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Green T.A.,Center for Addiction Research | Labate D.,University of Houston | Laezza F.,Center for Addiction Research | Laezza F.,Center for Biomedical Engineering | And 2 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2015

Background Phosphorylation plays an essential role in regulating voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. Results We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulates Nav channel function via its C-terminal tail. General significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders. © 2015 Published by Elsevier B.V.


Shavkunov A.S.,301 University Blvd | Wildburger N.C.,301 University Blvd | Wildburger N.C.,Sealy Center for Cancer Cell Biology | Nenov M.N.,301 University Blvd | And 10 more authors.
Journal of Biological Chemistry | Year: 2013

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na+ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14- dependent regulation of Na+ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novelGSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


PubMed | University of Texas Health Science Center at Houston and Center for Addiction Research
Type: | Journal: Addiction biology | Year: 2016

Cocaine use disorder is a chronic relapsing condition characterized by compulsive drug seeking and taking even after prolonged abstinence periods. Subsequent exposure to drug-associated cues can promote intense craving and lead to relapse in abstinent humans and rodent models. The responsiveness to these cocaine-related cues, or cue reactivity, can trigger relapse and cocaine-seeking behaviors; cue reactivity is measurable in cocaine-dependent humans as well as rodent models. Cue reactivity is thought to be predictive of cocaine craving and relapse. Here we report that PPAR agonism during abstinence from cocaine self-administration reduced previously active lever pressing in Sprague Dawley rats during cue-reactivity tests, while administration of the PPAR antagonist, GW9662, reversed this effect. PPAR agonism also normalized nuclear ERK activity in the medial prefrontal cortex and hippocampus which was reversed with GW9662. Our results support the utility of PPAR agonism as a relapse prevention strategy to maintain abstinence in the presence of cocaine-associated cues.


DeBeck K.,St Pauls Hospital | Kerr T.,St Pauls Hospital | Kerr T.,University of British Columbia | Bird L.,Western Aboriginal Harm Reduction Society | And 9 more authors.
Drug and Alcohol Dependence | Year: 2011

Background: Vancouver, Canada has a pilot supervised injecting facility (SIF), where individuals can inject pre-obtained drugs under the supervision of medical staff. There has been concern that the program may facilitate ongoing drug use and delay entry into addiction treatment. Methods: We used Cox regression to examine factors associated with the time to the cessation of injecting, for a minimum of 6 months, among a random sample of individuals recruited from within the Vancouver SIF. In further analyses, we evaluated the time to enrolment in addiction treatment. Results: Between December 2003 and June 2006, 1090 participants were recruited. In Cox regression, factors independently associated with drug use cessation included use of methadone maintenance therapy (Adjusted Hazard Ratio [AHR] = 1.57 [95% Confidence Interval [CI]: 1.02-2.40]) and other addiction treatment (AHR = 1.85 [95% CI: 1.06-3.24]). In subsequent analyses, factors independently associated with the initiation of addiction treatment included: regular SIF use at baseline (AHR = 1.33 [95% CI: 1.04-1.72]); having contact with the addiction counselor within the SIF (AHR = 1.54 [95% CI: 1.13-2.08]); and Aboriginal ancestry (AHR = 0.66 [95% CI: 0.47-0.92]). Conclusions: While the role of addiction treatment in promoting injection cessation has been well described, these data indicate a potential role of SIF in promoting increased uptake of addiction treatment and subsequent injection cessation. The finding that Aboriginal persons were less likely to enroll in addiction treatment is consistent with prior reports and demonstrates the need for novel and culturally appropriate drug treatment approaches for this population. © 2010 Elsevier Ireland Ltd.


Hernandez C.M.,University of Texas Medical Branch | Cortez I.,University of Texas Medical Branch | Gu Z.,U.S. National Institutes of Health | Colon-Saez J.O.,U.S. National Institutes of Health | And 6 more authors.
Journal of Physiology | Year: 2014

There is much interest in α7 nicotinic acetylcholine receptors (nAChRs) in CNS function since they are found throughout peripheral tissues as well as being highly expressed in brain regions implicated in attention, learning and memory. As such, the role of these receptors in many aspects of CNS function and disease is being actively investigated. To date, only one null mouse model (A7KO) is available which is non-conditional and constitutive. Since α7 nAChRs are present on neurons and glia (including astrocytes), as well as being developmentally regulated, there is an unmet need for the technical capability to control α7 nAChR gene expression. Therefore we have generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (B6-Chrna7LBDEx4007Ehs) which we refer to as floxed α7 nAChR conditional knockout or α7nAChRflox. We validated the chosen approach by mating α7nAChRflox with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChRflox, GFAP-A7KO and appropriate littermate controls performed equally in our standard Rodent In Vivo Assessment Core battery to assess general health, locomotion, emotional and cognitive behaviours. Neither α7nAChRflox nor GFAP-A7KO exhibited significant differences from littermate controls in any of the baseline behavioural assessments we conducted, similar to the 'first generation' non-conditional A7KO mice. We also determined that α7 nAChR binding sites were absent on GFAP-positive astrocytes in hippocampal slices obtained from GFAP-A7KO offspring from α7nAChRflox and GFAP-Cre crosses. Finally, we validated that Cre recombinase (Cre)-mediated excision led to functional, cell- and tissue-specific loss of α7 nAChRs by demonstrating that choline-induced α7 nAChR currents were present in Cre-negative, but not synapsin promoter-driven Cre-positive, CA1 pyramidal neurons. Additionally, electrophysiological characterization of α7 nAChR-mediated current traces was similar in terms of amplitude and time constants of decay (during desensitization) for the α7nAChRflox and wild-type (WT) mice. Thus, we have in vivo and in vitro evidence that the Chrna7 exon 4 targeting strategy does not alter behavioural, cognitive, or electrophysiological properties compared to WT and that Cre-mediated excision is an effective approach to delete α7 nAChR expression in a cell-specific manner. © 2014 The Physiological Society.


Semmler A.,University of Zürich | Heese P.,LVR Clinic Bonn | Stoffel-Wagner B.,University of Bonn | Muschler M.,Center for Addiction Research | And 9 more authors.
Alcohol | Year: 2015

Recent studies have shown that smoking and alcoholism may be associated with altered DNA methylation and that alcohol consumption might induce changes in DNA methylation by altering homocysteine metabolism. In this monocenter study, we included 363 consecutive patients referred for hospitalization for alcohol detoxification treatment. Blood samples were obtained on treatment days 1, 3, and 7 for measurement of global DNA methylation in leukocytes by liquid chromatography tandem mass spectrometry. Genomic DNA was used for genotyping the following seven genetic variants of homocysteine metabolism: cystathionine beta-synthase (CBS) c.844_855ins68, dihydrofolate-reductase (DHFR) c.594+59del19bp, methylenetetrahydrofolate-reductase (MTHFR) c.677C>T and c.1298A>C, methyltetrahydrofolate-transferase (MTR) c.2756A>G, reduced folate carrier 1 (RFC1) c.80G>A, and transcobalamin 2 c.776C>G. Multivariate linear regression showed a positive correlation of global DNA methylation with alcohol consumption and smoking on day 1 of hospitalization. DNA methylation was not correlated with homocysteine or vitamin plasma levels, nor with the tested genetic variants of homocysteine metabolism. This suggests a direct effect of alcohol consumption and smoking on DNA methylation, which is not mediated by effects of alcohol on homocysteine metabolism. © 2015 Elsevier Inc..


Anastasio N.C.,Center for Addiction Research | Anastasio N.C.,University of Texas Medical Branch | Stoffel E.C.,Center for Addiction Research | Stoffel E.C.,University of Texas Medical Branch | And 9 more authors.
Behavioural Pharmacology | Year: 2011

Alterations in the balance of functional activity within the serotonin [5-hydroxytryptamine (5-HT)] system are hypothesized to underlie impulse control. Cocaine-dependent subjects consistently show greater impulsivity relative to nondrug using control subjects. Preclinical studies suggest that the 5-HT2A receptor (5-HT2AR) contributes to the regulation of impulsive behavior and also mediates some of the behavioral effects of cocaine. We hypothesized that the selective 5-HT2AR antagonist M100907 would reduce inherent levels of impulsivity and attenuate impulsive responding induced by cocaine in two animal models of impulsivity, the differential reinforcement of low rate (DRL) task and the one-choice serial reaction time (1-CSRT) task. M100907 reduced rates of responding in the DRL task and premature responding in the 1-CSRT task. Conversely, cocaine disrupted rates of responding in the DRL task and increased premature responding in the 1-CSRT task. M100907 attenuated cocaine-induced increases in specific markers of behavioral disinhibition in the DRL and 1-CSRT tasks. These results suggest that the 5-HT2AR regulates inherent impulsivity, and that blockade of the 5-HT2AR alleviates specific aspects of elevated levels of impulsivity induced by cocaine exposure. These data point to the 5-HT 2AR as an important regulatory substrate in impulse control. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.


PubMed | University of Zürich, University of Bonn, Center for Addiction Research and LVR Clinic Bonn
Type: Journal Article | Journal: Alcohol (Fayetteville, N.Y.) | Year: 2015

Recent studies have shown that smoking and alcoholism may be associated with altered DNA methylation and that alcohol consumption might induce changes in DNA methylation by altering homocysteine metabolism. In this monocenter study, we included 363 consecutive patients referred for hospitalization for alcohol detoxification treatment. Blood samples were obtained on treatment days 1, 3, and 7 for measurement of global DNA methylation in leukocytes by liquid chromatography tandem mass spectrometry. Genomic DNA was used for genotyping the following seven genetic variants of homocysteine metabolism: cystathionine beta-synthase (CBS) c.844_855ins68, dihydrofolate-reductase (DHFR) c.594+59del19bp, methylenetetrahydrofolate-reductase (MTHFR) c.677C>T and c.1298A>C, methyltetrahydrofolate-transferase (MTR) c.2756A>G, reduced folate carrier 1 (RFC1) c.80G>A, and transcobalamin 2 c.776C>G. Multivariate linear regression showed a positive correlation of global DNA methylation with alcohol consumption and smoking on day 1 of hospitalization. DNA methylation was not correlated with homocysteine or vitamin plasma levels, nor with the tested genetic variants of homocysteine metabolism. This suggests a direct effect of alcohol consumption and smoking on DNA methylation, which is not mediated by effects of alcohol on homocysteine metabolism.


PubMed | University of Milan Bicocca and University of Texas Medical Branch
Type: | Journal: Neurotoxicology | Year: 2016

Pyrethroid insecticides are the most popular class of insecticides in the world, despite their near-ubiquity, their effects of delaying the onset of inactivation of voltage-gated sodium (NaHere we compare the well-studied NaWe used patch-clamp electrophysiology to record sodium currents encoded by either NaWe found that exposure of both isoforms to 10M deltamethrin significantly potentiated persistent and tail current densities without affecting peak transient current densities, and only NaCollectively, these data provide evidence that NaThese findings identify Na


Wilhelm J.,Center for Addiction Research | Wilhelm J.,Friedrich - Alexander - University, Erlangen - Nuremberg | Heberlein A.,Center for Addiction Research | Heberlein A.,Friedrich - Alexander - University, Erlangen - Nuremberg | And 10 more authors.
Alcoholism: Clinical and Experimental Research | Year: 2011

Background: Prolactin serum levels have been described to be elevated during alcohol withdrawal in alcohol-dependent patients and normalize during abstinence. Alterations in prolactin levels may reflect disturbances of dopaminergic neurotransmission which is of crucial importance for alcohol-seeking behavior. Methods: In this longitudinal observational study, we investigated prolactin serum levels in 99 male patients during the first 14days of alcohol withdrawal and early abstinence and in 43 healthy controls. To assess the severity of alcohol dependence, the extent of withdrawal symptoms, craving, depressive symptoms, and anxiety, we employed a structured interview including psychologic measurements. Results: Prolactin serum levels were elevated during the whole study period in alcohol-dependent patients compared to the healthy control group. Prolactin levels at admission (first day of alcohol withdrawal) were associated with the severity of alcohol withdrawal (CIWA-Ar) and of alcohol dependence (SESA) but not with the other assessed psychologic parameters. Conclusions: The presented findings confirm that prolactin is significantly elevated in alcohol-dependent patients during alcohol withdrawal and early abstinence, not showing a rapid decline after cessation of drinking. The association with the severity of withdrawal and dependence may reflect at least partially the individual alterations in the dopaminergic and glutamatergic pathways. © 2010 by the Research Society on Alcoholism.

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