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Each cerebral hemisphere is functionally connected to the contralateral side of the body through the decussating neural tracts. The crossed neural pathways set a basis for contralateral effects of brain injury such hemiparesis and hemiplegia as it has been already noted by Hippocrates. Recent studies demonstrated that, in addition to neural mechanisms, the contralateral effects of brain lesions are mediated through the humoral pathway by neurohormones that produce either the left or right side-specific effects. The side-specific humoral signaling defines whether the left or right limbs are affected after a unilateral brain injury. The hormonal signals are released by the pituitary gland and may operate through their receptors that are lateralized in the spinal cord and involved in the side-specific control of symmetric neurocircuits innervating the left and right limbs. Identification of features and a proportion of neurological deficits transmitted by neurohormonal signals vs. those mediated by neural pathways is essential for better understanding of mechanisms of brain trauma and stroke and development of new therapies. In a biological context, the left–right side-specific neuroendocrine signaling may be fundamental for the control of the left- and right-sided processes in bilaterally symmetric animals.

Disruption of neural tracts descending from the brain to the spinal cord after brain trauma and stroke causes postural and sensorimotor deficits. We previously showed that unilateral lesion to the sensorimotor cortex in rats with completely transected thoracic spinal cord produced asymmetry in hindlimb posture and withdrawal reflexes. Supraspinal signals to hindlimb muscles may be transmitted through the paravertebral chain of sympathetic ganglia that remain intact after the transection. We here demonstrated that prior transection of the spinal cord at the cervical level that was rostrally to segments with preganglionic sympathetic neurons, did not abolish formation of asymmetry in hindlimb posture and musculo-articular resistance to stretch after unilateral brain injury. Thus not the sympathetic system but humoral signals may mediate the effects of brain injury on the lumbar spinal circuits. The asymmetric responses in rats with transected spinal cords were eliminated by bilateral lumbar dorsal rhizotomy after the left-side brain injury, but resistant to deafferentation after the right-side brain lesion. Two mechanisms, one dependent on and one independent of afferent input may account for asymmetric hindlimb motor responses. Resistance to deafferentation may be due to sustained stretch- and effort-unrelated muscle contractions that is often observed in patients with central lesions. Left-right asymmetry is unusual feature of these mechanisms that both are activated by humoral signals.Graphical Abstract

Alcohol Use Disorder (AUD) is a major contributor to global mortality and morbidity. Postmortem human brain tissue enables the investigation of molecular mechanisms of AUD in the neurocircuitry of addiction. We aimed to identify differentially expressed (DE) genes in the ventral and dorsal striatum between individuals with AUD and controls, and to integrate the results with findings from genome- and epigenome-wide association studies (GWAS/EWAS) to identify functionally relevant molecular mechanisms of AUD. DNA-methylation and gene expression (RNA-seq) data was generated from postmortem brain samples of 48 individuals with AUD and 51 controls from the ventral striatum (VS) and the dorsal striatal regions caudate nucleus (CN) and putamen (PUT). We identified DE genes using DESeq2, performed gene-set enrichment analysis (GSEA), and tested enrichment of DE genes in results of GWASs using MAGMA. Weighted correlation network analysis (WGCNA) was performed for DNA-methylation and gene expression data and gene overlap was tested. Differential gene expression was observed in the dorsal (FDRARHGEF15 gene was upregulated in all three brain regions. GSEA in CN and VS pointed towards cell-structure associated GO-terms and in PUT towards immune pathways. The WGCNA modules most strongly associated with AUD showed strong enrichment for immune response and inflammation pathways. Our integrated analysis of multi-omics data sets provides further evidence for the importance of immune-and inflammation-related processes in AUD.

In spite of its apparent symmetry, the spinal cord is asymmetric in its reflexes and gene expression patterns including leftward expression bias of the opioid and glutamate genes. To examine whether this is a general phenomenon for neurotransmitter and neurohormonal genes, we here characterized expression and co-expression (transcriptionally coordinated) patterns of genes of the renin-angiotensin system (RAS) that is involved in neuroprotection and pathological neuroplasticity in the left and right lumbar spinal cord. We also tested whether the RAS expression patterns were affected by unilateral brain injury (UBI) that rewired lumbar spinal neurocircuits. The left and right halves of the lumbar spinal cord were analysed in intact rats, and rats with left- or right-sided unilateral cortical injury, and left- or right-sided sham surgery. The findings were (i) lateralized expression of the RAS genes Ace, Agtr2 and Ren with higher levels on the left side; (ii) the asymmetry in coordination of the RAS gene expression that was stronger on the right side; (iii) the decay in coordination of co-expression of the RAS and neuroplasticity-related genes induced by the right-side but not left-side sham surgery and UBI; and (iv) the UBI-induced shift to negative regulatory interactions between RAS and neuroplasticity-related genes on the contralesional spinal side. Thus, the RAS genes may be a part of lateralized gene co-expression networks and have a role in a side-specific regulation of spinal neurocircuits.

Traumatic brain injury and stroke result in hemiplegia, hemiparesis, and asymmetry in posture. The effects are mostly contralateral; however, ipsilesional deficits may also develop. We here examined whether ablation brain injury and controlled cortical impact (CCI), a rat model of clinical focal traumatic brain injury, both centered over the left or right sensorimotor cortex, induced hindlimb postural asymmetry (HL-PA) with contralesional or ipsilesional limb flexion. The contralesional hindlimb was flexed after left or right side ablation injury. In contrast, both the left and right CCI unexpectedly produced HL-PA with flexion on left side. The flexion persisted after complete spinal cord transection suggesting that CCI triggered neuroplastic processes in lumbar neural circuits enabling asymmetric muscle contraction. Left limb flexion was exhibited under pentobarbital anesthesia. However, under ketamine anesthesia, the body of the left and right CCI rats bent laterally in the coronal plane to the ipsilesional side suggesting that the left and right injury engaged mirror-symmetrical motor pathways. Thus, the effects of the left and right CCI on HL-PA were not mirror-symmetrical in contrast to those of the ablation brain injury, and to the left and right CCI produced body bending. Ipsilateral effects of the left CCI on HL-PA may be mediated by a lateralized motor pathway that is not affected by the left ablation injury. Alternatively, the left-side-specific neurohormonal mechanism that signals from injured brain to spinal cord may be activated by both the left and right CCI but not by ablation injury. Supplementary Information The online version contains supplementary material available at 10.1007/s00221-021-06118-4.

Effects of environmental factors may be transmitted to the following generation, and cause neuropsychiatric disorders including depression, anxiety, and posttraumatic stress disorder in the offspring. Enhanced synaptic plasticity induced by environmental enrichment may be also transmitted. We here test the hypothesis that the effects of brain injury in pregnant animals may produce neurological deficits in the offspring. Unilateral brain injury (UBI) by ablation of the hindlimb sensorimotor cortex in pregnant rats resulted in the development of hindlimb postural asymmetry (HL-PA), and impairment of balance and coordination in beam walking test in the offspring. The offspring of rats with the left UBI exhibited HL-PA before and after spinal cord transection with the contralesional (i.e., right) hindlimb flexion. The right UBI caused the offspring to develop HL-PA that however was cryptic and not-lateralized; it was evident only after spinalization, and was characterized by similar occurrence of the ipsi- and contralesional hindlimb flexion. The HL-PA persisted after spinalization suggesting that the asymmetry was encoded in lumbar spinal neurocircuits that control hindlimb muscles. Balance and coordination were affected by the right UBI but not the left UBI. Thus, the effects of a unilateral brain lesion in pregnant animals may be intergenerationally transmitted, and this process may depend on the side of brain injury. The results suggest the existence of left-right side-specific mechanisms that mediate transmission of the lateralized effects of brain trauma from mother to fetus.

Alcohol misuse is a major public health problem originating from genetic and environmental risk factors. Alterations in the brain epigenome may orchestrate changes in gene expression that lead to alcohol misuse and dependence. Through epigenome-wide association analysis of DNA methylation from human brain tissues, we identified a differentially methylated region, DMR-DLGAP2, associated with alcohol dependence. Methylation within DMR-DLGAP2 was found to be genotype-dependent, allele-specific and associated with reward processing in brain. Methylation at the DMR-DLGAP2 regulated expression of DLGAP2 in vitro, and Dlgap2-deficient mice showed reduced alcohol consumption compared with wild-type controls. These results suggest that DLGAP2 may be an interface for genetic and epigenetic factors controlling alcohol use and dependence.

Traumatic brain injury (TBI) is an established risk factor for neurodegenerative disorders and dementias. Epigenetic modifications, such as DNA methylation, may alter the expression of genes without altering the DNA sequence in response to environmental factors. We hypothesized that DNA methylation changes may occur in the injured human brain and be implicated in the neurodegenerative aftermath of TBI. The DNA methylation status of genes related to neurodegeneration; for example, amyloid beta precursor protein (APP), microtubule associated protein tau (MAPT), neurofilament heavy (NEFH), neurofilament medium (NEFM), and neurofilament light (NEFL), was analyzed in fresh, surgically resected human brain tissue from 17 severe TBI patients and compared with brain biopsy samples from 19 patients with idiopathic normal pressure hydrocephalus (iNPH). We also performed an epigenome-wide association study (EWAS) comparing TBI patients with iNPH controls. Thirty-eight CpG sites in the APP, MAPT, NEFH, and NEFL genes were differentially methylated by TBI. Among the top 20 differentially methylated CpG sites, 11 were in the APP gene. In addition, the EWAS evaluating 828,888 CpG sites revealed 308 differentially methylated CpG sites in genes related to cellular/anatomical structure development, cell differentiation, and anatomical morphogenesis. These preliminary findings provide the first evidence of an altered DNA methylome in the injured human brain, and may have implications for the neurodegenerative disorders associated with TBI.

Objective: Psychosocial stress is a key risk factor for substance abuse among adolescents. Recently, epigenetic processes such as DNA methylation have emerged as potential mechanisms that could mediate this relationship. The authors conducted a genome-wide methylation analysis to investigate whether differentially methylated regions are associated with psychosocial stress in an adolescent population. Methods: A methylome-wide analysis of differentially methylated regions was used to examine a sample of 1,287 14-year-old adolescents (50.7% of them female) from the European IMAGEN study. The Illumina 450k array was used to assess DNA methylation, pyrosequencing was used for technical replication, and linear regression analyses were used to identify associations with psychosocial stress and substance use (alcohol and tobacco). Findings were replicated by pyrosequencing a test sample of 413 participants from the IMAGEN study. Results: Hypermethylation in the sterile alpha motif/pointed domain containing the ETS transcription factor (SPDEF) gene locus was associated with a greater number of stressful life events in an allele-dependent way. Among individuals with the minor G-allele, SPDEF methylation moderated the association between psychosocial stress and substance abuse. SPDEF methylation interacted with lifetime stress in gray matter volume in the right cuneus, which in turn was associated with the frequency of alcohol and tobacco use. SPDEF was involved in the regulation of trans-genes linked to substance use. Conclusions: Taken together, the study findings describe a novel epigenetic mechanism that helps explain how psychosocial stress exposure influences adolescent substance abuse.

The aim was to assess the activation and association of the NF-κB system across synovial membrane (SM) and articular cartilage (AC) in patients with knee osteoarthritis (OA) and ascertain its potential effects on catabolic mediator expression in advanced OA. SM and AC were obtained from 40 OA patients undergoing total knee arthroplasty and from 19 postmortem control subjects. NF-κB subunit RelA in nuclear and cytosolic fractions and NF-κB1–DNA binding in nuclear extracts was assessed by ELISA, whereas NFKB1, RELA, IL-8, IL-6, and MMP3 gene expression were analyzed by reverse transcriptase–quantitative PCR in tissues. We observed higher SM nuclear RelA protein levels and upregulated NF-κB1–DNA binding in OA patients compared with postmortem controls. However, in AC, lower nuclear RelA levels were observed compared with cytosolic extracts in patients. Nuclear RelA levels correlated positively with NF-κB1–DNA binding in SM and AC in patients. SM RELA and MMP3 mRNA levels were upregulated, whereas IL-8 and IL-6 as well as AC RELA were downregulated in patients compared with controls. In SM, nuclear RelA levels correlated positively with MMP3 gene expression in patients. A negative correlation was observed between SM nuclear RelA levels and AC NF-κB1–DNA binding, and SM nuclear NF-κB1-DNA binding correlated negatively with AC MMP3 and NFKB1 mRNA levels in patients. These findings highlight NF-κB–triggered cross-talk and feedback mechanisms between SM and AC in OA. Further, our findings strongly support a role for an activated NF-κB system in the transcriptional mechanism of inflammatory processes, especially in SM of patients with advanced OA.

Molecular mechanisms that define patterns of neuropeptide expression are essential for the formation and rewiring of neural circuits. The prodynorphin gene (PDYN) gives rise to dynorphin opioid peptides mediating depression and substance dependence. We here demonstrated that PDYN is expressed in neurons in human dorsolateral prefrontal cortex (dlPFC), and identified neuronal differentially methylated region in PDYN locus framed by CCCTC-binding factor binding sites. A short, nucleosome size human-specific promoter CpG island (CGI), a core of this region may serve as a regulatory module, which is hypomethylated in neurons, enriched in 5-hydroxymethylcytosine, and targeted by USF2, a methylation-sensitive E-box transcription factor (TF). USF2 activates PDYN transcription in model systems, and binds to nonmethylated CGI in dlPFC. USF2 and PDYN expression is correlated, and USF2 and PDYN proteins are co-localized in dlPFC. Segregation of activatory TF and repressive CGI methylation may ensure contrasting PDYN expression in neurons and glia in human brain.

Spinal cord as a connection between brain and peripheral nervous system is an essential material for studying neural transmission, especially in pain-related research. This study was the first to investigate pain-related neuropeptide distribution in rat spinal cord using a matrix-assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI TOF MS) approach. The imaging workflow was evaluated and showed that MALDI TOF MS provides efficient resolution and robustness for neuropeptide imaging in rat spinal cord tissue. The imaging result showed that in naive rat spinal cord the molecular distribution of haeme, phosphatidylcholine, substance P and thymosin beta 4 were well in line with histological features. Three groups of pain-related neuropeptides, which are cleaved from prodynorphin, proenkephalin and protachykinin-1 proteins were detected. All these neuropeptides were found predominantly localized in the dorsal spinal cord and each group had unique distribution pattern. This study set the stage for future MALDI TOF MS application to elucidate signalling mechanism of pain-related diseases in small animal models.

A paradigm in neurology is that brain injury-induced motor deficits (e.g. hemiparesis and hemiplegia) arise due to aberrant activity of descending neural pathways. We discovered that a unilateral injury of the hindlimb sensorimotor cortex of rats with completely transected thoracic spinal cord produces hindlimb postural asymmetry with contralateral flexion, and asymmetric changes in nociceptive hindlimb withdrawal reflexes and gene expression patterns in lumbar spinal cord. The injury-induced postural effects were abolished by prior hypophysectomy and were mimicked by transfusion of serum from animals with unilateral brain injury. Antagonists of the opioid and vasopressin receptors blocked formation of hindlimb postural asymmetry suggesting that these neurohormones mediate effects of brain injury on lateralized motor responses. Our data indicate that descending neural control of spinal circuits is complemented by a previously unknown humoral signaling from injured brain to the contra- and ipsilesional hindlimbs, and suggest the existence of a body side-specific neuroendocrine regulation in bilaterally symmetric animals.

DNA methylation, which is modulated by both genetic factors and environmental exposures, may offer a unique opportunity to discover novel biomarkers of disease-related brain phenotypes, even when measured in other tissues than brain, such as blood. A few studies of small sample sizes have revealed associations between blood DNA methylation and neuropsychopathology, however, large-scale epigenome-wide association studies (EWAS) are needed to investigate the utility of DNA methylation profiling as a peripheral marker for the brain. Here, in an analysis of eleven international cohorts, totalling 3337 individuals, we report epigenome-wide meta-analyses of blood DNA methylation with volumes of the hippocampus, thalamus and nucleus accumbens (NAcc)—three subcortical regions selected for their associations with disease and heritability and volumetric variability. Analyses of individual CpGs revealed genome-wide significant associations with hippocampal volume at two loci. No significant associations were found for analyses of thalamus and nucleus accumbens volumes. Cluster-based analyses revealed additional differentially methylated regions (DMRs) associated with hippocampal volume. DNA methylation at these loci affected expression of proximal genes involved in learning and memory, stem cell maintenance and differentiation, fatty acid metabolism and type-2 diabetes. These DNA methylation marks, their interaction with genetic variants and their impact on gene expression offer new insights into the relationship between epigenetic variation and brain structure and may provide the basis for biomarker discovery in neurodegeneration and neuropsychiatric conditions.

Background: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the kappa-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain.Methods: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence activated nuclei sorting (FANS) from postmortem human striatal tissue. lmmunofluorescence staining and con focal microscopy was performed for human caudate nucleus.Results: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ASP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). Delta SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging.Conclusions and general significance: High levels of alternatively spliced Delta SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum. (C) 2016 Elsevier B.V. All rights reserved.

Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region-and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections. (C) 2016 The Authors. Published by Elsevier Inc.

Nociceptive discharges caused by the unilateral tissue damage are processed in the spinal cord by both ipsi- and contralateral neuronal circuits. The mechanisms of the neurotransmitter control of this bilateral excitation spread is poorly understood. Spinally administered opiates are known to suppress nociceptive transmission and nociceptive withdrawal reflexes. Here we investigated whether three major types of opioid receptors are involved in the bilateral control of the spinal nociceptive sensorimotor processing. Effects of the µ-, δ- and κ-opioid receptor agonists on the ipsi- and contralateral nociceptive reflexes were studied by recording slow ventral root potentials in an isolated spinal cord preparation of the new-born rat. Absolute levels of expression of the opioid genes were analyzed by the droplet digital PCR. Ipsi- and contralateral slow ventral root potentials were most strongly suppressed by the µ-opioid receptor agonist DAMGO, by 63% and 85%, followed by the κ-opioid receptor agonist U-50488H, by 44% and 73%, and δ-opioid receptor agonist leucine-enkephalin, by 27% and 49%, respectively. All these agonists suppressed stronger contra- than ipsilateral responses. Naloxone prevented effects of the agonists indicating that they act through opioid receptors, which, as we show, are expressed in the neonatal spinal cord at the levels similar to those in adults. Thus, opioid receptor agonists suppress the segmental nociceptive reflexes. Stronger contralateral effects suggest that the endogenous opioid system regulates sensorimotor processing in the spinal commissural pathways. These effects of opioids may be relevant for treatment of symmetric clinical pain symptoms caused by unilateral tissue injury.

DNA methylation, which is modulated by both genetic factors and environmental exposures, may offer a unique opportunity to discover novel biomarkers of disease-related brain phenotypes, even when measured in other tissues than brain, such as blood. A few studies of small sample sizes have revealed associations between blood DNA methylation and neuropsychopathology, however, large-scale epigenome-wide association studies (EWAS) are needed to investigate the utility of DNA methylation profiling as a peripheral marker for the brain. Here, in an analysis of eleven international cohorts, totalling 3,337 individuals, we report epigenome-wide meta-analyses of blood DNA methylation with volumes of the hippocampus, thalamus and nucleus accumbens (NAcc) –three subcortical regions selected for their associations with disease and heritability and volumetric variability. Analyses of individual CpGs revealed genome-wide significant associations with hippocampal volume at two loci. No significant associations were found for analyses of thalamus and nucleus accumbens volumes. CpG sites associated with hippocampus volume were significantly enriched within cancer-related genes and within regulatory elements containing the transcriptionally repressive histone H3K27 tri-methylation mark that is vital for stem cell fate specification. Cluster-based analyses revealed additional differentially methylated regions (DMRs) associated with hippocampal volume. DNA methylation at these loci affected expression of proximal genes involved in in learning and memory, stem cell maintenance and differentiation, fatty acid metabolism and type-2 diabetes. These DNA methylation marks, their interaction with genetic variants and their impact on gene expression offer new insights into the relationship between epigenetic variation and brain structure and may provide the basis for biomarker discovery in neurodegeneration and neuropsychiatric conditions.

Background Sex-related differences in the susceptibility, progression, and treatment response in alcohol-dependent subjects have been repeatedly reported. In this study, we aimed to investigate the associations of the sex-related hormone/protein levels with alcohol dependence (AD) and alcohol craving in male and female subjects. Methods Plasma sex-related hormones (estradiol, estrone, total testosterone, progesterone, follicle stimulated hormone [FSH], luteinizing hormone), and sex hormone binding globulin were measured by mass spectrometry or automated immunoassays from 44 recently-abstained subjects (29 males and 15 females; mean age = 45.9 ± 15.6) meeting DSM-IV-TR criteria for AD and 44 age-, sex- and race-matched non-AD controls. Conditional logistic regression was conducted to examine the association of sex-related hormone and protein levels with AD risk, accounting for matching variables. Their associations with alcohol craving scales (Penn Alcohol Craving Scale and Inventory of Drug-Taking Situations) were assessed in AD subjects. Results Plasma FSH level was significantly higher in AD males (10.3 ± 9.8 IU/L) than control males (8.0 ± 15.9 IU/L; p = 0.005, pcorrected = 0.035). We also found a significant inverse correlation of FSH level with propensity to drink in negative emotional situations (Spearman’s rho=-.540; p = 0.021) and positive correlations between progesterone level and craving intensity (Spearman’s rho=.464; p = 0.020) and between total testosterone level and propensity to drink under temptations (adjusted for no-drinking days; β=6.496; p = 0.041) in AD males. Conclusions These results suggest that FSH, progesterone, and testosterone levels may be associated with AD and alcohol craving in AD males. Future research is needed to replicate these findings and investigate the underlying biological mechanisms.

The endogenous opioid system (EOS) controls the processing of nociceptive stimuli and is a pharmacological target for opioids. Alterations in expression of the EOS genes under neuropathic pain condition may account for low efficacy of opioid drugs. We here examined whether EOS expression patterns are altered in the lumbar spinal cord of the rats with spinal nerve ligation (SNL) as a neuropathic pain model. Effects of the left- and right-side SNL on expression of EOS genes in the ipsi- and contralateral spinal domains were analysed. The SNL-induced changes were complex and different between the genes; between the dorsal and ventral spinal domains; and between the left and right sides of the spinal cord. Prodynorphin (Pdyn) expression was upregulated in the ipsilateral dorsal domains by each the left and right-side SNL, while changes in expression of μ-opioid receptor (Oprm1) and proenkephalin (Penk) genes were dependent on the SNL side. Changes in expression of the Pdyn and κ-opioid receptor (Oprk1) genes were coordinated between the ipsi- and contralateral sides. Withdrawal response thresholds, indicators of mechanical allodynia correlated negatively with Pdyn expression in the right ventral domain after right side SNL. These findings suggest multiple roles of the EOS gene products in spinal sensitization and changes in motor reflexes, which may differ between the left and right sides.

OBJECTIVE: The genetic component of alcohol use disorder is substantial, but monozygotic twin discordance indicates a role for nonheritable differences that could be mediated by epigenetics. Despite growing evidence associating epigenetics and psychiatric disorders, it is unclear how epigenetics, particularly DNA methylation, relate to brain function and behavior, including drinking behavior. METHOD: The authors carried out a genome-wide analysis of DNA methylation of 18 monozygotic twin pairs discordant for alcohol use disorder and validated differentially methylated regions. After validation, the authors characterized these differentially methylated regions using personality trait assessment and functional MRI in a sample of 499 adolescents. RESULTS: Hypermethylation in the 3'-protein-phosphatase-1G (PPM1G) gene locus was associated with alcohol use disorder. The authors found association of PPM1G hypermethylation with early escalation of alcohol use and increased impulsiveness. They also observed association of PPM1G hypermethylation with increased blood-oxygen-level-dependent response in the right subthalamic nucleus during an impulsiveness task. CONCLUSIONS: Overall, the authors provide first evidence for an epigenetic marker associated with alcohol consumption and its underlying neurobehavioral phenotype.

Molecular changes induced by excessive alcohol consumption may underlie formation of dysphoric state during acute and protracted alcohol withdrawal which leads to craving and relapse. A main molecular addiction hypothesis is that the upregulation of the dynorphin (DYN)/κ-opioid receptor (KOR) system in the nucleus accumbens (NAc) of alcohol-dependent individuals causes the imbalance in activity of D1- and D2 dopamine receptor (DR) expressing neural circuits that results in dysphoria. We here analyzed post-mortem NAc samples of human alcoholics to assess changes in prodynorphin (PDYN) and KOR (OPRK1) gene expression and co-expression (transcriptionally coordinated) patterns. To address alterations in D1- and D2-receptor circuits, we studied the regulatory interactions between these pathways and the DYN/KOR system. No significant differences in PDYN and OPRK1 gene expression levels between alcoholics and controls were evident. However, PDYN and OPRK1 showed transcriptionally coordinated pattern that was significantly different between alcoholics and controls. A downregulation of DRD1 but not DRD2 expression was seen in alcoholics. Expression of DRD1 and DRD2 strongly correlated with that of PDYN and OPRK1 suggesting high levels of transcriptional coordination between these gene clusters. The differences in expression and co-expression patterns were not due to the decline in neuronal proportion in alcoholic brain and thereby represent transcriptional phenomena. Dysregulation of DYN/KOR system and dopamine signaling through both alterations in co-expression patterns of opioid genes and decreased DRD1 gene expression may contribute to imbalance in the activity of D1- and D2-containing pathways which may lead to the negative affective state in human alcoholics. Electronic supplementary material The online version of this article (10.1007/s12035-017-0844-4) contains supplementary material, which is available to authorized users.

Here, we describe a molecular switch associated with opioid receptors-linked signalling cascades that provides a dual opioid control over P2X3 purinoceptor in sensory neurones. Leu-enkephalin inhibited P2X3-mediated currents with IC50 ~10 nM in ~25 % of small nociceptive rat dorsal root ganglion (DRG) neurones. In contrast, in neurones pretreated with pertussis toxin leu-enkephalin produced stable and significant increase of P2X3 currents. All effects of opioid were abolished by selective μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), nonselective inhibitor naloxone, and by PLC inhibitor U73122. Thus, we discovered a dual link between purinoceptors and μ-opioid receptors: the latter exert both inhibitory (pertussis toxin-sensitive) and stimulatory (pertussis toxin-insensitive) actions on P2X3 receptors through phospholipase C (PLC)-dependent pathways. This dual opioid control of P2X3 receptors may provide a molecular explanation for dichotomy of opioid therapy. Pharmacological control of this newly identified facilitation/inhibition switch may open new perspectives for the adequate medical use of opioids, the most powerful pain-killing agents known today.

BACKGROUND: Nociceptin is a key regulator linking environmental stress and alcohol drinking. In a genome-wide methylation analysis, we recently identified an association of a methylated region in the OPRL1 gene with alcohol-use disorders. METHODS: Here, we investigate the biological basis of this observation by analysing psychosocial stressors, methylation of the OPRL1 gene, brain response during reward anticipation and alcohol drinking in 660 fourteen-year-old adolescents of the IMAGEN study. We validate our findings in marchigian sardinian (msP) alcohol-preferring rats that are genetically selected for increased alcohol drinking and stress sensitivity. RESULTS: We found that low methylation levels in intron 1 of OPRL1 are associated with higher psychosocial stress and higher frequency of binge drinking, an effect mediated by OPRL1 methylation. In individuals with low methylation of OPRL1, frequency of binge drinking is associated with stronger BOLD response in the ventral striatum during reward anticipation. In msP rats, we found that stress results in increased alcohol intake and decreased methylation of OPRL1 in the nucleus accumbens. CONCLUSIONS: Our findings describe an epigenetic mechanism that helps to explain how psychosocial stress influences risky alcohol consumption and reward processing, thus contributing to the elucidation of biological mechanisms underlying risk for substance abuse.

The dynorphin κ-opioid receptor system is implicated in mental health and brain/mental disorders. However, despite accumulating evidence that PDYN and/or dynorphin peptide expression is altered in the brain of individuals with brain/mental disorders, little is known about transcriptional control of PDYN in humans. In the present study, we show that PDYN is targeted by the transcription factor REST in human neuroblastoma SH-SY5Y cells and that that interfering with REST activity increases PDYN expression in these cells. We also show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain and may have implications for mental health and brain/mental disorders.

The myriad proteins may be involved in the mechanisms underlying the development and maintenance of neuropathic pain, an extremely disabling condition that originates from pathology of the nervous system. To address the mechanisms, we here analyzed proteins and cellular networks in the dorsal spinal cord mediating pain processing in a well-established rat model of neuropathic pain induced by spinal nerve ligation (SNL). Labeling-based proteomic methods together with high-resolution mass spectrometry for proteome analysis were applied. 38 proteins including synapsin 1 and microtubule-associated protein 2 were identified as differently expressed in the SNL group. Pathway analysis suggests that maladaptive changes in the levels of these proteins may contribute to abnormal synaptic transmission and neuronal intracellular signaling underlying the onset and development of neuropathic pain.

Aims In rheumatoid arthritis (RA), pain and inflammation are initial symptoms followed by various degrees of bone and cartilage destruction. Previously, we have shown that reversible proteasome inhibitor MG132 attenuates pain and joint inflammation in a rat model of adjuvant-arthritis. Our present study aims to study the effects of MG132 on molecular changes in the dorsal root ganglia (DRG) and in the spinal cord (SC) using the same animal model. Methods Arthritis was induced by heat-killed Mycobacterium butyricum in rats. The expression of substance P (SP) was analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in DRG and in the SC. The nuclear factor-κB (NF-κB) DNA-binding activity in the SC was analyzed by electromobility shift assay. Results Arthritic rats treated daily with MG132 demonstrated a marked reduction of SP gene expression in the DRG and number of SP-positive cells was reduced. In the spinal cord of arthritic rats elevated SP messenger RNA levels were normalized and NF-κB-DNA-binding activity was down-regulated in arthritic rats treated with MG132. Conclusion Our results indicate that proteasome inhibitor MG132 attenuates pain in adjuvant arthritis by targeting the sensory neuropeptide substance P in the peripheral and central nervous systems. These effects may be mediated through the inhibition of NF-κB activation.

We have recently identified missense mutations in prodynorphin (PDYN), the precursor to dynorphin opioid peptides, as the cause for spinocerebellar ataxia (SCA23) in Dutch ataxia cases. We report a screen of PDYN for mutations in 371 cerebellar ataxia cases, which had a positive family history; most are of French origin. Sequencing revealed three novel putative missense mutations and one heterozygous two-base pair deletion in four independent SCA patients. These variants were absent in 400 matched controls and are located in the highly conserved dynorphin domain. To resolve the pathogenicity of the heterozygous variants, we assessed the peptide production of the mutant PDYN proteins. Two missense mutations raised dynorphin peptide levels, the two-base pair deletion terminated dynorphin synthesis, and one missense mutation did not affect PDYN processing. Given the outcome of our functional analysis, we may have identified at least two novel PDYN mutations in a French and a Moroccan SCA patient. Our data corroborates recent work that also showed that PDYN mutations only account for a small percentage (similar to 0.1 %) of European SCA cases.

Synthetic κ-opioid receptor (KOR) agonists induce dysphoric and pro-depressive effects and variations in the KOR (OPRK1) and prodynorphin (PDYN) genes have been shown to be associated with alcohol dependence. We genotyped 23 single nucleotide polymorphisms (SNPs) in the PDYN and OPRK1 genes in 816 alcohol-dependent subjects and investigated their association with: (1) negative craving measured by a subscale of the Inventory of Drug Taking Situations; (2) a self-reported history of depression; (3) the intensity of depressive symptoms measured by the Beck Depression Inventory-II. In addition, 13 of the 23 PDYN and OPRK1 SNPs, which were previously genotyped in a set of 1248 controls, were used to evaluate association with alcohol dependence. SNP and haplotype tests of association were performed. Analysis of a haplotype spanning the PDYN gene (rs6045784, rs910080, rs2235751, rs2281285) revealed significant association with alcohol dependence (p = 0.00079) and with negative craving (p = 0.0499). A candidate haplotype containing the PDYN rs2281285-rs1997794 SNPs that was previously associated with alcohol dependence was also associated with negative craving (p = 0.024) and alcohol dependence (p = 0.0008) in this study. A trend for association between depression severity and PDYN variation was detected. No associations of OPRK1 gene variation with alcohol dependence or other studied phenotypes were found. These findings support the hypothesis that sequence variation in the PDYN gene contributes to both alcohol dependence and the induction of negative craving in alcohol-dependent subjects.

Damaged reward areas in human alcoholics : neuronal proportion decline and astrocyte activation

Excessive alcohol consumption is a major public health problem worldwide. Although drinking habits are known to be inherited, few genes have been identified that are robustly linked to alcohol drinking. We conducted a genome-wide association metaanalysis and replication study among >105,000 individuals of European ancestry and identified beta-Klotho (KLB) as a locus associated with alcohol consumption (rs11940694; P = 9.2 x 10(-12)). beta-Klotho is an obligate coreceptor for the hormone FGF21, which is secreted from the liver and implicated in macronutrient preference in humans. We show that brain-specific beta-Klotho KO mice have an increased alcohol preference and that FGF21 inhibits alcohol drinking by acting on the brain. These data suggest that a liver-brain endocrine axis may play an important role in the regulation of alcohol drinking behavior and provide a unique pharmacologic target for reducing alcohol consumption.

Regulation of the formation and rewiring of neural circuits by neuropeptides may require coordinated production of these signaling molecules and their receptors that may be established at the transcriptional level. Here, we address this hypothesis by comparing absolute expression levels of opioid peptides with their receptors, the largest neuropeptide family, and by characterizing coexpression (transcriptionally coordinated) patterns of these genes. We demonstrated that expression patterns of opioid genes highly correlate within and across functionally and anatomically different areas. Opioid peptide genes, compared with their receptor genes, are transcribed at much greater absolute levels, which suggests formation of a neuropeptide cloud that covers the receptor-expressed circuits. Surprisingly, we found that both expression levels and the proportion of opioid receptors are strongly lateralized in the spinal cord, interregional coexpression patterns are side specific, and intraregional coexpression profiles are affected differently by left- and right-side unilateral body injury. We propose that opioid genes are regulated as interconnected components of the same molecular system distributed between distinct anatomic regions. The striking feature of this system is its asymmetric coexpression patterns, which suggest side-specific regulation of selective neural circuits by opioid neurohormones.-Kononenko, O., Galatenko, V., Andersson, M., Bazov, I., Watanabe, H., Zhou, X. W., Iatsyshyna, A., Mityakina, I., Yakovleva, T., Sarkisyan, D., Ponomarev, I., Krishtal, O., Marklund, N., Tonevitsky, A., Adkins, D. L., Bakalkin, G. Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits.

This review represents the focus of a symposium that was presented at the “Alcoholism and Stress: A Framework for Future Treatment Strategies” conference in Volterra, Italy on May 3–6, 2011 and organized / chaired by Dr. Brendan M. Walker. The primary goal of the symposium was to evaluate and disseminate contemporary findings regarding the emerging role of kappa-opioid receptors (KORs) and their endogenous ligands dynorphins (DYNs) in the regulation of escalated alcohol consumption, negative affect and cognitive dysfunction associated with alcohol dependence, as well as DYN / KOR mediation of the effects of chronic stress on alcohol reward and seeking behaviors. Dr. Glenn Valdez described a role for KORs in the anxiogenic effects of alcohol withdrawal. Dr. Jay McLaughlin focused on the role of KORs in repeated stress-induced potentiation of alcohol reward and increased alcohol consumption. Dr. Brendan Walker presented data characterizing the effects of KOR antagonism within the extended amygdala on withdrawal-induced escalation of alcohol self-administration in dependent animals. Dr. Georgy Bakalkin concluded with data indicative of altered DYNs and KORs in the prefrontal cortex of alcohol dependent humans that could underlie diminished cognitive performance. Collectively, the data presented within this symposium identified the multifaceted contribution of KORs to the characteristics of acute and chronic alcohol-induced behavioral dysregulation and provided a foundation for the development of pharmacotherapeutic strategies to treat certain aspects of alcohol use disorders.

Osteoarthritis is a degenerative joint disease with pain and loss of joint function as major pathological features. Recent studies show that proteasome inhibitors reduce pain in various pathological conditions. We evaluated the effects of MG132, a reversible proteasome inhibitor on pain and joint destruction in a rat model of osteoarthritis. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate into the rat knee. Knee joint stiffness was scored and nociception was evaluated by mechanical pressure applied to the respective hind paw. Knee joint destruction was assessed by radiological and histological analyses. Expression of matrix metalloproteinase-3 (MMP-3) was analyzed by quantitative reverse transcription polymerase chain reaction in the knee articular cartilage. Expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the dorsal root ganglia (L4-L6) by quantitative reverse transcription polymerase chain reaction and in the knee joints by immunohistochemistry. Our results indicate that daily treatment of osteoarthritic rats with MG132 significantly increases their mobility while the swelling, pain thresholds, and pathological features of the affected joints were reduced. Furthermore, the upregulated expression of MMP-3, SP, and CGRP in the arthritic rats was normalized by MG132 administration. We conclude that the proteasome inhibitor MG132 reduces pain and joint destruction, probably by involving the peripheral nervous system, and that changes in SP and CGRP expression correlate with alterations in behavioural responses. Our findings suggest that nontoxic proteasome inhibitors may represent a novel pharmacotherapy for osteoarthritis.

The effects of repeated, intermittent administration of a moderate dose of ethanol (3.4 g/kg/day × 6 days, intragastrically via gavages) on cognitive function were examined in male Wistar rats. No significant differences in weight gain between the ethanol- and water-treated rats were found. Analysis of physical dependence revealed no signs of spontaneous withdrawal, whereas withdrawal signs exacerbated by Ro15-4513, an inverse benzodiazepine agonist, were apparent 5 hours but not 24 hours after the cessation of ethanol treatment. Spatial learning and memory, as assessed in the Barnes maze, were impaired 3-6 days following the treatment but recovered by the 11th-14th days. Reversal learning, however, was impaired throughout the 2-week observation period. Thus, bouts of moderate-dose ethanol administration transiently impair spatial learning and memory, and promote cognitive inflexibility. The employed ethanol exposure paradigm may provide a model of human cognitive deficits associated with alcohol binge drinking.

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries ...