The application of proteomics to the human alcoholic brain

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.

Genetic polymorphisms in post-mortem alcoholics

We investigated the hypothesis that alcoholism risk may be mediated by genes for neurotransmitters (dopamine, serotonin, opioid, GABAA and glutamate) associated with the dopamine reward system, and with genes involved in ethanol metabolism and fibrogenesis (ADH2, ADH3, ALDH2, CYP2E1, COL1A2, and ApoE). DNA was extracted from brain tissue collected at autopsy from pathologically characterised alcoholics and controls. PCR-based studies showed that alcoholism was associated with polymorphisms of the dopamine D2 receptor (DRD2) Taq1 B (p 0.005) and the GABAA 2 subunit C1412T (p 0.007) genes but not with the glutamate receptor subunit gene NR2B (366C/G), the serotonin transporter gene (5HTTL-PR), the dopamine transporter gene DAT1(SLC6A3), the Mu opioid receptor gene MOR1 (A118G and C1031G), the dopamine D2 receptor gene DRD2 Taq1 A or the GABAA 1(A15G), 6(T1519C) and 2(G3145A) subunit genes. The glial glutamate transporter gene EAAT2 polymorphism G603A was associated with alcoholic cirrhosis (p 0.024). The genotype for the most active alcohol dehydrogenase ADH3 was associated with a lower risk of alcoholism (p 0.027) and was less prevalent in alcoholics with DRD2 Taq1 A2/A2 (p 0.007), Taq1 B2/B2 (p 0.038) and GABAA-2 1412C/C (p 0.005) and EAAT2 603G/A (p 0.020) genotypes. Combined genotypes of DRD2 Taq1 A and B, GABAA-2, and EAAT2 G603A polymorphisms suggested a concerted influence of dopamine, GABAA and glutamatergic neurotransmitters in the predisposition to alcoholism.

The effect of genotype on receptor expression in human alcoholic brain

Severe 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.

EAAT2 splice variants in Alzheimer disease

Regional atrophy caused by neuronal loss is a characteristic of Alzheimer Disease (AD). Excitatory amino acid transporter-2 (EAAT2) is the major carrier responsible for clearing glutamate from the synaptic cleft in mammalian CNS. A localized attenuation of glutamate transport via reduced expression of functional forms of EAAT2 might contribute to regional excitotoxicity. The EAAT2 gene spans over 100 kb and encodes a 12-kb message. Several groups have identified alternative splice variants of EAAT2 in human brain tissue. These variants can affect transport by altering wild-type EAAT2 protein expression, localization, or transport efficiency. Alternative EAAT2 mRNA transcripts reportedly elicit a dominant-negative effect on glutamate uptake in cell culture. A 50% reduction in the expression in AD cortex of the truncated EAAT2 C-terminal isoform, EAAT2b, has been reported. We obtained cerebral cortex tissue, under informed written consent from the next of kin, from pathologically confirmed control, AD, and non-AD dementia cases. We aimed to determine the distribution and expression patterns of EAAT2 subtypes in susceptible and spared brain regions. We detected five alternate transcripts of EAAT2, two of which had not previously been reported. The relative contributions of novel variants, wild-type EAAT2, and previously discovered splice variants was investigated using Real-time PCR in AD, non-AD dementia, and age-matched control cortex. Our aim is to survey the relationship between these expression patterns and those of markers such as tau, GFAP, and b-amyloid, and to assess the correlation between variant-transporter expression and the level of cell loss.