Variation at candidate gene loci and their fuctional importance in rodent models of ethanol dependence

Abstract: As genetic research has progressed, psychiatric disorders, including the addictions have emerged as moderately heritable. For alcoholism, the overall heritability has been estimated from twin studies to range between 50 to 60%. In spite of efforts to identify candidate genes for alcoholism, thus far only very few genes have been consistently replicated. This thesis work was aimed at identifying and characterizing novel candidate genes using genetic rat models of alcoholism. An initial study applied oligonucleotide DNA based microarrays to identify genes that are differentially expressed in key brain regions for addiction (i.e. nucleus accumbens, amygdala, hippocampus and medial prefrontal cortex) of alcohol-preferring AA rats compared to non-preferring ANA rats. The main finding of this study was differential expression of a cluster of genes involved in the mitogen-activated protein kinase signaling pathways and the G-protein coupled receptor regulator, beta-arrestin 2 (ARRB2). In the next study we followed up on the latter finding. Elevated expression and genetic variation of the ARRB2 gene was found in the striatum and the hippocampus of AA rats compared to the ANA line. The increased expression was associated with a 7-marker haplotype in complete linkage dysequilibrium, which segregated fully between the lines, and was unique to the preferring line. These findings were functionally validated using mice lacking ARRB2, which displayed both reduced voluntary alcohol consumption and alcohol induced psychomotor stimulation. The results suggest that ARRB2 modulates acute responses to alcohol and is an important mediator of alcohol reward. Next, gene expression in the medial prefrontal cortex of AA and ANA rats was assessed. Microarray analysis revealed that glutathione S-transferase alpha 4 (GSTA4) expression levels were increased in the AA line compared to the ANA line. The results suggested that ANA rats seem to be impaired in metabolic pathways that may limit their capacity to metabolize alcohol, compromise their defense against reactive oxygen species and reduce longevity. Finally, corticotrophin releasing hormone receptor subtype 1 (CRHR1) expression was analyzed in search of genes underlying the phenotype of alcohol-preferring msP rats. Increased CRHR1 mRNA levels were observed in several brain regions of msP rats, including the amygdala, compared to non-preferring Wistar rats. This finding was accompanied by genetic variation at the CRHR1 locus and increased sensitivity to reinstatement of alcohol following environmental stress. Taken together, the results presented here shows the combined use of genetic models and high throughput screening is a viable approach for identifying novel candidate genes for alcoholism. In addition, CRHR1 finding also suggests that this strategy can be used to discover genes that exert their influence in a gene x environment dependent manner.