A. L. Eckert

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.

Expression and distribution of GABAA receptor subtypes in human alcoholic cerebral cortex

Long‐term alcohol abuse is known to target specific areas of the brain such as the superior frontal cortex (SFC), resulting in neuronal cell loss. Abnormal transmission of the inhibitory neurotransmitter GABA may contribute to this damage. Previous work in our laboratory has found differential expression and distribution of certain α subunit genes of the GABAA receptor in the SFC of human alcoholic brain, suggesting that differences in GABAA receptor subunit expression could give rise to the locally altered GABAA pharmacology which is associated with alcohol abuse. A competitive RT‐PCR assay has been developed to study the expression of the GABAA receptor β‐subunit genes β1, β2, and β3. A single set of primers homologous to all three β isoform sequences has been shown to amplify each of the β isoforms from mRNA isolated from human brain tissue obtained at autopsy. An internal standard has been designed which is identical to the target except for a 61‐bp deletion and a unique restriction enzyme (RE) site. This is co‐amplified with the target sequences to allow amplification efficiency to be assessed and thus enable the quantitation of gene expression. A range of GABAA receptor ligands were used to look at differential distribution of receptor subtypes in the cortical laminae by autoradiography. Differences in distribution of the ligands were demonstrated, consistent with a hypothesis of alcohol‐induced variations in the expression of receptor subunits.

Comorbidity and genotype modify receptor expression in human alcoholics

RODGER BROUGH: Next we have a joint presentation Coralie Ober who currently works as a research fellow, Queensland Alcohol and Drug Research and Education Centre at the University of Queensland School of Population Health. She has a very broad experience in clinical nursing through to teaching, advocacy and advisory roles. Her work with Indigenous communities is widely recognised inside and outside this country and her role with the WHO is testimony to that.Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic aetiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelae to the liver damage.

The effect of genotype on receptor expression in human alcoholic brain

trafficking of APP into either the aor b-secretase compartments (statins and cholesterol, for example) may also prove of value. Downstream, pathways which promote the degradation of Ab (insulin degrading enzyme and neprolysin) or modulate its clearance from the brain (immunization with native or modified forms of Ab) also offer windows for therapeutic opportunity. At the center, however, lies the mechanism through which Ab undergoes a toxic gain-of-function to induce neuronal damage. Logically, this should provide the most direct route for therapeutic intervention, with the least risk of therapeutic sideeffects, since the toxicity of Ab is unlikely to mimic any normal function. Two principal hypotheses have emerged to explain the toxicity of Ab: redox chemistry associated with the Cu/Zn metal binding sites on Ab (His 6, 13/14, Tyr 10, with an influence of Met 35) and lipid interactions associated with the a/b conformation of the hydrophobic Cterminus. Drugs targeting either or both of these mechanisms are now in clinical development with encouraging preliminary results. PL3 ALZHEIMER’S DISEASE: Ab-AMYLOID AS THE MOLECULAR THERAPEUTIC AND DIAGNOSTIC TARGET Masters, C.L. Department of Pathology and the Mental Health Research Institute, The University of Melbourne, Melbourne, AustraliaSevere long-term alcohol misuse leads to localized brain damage that is prominent in superior frontal cortex but less so in other cortical areas e.g. primary motor. Alcohol dependence is also associated with several genetic markers. GABAA receptor expression differs selectively between alcoholics and controls in a manner that conforms to the pathology, whereas glutamate receptors are much less regionally variable in these subjects. We determined whether genotype differentiated the pharmacology of glutamate-NMDA receptors and the expression GABAA receptor subunits transcripts in a locally appropriate way so as to influence the severity of alcohol-induced brain damage.