J. M. Lewohl

Patterns of gene expression are altered in the frontal and motor cortices of human alcoholics

Alcoholism is a major health problem in Western countries, yet relatively little is known about the mechanisms by which chronic alcohol abuse causes the pathologic changes associated with the disease. It is likely that chronic alcoholism affects a number of signaling cascades and transcription factors, which in turn result in distinct gene expression patterns. These patterns are difficult to detect by traditional experiments measuring a few mRNAs at a time, but are well suited to microarray analyses. We used cDNA microarrays to analyze expression of approximately 10 000 genes in the frontal and motor cortices of three groups of chronic alcoholic and matched control cases. A functional hierarchy was devised for classification of brain genes and the resulting groups were compared based on differential expression. Comparison of gene expression patterns in these brain regions revealed a selective reprogramming of gene expression in distinct functional groups. The most pronounced differences were found in myelin‐related genes and genes involved in protein trafficking. Significant changes in the expression of known alcohol‐responsive genes, and genes involved in calcium, cAMP, and thyroid signaling pathways were also identified. These results suggest that multiple pathways may be important for neuropathology and altered neuronal function observed in alcoholism.

G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action

G-protein-coupled inwardly rectifying potassium channels (GIRKs) are important for regulation of synaptic transmission and neuronal firing rates. Because of their key role in brain function, we asked if these potassium channels are targets of alcohol action. Ethanol enhanced function of cerebellar granule cell GIRKs coupled to GABAB receptors. Enhancement of GIRK function by ethanol was studied in detail using Xenopus oocytes expressing homomeric or heteromeric channels. Function of all GIRK channels was enhanced by intoxicating concentrations of ethanol, but other, related inwardly rectifying potassium channels were not affected. GIRK2/IRK1 chimeras and GIRK2 truncation mutants were used to identify a region of 43 amino acids in the carboxyl (C) terminus that is critical for the action of ethanol on these channels.

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT‐PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post‐mortem. However, their abundance and integrity can exhibit marked intra‐ and intercase variability, making comparisons between case‐groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante‐mortem status, agonal state and post‐mortem and post‐autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante‐ and post‐mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

Patterns of gene expression in the frontal cortex discriminate alcoholic from nonalcoholic individuals.

Alcohol dependence is characterized by tolerance, physical dependence, and craving. The neuroadaptations underlying these effects of chronic alcohol abuse are likely due to altered gene expression. Previous gene expression studies using human post-mortem brain demonstrated that several gene families were altered by alcohol abuse. However, most of these changes in gene expression were small. It is not clear if gene expression profiles have sufficient power to discriminate control from alcoholic individuals and how consistent gene expression changes are when a relatively large sample size is examined. In the present study, microarray analysis (∼47 000 elements) was performed on the superior frontal cortex of 27 individual human cases (14 well characterized alcoholics and 13 matched controls). A partial least squares statistical procedure was applied to identify genes with altered expression levels in alcoholics. We found that genes involved in myelination, ubiquitination, apoptosis, cell adhesion, neurogenesis, and neural disease showed altered expression levels. Importantly, genes involved in neurodegenerative diseases such as Alzheimers disease were significantly altered suggesting a link between alcoholism and other neurodegenerative conditions. A total of 27 genes identified in this study were previously shown to be changed by alcohol abuse in previous studies of human post-mortem brain. These results revealed a consistent re-programming of gene expression in alcohol abusers that reliably discriminates alcoholic from non-alcoholic individuals.

Gene expression profiling of individual cases reveals consistent transcriptional changes in alcoholic human brain

Chronic alcohol exposure induces lasting behavioral changes, tolerance, and dependence. This results, at least partially, from neural adaptations at a cellular level. Previous genome‐wide gene expression studies using pooled human brain samples showed that alcohol abuse causes widespread changes in the pattern of gene expression in the frontal and motor cortices of human brain. Because these studies used pooled samples, they could not determine variability between different individuals. In the present study, we profiled gene expression levels of 14 postmortem human brains (seven controls and seven alcoholic cases) using cDNA microarrays (46 448 clones per array). Both frontal cortex and motor cortex brain regions were studied. The list of genes differentially expressed confirms and extends previous studies of alcohol responsive genes. Genes identified as differentially expressed in two brain regions fell generally into similar functional groups, including metabolism, immune response, cell survival, cell communication, signal transduction and energy production. Importantly, hierarchical clustering of differentially expressed genes accurately distinguished between control and alcoholic cases, particularly in the frontal cortex.

Up-regulation of MicroRNAs in Brain of Human Alcoholics

BACKGROUND MicroRNAs (miRNAs) are small, noncoding oligonucleotides with an important role in posttranscriptional regulation of gene expression at the level of translation and mRNA degradation. Recent studies have revealed that miRNAs play important roles in a variety of biological processes, such as cell proliferation, neuronal differentiation, developmental timing, synapse function, and neurogenesis. A single miRNA can target hundreds of mRNA transcripts for either translation repression or degradation, but the function of many human miRNAs is not known. METHODS miRNA array analysis was performed on the prefrontal cortex of 27 individual human cases (14 alcoholics and 13 matched controls). Target genes for differentially expressed miRNAs were predicted using multiple target prediction algorithms and a consensus approach, and predicted targets were matched against differentially expressed mRNAs from the same samples. Over- and under-representation analysis was performed using hypergeometric probability and z-score tests. RESULTS Approximately 35 miRNAs were significantly up-regulated in the alcoholic group compared with controls. Target prediction showed a large degree of overlap with our published cDNA microarray data. Functional classification of the predicted target genes of the regulated miRNAs includes apoptosis, cell cycle, cell adhesion, nervous system development, and cell-cell signaling. CONCLUSIONS These data suggest that the reduced expression of genes in human alcoholic cases may be because of the up-regulated miRNAs. Cellular processes fundamental to neuronal plasticity appear to represent major targets of the suggested miRNA regulation.

Expression of the α1, α2 and α3 isoforms of the GABAA receptor in human alcoholic brain

The expression of the α1, α2 and α3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the α class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patients age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because α1 expression was raised in both groups of alcoholics cf controls. The two groups of alcoholics could be differentiated on the basis of regional variations in α1 expression. In frontal cortex, α1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, α1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform.

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.

Differential effects of general anesthetics on G-protein coupled inwardly rectifying and other potassium channels

BackgroundGeneral anesthetics differentially affect various families of potassium channels, and some potassium channels are suggested to be potential targets for anesthetics and alcohols. MethodsThe voltage-gated (ERG1, ELK1, and KCNQ2/3) and inwardly rectifying (GIRK1/2, GIRK1/4, GIRK2, IRK1, and ROMK1) potassium channels were expressed in Xenopus oocytes. Effects of volatile agents [halothane, isoflurane, enflurane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and the structurally related nonimmobilizer F6 (1,2-dichlorohexafluoro-cyclobutane)], as well as intravenous (pentobarbital, propofol, etomidate, alphaxalone, ketamine), and gaseous (nitrous oxide) anesthetics and alcohols (ethanol and hexanol) on channel function were studied using a two-electrode voltage clamp. ResultsERG1, ELK1, and KCNQ2/3 channels were either inhibited slightly or unaffected by concentrations corresponding to twice the minimum alveolar concentrations or twice the anesthetic EC50 of volatile and intravenous anesthetics and alcohols. In contrast, G protein–coupled inwardly rectifying potassium (GIRK) channels were inhibited by volatile anesthetics but not by intravenous anesthetics. The neuronal-type GIRK1/2 channels were inhibited by 2 minimum alveolar concentrations of halothane or F3 by 45 and 81%, respectively, whereas the cardiac-type GIRK1/4 channels were inhibited only by F3. Conversely, IRK1 and ROMK1 channels were completely resistant to all anesthetics tested. Current responses of GIRK2 channels activated by &mgr;-opioid receptors were also inhibited by halothane. Nitrous oxide (∼0.6 atmosphere) slightly but selectively potentiated GIRK channels. Results of chimeric and multiple amino acid mutations suggest that the region containing the transmembrane domains, but not the pore-forming domain, may be involved in determining differences in anesthetic sensitivity between GIRK and IRK channels. ConclusionsG protein–coupled inwardly rectifying potassium channels, especially those composed of GIRK2 subunits, were inhibited by clinical concentrations of volatile anesthetics. This action may be related to some side effects of these agents.

Gene Expression in Brain: A Window on Ethanol Dependence, Neuroadaptation, and Preference

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.