Fyn-Dependent Gene Networks in Acute Ethanol Sensitivity

Studies in humans and animal models document that acute behavioral responses to ethanol are predisposing factor for the risk of long-term drinking behavior. Prior microarray data from our laboratory document strain- and brain region-specific variation in gene expression profile responses to acute ethanol that may be underlying regulators of ethanol behavioral phenotypes. The non-receptor tyrosine kinase Fyn has previously been mechanistically implicated in the sedative-hypnotic response to acute ethanol. To further understand how Fyn may modulate ethanol behaviors, we used whole-genome expression profiling. We characterized basal and acute ethanol-evoked (3 g/kg) gene expression patterns in nucleus accumbens (NAC), prefrontal cortex (PFC), and ventral midbrain (VMB) of control and Fynknockout mice. Bioinformatics analysis identified a set of Fyn-related gene networks differently regulated by acute ethanol across the three brain regions. In particular, our analysis suggested a coordinate basal decrease in myelin-associated gene expression within NAC and PFC as an underlying factor in sensitivity of Fyn null animals to ethanol sedation. An in silico analysis across the BXD recombinant inbred (RI) strains of mice identified a significant correlation between Fyn expression and a previously published ethanol loss-of-righting-reflex (LORR) phenotype. By combining PFC gene expression correlates to Fyn and LORR across multiple genomic datasets, we identified robust Fyn-centric gene networks related to LORR. Our results thus suggest that multiple system-wide changes exist within specific brain regions of Fynknockout mice, and that distinct Fyn-dependent expression networks within PFC may be important determinates of the LORR due to acute ethanol. These results add to the interpretation of acute ethanol behavioral sensitivity in Fyn kinase null animals, and identifyFyn-centric gene networks influencing variance in ethanol LORR. Such networks may also inform future design of pharmacotherapies for the treatment and prevention of alcohol use disorders

Phosducin-like protein (PhLP), a regulator of Gβγ function, interacts with the proteasomal protein SUG1

AbstractPhosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the βγ subunits of guanine nucleotide binding proteins. While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear. To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system. PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription. This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies. Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum. We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation

Empirical validation of the S-Score algorithm in the analysis of gene expression data

BACKGROUND: Current methods of analyzing Affymetrix GeneChip(® )microarray data require the estimation of probe set expression summaries, followed by application of statistical tests to determine which genes are differentially expressed. The S-Score algorithm described by Zhang and colleagues is an alternative method that allows tests of hypotheses directly from probe level data. It is based on an error model in which the detected signal is proportional to the probe pair signal for highly expressed genes, but approaches a background level (rather than 0) for genes with low levels of expression. This model is used to calculate relative change in probe pair intensities that converts probe signals into multiple measurements with equalized errors, which are summed over a probe set to form the S-Score. Assuming no expression differences between chips, the S-Score follows a standard normal distribution, allowing direct tests of hypotheses to be made. Using spike-in and dilution datasets, we validated the S-Score method against comparisons of gene expression utilizing the more recently developed methods RMA, dChip, and MAS5. RESULTS: The S-score showed excellent sensitivity and specificity in detecting low-level gene expression changes. Rank ordering of S-Score values more accurately reflected known fold-change values compared to other algorithms. CONCLUSION: The S-score method, utilizing probe level data directly, offers significant advantages over comparisons using only probe set expression summaries

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC

Adolescents primarily consume alcohol in binges, which can be particularly harmful to the developing frontal cortex and increase risk for an adult alcohol use disorder. We conducted a study investigating immediate and long lasting changes to the prefrontal cortex (PFC) transcriptome to determine the molecular mechanisms underlying adult ethanol behavioral sensitivity following binge ethanol in adolescence. DBA/2J mice were orally dosed with 4 g/kg ethanol intermittently from day 29 to 42. Adolescent mice were tested for anxiety-like behavior and ethanol sensitivity using the loss of righting reflex task. As adults, mice were tested for cognitive changes using the novel object recognition task, ethanol-induced anxiolysis and ethanol sensitivity. Adolescent binge ethanol altered ethanol sensitivity in young mice and led to lasting memory deficits in the object recognition test and greater ethanol sensitivity in adulthood. Using genomic profiling of transcripts in the PFC, we found that binge ethanol reduced myelin-related gene expression and altered chromatin modifying genes involved in histone demethylation at H3K9 and H3K36. We hypothesize that ethanol’s actions on histone methylation may be a switch for future transcriptional changes that underlie the behavioral changes lasting into adulthood

Selective GSK3B Deletion in Camk2a+ Forebrain Neurons or Inhibition Via Tideglusib, Decreases Ethanol Consumption in C57BL/6J Mice

Purpose: We previously identified glycogen synthase kinase-3 beta (Gsk3b) as a central member of a gene network highly regulated by acute ethanol in medial prefrontal cortex (mPFC) and associated with risk for alcohol dependence in humans. Further, we have demonstrated modulation of Gsk3b alters ethanol consumption in rodent models. GSK3B could thus represent a potential new therapeutic target for the treatment of alcohol use disorder (AUD). Here, we investigate the mechanisms of Gsk3b action in ethanol consumption and report preclinical evidence for the selective GSK3B inhibitor, tideglusib, as a therapeutic agent for AUD. Methods: (1) Selective Cre-induced Gsk3b deletion in Camk2a-neurons within the forebrain using transgenic Camk2a-CreER/Gsk3b floxed mice bred with Gsk3b fl/fl mice to produce Cre/Gsk3b fl/fl mice, which were injected with tamoxifen to induce Gsk3b deletion or (2) selective pharmacological antagonism of GSK3B using Tideglusib delivered via gavage in a corn oil vehicle. Actions on drinking behavior were measured using mouse intermittent ethanol, two-bottle choice self-administration models in C57BL/6J mice. Results: Deletion of Gsk3b in Camk2a-neurons decreased ethanol consumption and preference. There was no significant effects of sex or sex*genotype on either consumption or preference, so sexes were pooled. Gsk3b deletion did not alter basal locomotor activity, anxiety-like behavior (light-dark box), taste preference for quinine or saccharin, or ethanol pharmacokinetics. Initial administration of tideglusib (100mg/kg twice daily) or corn oil vehicle via gavage decreased total fluid consumption in all groups, regardless of ethanol drinking history or tideglusib treatment. However, following prolonged tideglusib, mice decreased binge (2hr) and daily (24hr) ethanol consumption and preference after three weeks of administration relative to vehicle controls. Tideglusib studies were only performed in male mice. Control studies showed no effect of tideglusib on liver fat accumulation in ethanol consuming animals. Ongoing work is assessing alternative oral tideglusib delivery methods in decreasing ethanol consumption. Conclusion: These results suggest GSK3B may be a therapeutic target for treatment of AUD. Deletion of Gsk3b in forebrain Camk2a-neurons showed a regional and cell-type specificity in GSK3B’s modulation of ethanol consumption and preference, providing insight into the mechanisms of Gsk3b action in ethanol consumption. Targeting GSK3B using tideglusib, a selective GSK3B inhibitor, also produced a decrease in ethanol consumption and preference over water during the fourth week of treatment. These findings were consistent with previous work in our lab investigating the delivery of tideglusib through intraperitoneal injections, though these studies were limited to a shorter drug-administration period. Here we have used a more therapeutically translatable route of administration via oral gavage and begun to investigate the longer-term effects of tideglusib on ethanol behaviors and toxicity. Tideglusib is a clinically available agent that warrants investigation in the treatment of AUD. Supported by NIAAA grants P50AA022537 and R01AA027581.https://scholarscompass.vcu.edu/gradposters/1161/thumbnail.jp

Time-Course Analysis of Brain Regional Expression Network Responses to Chronic Intermittent Ethanol and Withdrawal: Implications for Mechanisms Underlying Excessive Ethanol Consumption

Long lasting abusive consumption, dependence, and withdrawal are characteristic features of alcohol use disorders (AUD). Mechanistically, persistent changes in gene expression are hypothesized to contribute to brain adaptations leading to ethanol toxicity and AUD. We employed repeated chronic intermittent ethanol (CIE) exposure by vapor chamber as a mouse model to simulate the cycles of ethanol exposure and withdrawal commonly seen with AUD. This model has been shown to induce progressive ethanol consumption in rodents. Brain CIE-responsive expression networks were identified by microarray analysis across five regions of the mesolimbic dopamine system and extended amygdala with tissue harvested from 0-hours to 7-days following CIE. Weighted Gene Correlated Network Analysis (WGCNA) was used to identify gene networks over-represented for CIE-induced temporal expression changes across brain regions. Differential gene expression analysis showed that long-lasting gene regulation occurred 7-days after the final cycle of ethanol exposure only in prefrontal cortex (PFC) and hippocampus. Across all brain regions, however, ethanol-responsive expression changes occurred mainly within the first 8-hours after removal from ethanol. Bioinformatics analysis showed that neuroinflammatory responses were seen across multiple brain regions at early time-points, whereas co-expression modules related to neuroplasticity, chromatin remodeling, and neurodevelopment were seen at later time-points and in specific brain regions (PFC or HPC). In PFC a module containing Bdnf was identified as highly CIE responsive in a biphasic manner, with peak changes at 0 hours and 5 days following CIE, suggesting a possible role in mechanisms underlying long-term molecular and behavioral response to CIE. Bioinformatics analysis of this network and several other modules identified Let-7 family microRNAs as potential regulators of gene expression changes induced by CIE. Our results suggest a complex temporal and regional pattern of widespread gene network responses involving neuroinflammatory and neuroplasticity related genes as contributing to physiological and behavioral responses to chronic ethanol

Nonlinear Competition Between Small and Large Hexagonal Patterns

Recent experiments by Kudrolli, Pier and Gollub on surface waves, parametrically excited by two-frequency forcing, show a transition from a small hexagonal standing wave pattern to a triangular ``superlattice'' pattern. We show that generically the hexagons and the superlattice wave patterns bifurcate simultaneously from the flat surface state as the forcing amplitude is increased, and that the experimentally-observed transition can be described by considering a low-dimensional bifurcation problem. A number of predictions come out of this general analysis.Comment: 4 pages, RevTex, revised, to appear in Phys. Rev. Let

Effects of the Selective GSK3B Inhibitor, Tideglusib, on Ethanol Consumption, Anxiety-like Behavior, Taste Preference, and Downstream Proteins

Background: We have shown modulations in glycogen synthase kinase 3 beta (GSK3B) abundance or activity regulate ethanol consumption, suggesting potential as a therapeutic target for alcohol use disorder (AUD). Here we report the GSK3B inhibitor tideglusib’s actions on ethanol consumption, basal behaviors, and modulation of GSK3B targets. Methods: C57BL/6J males and females received i.g. 200mg/kg tideglusib, except drinking-in-the-dark (males;100mg/kg i.p.). Drinking-in-the-dark (DID): Mice given 20% ethanol 4-hours, 4-days/week x 3 weeks and then i.p. tideglusib or vehicle x 4 days in a Latin Square design with ethanol consumption measured daily. Light/Dark Box: Mice gavaged with tideglusib or vehicle and i.p. injected with 1.8g/kg ethanol or saline then tested for 10-min. Taste Preference: Mice received tideglusib x 6 days and then tested daily for saccharin or quinine taste preference. Western Blots: Mice received tideglusib or vehicle i.g. 3x/week for 2-weeks and mPFC assayed for phosphorylated and total GSK3B, Dynamin1, and PSD-95. Results: Tideglusib decreased ethanol DID consumption, transiently increased locomotion, and had no effect on anxiety-like behaviors or taste preference. Only total Dynamin1 showed tideglusib-induced modulation where females had increased Dynamin1 and decreased pDynamin1/total Dynamin1. Conclusion: Tideglusib is a promising AUD therapeutic, rapidly decreasing ethanol consumption in a binge-drinking model. Tideglusib is likely not reducing consumption by altering taste or anxiety-like behaviors. Dynamin1 is integral in activity-dependent bulk endocytosis and requires GSK3B-induced rephosphorylation. Tideglusib increased Dynamin1 levels likely represent a compensatory response to decreased GSK3B activity, providing insight to tideglusib’s mechanism in ethanol behaviors. Funded by NIAAA grant R01AA027581.https://scholarscompass.vcu.edu/gradposters/1177/thumbnail.jp

The neural correlates of emotion regulation by implementation intentions

Several studies have investigated the neural basis of effortful emotion regulation (ER) but the neural basis of automatic ER has been less comprehensively explored. The present study investigated the neural basis of automatic ER supported by ‘implementation intentions’. 40 healthy participants underwent fMRI while viewing emotion-eliciting images and used either a previously-taught effortful ER strategy, in the form of a goal intention (e.g., try to take a detached perspective), or a more automatic ER strategy, in the form of an implementation intention (e.g., “If I see something disgusting, then I will think these are just pixels on the screen!”), to regulate their emotional response. Whereas goal intention ER strategies were associated with activation of brain areas previously reported to be involved in effortful ER (including dorsolateral prefrontal cortex), ER strategies based on an implementation intention strategy were associated with activation of right inferior frontal gyrus and ventro-parietal cortex, which may reflect the attentional control processes automatically captured by the cue for action contained within the implementation intention. Goal intentions were also associated with less effective modulation of left amygdala, supporting the increased efficacy of ER under implementation intention instructions, which showed coupling of orbitofrontal cortex and amygdala. The findings support previous behavioural studies in suggesting that forming an implementation intention enables people to enact goal-directed responses with less effort and more efficiency

Hepatic Endothelial CCL25 Mediates the Recruitment of CCR9+ Gut-homing Lymphocytes to the Liver in Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433–1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150–157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates α4β7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD