RSA 2005 Abstracts

RSA 2005 Abstracts

UNIVERSITY OF COLORADO HEALTH SCIENCES CENTER

ASSOCIATION OF ADENYLATE CYCLASE TYPE VII WITH FAMILIAL DEPRESSION: A HAPLOTYPE ANALYSIS

L. Hines; A.L. Kaiser; B. Gonchig; R. Anton; B. Tabakoff; on behalf of the WHO/ISBRA Study on State and Trait Markers of Alcohol Use and Dependence Investigators

University of Colorado Health Sciences Center, Denver, CO 80262

Alcoholism is often associated with comorbid affective disorders, such as major depression. Both are complex disorders attributed to the interaction of genetic and environmental factors. Previous studies have shown that certain measures of platelet adenylyl cyclase activity are lower in alcohol dependent subjects and in subjects diagnosed with major depression. The type VII isoform of adenylyl cyclase (AC7) is a major isoform in platelets. The human AC 7 gene (ADCY7) has a tetranucleotide repeat polymorphism (AACA) in the 3’ untranslated region. The 7-repeat polymorphism (ADCY7R7) was previously reported to be associated with the phenotype of familial depression (major depression in the subject and in a first-degree relative) in Caucasian subjects from Montreal, Canada, who were recruited as part of the WHO/ISBRA Study on State and Trait Markers of Alcohol Use and Dependence. The present study sought to further examine the relationship between ADCY7 and depression in the Caucasian Montreal population (n=497). Haplotype analysis was carried out in the region of ADCY7 on human chromosome 16. A total of 10 single nucleotide polymorphisms (SNPs) were selected based on the pattern of linkage disequilibrium blocks in the ADCY7 region, and covered a region of 330Kb. Using Haploview version 2.05, a haplotype block which included 2 SNPs and the tetranucleotide repeat (20Kb) was identified. This haplotype block was confirmed in an analysis of 841 Caucasian subjects from the Project MATCH study population, recruited from 9 different clinical research centers across the US. Haplotypes for the Montreal population were generated using PHASE version 2.1. Among the common haplotypes, the TG7 haplotype was overrepresented among individuals diagnosed with familial depression. The relative risk for individuals with this haplotype was 3.51 (P=0.03). The estimated risk was similar to that observed for the ADCY7R7 allele alone (3.31, P=0.02), suggesting a potentially functional link between the ADCY7R7 allele and familial depression. This association appeared to be stronger among females. Supported by NIAAA (U10AA08428, R.A.); University of Connecticut GCRC (M01RR061 92); and the Banbury Fund.

“MAGIC-B” IDENTIFIES CANDIDATE GENES FOR ALCOHOL PREFERENCE IN HAP AND LAP MICE

S.V. Bhave; N. Grahame; P.L. Hoffman; B. Tabakoff

University of Colorado School of Medicine, Aurora, CO, 80010

The “MAGIC-B” (microarray analysis of genes involved in complex behaviors) (Tabakoff et al, J. Neurosci. 29:4491, 2003) procedure was used to identify candidate genes associated with alcohol preference. In this procedure, gene expression profiles are determined in brains of selected lines of mice to identify genes that are differentially expressed between the lines. The differentially expressed genes are localized to QTLs determined for the selected trait, providing a list of genes that are likely to contribute to the trait via differences in expression levels. HAP and LAP mice were selectively bred (Grahame et al, Behav. Gen. 29:47, 1999) for differences in alcohol preference based on two-bottle choice alcohol consumption. We analyzed gene expression differences in whole brain of mice from two replicate lines (n = 6 per line; per replicate) using Affymetrix whole genome oligonucleotide arrays. The array data were normalized, and gene expression was determined, using MAS 5 (Affymetrix) and robust microarray averaging (RMA). Differentially expressed genes were identified using two statistical analyses, i.e., permutation and the t-test noise distribution (Tabakoff et al., J. Neurosci. 29:4491, 2003). The chromosomal localization of genes found to be differentially expressed by all of these criteria, and in the same direction in both replicate lines of mice, was determined, and genes localized within QTLs determined for two-bottle choice alcohol preference measures (Belknap and Atkinson, Mamm. Genome 12:893, 2001) were identified. The known genes were assembled into signaling pathways using PathwayAssist (Stratagene). The results indicate candidate genes that influence differences in development and maintenance of dopamine signaling between the lines, as well as differences in opiate pathways (e.g., POMC) that may contribute to alcohol preference. Supported by the National Institute on Alcohol Abuse and Alcoholism, NIH (AA U01 13489 – INIA Project; AA R01 13162) and The Banbury Fund.

MODIFICATION OF ALCOHOL PREFERENCE -WITH TRANSPLANTATION OF STEM CELLS EXPRESSING THE HUMAN DOPAMINE TRANSPORTER INTO THE NUCLEUS ACCUMBENS OF MICE

S. M. Jones; T. Grammatopoulos; M. Yoshimura; B.R. Hoover; E. Snyder; N. R. Zahniser; B. Tabakoff; W.M. Zawada

University of Colorado Health Sciences Center, Denver, CO 80262 and The Burnham Institute, La Jolla, CA 92037

To determine whether dopamine (DA) levels in the mesolimbic system modulate the preference for alcohol, we have stably transfected C17.2 neural stems cells with the human dopamine transporter (hDAT) gene to generate the C17.hDAT stem cell line. We hypothesized that reduction of the extracellular DA levels via hDAT will alter alcohol preference. Several clones exhibited constitutive [3H]-DA uptake. A clone that showed good survival after transplantation into one-day-old C57BL/6 mouse pups, functional [3H]-DA uptake, and robust _-galactosidase expression was transplanted bilaterally into nucleus accumbens of adult C57BL/6 mice, one of the nuclei thought to affect ethanol preference. Prior to transplantation, mice were tested for ethanol preference using a two bottle choice paradigm. After 15 days of exposure, 90% of mice consumed more ethanol than water, with an average preference ratio of 0.77. Mice were split into three groups and transplanted with C17.hDAT cells, a mock-transfected C17.2 clone, or saline (sham transplant). After a 3-day recovery, mice were again tested for ethanol preference for an additional 7 days. Mice receiving the C17.hDAT clone exhibited 20 % decrease in the ethanol preference at the first time point tested (6 days after surgery), but their preference gradually increased to match that of the sham or mock transfected controls by the termination of the study. Surviving C17.hDAT cells were identified in the transplants by X-gal staining. These preliminary data suggest that transplantation of hDAT-expressing stem cells into brain nuclei known for their role in substance seeking should be useful for exploring the role of DA in alcohol dependence.
Supported by NIH, National Institute of Alcohol Abuse and Alcoholism, U01 AA13473 – INIA Project and National Institute of Drug Abuse DA 015050 and DA 016860

ROLE OF BRAIN ADENYLYL CYCLASE TYPE 7 IN AN ANIMAL MODEL OF DEPRESSION

L.D. Snell; M. Yoshimura; P.L. Hoffman; B. Tabakoff

Lohocla Research Corporation and University of Colorado Health Sciences Center, Aurora, CO 80010

In several large national surveys, alcohol dependence has been significantly associated with higher rates of anxiety and affective disorders such as major depression (comorbidity). The cyclic AMP/PKA/CREB signaling cascade has been linked to the etiology of anxiety, depression and alcoholism. Platelet adenylyl cyclase (AC) activity is lower in alcoholics and in depressed individuals than in controls, and a major isoform of AC in platelets is Type VII AC (AC7). We have developed transgenic mice (TG) expressing the human AC7 gene in brain and heterozygous knockout mice (HET) that lack one copy of the endogenous mouse AC7 gene, both on inbred C57BL/6 backgrounds. We have previously found that these mice show gender- and genotype-specific differences in anxiety, evaluated on the elevated plus-maze, compared to wild-type (WT) mice. Our current studies are examining the response of the TG and HET mice to “inescapable stress” in the Porsolt forced-swim animal model of depression. Mice were placed in a glass cylinder filled with water to a depth such that they were unable to touch the bottom and behavior was videotaped for 10 min. The duration of immobility, which was defined as floating in an upright position without any additional activity other than that necessary for the animal to keep its head above water, was rated from the videotapes by a trained observer who was blind to the genotype and gender of the animals. The duration of immobility has been interpreted as a measure of depression, i.e., more immobility reflects greater depression. We have found that female AC7 TG mice spend significantly less time immobile than their WT littermates (i.e., less depression in TG), while immobility durations were not significantly different in male AC7 TG mice compared to their WT littermates. These data indicate a gender-specific antidepressant-like effect of AC7 over-expression. We are currently evaluating the behavior of HET male and female mice in the forced-swim test. We are also currently using these mice to examine the role of AC7 in the antidepressant actions of neurotransmitter re-uptake inhibitors (fluoxetine and desipramine), and the cAMP phosphodiesterase type 4D inhibitor, rolipram. Supported by the National Institute on Alcohol Abuse and Alcoholism, NIH (AA U01 13489 – INIA Project) and The Banbury Fund.

TRANSCRIPTIONAL CONTROL AND BEHAVIORAL CHANGES IN SELECTIVELY BRED MICE

R. Lapadat; S.V. Bhave; B. Tabakoff; P.L. Hoffman; L. Hunter

University of Colorado Health Sciences Center, Department of Pharmacology, Aurora, CO, 80045

Comprehensive delineation of functional noncoding DNA sequences in complex genomes is a major goal of modern biology. The interaction between the cellular environment, genetic background and ethanol triggers neuroadaptive events manifested as behavioral changes. In this study we focus on understanding the transcriptional changes responsible for preference and acute functional tolerance to ethanol, respectively. By itself, gene expression profiling fails to identify control elements in the context of the effects of different genotypes on behavioral patterns. We describe a method combining promoter sequence similarity in differentially expressed genes, cross species transcription factor mapping and in silico signaling pathway and literature mining. We have added to the repertoire of our analytical techniques sRNA and miRNA binding prediction tools. Transcriptional profiles of whole brain extracts from mice selectively bred for either acute functional tolerance (HIGH/LOW) or ethanol preference (HIGH/LOW) were measured using Affymetrix microarrays. Differentially expressed genes were analyzed using a positional scoring matrix algorithm for the first two kilobases upstream regions to identify conserved sequence patterns. Independently, the same regions were analyzed for the most conserved transcription factor binding sites based on a cross-species model. Resulting predictions were used together with the differentially expressed genes as input for signal transduction pathways and literature mining in order to establish a model of the interaction network modulating the gene expression events. <BR><BR>The identified processes synthesize the integrated neuronal response of cells bearing different genotypic fingerprints, including signal transduction, transcriptional regulation, ion channel activity and neuronal activity modulation. This work strongly suggests that combining transcription control module discovery with interaction network data mining represent a powerful approach for cis-regulation of gene expression and the involvement of signal transduction mechanisms using high-throughput techniques.<BR>Acknowledgements: This research was supported by grants from the National Institute on Alcohol Abuse and Alcoholism, NIH (AA U01 13524 – INIA Project; AA R01 13162); and The Banbury Fund.

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