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Opioid craving and the vulnerability to relapse is associated with severe and persistent disruptions to sleep and circadian rhythms. Investigations into the cellular and molecular pathways in the human brain underlying the relationship between circadian rhythms and OUD remain limited. In human subjects with OUD, previous transcriptomics work implicated a role for circadian regulation of synaptic processes in key cognitive- and reward-related brain regions, dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc). To provide further insights into the synaptic alterations associated with OUD, we used mass-spectrometry based proteomics to deeply profile protein alterations in tissue homogenates and synaptosomes from both NAc and DLPFC of unaffected and OUD subjects. Between unaffected and OUD subjects, we identified 43 differentially expressed (DE) proteins in NAc homogenates and 55 DE proteins in DLPFC homogenates. In synaptosomes, we found 56 DE proteins in NAc of OUD subjects and 161 DE proteins in DLPFC. Examining synaptosome enrichment of specific proteins enabled us to identify brain region- and synapse-specific pathway alterations in NAc and DLPFC associated with OUD. Across both regions, we found OUD-associated protein alterations primarily in pathways involved in GABAergic and glutamatergic synaptic functions, as well as circadian rhythms. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24-hour cycle, we were able to map circadian-related changes in the synaptic proteomes in NAc and DLPFC associated with OUD. In OUD, TOD analysis revealed significant circadian changes in endoplasmic reticulum to Golgi vesicle-mediated transport and protein membrane trafficking in NAc synapses, accompanied by changes in platelet derived growth factor receptor beta signaling in DLPFC synapses. Together, our results lend further support for molecular disruption of circadian regulation of synaptic signaling in the human brain as a key factor in opioid addiction.

Severe and persistent disruptions to sleep and circadian rhythms are common in people with opioid use disorder (OUD). Preclinical evidence suggests altered molecular rhythms in the brain modulate opioid reward and relapse. However, whether molecular rhythms are disrupted in the brains of people with OUD remained an open question, critical to understanding the role of circadian rhythms in opioid addiction. Using subjects' times of death as a marker of time of day, we investigated transcriptional rhythms in the brains of subjects with OUD compared to unaffected comparison subjects. We discovered rhythmic transcripts in both the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), key brain areas involved in OUD, that were largely distinct between OUD and unaffected subjects. Fewer rhythmic transcripts were identified in DLPFC of subjects with OUD compared to unaffected subjects, whereas in the NAc, nearly double the number of rhythmic transcripts was identified in subjects with OUD. In NAc of subjects with OUD, rhythmic transcripts peaked either in the evening or near sunrise, and were associated with an opioid, dopamine, and GABAergic neurotransmission. Associations with altered neurotransmission in NAc were further supported by co-expression network analysis which identified OUD-specific modules enriched for transcripts involved in dopamine, GABA, and glutamatergic synaptic functions. Additionally, rhythmic transcripts in DLPFC and NAc of subjects with OUD were enriched for genomic loci associated with sleep-related GWAS traits, including sleep duration and insomnia. Collectively, our findings connect transcriptional rhythm changes in opioidergic, dopaminergic, GABAergic signaling in the human brain to sleep-related traits in opioid addiction.

Background Prevalence rates of opioid use disorder (OUD) have increased dramatically, accompanied by a surge of overdose deaths. While opioid dependence has been extensively studied in preclinical models, an understanding of the biological alterations that occur in the brains of people who chronically use opioids and who are diagnosed with OUD remains limited. To address this limitation, RNA sequencing was conducted on the dorsolateral prefrontal cortex and nucleus accumbens, regions heavily implicated in OUD, from postmortem brains in subjects with OUD. Methods We performed RNA sequencing on the dorsolateral prefrontal cortex and nucleus accumbens from unaffected comparison subjects (n = 20) and subjects diagnosed with OUD (n = 20). Our transcriptomic analyses identified differentially expressed transcripts and investigated the transcriptional coherence between brain regions using rank-rank hypergeometric orderlap. Weighted gene coexpression analyses identified OUD-specific modules and gene networks. Integrative analyses between differentially expressed transcripts and genome-wide association study datasets using linkage disequilibrium scores assessed the genetic liability of psychiatric-related phenotypes in OUD. Results Rank-rank hypergeometric overlap analyses revealed extensive overlap in transcripts between the dorsolateral prefrontal cortex and nucleus accumbens in OUD, related to synaptic remodeling and neuroinflammation. Identified transcripts were enriched for factors that control proinflammatory cytokine, chondroitin sulfate, and extracellular matrix signaling. Cell-type deconvolution implicated a role for microglia as a potential driver for opioid-induced neuroplasticity. Linkage disequilibrium score analysis suggested genetic liabilities for risky behavior, attention-deficit/hyperactivity disorder, and depression in subjects with OUD. Conclusions Overall, our findings suggest connections between the brain’s immune system and opioid dependence in the human brain.

BackgroundPrevalence rates of opioid use disorder (OUD) have increased dramatically, accompanied by a surge of overdose deaths. While opioid dependence has been extensively studied in preclinical models, an understanding of the biological alterations that occur in the brains of people who chronically use opioids and who are diagnosed with OUD remains limited. To address this limitation, RNA-sequencing (RNA-seq) was conducted on the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), regions heavily implicated in OUD, from postmortem brains in subjects with OUD.MethodsWe performed RNA-seq on the DLPFC and NAc from unaffected comparison subjects (n=20) and subjects diagnosed with OUD (n=20). Our transcriptomic analyses identified differentially expressed (DE) transcripts and investigated the transcriptional coherence between brain regions using rank-rank hypergeometric ordering (RRHO). Weighted gene co-expression analyses (WGCNA) also identified OUD-specific modules and gene networks. Integrative analyses between DE transcripts and GWAS datasets using linkage disequilibrium score (LDSC) assessed the genetic liability psychiatric-related phenotypes.ResultsRRHO analyses revealed extensive overlap in transcripts between DLPFC and NAc in OUD, primarily relating to synaptic remodeling and neuroinflammation. Identified transcripts were enriched for factors that control pro-inflammatory cytokine-mediated, chondroitin sulfate, and extracellular matrix signaling. Cell-type deconvolution implicated a role for microglia as a critical driver for opioid-induced neuroplasticity. Using LDSC, we discovered genetic liabilities for risky behavior, attention deficit hyperactivity disorder, and depression.ConclusionsOverall, our findings reveal new connections between the brain’s immune system and opioid dependence in the human brain.

Circadian variability is driven by genetics and Diversity Outbred (DO) mice is a powerful tool for examining the genetics of complex traits because their high genetic and phenotypic diversity compared to conventional mouse crosses. The DO population combines the genetic diversity of eight founder strains including five common inbred and three wild-derived strains. In DO mice and their founders, we established a high-throughput system to measure cellular rhythms using in vitro preparations of skin fibroblasts. Among the founders, we observed strong heritability for rhythm period, robustness, phase and amplitude. We also found significant sex and strain differences for these rhythms. Extreme differences in period for molecular and behavioral rhythms were found between the inbred A/J strain and the wild-derived CAST/EiJ strain, where A/J had the longest period and CAST/EiJ had the shortest. In addition, we measured cellular rhythms in 329 DO mice, which displayed far greater phenotypic variability than the founders—80% of founders compared to only 25% of DO mice had periods of ~ 24 h. Collectively, our findings demonstrate that genetic diversity contributes to phenotypic variability in circadian rhythms, and high-throughput characterization of fibroblast rhythms in DO mice is a tractable system for examining the genetics of circadian traits.

Background The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro. Methods The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg–Marquardt algorithm. Nonlinear regression and Akaike’s information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests. Results We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness. Conclusions Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.

Concerns about external validity of rodent models and translation of findings across species are often based on narrow investigations of populations with limited diversity. Sources of individual variation – including genetics and sex – are only infrequently encompassed in model organism studies. As with most complex diseases, risk for cocaine use disorder is subject to considerable inter-individual variation. Explicit inclusion of individual differences in rodent research may reveal conserved phenotypes and molecular systems relevant to human addiction. We surveyed cocaine-related traits in both males and females of eight inbred mouse strains whose genomes collectively capture 90% of the genetic diversity of the mouse species. Across these strains, individual differences explained a substantial proportion of variance in cocaine-responsive or cocaine response-predictive behavioral and physiological phenotypes. Wild-derived mouse strains often extended the phenotypic ranges of these behaviors beyond what is observed in conventional laboratory strains. Striatum transcriptional responses to cocaine were also highly dependent upon strain and sex differences; most cocaine-responsive genes were differentially expressed in a manner moderated by strain, sex, or their combination. We compared the strain- and sex-mediated transcriptional responses to cocaine in mice to transcriptomic analysis of people with cocaine use disorder and found that mouse similarity to humans was highly dependent upon mouse genetic background and sex. Specifically, male WSB/EiJ mice and female NOD/ShiLtJ mice exhibited the greatest degree of neural transcriptional consilience with humans with cocaine use disorder. Model organism diversity thus represents a crucial source of biological information that can substantially improve external validity of neuropsychiatric research.Significance StatementLaboratory mice are widely used in research on neurobiological mechanisms of addiction, but most studies use a single strain and often sex of mice. To assess how individual differences in mice modulate addiction-related traits and how this impacts comparative analysis with humans, we studied cocaine-relevant behaviors and brain molecular correlates in both males and females of genetically diverse mouse strains. In this population, individual differences related to sex and/or genetics explain large proportions of differences in cocaine-related traits. Importantly, brain gene expression data demonstrated that some strains mimic human genomic states more readily than others. Individual differences thus represent a crucial and underdeveloped source of biological information about addiction mechanisms that may influence the translational utility of such studies.

RATIONALE: Few effective treatments exist for cocaine use disorders due to gaps in knowledge about the complex etiology. Genetically defined animal models provide a useful tool for advancing our understanding of the biological and genetic underpinnings of addiction-related behavior and evaluating potential treatments. However, many attempts at developing mouse models of behavioral disorders were based on overly simplified single gene perturbations, often leading to inconsistent and misleading results in pre-clinical pharmacology studies. A genetically complex mouse model may better reflect disease related behaviors. OBJECTIVES: Screening defined, yet genetically complex, intercrosses of the Collaborative Cross (CC) mice revealed two lines, RIX04/17 and RIX41/51, with extreme high and low behavioral responses to cocaine. We characterized these lines as well as their CC parents, CC004/TauUnc and CC041/TauUnc, to evaluate their utility as novel model systems for studying the biological and genetic mechanisms underlying behavioral responses to cocaine. METHODS: Behavioral responses to acute (initial locomotor sensitivity) and repeated (behavioral sensitization, conditioned place preference, intravenous self-administration) exposures to cocaine were assessed. We also examined the monoaminergic system (striatal tissue content and in vivo fast scan cyclic voltammetry), HPA axis reactivity and circadian rhythms as potential mechanisms for the divergent phenotypic behaviors observed in the two strains, as these systems have a previously known role in mediating addiction-related behaviors. RESULTS: RIX04/17 and 41/51 show strikingly divergent initial locomotor sensitivity to cocaine with RIX04/17 exhibiting very high and RIX41/51 almost no response. The lines also differ in the emergence of behavioral sensitization with RIX41/51 requiring more exposures to exhibit a sensitized response. Both lines show conditioned place preference for cocaine. We determined that the cocaine sensitivity phenotype in each RIX line was largely driven by the genetic influence of one CC parental strain, CC004/TauUnc and CC041/TauUnc. CC004 demonstrates active operant cocaine self-administration and CC041 is unable to acquire under the same testing conditions, a deficit which is specific to cocaine as both strains show operant response for a natural food reward. Examination of potential mechanisms driving differential responses to cocaine show strain differences in molecular and behavioral circadian rhythms. Additionally, while there is no difference in striatal dopamine tissue content or dynamics, there are selective differences in striatal norepinephrine and serotonergic tissue content. CONCLUSIONS: These CC strains offer a complex polygenic model system to study underlying mechanisms of cocaine response. We propose that CC041/TauUnc and CC004/TauUnc will be useful for studying genetic and biological mechanisms underlying resistance or vulnerability to the stimulatory and reinforcing effects of cocaine.

Background The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro. Methods The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg–Marquardt algorithm. Nonlinear regression and Akaike’s information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests. Results We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness. Conclusions Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day. Electronic supplementary material The online version of this article (10.1186/s12885-017-3896-y) contains supplementary material, which is available to authorized users.

Background Previous studies indicated that cadmium (Cd) increases PI3-kinase/Akt phosphorylation, resulting in an alteration in GSK-3β activity. However, the mechanism of Cd-induced endoplasmic reticulum (ER) stress in neuronal cells has yet to be studied in needs further elucidation. We examined the role of GSK-3β in Cd-induced neuronal cell death and the related downstream signaling pathways. Methods SH-SY5Y human neuroblastoma cells were treated with 10 or 20 μM BAPTA-AM and 1 μM wortmannin for 30 min and then incubated with 25 μM Cd for 12 h. Apoptotic cells were visualized via DAPI and PI staining. Data were evaluated with one-way analysis of variance (ANOVA) followed by Student’s t-test. Data are expressed as the means ± SD of experiments performed at least three times. Results Treatment of human neuronal SH-SY5Y cells with Cd induced ER, stress as evidenced by the increased expression of GRP78, which is a marker of ER stress. Cd exposure significantly increased the phosphorylation of Akt at thr308 and ser473 and that of GSK-3β at ser9 in a time-dependent manner, while the total protein levels of GSK-3β and Akt did not change. Cd-induced apoptosis was higher in GSK-3β-knockdown cells than in normal cells. Conclusions Our data suggest that Akt/GSK-3β signaling activated by Cd is involved in neuronal cell survival.

Based on genetic models with mutation or deletion of core clock genes, circadian disruption has been implicated in the pathophysiology of metabolic disorders. Thus, we examined whether circadian desynchronization in response to shift work–type schedules is sufficient to compromise metabolic homeostasis and whether inflammatory mediators provide a key link in the mechanism by which alterations of circadian timekeeping contribute to diet-induced metabolic dysregulation. In high-fat diet (HFD)-fed mice, exposure to chronic shifts of the light–dark cycle (12 h advance every 5 d): 1) disrupts photoentrainment of circadian behavior and modulates the period of spleen and macrophage clock gene rhythms; 2) potentiates HFD-induced adipose tissue infiltration and activation of proinflammatory M1 macrophages; 3) amplifies macrophage proinflammatory cytokine expression in adipose tissue and bone marrow–derived macrophages; and 4) exacerbates diet-induced increases in body weight, insulin resistance, and glucose intolerance in the absence of changes in total daily food intake. Thus, complete disruption of circadian rhythmicity or clock gene function as transcription factors is not requisite to the link between circadian and metabolic phenotypes. These findings suggest that macrophage proinflammatory activation and inflammatory signaling are key processes in the physiologic cascade by which dysregulation of circadian rhythmicity exacerbates diet-induced systemic insulin resistance and glucose intolerance.—Kim, S.-M., Neuendorff, N., Alaniz, R. C., Sun, Y., Chapkin, R. S., Earnest, D. J. Shift work cycle-induced alterations of circadian rhythms potentiate the effects of high-fat diet on inflammation and metabolism.

The circadian clockworks gate macrophage inflammatory responses. Given the association between clock dysregulation and metabolic disorders, we conducted experiments to determine the extent to which over-nutrition modulates macrophage clock function and whether macrophage circadian dysregulation is a key factor linking over-nutrition to macrophage proinflammatory activation, adipose tissue inflammation, and systemic insulin resistance. Our results demonstrate that 1) macrophages from high fat diet-fed mice are marked by dysregulation of the molecular clockworks in conjunction with increased proinflammatory activation, 2) global disruption of the clock genes Period1 (Per1) and Per2 recapitulates this amplified macrophage proinflammatory activation, 3) adoptive transfer of Per1/2-disrupted bone marrow cells into wild-type mice potentiates high fat diet-induced adipose and liver tissue inflammation and systemic insulin resistance, and 4) Per1/2-disrupted macrophages similarly exacerbate inflammatory responses and decrease insulin sensitivity in co-cultured adipocytes in vitro. Furthermore, PPARγ levels are decreased in Per1/2-disrupted macrophages and PPARγ2 overexpression ameliorates Per1/2 disruption-associated macrophage proinflammatory activation, suggesting that this transcription factor may link the molecular clockworks to signaling pathways regulating macrophage polarization. Thus, macrophage circadian clock dysregulation is a key process in the physiological cascade by which diet-induced obesity triggers macrophage proinflammatory activation, adipose tissue inflammation, and insulin resistance.

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p+miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.

Background Multiple pathogenic factors may contribute to the pathophysiology of Alzheimer's disease (AD). Peripheral blood markers have been used to assess biochemical changes associated with AD and mild cognitive impairment (MCI) and involved in their pathophysiology. Methods Plasma samples and clinical data were obtained from participants in the Ansan Geriatric Study (AGE study). Plasma concentrations of four candidate biomarkers were measured in the normal control (NC), MCI, and AD group: interleukin-8 (IL-8), IL-10, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α). Body mass index (BMI), MMSE (Mini Mental State Examination), CDR(Clinical Dementia Rating) score and homocystein level were recorded with social and demographic information. Results Total of 59 subjects were randomly selected for this analysis [NC (n = 21), MCI(n = 20) and AD(n = 18)]. In demographic data, educational year was correlated with the diagnosis states (p < 0.0001). No significant differences in cardiovascular disease, BMI and use of NSAIDs were found in MCI or AD group compared with NC group, respectively. The involvement of inflammatory illness or conditions in subjects, WBC count, fibrinogen and homocystein of the three groups, but no significant differences were found in each groups. The plasma IL-8 level was lower in MCI and AD patients compared with the normal control group (respectively, p < 0.0001). The MCI and AD patients had similar MCP-1, IL-10, and TNF-α level. Conclusions Our study suggests the existence of an independent and negative relationship between plasma IL-8 levels and functional status in MCI and AD patients.