Tiebing Liang

α-Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats

Total gene expression analysis (TOGA) was used to identify genes that are differentially expressed in brain regions between the alcohol-naïve, inbred alcohol-preferring (iP), and -nonpreferring (iNP) rats. α-Synuclein, expressed at >2-fold higher levels in the hippocampus of the iP than the iNP rat, was prioritized for further study. In situ hybridization was used to determine specific brain regions and cells expressing α-synuclein in the iP and iNP rats. Similar to α-synuclein mRNA levels, protein levels in the hippocampus were higher in iP rats than iNP rats. Higher protein levels were also observed in the caudate putamen of iP rats compared with iNP rats. Sequence analysis identified two single nucleotide polymorphisms in the 3′ UTR of the cDNA. The polymorphism was used to map the gene, by using recombination-based methods, to chromosome 4, within a quantitative trait locus for alcohol consumption that was identified in the iP and iNP rats. A nucleotide exchange in the iNP 3′ UTR reduced expression of the luciferase reporter gene in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the α-synuclein gene may contribute to alcohol preference in the iP rats.

Gene expression changes in the nucleus accumbens of alcohol-preferring rats following chronic ethanol consumption.

The objective of this study was to determine the effects of binge-like alcohol drinking on gene expression changes in the nucleus accumbens (ACB) of alcohol-preferring (P) rats. Adult male P rats were given ethanol under multiple scheduled access (MSA; three 1-h dark cycle sessions/day) conditions for 8 weeks. For comparison purposes, a second ethanol drinking group was given continuous/daily alcohol access (CA; 24h/day). A third group was ethanol-naïve (W group). Average ethanol intakes for the CA and MSA groups were approximately 9.5 and 6.5 g/kg/day, respectively. Fifteen hours after the last drinking episode, rats were euthanized, the brains extracted, and the ACB dissected. RNA was extracted and purified for microarray analysis. The only significant differences were between the CA and W groups (p<0.01; Storey false discovery rate=0.15); there were 374 differences in named genes between these 2 groups. There were 20 significant Gene Ontology (GO) categories, which included negative regulation of protein kinase activity, anti-apoptosis, and regulation of G-protein coupled receptor signaling. Ingenuity analysis indicated a network of transcription factors, involving oncogenes (Fos, Jun, Junb had higher expression in the ACB of the CA group), suggesting increased neuronal activity. There were 43 genes located within rat QTLs for alcohol consumption and preference; 4 of these genes (Tgfa, Hspa5, Mtus1 and Creb3l2) are involved in anti-apoptosis and increased transcription, suggesting that they may be contributing to cellular protection and maintaining high alcohol intakes. Overall, these findings suggest that chronic CA drinking results in genomic changes that can be observed during the early acute phase of ethanol withdrawal. Conversely, chronic MSA drinking, with its associated protracted withdrawal periods, results in genomic changes that may be masked by tight regulation of these genes following repeated experiences of ethanol withdrawal.

Alteration of gene expression by alcohol exposure at early neurulation

BackgroundWe have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables.ResultAlcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos.ConclusionThis study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.

Neuropeptide Y Receptor Genes Are Associated with Alcohol Dependence, Alcohol Withdrawal Phenotypes and Cocaine Dependence

BACKGROUND Several lines of evidence in both human and animal studies suggest that variation in neuropeptide Y (NPY) or its receptor genes (NPY1R, NPY2R and NPY5R) is associated with alcohol dependence as well as alcohol withdrawal symptoms. Additional studies suggest that cocaine may affect NPY expression. METHODS A total of 39 single nucleotide polymorphisms (SNPs) were genotyped across NPY and its 3 receptor genes in a sample of 1,923 subjects from 219 multiplex alcoholic families of European American descent recruited as part of the Collaborative Studies on the Genetics of Alcoholism (COGA) study. Family-based association analysis was performed to test the primary hypothesis that variation in these genes is associated with alcohol dependence. Secondary analyses evaluated whether there was an association of these SNPs with symptoms of alcohol withdrawal, cocaine dependence, or comorbid alcohol and cocaine dependence. RESULTS Although variations in NPY itself were not associated with these phenotypes, variations in 2 NPY-receptor genes were. SNPs in NPY2R provided significant evidence of association with alcohol dependence, alcohol withdrawal symptoms, comorbid alcohol and cocaine dependence, and cocaine dependence (all p < 0.03). Haplotype analyses strengthened the evidence for these phenotypes (global 0.0004 < p < 0.005). SNPs in NPY5R demonstrated significant association with alcohol withdrawal characterized by seizures (p < 0.05). CONCLUSION These results indicate that sequence variations in NPY receptor genes are associated with alcohol dependence, particularly a severe subtype of alcohol dependence characterized by withdrawal symptoms, comorbid alcohol and cocaine dependence, and cocaine dependence.

Loss of metabotropic glutamate receptor 2 escalates alcohol consumption

Significance Gene identification for complex behavioral traits, alcoholism in particular, has been largely unsuccessful, in part because of the rarity of many causative variants and the heterogeneity and small effect size of the causal loci. Artificially selected animals may be valuable in gene identification. We identified a causal role of metabotropic glutamate receptor 2 (mGluR2) in altered alcohol preference via genomic sequencing of selectively bred rats, linkage analysis in the F2 rats, and function validation in mGluR2 null mice. Our findings represent a valuable contribution to understanding the neurobiology of alcoholism and a strategy for gene identification for complex behavior traits. Identification of genes influencing complex traits is hampered by genetic heterogeneity, the modest effect size of many alleles, and the likely involvement of rare and uncommon alleles. Etiologic complexity can be simplified in model organisms. By genomic sequencing, linkage analysis, and functional validation, we identified that genetic variation of Grm2, which encodes metabotropic glutamate receptor 2 (mGluR2), alters alcohol preference in animal models. Selectively bred alcohol-preferring (P) rats are homozygous for a Grm2 stop codon (Grm2 *407) that leads to largely uncompensated loss of mGluR2. mGluR2 receptor expression was absent, synaptic glutamate transmission was impaired, and expression of genes involved in synaptic function was altered. Grm2 *407 was linked to increased alcohol consumption and preference in F2 rats generated by intercrossing inbred P and nonpreferring rats. Pharmacologic blockade of mGluR2 escalated alcohol self-administration in Wistar rats, the parental strain of P and nonpreferring rats. The causal role of mGluR2 in altered alcohol preference was further supported by elevated alcohol consumption in Grm2 −/− mice. Together, these data point to mGluR2 as an origin of alcohol preference and a potential therapeutic target.

A Polymorphism in GABRA2 Is Associated With the Medial Frontal Response to Alcohol Cues in an fMRI Study

BACKGROUND Significant evidence has accumulated to suggest an association between single-nucleotide polymorphisms (SNPs) in the GABRA2 gene and alcoholism. However, research has yet to show an association between these polymorphisms and the human brains reward system function. In this study, we stratified subjects who had participated in an fMRI study of alcohol cue responses according to their genotype at a SNP in GABRA2 (rs279871) shown to be associated with alcohol dependence (Edenberg et al., 2004). METHODS Genotyping showed 13 subjects to be homozygous for the high-risk allele (AA), and 23 subjects to be heterozygous (AG). In fMRI, subjects were exposed to the aromas of their preferred alcoholic drink odors (AO), as well as to appetitive control odors (ApCO) under both alcohol intoxication and placebo control conditions. RESULTS Homozygous AA subjects had a larger [AO > ApCO] response than did AG subjects in medial frontal cortical areas thought to code reward value. However, AG subjects had a larger [AO > ApCO] effect in the ventral tegmental area. Alcohol intoxication did not alter these group differences. CONCLUSIONS These are the first data to suggest that GABRA2 genotype could affect the brains responses to cues associated with alcohol.

Evaluation of Aldehyde Dehydrogenase 1 Promoter Polymorphisms Identified in Human Populations

BACKGROUND Cytosolic aldehyde dehydrogenase, or ALDH1A1, functions in ethanol detoxification, metabolism of neurotransmitters, and synthesis of retinoic acid. Because the promoter region of a gene can influence gene expression, the ALDH1A1 promoter regions were studied to identify polymorphism, to assess their functional significance, and to determine whether they were associated with a risk for developing alcoholism. METHODS Sequence analysis was performed in the promoter region by using Asian, Caucasian, and African American subjects. The resulting polymorphisms were assessed for frequency in Asian, Caucasian, Jewish, and African American populations and tested for associations with alcohol dependence in Asian and African American populations of alcoholics and controls. The functional significance of each polymorphism was determined through in vitro expression analysis by using HeLa and HepG2 cells. RESULTS Two polymorphisms, a 17 base pair (bp) deletion (-416/-432) and a 3 bp insertion (-524), were discovered in the ALDH1A1 promoter region: ALDH1A1*2 and ALDH1A1*3, respectively. ALDH1A1*2 was observed at frequencies of 0.035, 0.023, 0.023, and 0.012 in the Asian, Caucasian, Jewish, and African American populations, respectively. ALDH1A1*3 was observed only in the African American population, at a frequency of 0.029. By using HeLa and HepG2 cells for in vitro expression, the activity of the luciferase reporter gene was significantly decreased after transient transfection of ALDH1A1*3-luciferase compared with the wild-type construct ALDH1A1*1-luciferase. In an African American population, a trend for higher frequencies of the ALDH1A1*2 and ALDH1A1*3 alleles was observed in a population of alcoholics (p = 0.03 and f = 0.12, respectively) compared with the control population. CONCLUSIONS ALDH1A1*2 and ALDH1A1*3 may influence ALDH1A1 gene expression. Both ALDH1A1*2 and ALDH1A1*3 produce a trend in an African American population that may be indicative of an association with alcoholism; however, more samples are required to validate this observation. The underlying mechanisms contributing to these trends are still unknown.

Associations of ALDH2 and ADH1B genotypes with alcohol-related phenotypes in Asian young adults.

BACKGROUND Associations of ALDH2 and ADH1B genotypes with alcohol use have been evaluated largely using case-control studies, which typically focus on adult samples and dichotomous diagnostic outcomes. Relatively fewer studies have evaluated ALDH2 and ADH1B in relation to continuous drinking outcomes or at different developmental stages. This study examined additive and interactive effects of ALDH2 and ADH1B genotypes on drinking behavior in a mixed-gender sample of Asian young adults, focusing on continuous phenotypes (e.g., heavy episodic and hazardous drinking, alcohol sensitivity, drinking consequences) whose expression is expected to precede the onset of alcohol use disorders. METHODS The sample included 182 Chinese- and Korean-American young adults ages 18 years and older (mean age = 20 years). Effects of ALDH2, ADH1B and ethnicity were estimated using generalized linear modeling. RESULTS The ALDH2*2 allele predicted lower reported rates of alcohol use and drinking consequences as well as greater reported sensitivity to alcohol. There were significant ethnic group differences in drinking outcomes, such that Korean ethnicity predicted higher drinking rates and lower alcohol sensitivity. ADH1B status was not significantly related to drinking outcomes. CONCLUSIONS Ethnicity and ALDH2 status, but not ADH1B status, consistently explained significant variance in alcohol consumption in this relatively young sample. Results extend previous work by showing an association of ALDH2 genotype with drinking consequences. Findings are discussed in the context of possible developmental and population differences in the influence of ALDH2 and ADH1B variations on alcohol-related phenotypes.

ADH and ALDH Polymorphisms and Alcohol Dependence in Mexican and Native Americans

Background: Ethanol is primarily metabolized in the liver by two rate-limiting reactions: conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) and subsequent conversion of acetaldehyde to acetate by aldehyde dehydrogenase (ALDH). ADH and ALDH exist in multiple isozymes that differ in their kinetic properties. Notably, polymorphisms within the genes that encode for these isozymes vary in their allele frequencies between ethnic groups, and thus, they have been considered as candidate genes that may differentially influence risk for the development of alcohol dependence across ethnic groups. Objectives and methods: Associations between alcohol dependence and polymorphisms in ADH1B, ADH1C, and ALDH2 were compared in a community sample of Native Americans (n 791) living on reservations and Mexican Americans (n 391) living within the same county. Results: Two Mexican Americans and no Native Americans possessed one ALDH2*2 allele. Presence of at least one ADH1B*2 allele was found in 7% of the Native Americans and 13% of the Mexican Americans, but was only associated with protection against alcohol dependence in the Mexican Americans. Presence of at least one ADH1B*3 allele was found in 4% of the Native Americans and 2% of the Mexican Americans, but was associated with protection against alcohol dependence only in the Native Americans. No associations between alcohol dependence and polymorphisms in ADH1C were found. Conclusions and Scientific Significance: Polymorphisms in ADH1B are protective against alcoholism in these two populations; however, these findings do not explain the high prevalence of alcoholism in these populations.

ALDH2, ADH1B and alcohol expectancies: Integrating genetic and learning perspectives.

The present study evaluated associations of ALDH2 and ADH1B genotypes with alcohol expectancies and drinking behavior in a sample of Asian American young adults. In addition to assessing global alcohol expectancies, the authors developed a measure of physiological expectancies to evaluate an expectancy phenotype specific to the mechanism by which ALDH2 and ADH1B variations presumably influence drinking behavior. Compared with individuals with the ALDH2*1/*1 genotype, those with the ALDH2*2 allele reported greater negative alcohol expectancies, greater expectancies for physiological effects of alcohol and lower rates of alcohol use. ADH1B was not associated with alcohol expectancies or drinking behavior. Hierarchical models showed that demographic factors, ALDH2 genotype, and expectancy variables explained unique variance in drinking outcomes. Mediation tests showed significant indirect effects of ALDH2 on drinking frequency and peak lifetime consumption through expectancies. These results provide support for influences of genetic factors and alcohol sensitivity on alcohol-related learning and suggest the importance of developing biopsychosocial models of drinking behavior in Asian Americans.