility, shrinkage, and inhibition of spine proliferation in these rats. These findings show that contextual stimuli previously associated with either the presence or absence of amphetamine differentially regulate cytoskeletal signaling pathways in the NAcc.

Parental binge alcohol abuse alters F1 generation hypothalamic gene expression in the absence of direct fetal alcohol exposure.

Adolescent binge alcohol exposure has long-lasting effects on the expression of hypothalamic genes that regulate the stress response, even in the absence of subsequent adult alcohol exposure. This suggests that alcohol can induce permanent gene expression changes, potentially through epigenetic modifications to specific genes. Epigenetic modifications can be transmitted to future generations therefore, and in these studies we investigated the effects of adolescent binge alcohol exposure on hypothalamic gene expression patterns in the F1 generation offspring. It has been well documented that maternal alcohol exposure during fetal development can have devastating neurological consequences. However, less is known about the consequences of maternal and/or paternal alcohol exposure outside of the gestational time frame. Here, we exposed adolescent male and female rats to a repeated binge EtOH exposure paradigm and then mated them in adulthood. Hypothalamic samples were taken from the offspring of these animals at postnatal day (PND) 7 and subjected to a genome-wide microarray analysis followed by qRT-PCR for selected genes. Importantly, the parents were not intoxicated at the time of mating and were not exposed to EtOH at any time during gestation therefore the offspring were never directly exposed to EtOH. Our results showed that the offspring of alcohol-exposed parents had significant differences compared to offspring from alcohol-naïve parents. Specifically, major differences were observed in the expression of genes that mediate neurogenesis and synaptic plasticity during neurodevelopment, genes important for directing chromatin remodeling, posttranslational modifications or transcription regulation, as well as genes involved in regulation of obesity and reproductive function. These data demonstrate that repeated binge alcohol exposure during pubertal development can potentially have detrimental effects on future offspring even in the absence of direct fetal alcohol exposure.

Long-term effects of peripubertal binge EtOH exposure on hippocampal microRNA expression in the rat.

Adolescent binge alcohol abuse induces long-term changes in gene expression, which impacts the physiological stress response and memory formation, two functions mediated in part by the ventral (VH) and dorsal (DH) hippocampus. microRNAs (miRs) are small RNAs that play an important role in gene regulation and are potential mediators of long-term changes in gene expression. Two genes important for regulating hippocampal functions include brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT1), which we identified as putative gene targets of miR-10a-5p, miR-26a, miR-103, miR-495. The purpose of this study was to quantify miR-10a-5p, miR-26a, miR-103, miR-495 expression levels in the dorsal and ventral hippocampus of male Wistar rats during normal pubertal development and then assess the effects of repeated binge-EtOH exposure. In addition, we measured the effects of binge EtOH-exposure on hippocampal Drosha and Dicer mRNA levels, as well as the putative miR target genes, BDNF and SIRT1. Overall, mid/peri-pubertal binge EtOH exposure altered the normal expression patterns of all miRs tested in an age- and brain region-dependent manner and this effect persisted for up to 30 days post-EtOH exposure. Moreover, our data revealed that mid/peri-pubertal binge EtOH exposure significantly affected miR biosynthetic processing enzymes, Drosha and Dicer. Finally, EtOH-induced significant changes in the expression of a subset of miRs, which correlated with changes in the expression of their predicted target genes. Taken together, these data demonstrate that EtOH exposure during pubertal development has long-term effects on miRNA expression in the rat hippocampus.

Effect of repetitive daily ethanol intoxication on adult rat brain: significant changes in phospholipase A2 enzyme levels in association with increased PARP-1 indicate neuroinflammatory pathway activation.

Collaborating on studies of subchronic daily intoxication in juvenile and adult rats, we examined whether the repetitive ethanol treatments at these two life stages altered levels of key neuroinflammation-associated proteins-aquaporin-4 (AQP4), certain phospholipase A2 (PLA2) enzymes, PARP-1 and caspase-3-in hippocampus (HC) and entorhinal cortex (EC). Significant changes in the proteins could implicate activation of specific neuroinflammatory signaling pathways in these rats as well as in severely binge-intoxicated adult animals that are reported to incur degeneration of vulnerable neurons in HC and EC. Male Wistar rats, ethanol-intoxicated (3 g/kg i.p.) once daily for 6 days over an 8-day interval beginning at 37 days old and repeated at age 68-75 days, were sacrificed 1 h after the day 75 dose (blood ethanol, 200- 230 mg/dl). Analysis of HC with an immunoblot technique showed that AQP4, Ca(+2)-dependent PLA2 (cPLA2 IVA), phosphorylated (activated) p-cPLA2, cleaved (89 kD) PARP (c-PARP), and caspase-3 levels were significantly elevated over controls, whereas Ca(+2)-independent PLA2 (iPLA2 VIA) was reduced ∼70%; however, cleaved caspase-3 was undetectable. In the EC, AQP4 was unchanged, but cPLA2 and p-cPLA2 were significantly increased while iPLA2 levels were diminished (∼40%) similar to HC, although just outside statistical significance (p = 0.06). In addition, EC levels of PARP-1 and c-PARP were significantly increased. The ethanol-induced activation of cPLA2 in association with reduced iPLA2 mirrors PLA2 changes in reports of neurotrauma and also of dietary omega-3 fatty acid depletion. Furthermore, the robust PARP-1 elevations accompanied by negligible caspase-3 activation indicate that repetitive ethanol intoxication may be potentiating non-apoptotic neurodegenerative processes such as parthanatos. Overall, the repetitive ethanol treatments appeared to instigate previously unappreciated neuroinflammatory pathways in vivo. The data provide insights into mechanisms of binge ethanol abuse that might suggest new therapeutic approaches to counter neurodegeneration and dementia.

17ß-Estradiol is required for the sexually dimorphic effects of repeated binge-pattern alcohol exposure on the HPA axis during adolescence.

Alcohol consumption during adolescence has long-term sexually dimorphic effects on anxiety behavior and mood disorders. We have previously shown that repeated binge-pattern alcohol exposure increased the expression of two critical central regulators of stress and anxiety, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), in adolescent male rats. By contrast, there was no effect of alcohol on these same genes in adolescent females. Therefore, we tested the hypothesis that 17ß-estradiol (E(2)), the predominant sex steroid hormone in females, prevents alcohol-induced changes in CRH and AVP gene expression in the paraventricular nucleus (PVN) of the hypothalamus. To test this hypothesis, postnatal day (PND) 26 females were ovariectomized and given E(2) replacement or cholesterol as a control. Next, they were given an alcohol exposure paradigm of 1) saline alone, 2) acute (single dose) or 3) a repeated binge-pattern. Our results showed that acute and repeated binge-pattern alcohol treatment increased plasma ACTH and CORT levels in both E(2)- and Ch-treated groups, however habituation to repeated binge-pattern alcohol exposure was evident only in E(2)-treated animals. Further, repeated binge-pattern alcohol exposure significantly decreased CRH and AVP mRNA in Ch-, but not E(2)-treated animals, which was consistent with our previous observations in gonad intact females. We further tested the effects of E(2) and alcohol treatment on the activity of the wild type CRH promoter in a PVN-derived neuronal cell line. Alcohol increased CRH promoter activity in these cells and concomitant treatment with E(2) completely abolished the effect. Together our data suggest that E(2) regulates the reactivity of the HPA axis to a repeated stressor through modulation of the habituation response and further serves to maintain normal steady state mRNA levels of CRH and AVP in the PVN in response to a repeated alcohol stressor.

Alcohol dysregulates corticotropin-releasing-hormone (CRH) promoter activity by interfering with the negative glucocorticoid response element (nGRE).

EtOH exposure in male rats increases corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN), a brain region responsible for coordinating stress and anxiety responses. In this study we identified the molecular mechanisms involved in mediating these effects by examining the direct effects of EtOH on CRH promoter activity in a neuronal cell line derived from the PVN (IVB). In addition, we investigated the potential interactions of EtOH and glucocorticoids on the CRH promoter by concomitantly treating cells with EtOH and the glucocorticoid receptor (GR) antagonist RU486, and by sequentially deleting GR binding sites within glucocorticoid response element (GRE) on the CRH promoter. Cells were transiently transfected with a firefly luciferase reporter construct containing 2.5 kb of the rat wild type (WT) or mutated CRH promoter. Our results showed that EtOH treatment induced a biphasic response in CRH promoter activity. EtOH exposure for 0.5 h significantly decreased promoter activity compared to vehicle treated controls, whereas promoter activity was significantly increased after 2.0 h of EtOH exposure. Treatment with RU486, or deletion of the GR binding sites 1 and 2 within the GRE, abolished the EtOH-induced increase in the promoter activity, however did not affect EtOH-induced decrease in CRH promoter activity at an earlier time point. Overall, our data suggest that alcohol exposure directly regulates CRH promoter activity by interfering with the normal feedback mechanisms of glucocorticoids mediated by GR signaling at the GRE site of the CRH promoter.

Binge-pattern alcohol exposure during puberty induces long-term changes in HPA axis reactivity.

Adolescence is a dynamic and important period of brain development however, little is known about the long-term neurobiological consequences of alcohol consumption during puberty. Our previous studies showed that binge-pattern ethanol (EtOH) treatment during pubertal development negatively dysregulated the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis, as manifested by alterations in corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP), and corticosterone (CORT) during this time period. Thus, the primary goal of this study was to determine whether these observed changes in important central regulators of the stress response were permanent or transient. In this study, juvenile male Wistar rats were treated with a binge-pattern EtOH treatment paradigm or saline alone for 8 days. The animals were left undisturbed until adulthood when they received a second round of treatments consisting of saline alone, a single dose of EtOH, or a second binge-pattern treatment paradigm. The results showed that pubertal binge-pattern EtOH exposure induced striking long-lasting alterations of many HPA axis parameters. Overall, our data provide strong evidence that binge-pattern EtOH exposure during pubertal maturation has long-term detrimental effects for the healthy development of the HPA axis.