D. J. Innes

Association Studies of Neurotransmitter Gene Polymorphisms in Alcoholic Caucasians

Abstract: Ethanol enhances mesolimbic/cortical dopamine activity in reward and reinforcement circuits. We investigated the hypothesis that risk for alcoholism may be mediated by genes for neurotransmitters associated with the dopamine reward system as well as genes for enzymes involved in ethanol metabolism. DNA was extracted from brain tissue collected at autopsy from pathologically characterized alcoholics and controls. PCR‐based assays showed that alcoholism was associated with polymorphisms of the dopamine D2 receptor (DRD2) TaqI B (P= .029) and the GABAA‐β2 subunit C1412T (P= .012) genes, but not with the glutamate receptor subunit gene NMDAR2B (366C/G), the serotonin transporter gene (5HTTL‐PR), the dopamine transporter gene DAT1(SLC6A3), the dopamine D2 receptor gene DRD2 TaqI A, or the GABAAα1(A15G), α6(T1519C), and γ2(G3145A) subunit genes. The glial glutamate transporter gene EAAT2 polymorphism G603A was associated with alcoholic cirrhosis (P= .048). The genotype for the most active alcohol dehydrogenase enzyme ADH1C was associated with a lower risk of alcoholism (P= .026) and was less prevalent in alcoholics with DRD2TaqIA2/A2 (P= .047), GABAA‐β2 1412C/C (P= .01), or EAAT2 603G/A (P= .022) genotypes. Combined DRD2TaqI A or B with GABAA‐β2 or EAAT2 G603A genotypes may have a concerted influence in the predisposition to alcoholism.

The application of proteomics to the human alcoholic brain

Abstract: Alcoholism results in changes in the human brain that reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of proteins in key cells and brain areas. Described here is a proteomics‐based approach aimed at determining the identity of proteins in the superior frontal cortex (SFC) of the human brain that show different levels of expression in autopsy samples taken from healthy and long‐term alcohol abuse subjects. Soluble protein fractions constituting pooled samples combined from SFC biopsies of four well‐characterized chronic alcoholics (mean consumption > 80 g ethanol/day throughout adulthood) and four matched controls (<20 g/day) were generated. Two‐dimensional electrophoresis was performed in triplicate on alcoholic and control samples and the resultant protein profiles analyzed for differential expression. Overall, 182 proteins differed by the criterion of twofold or more between case and control samples. Of these, 139 showed significantly lower expression in alcoholics, 35 showed significantly higher expression, and 8 were new or had disappeared. To date, 63 proteins have been identified using MALDI‐MS and MS‐MS. The finding that the expression level of differentially expressed proteins is preponderantly lower in the alcoholic brain is supported by recent results from parallel studies using microarray mRNA transcript.

The expression of NMDA receptor subunit mRNA in human chronic alcoholics.

Ethanol is a modulator at the N‐methyl‐d‐aspartate class of glutamate receptors in the brain. In animal studies the receptor adapts to sustained ethanol exposure through altered expression of the subunits that make up the receptor complex. We used real‐time RT‐PCR normalized to GAPDH to assay NR1, NR2A, and NR2B subunit mRNA in superior frontal and primary motor cortex tissue obtained at autopsy from chronic alcoholics with and without co‐morbid cirrhosis of the liver, and from matched controls. The expression of all three subunits was significantly lower in both areas of cirrhotic alcoholics than in the corresponding areas in both controls and alcoholics without co‐morbid disease, who did not differ significantly from each other. The decrease was area‐dependent when cases were partitioned by the 5‐HTTLPR allele. Thus, polymorphisms in one gene can have a significant effect on the expression of a second, unrelated, gene. The expression of the N‐methyl‐d‐aspartate glutamate receptor complex is under multifactorial control.

Analysis of multiple exon-skipping mRNA splice variants using SYBR Green real-time RT-PCR

Fluorescence-based PCR techniques are becoming an increasingly popular method for measuring low-abundance alternatively spliced mRNA transcripts. The dynamic range of real-time RT-PCR affords high sensitivity for the measurement of gene expression, but this mandates the need for strict controls to ensure assay validity. Primer design, reverse transcription, and cycling conditions need to be optimized to ensure an accurate and reproducible assay. Here, we describe a procedure for creating a cost effective and reliable method for the absolute quantification of several exon-skipping variants of human excitatory amino acid transporter-2 (EAAT2). We show that the cycling conditions can be adjusted to increase the specificity of primers that span exon-exon junctions, and that differences in the reverse transcription reaction can be minimized. Standard curves are stable and produce accurate absolute copy number data. We report that exon-skipping transcripts, EAAT2Delta7 and EAAT2Delta9, account for 5.8% of EAAT2 mRNA in autopsy human neocortex.

GABAA receptor β isoform protein expression in human alcoholic brain : interaction with genotype

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex. Reduced GABA transmission may mediate this. The expression of GABA(A) receptor beta(1), beta(2), and beta(3) isoform proteins was analyzed by western blotting in vulnerable (superior frontal cortex) and spared (primary motor cortex) cortical tissue obtained at autopsy from Caucasian subjects, and the effect of genotypes of candidate genes for alcoholism assessed. There was a significant regional difference in global isoform expression, but no significant overall group difference in beta(2) or beta(3)expression between controls and alcoholics undifferentiated by genotype in either cortical region. There were significant, regionally selective, interactions of DRD2B, SLC1A2 and APOE genotypes with beta protein expression when alcoholics were compared with controls. In each instance possession of the alcoholism-associated allele increased the beta(2):beta(3) ratio in the pathologically vulnerable region, by two distinct mechanisms. The SFC beta(2):beta(3) ratio in DRD2B-B2,B2 alcoholics was 22% higher than that in DRD2B-B1,B1 alcoholics, and 17% higher than that in DRD2B-B2,B2 controls. The SFC beta(2):beta(3) ratio in SLC1A2A603 homozygote alcoholics was 25% higher than that in alcoholics with at least one 603G allele, and 75% higher than that in SLC1A2A603 homozygote controls. The SFC beta(2):beta(3) ratio in alcoholics lacking an APOE epsilon3 allele was 73% higher than that in alcoholics with at least one epsilon3 allele, and 70% higher than that in controls without an epsilon3 allele. ADH1C genotype also differentiated cases and controls, but the effect was not localized. GABRB2 and GRIN2B genotypes were associated with significant regional differences in the pattern of beta subunit expression, but this was not influenced by alcoholism status. DRD2A and SLC6A4 genotypes were without significant effect. A restricted set of genotypes may influence subunit expression in this group of high-consumption alcoholics.

Genes and Gene Expression in the Brains of Human Alcoholics

Abstract:  Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex (SFC). Propensity to alcoholism is associated with several genes. γ‐Aminobutyric acid (GABA)A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the regional presentation of GABAA and glutamate‐NMDA (N‐methyl‐d‐aspartate) receptors in SFC. Autopsy tissue was obtained from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and matched controls. ADH1C, DRD2B, EAAT2, and APOE genotypes modulated GABAA‐β subunit protein expression in SFC toward a less‐effective form of the receptor. Most genotypes did not divide alcoholics and controls on glutamate‐NMDA receptor pharmacology, although gender and cirrhosis did. Genotype may affect amino acid transmission locally to influence neuronal vulnerability.

Genetic map of mango: a tool for mango breeding

Mango (Mangifera indica) is an economically and nutritionally important tropical/subtropical tree fruit crop. Most of the current commercial cultivars are selections rather than the products of breeding programs. To improve the efficiency of mango breeding, molecular markers have been used to create a consensus genetic map that identifies all 20 linkage groups in seven mapping populations. Polyembryony is an important mango trait, used for clonal propagation of cultivars and rootstocks. In polyembryonic mango cultivars, in addition to a zygotic embryo, several apomictic embryos develop from maternal tissue surrounding the fertilized egg cell. This trait has been associated with linkage group 8 in our consensus genetic map and has been validated in two of the seven mapping populations. In addition, we have observed a significant association between trait and single nucleotide polymorphism (SNP) markers for the vegetative trait of branch habit and the fruit traits of bloom, ground skin color, blush intensity, beak shape, and pulp color.

Genetic modulation of amino acid transmitter receptor subunit expression in alcoholic human brain

Early P MRS studies using a novel superfusion system demonstrated that cortical slices respond differently to the two principal elements of ischaemic challenge, hypoxia and hypoglycaemia. In the former, ATP levels are maintained as phosphocreatine (PCr) levels fall, whereas in the latter ATP and PCr levels fall in concert. In both cases P saturation transfer NMR demonstrates an increase in the creatine kinase mediated exchange between ATP and PCr. The use of F NMR in conjunction with the selective calcium chelator 5FBAPTA enables levels of intracellular Ca to be determined in the same preparation. Neither hypoxia nor hypoglycaemia lead to an increase in intracellular Ca whereas in combination a large increase is observed. Previous exposure to the combination of hypoxia and hypoglycaemia (ischaemic pre-conditioning) abolishes the increase in intracellular Ca during subsequent ischaemic challenge. Interestingly, excitotoxic challenge both increases intracellular Ca and releases endogenous Zn . C MRS has demonstrated that, in severe hypoxia, glycerol 3-phosphate is produced as an alternative to the regeneration of NAD by conversion of pyruvate to lactate. C MRS has also proved an ideal tool to study neuronal/glial metabolic interactions in model systems and in man. An increase in TCA cycle rate on visual activation of about 50% has been found similar to the increases in CMRglc reported in PET studies, but suggests in contrast to these studies, that cerebral glucose is metabolised oxidatively. The data have also been used to determine the rate of the glutamate/glutamine cycle via which much of the neurotransmitter glutamate is recycled. Such measurements will be substantially improved at higher fields (7T and up). PL2 Magnetic resonance neurospectroscopy, from test tube to clinic

Proteomic analysis of excitatory amino acid transporter 2 isoforms in Alzheimer's disease

therefore provide multiple inroads for the analysis of the GABAergic contributions to the regulation of behaviour. Drugs which selectively modulate GABA-A receptor function, such as benzodiazepines, have been uniquely helpful in analysing the role of GABAergic transmission in brain function. By rendering particular GABA-A receptors benzodiazepine-insensitive by a point mutation, the contribution of the respective neuronal ensemble to the drug-induced behaviour can be determined Ann.Rev.Pharmacol.Toxicol. pp. 475(2004). Thus, the genetic dissection of GABA-A receptor pathways provides new insights into the regulation of complex behaviour in particular for sedation Nature 401, 796(1999), Nature Neurosci. 3, 587(2000), anxiolytic activity Nature 401, 796(1999), Science 290, 131(2000), Proc. Natl. Acad. Sci. USA 100, 15218(2003), anaesthetic drug actions FASEB J.17, 250(2003), memory performance Proc. Natl. Acad. Sci. USA 99, 8980(2002), J.Neurosci. 22, 5572(2002), and also provides new views on the control of brain development as shown for visual cortical plasticity Science 303, 1681(2004). PL4 THE INHIBITED BRAIN IN ACTION Mohler, H. 1 Institute of Pharmacology, University of Zurich, Zurich, Switzerland 2 Swiss Federal Institute of Technology (ETH), Zurich, Switzerland

Phenotype/genotype studies in alcohol misuse

Methamphetamine has been a most popular drug in Japan after the World War II, and estimated methamphetamine abusers are more than 200,000. Many lines of evidence from twin and family studies have clearly showed that genetic factors play major roles in susceptibility of substance-related disorders, including methamphetamine. To assess individual genetic risk factors for methamphetamine use disorders, JGIDA (Japanese Genetics Initiative for Drug Abuse), a multi-center collaboration group, has been established in 2001. To date, JGIDA have examined case-control association of more than 100 genes of diverse molecules, e.g. several neurotransmitter receptors and transporters, ion channels, enzymes, nerve growth factors, cell adhesion, neurotoxicity-related molecule and candidate genes for schizophrenia. JGIDA studies have confirmed several genetic risk or negative risk factors for methamphetamine dependence and psychosis. For example, a certain genetic variant of the CYP2D6, metabolizing enzyme of methamphetamine at the first step, Frizzled-3, a Wnt signal receptor, and dysbindin (DTNBP1) gene produce susceptibility to methamphetamine dependence and psychosis. In addition, genetic variants of dopamine D2 receptors, dopamine transporters, COMT and MAO-A genes affect prognosis and some clinical phenotypes of methamphetamine psychosis. JGIDA studies have revealed that many genes are involved in regulation of individual susceptibility or resistance to methamphetamine dependence and psychosis, and individual variation of those prognosis and clinical phenotypes.