Gene expression profiling in whole blood stimulated ex vivo with lipopolysaccharide as a tool to predict post-stroke depressive symptoms: Proof-of-concept study.

Prediction of post-stroke depressive symptoms (DSs) is challenging in patients without a history of depression. Gene expression profiling in blood cells may facilitate the search for biomarkers. The use of an ex vivo stimulus to the blood helps to reveal differences in gene profiles by reducing variation in gene expression. We conducted a proof-of-concept study to determine the usefulness of gene expression profiling in lipopolysaccharide (LPS)-stimulated blood for predicting post-stroke DS. Out of 262 enrolled patients with ischemic stroke, we included 96 patients without a pre-stroke history of depression and not taking any anti-depressive medication before or during the first 3 months after stroke. We assessed DS at 3 months after stroke using the Patient Health Questionnaire-9. We used RNA sequencing to determine the gene expression profile in LPS-stimulated blood samples taken on day 3 after stroke. We constructed a risk prediction model using a principal component analysis combined with logistic regression. We diagnosed post-stroke DS in 17.7% of patients. Expression of 510 genes differed between patients with and without DS. A model containing 6 genes (PKM, PRRC2C, NUP188, CHMP3, H2AC8, NOP10) displayed very good discriminatory properties (area under the curve: 0.95) with the sensitivity of 0.94 and specificity of 0.85. Our results suggest the potential utility of gene expression profiling in whole blood stimulated with LPS for predicting post-stroke DS. This method could be useful for searching biomarkers of post-stroke depression.

Genomic variants and inferred biological processes in multiplex families with Tourette syndrome.

BACKGROUND: Tourette syndrome is a developmental neuropsychiatric disorder. Its etiology is complex and elusive, although an important role of genetic factors has been established. The aim of the present study was to identify the genomic basis of Tourette syndrome in a group of families with affected members in 2 or 3 generations. METHODS: Whole-genome sequencing was performed followed by co-segregation and bioinformatic analyses. Identified variants were used to select candidate genes, which were then subjected to gene ontology and pathway enrichment analysis. RESULTS: The study group included 17 families comprising 80 patients with Tourette syndrome and 44 healthy family members. Co-segregation analysis and subsequent prioritization of variants pinpointed 37 rare and possibly pathogenic variants shared among affected individuals within a single family. Three such variants, in the ALDH2, DLD and ALDH1B1 genes, could influence oxidoreductase activity in the brain. Two variants, in SLC17A8 and BSN genes, were involved in sensory processing of sound by inner hair cells of the cochlea. Enrichment analysis of genes whose rare variants were present in all patients from at least 2 families identified significant gene sets implicated in cell-cell adhesion, cell junction assembly and organization, processing of sound, synapse assembly, and synaptic signalling processes. LIMITATIONS: We did not examine intergenic variants, but they still could influence clinical phenotype. CONCLUSION: Our results provide a further argument for a role of adhesion molecules and synaptic transmission in neuropsychiatric diseases. Moreover, an involvement of processes related to oxidative stress response and sound-sensing in the pathology of Tourette syndrome seems likely.

Genetic Profiling in Children With Acute Lymphoblastic Leukemia Referred for Allogeneic Hematopoietic Stem Cell Transplantation.

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is the essential and often the only curative therapeutic option in high risk and relapsed pediatric acute lymphoblastic leukemia (ALL). METHODS: The objective of the study was to investigate whole-genome expression in children with high risk or relapsed ALL referred for HSCT. Gene expression was assessed in 18 children with ALL referred for HSCT (10 high risk, 8 relapsed; median age of 9.4 years) and in a control group of 38 obese children (median age of 14.1 years). Whole-genome expression was assessed in leukocytes using GeneChip® HumanGene 1.0 ST microarray. RESULTS: The analysis of genomic profiles revealed a significantly lower expression of 21 genes with a defined function, involved in immunoglobulin production, lymphocyte function, or regulation of DNA processing in ALL patients referred for HSCT compared with the control group. CONCLUSION: Genome expression of patients with ALL in remission referred to HSCT revealed deep immunosuppression of both B-cell and T-cell lineages, which may increase the probability of donor cell engraftment.

Glucocorticoid-Regulated Kinase CAMKIγ in the Central Amygdala Controls Anxiety-like Behavior in Mice.

The expression of the Calcium/Calmodulin-Dependent Protein Kinase I gamma (encoded by the Camk1g gene) depends on the activation of glucocorticoid receptors (GR) and is strongly regulated by stress. Since Camk1g is primarily expressed in neuronal cells of the limbic system in the brain, we hypothesized that it could be involved in signaling mechanisms that underlie the adaptive or maladaptive responses to stress. Here, we find that restraint-induced stress and the GR agonist dexamethasone robustly increase the expression of Camk1g in neurons of the amygdalar nuclei in the mouse brain. To assess the functional role of Camk1g expression, we performed a virally induced knock-down of the transcript. Mice with bilateral amygdala-specific Camk1g knock-down showed increased anxiety-like behaviors in the light-dark box, and an increase in freezing behavior after fear-conditioning, but normal spatial working memory during exploration of a Y-maze. Thus, we confirm that Camk1g is a neuron-specific GR-regulated transcript, and show that it is specifically involved in behaviors related to anxiety, as well as responses conditioned by aversive stimuli.

Cocaine attenuates acid sphingomyelinase activity during establishment of addiction-related behavior-A translational study in rats and monkeys.

Cocaine addiction is a severe psychiatric condition for which currently no effective pharmacotherapy is available. Brain mechanisms for the establishment of addiction-related behaviors are still not fully understood, and specific biomarkers for cocaine use are not available. Sphingolipids are major membrane lipids, which shape neuronal membrane composition and dynamics in the brain. Here, we investigated how chronic cocaine exposure during establishment of addiction-related behaviors affects the activity of the sphingolipid rheostat controlling enzymes in the brain of rats. As we detected specific effects on several enzymes in the brain, we tested whether the activity of selected enzymes in the blood may serve as potential biomarker for cocaine exposure in non-human primates (Callithrix penicillata). We found that intravenous cocaine self-administration led to a reduced mRNA expression of Cers1, Degs1 and Degs2, and Smpd1 in the prefrontal cortex of rats, as well as a reduction of Cers4 expression in the striatum. These effects reversed after 10 days of abstinence. Monkeys showed a robust cocaine-induced place preference (CPP). This coincided with a reduction in blood acid sphingomyelinase (ASM) activity after CPP establishment. This effect normalized after 15 days of abstinence. Altogether, these findings suggest that the establishment of cocaine addiction-related behaviors coincides with changes in the activity of sphingolipid controlling enzymes. In particular, blood ASM levels may serve as a translational biomarker for recent cocaine exposure.

Role of Non-Coding Regulatory Elements in the Control of GR-Dependent Gene Expression.

The glucocorticoid receptor (GR, also known as NR3C1) coordinates molecular responses to stress. It is a potent transcription activator and repressor that influences hundreds of genes. Enhancers are non-coding DNA regions outside of the core promoters that increase transcriptional activity via long-distance interactions. Active GR binds to pre-existing enhancer sites and recruits further factors, including EP300, a known transcriptional coactivator. However, it is not known how the timing of GR-binding-induced enhancer remodeling relates to transcriptional changes. Here we analyze data from the ENCODE project that provides ChIP-Seq and RNA-Seq data at distinct time points after dexamethasone exposure of human A549 epithelial-like cell line. This study aimed to investigate the temporal interplay between GR binding, enhancer remodeling, and gene expression. By investigating a single distal GR-binding site for each differentially upregulated gene, we show that transcriptional changes follow GR binding, and that the largest enhancer remodeling coincides in time with the highest gene expression changes. A detailed analysis of the time course showed that for upregulated genes, enhancer activation persists after gene expression changes settle. Moreover, genes with the largest change in EP300 binding showed the highest expression dynamics before the peak of EP300 recruitment. Overall, our results show that enhancer remodeling may not directly be driving gene expression dynamics but rather be a consequence of expression activation.

MicroRNA Let-7e in the Mouse Prefrontal Cortex Differentiates Restraint-Stress-Resilient Genotypes from Susceptible Genotype.

Three strains of mice with various susceptibilities to restraint stress (RS), i.e., mice with a knocked out norepinephrine transporter gene (NET-KO), SWR/J and C57BL/6J (WT) mice were shown to serve as a good model to study the molecular mechanisms underlying different stress-coping strategies. We identified 14 miRNAs that were altered by RS in the PFC of these mice in a genotype-dependent manner, where the most interesting was let-7e. Further in silico analysis of its potential targets allowed us to identify five mRNAs (Bcl2l11, Foxo1, Pik3r1, Gab1 and Map2k4), and their level alterations were experimentally confirmed. A next-generation sequencing (NGS) approach, which was employed to find transcripts differentially expressed in the PFC of NET-KO and WT mice, showed that, among others, two additional mRNAs were regulated by mmu-let-7e, i.e., mRNAs that encode Kmt2d and Inf2. Since an increase in Bcl2l11 and Pik3r1 mRNAs upon RS in the PFC of WT mice resulted from the decrease in mmu-let-7e and mmu-miR-484 regulations, we postulated that MAPK, FoxO and PI3K-Akt signaling pathways were associated with stress resilience, although via different, genotype-dependent regulation of various mRNAs by let-7e and miR-484. However, a higher level of Kmt2d mRNA (regulated by let-7e) that was found with NGS analysis in the PFC of NET-KO mice indicated that histone methylation was also important for stress resilience.

The specific ex vivo released cytokine profile is associated with ischemic stroke outcome and improves its prediction.

BACKGROUND: Inflammation is associated with poor outcome after stroke. A relationship between ex vivo cytokine synthesis and stroke outcome remains unclear. We explored an association between ex vivo cytokine release, circulating interleukin (IL)-6 as a marker of systemic inflammation, and stroke prognosis. We assessed the utility of ex vivo synthesized cytokines for predicting stroke outcome. METHODS: We collected blood from 248 ischemic stroke patients and stimulated it ex vivo with lipopolysaccharide. We measured concentration of synthesized cytokines (TNFα, IP-10, IL-1ß, IL-6, IL-8, IL-10, and IL-12) and plasma IL-6. We assessed functional outcome 3 months after stroke using the modified Rankin Scale. To assess the prognostic ability of cytokines, we applied multivariate logistic regression, cluster analysis, and construction of multimarker score. RESULTS: Decreased release of IP-10, TNFα, IL-1ß, and IL-12; increased release of IL-10 and IL-8; and higher plasma IL-6 level were associated with poor outcome. Cluster analysis identified three groups of patients with distinct cytokine profiles. The group with the worst outcome demonstrated high synthesis of IL-10, IL-8, IL-1ß, and IL-6 and low synthesis of IL-12, IP-10, and TNFα accompanied by high circulating IL-6 level. The group with the best prognosis showed high synthesis of TNFα, IP-10, IL-12, IL-1ß, and IL-6; low synthesis of IL-10 and IL-8; and low plasma IL-6. Patients with intermediate outcome had low synthesis of all cytokines accompanied by low circulating IL-6. We constructed a multimarker score composed of ex vivo released IL-12, IL-10, TNFα, and plasma IL-6. Addition of this score to clinical variables led to significant increase in c-statistic (0.81 vs 0.73, p = 0.02) and net reclassification improvement. CONCLUSION: The decreased ex vivo release of pro-inflammatory cytokines and increased release of IL-10 and IL-8 are related to poor outcome after stroke. Cytokine-based multimarker score adds prognostic value to clinical model for predicting stroke outcome.

Gastrointestinal peptides in children before and after hematopoietic stem cell transplantation.

BACKGROUND: Gastrointestinal tract function and it's integrity are controlled by a number of peptides whose secretion is influenced by severe inflammation. In stomach the main regulatory peptide is ghrelin. For upper small intestine cholecystokinin and lower small intestine glucagon-like peptide- 1 are secreted, while fibroblast growth factor-21 is secreted by several organs, including the liver, pancreas, and adipose tissue [12]. Hematopoietic stem cell transplantation causes serious mucosal damage, which can reflect on this peptides. METHODS: The aim of the study was to determine fasting plasma concentrations of ghrelin, cholecystokinin, glucagon- like peptide-1, and fibroblast growth factor-21, and their gene expressions, before and 6 months after hematopoietic stem cell transplantation.27 children were studied, control group included 26 healthy children. RESULTS: Acute graft versus host disease was diagnosed in 11 patients (41%, n = 27). Median pre-transplantation concentrations of gastrointestinal peptides, as well as their gene expressions, were significantly lower in studied group compared with the control group. Only median of fibroblast growth factor-21 concentration was near-significantly higher before stem cell transplantation than in the control group. The post-hematopoietic transplant results revealed significantly higher concentrations of the studied peptides (except fibroblast growth factor-21) and respective gene expressions as compare to pre transplant results. Median glucagone like peptide-1 concentrations were significantly decreased in patients with features of acute graft versus host disease. Moreover, negative correlation between glucagone like peptide-1 concentrations and acute graft versus host disease severity was found. CONCLUSIONS: Increased concentrations and gene expressions of gastrointestinal tract regulation peptides can be caused by stimulation of regeneration in the severe injured organ. Measurement of these parameters may be a useful method of assessment of severity of gastrointestinal tract complications of hematopoietic stem cell transplantation.

Cocaine Administration and Its Abstinence Conditions Modulate Neuroglia.

Cocaine induces neuronal changes as well as non-neuronal (astrocytes, microglia, oligodendroglia) mechanisms, but these changes can also be modulated by various types of drug abstinence. Due to the very complex and still incompletely understood nature of cocaine use disorder, understanding of the mechanisms involved in addictive behavior is necessary to further search for effective pharmacotherapy of this disease. The aim of this study was to investigate changes at the gene and protein levels associated with glial cell activity after cocaine exposure, as well as during early cocaine abstinence (3 days) with extinction training or in home cage isolation. Cocaine self-administration significantly decreased myelin regulatory factor (MYRF) and cyclic nucleotide phosphodiesterase (CNP) expression in the hippocampus as well as pleckstrin (PLEK) and T-lymphocyte activation antigen (CD86) in the rat striatum. Depending on cocaine abstinence conditions, microglial PLEK expression was increased through extinction training but did not change in the home cage isolation. In addition, downregulation of gene expression associated with oligodendrocytes (CNP, MYRF) and microglia regulator of G protein signaling 1 (RGS1) was observed in the hippocampus, regardless of the type of drug abstinence, while downregulation of myelin and lymphocyte protein (MAL) expression was found only in rats exposed to abstinence in the home cage. Taken together, the presented results strongly suggest that cocaine abstinence evokes significant changes in gene expression associated with the proper functioning of glial cells, suggesting their significant involvement in adaptive changes in the brain associated with cocaine exposure. Interestingly, drug abstinence conditions are important factors influencing observed changes at the transcript levels of selected genes, which may be of clinical interest.

Systemic response to rupture of intracranial aneurysms involves expression of specific gene isoforms.

BACKGROUND: Rupture of an intracranial aneurysm (IA) causes a systemic response that involves an immune/inflammatory reaction. Our previous study revealed a downregulation of genes related to T lymphocytes and an upregulation of genes related to monocytes and neutrophils after IA rupture. It remains unknown whether that resulted from alterations in transcription or cell count. We sought to characterize the systemic response to IA rupture through analysis of transcript expression profiles in peripheral blood cells. We also investigated effects of IA rupture on the composition of mononuclear cells in peripheral blood. METHODS: We included 19 patients in the acute phase of IA rupture (RAA, first 72 h), 20 patients in the chronic phase (RAC, 3-15 months), and 20 controls. Using deep transcriptome sequencing, we analyzed the expression of protein-coding and noncoding RNAs. Expression levels, transcript biotypes, alternative splicing and other features of the regulated transcripts were studied. A functional analysis was performed to determine overrepresented ontological groups among gene expression profiles. Flow cytometry was used to analyze alterations in the level of mononuclear leukocyte subpopulations. RESULTS: Comparing RAA and controls, we identified 491 differentially expressed transcripts (303 were downregulated, and 188 were upregulated in RAA). The results indicate that the molecular changes in response to IA rupture occur at the level of individual transcripts. Functional analysis revealed that the most impacted biological processes are related to regulation of lymphocyte activation and toll-like receptor signaling pathway. Differences between RAC and controls were less prominent. Analysis of leukocyte subsets revealed a significantly decreased number of CD4+ lymphocytes and increase of classical and intermediate monocytes in RAA patients compared to controls. CONCLUSIONS: IA rupture in the acute phase strongly influences the transcription profiles of peripheral blood cells as well as the composition of mononuclear cells. A specific pattern of gene expression alteration was found, suggesting a depression of lymphocyte response and enhancement of monocyte activity.

Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients.

BACKGROUND: Limb girdle muscular dystrophies (LGMD) are a group of heterogeneous hereditary myopathies with similar clinical symptoms. Disease onset and progression are highly variable, with an elusive genetic background, and around 50% cases lacking molecular diagnosis. METHODS: Whole exome sequencing (WES) was performed in 73 patients with clinically diagnosed LGMD. A filtering strategy aimed at identification of variants related to the disease included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of genes expressed in muscle, analysis of the disease-associated interactome and copy number variants analysis. RESULTS: Genetic diagnosis was possible in 68.5% of cases. On average, 36.3 rare variants in genes associated with various muscle diseases per patient were found that could relate to the clinical phenotype. The putative causative mutations were mostly in LGMD-associated genes, but also in genes not included in the current LGMD classification (DMD, COL6A2, and COL6A3). In three patients, mutations in two genes were suggested as the joint cause of the disease (CAPN3+MYH7, COL6A3+CACNA1S, DYSF+MYH7). Moreover, a variety of phenotype-influencing variants were postulated, including in patients with an identified already known primary pathogenic mutation. CONCLUSIONS: We hypothesize that LGMD could be better described as oligogenic disorders in which dominant clinical presentation can result from the combined effect of mutations in a set of genes. In this view, the inter- and intrafamilial variability could reflect a specific genetic background and the presence of sets of phenotype-influencing or co-causative mutations in genes that either interact with the known LGMD-associated genes or are a part of the same pathways or structures.

Cell-type-specific gene expression patterns in the knee cartilage in an osteoarthritis rat model.

Osteoarthritis (OA) is a chronic degenerative disease that leads to joint failure, pain, and disability. Gene regulation is implicated as a driver of the imbalance between the expression of catabolic and anabolic factors that eventually leads to the degeneration of osteoarthritic cartilage. In our model, knee-joint OA was induced in male Wistar rats by intra-articular sodium monoiodoacetate (MIA) injections. Whole-genome microarrays were used to analyse the alterations in gene expression during the time-course of OA development (at 2, 14, and 28 days post-injection) in rat knee joints. The identified co-expressed groups of genes were analysed for enriched regulatory mechanisms, functional classes, and cell-type-specific expression. This analysis revealed 272 regulated transcripts (ANOVA FDR < 0.1% and fold > 2). Functionally, the five major gene expression patterns (A-E) were connected to PPAR signalling and adipogenesis (in cluster A), WNT signalling (in cluster B), endochondral ossification (in cluster C), matrix metalloproteinases and the ACE/RAGE pathway (in cluster D), and the Toll-like receptor, and IL-1 signalling pathways (in cluster E). Moreover, the dynamic profiles of these transcriptional changes were assigned to cellular compartments of the knee joint. Classifying the molecular processes associated with the development of cartilage degeneration provides novel insight into the OA disease process. Our study identified groups of co-regulated genes that share functional relationships and that may play an important role in the early and intermediate stages of OA.

Expression of alternatively spliced variants of the Dclk1 gene is regulated by psychotropic drugs.

BACKGROUND: The long-term effects of psychotropic drugs are associated with the reversal of disease-related alterations through the reorganization and normalization of neuronal connections. Molecular factors that trigger drug-induced brain plasticity remain only partly understood. Doublecortin-like kinase 1 (Dclk1) possesses microtubule-polymerizing activity during synaptic plasticity and neurogenesis. However, the Dclk1 gene shows a complex profile of transcriptional regulation, with two alternative promoters and exon splicing patterns that suggest the expression of multiple isoforms with different kinase activities. RESULTS: Here, we applied next-generation sequencing to analyze changes in the expression of Dclk1 gene isoforms in the brain in response to several psychoactive drugs with diverse pharmacological mechanisms of action. We used bioinformatics tools to define the range and levels of Dclk1 transcriptional regulation in the mouse nucleus accumbens and prefrontal cortex. We also sought to investigate the presence of DCLK1-derived peptides using mass spectrometry. We detected 15 transcripts expressed from the Dclk1 locus (FPKM > 1), including 2 drug-regulated variants (fold change > 2). Drugs that act on serotonin receptors (5-HT2A/C) regulate a subset of Dclk1 isoforms in a brain-region-specific manner. The strongest influence was observed for the mianserin-induced expression of an isoform with intron retention. The drug-activated expression of novel alternative Dclk1 isoforms was validated using qPCR. The drug-regulated isoform contains genetic variants of DCLK1 that have been previously associated with schizophrenia and hyperactivity disorder in humans. We identified a short peptide that might originate from the novel DCLK1 protein product. Moreover, protein domains encoded by the regulated variant indicate their potential involvement in the negative regulation of the canonical DCLK1 protein. CONCLUSIONS: In summary, we identified novel isoforms of the neuroplasticity-related gene Dclk1 that are expressed in the brain in response to psychotropic drug treatments.

Transcriptional signatures of steroid hormones in the striatal neurons and astrocytes.

BACKGROUND: The mechanisms of steroids actions in the brain mainly involve the binding and nuclear translocation of specific cytoplasmic receptors. These receptors can act as transcription factors and regulate gene expression. However, steroid-dependent transcriptional regulation in different types of neural cells is not yet fully understood. The aim of this study was to evaluate and compare transcriptional alterations induced by various steroid receptor agonists in primary cultures of astrocytes and neurons from mouse brain. RESULTS: We utilized whole-genome microarrays (Illumina Mouse WG-6) and quantitative PCR analyses to measure mRNA abundance levels. To stimulate gene expression we treated neuronal and astroglial cultures with dexamethasone (100 nM), aldosterone (200 nM), progesterone (200 nM), 5α-dihydrotestosterone (200 nM) and ß-Estradiol (200 nM) for 4 h. Neurons were found to exhibit higher levels of expression of mineralocorticoid receptor, progesterone receptor and estrogen receptor 2 than astrocytes. However, higher mRNA level of glucocorticoid receptor mRNA was observed in astrocytes. We identified 956 genes regulated by steroids. In astrocytes we found 381 genes altered by dexamethasone and 19 altered by aldosterone. Functional classification of the regulated genes indicated their putative involvement in multiple aspects of cell metabolism (up-regulated Slc2a1, Pdk4 and Slc45a3) and the inflammatory response (down-regulated Ccl3, Il1b and Tnf). Progesterone, dihydrotestosterone and estradiol did not change gene expression in astrocytes. We found no significant changes in gene expression in neurons. CONCLUSIONS: The obtained results indicate that glial cells might be the primary targets of transcriptional action of steroids in the central nervous system. Substantial changes in gene expression driven by the glucocorticoid receptor imply an important role for the hypothalamic-pituitary-adrenal axis in the hormone-dependent regulation of brain physiology. This is an in vitro study. Hence, the model may not accurately reflect all the effects of steroids on gene expression in neurons in vivo.

Neuropsin cleaves EphB2 in the amygdala to control anxiety.

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

Behavioral and transcriptional patterns of protracted opioid self-administration in mice.

Chronic exposure to opioids induces adaptations in brain function that lead to the formation of the behavioral and physiological symptoms of drug dependence and addiction. Animal models commonly used to test these symptoms typically last less than two weeks, which is presumably too short to observe the alterations in the brain that accompany drug addiction. Here, we analyzed the phenotypic and molecular effects of nearly lifelong morphine or saccharin intake in C57BL/6J mice. We used multiple paradigms to evaluate the symptoms of compulsive drug intake: a progressive ratio schedule, intermittent access and a schedule involving a risk of punishment were programmed into an automated IntelliCage system. Gene expression profiles were evaluated in the striatum using whole-genome microarrays and further validated using quantitative polymerase chain reaction in the striatum and the prefrontal cortex. Mice voluntary self-administering morphine showed addiction-related behavioral pattern that included: higher motivation to work for a drug reward, increased reward seeking and increased craving. The analysis of molecular changes revealed a tolerance effect in the transcriptional response to morphine injection (20 mg/kg, ip), as well as some long-lasting alterations in gene expression profiles between the analyzed groups of animals. Interestingly, among the morphine-drinking animals, certain transcriptional profiles were found to be associated with alterations in behavior. In conclusion, our model represents a novel approach for investigating the behavioral and molecular mechanisms underlying opioid addiction. Prolonged morphine intake caused adaptive processes in the brain that manifested as altered behavior and transcriptional sensitivity to opioids.

Seqinspector: position-based navigation through the ChIP-seq data landscape to identify gene expression regulators.

BACKGROUND: The regulation of gene expression in eukaryotic cells is a complex process that involves epigenetic modifications and the interaction of DNA with multiple transcription factors. This process can be studied with unprecedented sensitivity using a combination of chromatin immunoprecipitation and next-generation DNA sequencing (ChIP-seq). Available ChIP-seq data can be further utilized to interpret new gene expression profiling experiments. RESULTS: Here, we describe seqinspector, a tool that accepts any set of genomic coordinates from ChIP-seq or RNA-seq studies to identify shared transcriptional regulators. The presented web resource includes a large collection of publicly available ChIP-seq and RNA-seq experiments (>1300 tracks) performed on transcription factors, histone modifications, RNA polymerases, enhancers and insulators in humans and mice. Over-representation is calculated based on the coverage computed directly from indexed files storing ChIP-seq data (bigwig). Therefore, seqinspector is not limited to pre-computed sets of gene promoters. CONCLUSION: The tool can be used to identify common gene expression regulators for sets of co-expressed transcripts (including miRNAs, lncRNAs or any novel unannotated RNAs) or for sets of ChIP-seq peaks to identify putative protein-protein interactions or transcriptional co-factors. The tool is available at http://seqinspector.cremag.org.

Molecular profile of dissociative drug ketamine in relation to its rapid antidepressant action.

BACKGROUND: The NMDA receptor antagonist ketamine was found to act as a fast-acting antidepressant. The effects of single treatment were reported to persist for days to weeks, even in otherwise treatment-refractory cases. Identification of the mechanisms underlying ketamine's antidepressant action may permit development of novel drugs, with similar clinical properties but lacking psychotomimetic, sedative and other side effects. METHODS: We applied whole-genome microarray profiling to analyze detailed time-course (1, 2, 4 and 8 h) of transcriptome alterations in the striatum and hippocampus following acute administration of ketamine, memantine and phencyclidine in C57BL/6 J mice. The transcriptional effects of ketamine were further analyzed using next-generation sequencing and quantitative PCR. Gene expression alterations induced by the NMDA antagonists were compared to the molecular profiles of psychotropic drugs: antidepressants, antipsychotics, anxiolytics, psychostimulants and opioids. RESULTS: We identified 52 transcripts (e.g. Dusp1, Per1 and Fkbp5) with altered expression (FDR < 1 %) in response to treatment with NMDA receptor antagonists. Functional links that connect expression of the regulated genes to the MAPK, IL-6 and insulin signaling pathways were indicated. Moreover, ketamine-regulated expression of specific gene isoforms was detected (e.g. Tsc22d3, Sgk1 and Hif3a). The comparison with other psychotropic drugs revealed that the molecular effects of ketamine are most similar to memantine and phencyclidine. Clustering based on expression profiles placed the NMDA antagonists among fluoxetine, tianeptine, as well as opioids and ethanol. CONCLUSIONS: The identified patterns of gene expression alteration in the brain provided novel molecular classification of ketamine. The transcriptional profile of ketamine reflects its multi-target pharmacological nature. The results reveal similarities between the effects of ketamine and monoaminergic antidepressants that may explain the mechanisms of its rapid antidepressant action.

Astrocytes are a neural target of morphine action via glucocorticoid receptor-dependent signaling.

Chronic opioid use leads to the structural reorganization of neuronal networks, involving genetic reprogramming in neurons and glial cells. Our previous in vivo studies have revealed that a significant fraction of the morphine-induced alterations to the striatal transcriptome included glucocorticoid (GC) receptor (GR)-dependent genes. Additional analyses suggested glial cells to be the locus of these changes. In the current study, we aimed to differentiate the direct transcriptional effects of morphine and a GR agonist on primary striatal neurons and astrocytes. Whole-genome transcriptional profiling revealed that while morphine had no significant effect on gene expression in both cell types, dexamethasone significantly altered the transcriptional profile in astrocytes but not neurons. We obtained a complete dataset of genes undergoing the regulation, which includes genes related to glucose metabolism (Pdk4), circadian activity (Per1) and cell differentiation (Sox2). There was also an overlap between morphine-induced transcripts in striatum and GR-dependent transcripts in cultured astrocytes. We further analyzed the regulation of expression of one gene belonging to both groups, serum and GC regulated kinase 1 (Sgk1). We identified two transcriptional variants of Sgk1 that displayed selective GR-dependent upregulation in cultured astrocytes but not neurons. Moreover, these variants were the only two that were found to be upregulated in vivo by morphine in a GR-dependent fashion. Our data suggest that the morphine-induced, GR-dependent component of transcriptome alterations in the striatum is confined to astrocytes. Identification of this mechanism opens new directions for research on the role of astrocytes in the central effects of opioids.