Peter A. Wilce

Egr transcription factors in the nervous system

The Egr proteins, Egr-1, Egr-2, Egr-3 and Egr-4, are closely related members of a subclass of immediate early gene-encoded, inducible transcription factors. They share a highly homologous DNA-binding domain which recognises an identical DNA response element. In addition, they have several less-well conserved structural features in common. As immediate early proteins, the Egr transcription factors are rapidly induced by diverse extracellular stimuli within the nervous system in a discretely controlled manner. The basal expression of the Egr proteins in the developing and adult rat brain and the induction of Egr proteins by neurotransmitter analogue stimulation, physiological mimetic and brain injury paradigms is reviewed. We review evidence indicating that Egr proteins are subject to tight differential control through diverse mechanisms at several levels of regulation. These include transcriptional, translational and post-translational (including glycosylation, phosphorylation and redox) mechanisms and protein-protein interaction. Ultimately the differentially co-ordinated Egr response may lead to discrete effects on target gene expression. Some of the known target genes of Egr proteins and functions of the Egr proteins in different cell types are also highlighted. Future directions for research into the control and function of the different Egr proteins are also explored.

Glutamate-mediated transmission, alcohol, and alcoholism

Glutamate-mediated neurotransmission may be involved in the range of adaptive changes in brain which occur after ethanol administration in laboratory animals, and in chronic alcoholism in human cases. Excitatory amino acid transmission is modulated by a complex system of receptors and other effectors, the efficacy of which can be profoundly affected by altered gene or protein expression. Local variations in receptor composition may underlie intrinsic regional variations in susceptibility to pathological change. Equally, ethanol use and abuse may bring about alterations in receptor subunit expression as the essence of the adaptive response. Such considerations may underlie the regional localization characteristic of the pathogenesis of alcoholic brain damage, or they may form part of the homeostatic change that constitutes the neural substrate for alcohol dependence.

Alcohol‐responsive genes in the frontal cortex and nucleus accumbens of human alcoholics

The molecular processes underlying alcohol dependence are not fully understood. Many characteristic behaviours result from neuroadaptations in the mesocorticolimbic system. In addition, alcoholism is associated with a distinct neuropathology. To elucidate the molecular basis of these features, we compared the RNA expression profile of the nucleus accumbens and prefrontal cortex of human brain from matched individual alcoholic and control cases using cDNA microarrays. Approximately 6% of genes with a marked alcohol response were common to the two brain regions. Alcohol-responsive genes were grouped into 11 functional categories. Predominant alcohol-responsive genes in the prefrontal cortex were those encoding DNA-binding proteins including transcription factors and repair proteins. There was also a down-regulation of genes encoding mitochondrial proteins, which could result in disrupted mitochondrial function and energy production leading to oxidative stress. Other alcohol-responsive genes in the prefrontal cortex were associated with neuroprotection/apoptosis. In contrast, in the nucleus accumbens, alcohol-responsive genes were associated with vesicle formation and regulation of cell architecture, which suggests a neuroadaptation to chronic alcohol exposure at the level of synaptic structure and function. Our data are in keeping with the previously reported alcoholism-related pathology characteristic of the prefrontal cortex, but suggest a persistent decrease in neurotransmission and changes in plasticity in the nucleus accumbens of the alcoholic.

Alcohol-responsive genes in the frontal cortex and nucleus accumbens of human alcoholics: Alcohol responsive genes in human brain

The molecular processes underlying alcohol dependence are not fully understood. Many characteristic behaviours result from neuroadaptations in the mesocorticolimbic system. In addition, alcoholism is associated with a distinct neuropathology. To elucidate the molecular basis of these features, we compared the RNA expression profile of the nucleus accumbens and prefrontal cortex of human brain from matched individual alcoholic and control cases using cDNA microarrays. Approximately 6% of genes with a marked alcohol response were common to the two brain regions. Alcohol‐responsive genes were grouped into 11 functional categories. Predominant alcohol‐responsive genes in the prefrontal cortex were those encoding DNA‐binding proteins including transcription factors and repair proteins. There was also a down‐regulation of genes encoding mitochondrial proteins, which could result in disrupted mitochondrial function and energy production leading to oxidative stress. Other alcohol‐responsive genes in the prefrontal cortex were associated with neuroprotection/apoptosis. In contrast, in the nucleus accumbens, alcohol‐responsive genes were associated with vesicle formation and regulation of cell architecture, which suggests a neuroadaptation to chronic alcohol exposure at the level of synaptic structure and function. Our data are in keeping with the previously reported alcoholism‐related pathology characteristic of the prefrontal cortex, but suggest a persistent decrease in neurotransmission and changes in plasticity in the nucleus accumbens of the alcoholic.

Increased NMDA-induced excitability during ethanol withdrawal: a behavioural and histological study

Intrahippocampal injections of N-methyl-D-aspartic acid (NMDA) leads to neurodegeneration in a dose-dependent manner. Chronic administration of ethanol to animals leads to CNS tolerance and dependence. Hyperexcitability following ethanol withdrawal is thought to be related to increased sensitivity of the NMDA receptors. The purpose of this study was to investigate this predisposition to hyperexcitability by intrahippocampal injection of low dose of NMDA. Using control and ethanol-withdrawn male Wistar rats, behavioural indices were determined immediately after injection and morphological damage was assessed after a period of recovery. There was significantly increased hyperactivity in the ethanol-treated rats immediately after injection. Morphological damage resulting from 5 nmol of NMDA was significantly greater in the CA3 region of the hippocampus in these animals. These data support the hypothesis that ethanol dependence and subsequent withdrawal is associated with increased sensitivity to NMDA which may underlie ethanol withdrawal-associated brain damage.

Increased sensitivity of the hippocampus in ethanol-dependent rats to toxic effect of N-methyl-D-aspartic acid in vivo

Chronic administration of ethanol in animals leads to CNS tolerance and physical dependence. Subsequent withdrawal of ethanol causes hyperexcitability which is thought to be related to increased sensitivity of N-methyl-D-aspartic acid (NMDA) receptors. The purpose of this study was to investigate sensitivity to NMDA in ethanol-treated animals by detecting damage after intrahippocampal injection of NMDA. Choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) specific activity was used as markers of cholinergic and gamma-aminobutyric acid neurons, respectively. Ethanol-dependent animals were more liable to die following intrahippocampal injection of either 120 or 240 nmol of NMDA. There was a significantly greater decrease in hippocampal GAD but not ChAT specific activity in the surviving animals. These data support the hypothesis that ethanol dependence is associated with increased sensitivity to NMDA which may be responsible for excitotoxic brain damage and death.

Increased TUNEL positive cells in human alcoholic brains

Alcohol-sensitive neuronal cell loss, which has been reported in the superior frontal cortex and hippocampus, may underlie the pathogenesis of subsequent cognitive deficits. In the present study, we have used the TUNEL labeling to detect the DNA damage in human alcoholic brains. Seven out of eleven alcoholics exhibited TUNEL-positive cells in both superior frontal cortex and hippocampus, which were co-localized with GFAP immunoreactivity. In contrast, almost no positive cells were detected in the non-alcoholic controls. None of the TUNEL-positive cells showed any typical morphological features of apoptosis or necrosis. TUNEL-positive cells observed in the present study may indicate DNA damage induced by ethanol-related overproduction of reactive oxygen species.

Modification of proteins and other biological molecules by acetaldehyde: Adduct structure and functional significance

1. Chronic ethanol consumption is a major cause of liver disease. The modification of hepatic proteins by acetaldehyde (AcH), the primary metabolite of ethanol, has for some time been suggested as one of the major events initiating alcoholic liver disease. 2. These alterations in protein structure are believed to affect liver cell function, and may serve to activate the immune system. 3. This review considers the interaction between AcH and macromolecules and its functional implications.

Antibodies against acetaldehyde-modified epitopes: an elevated IgA response in alcoholics

Abstract. Several recent reports have shown that antibodies reactive with acetaldehyde (AcH)‐modified epitopes are present in alcoholics. However, similar antibodies have also been found in patients with nonalcoholic liver disease and control subjects. In each of these studies total immunoglobulin binding to the AcH‐modified proteins was measured, with no attempt being made to identify the classes of immunoglobulin involved. In the present study we employed an enzyme‐linked immunosorbent assay (ELISA) to assess the classes of immunoglobulin involved in this response, using plasma samples from 97 alcoholics with varying degrees of liver disease, 35 patients with non‐alcoholic liver disease and 33 control subjects. All three groups exhibited a large IgM response and a negligible IgG response. However, the alcoholics exhibited a significantly higher IgA response than either of the other groups. This suggests that the measurement of the IgA response to AcH‐modified epitopes may be a specific marker of ethanol abuse.

Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis

Elevated concentrations of plasma tumour necrosis factor (TNF)‐α, interleukin (IL)‐1 and IL‐6 have been detected in patients with alcoholic hepatitis and have been implicated in the pathogenesis of hepatocyte necrosis. The present study used a rat model to conduct a detailed histological and biochemical examination of the expression of various pro‐inflammatory cytokines and associated liver pathology in ethanol‐potentiated lipopolysaccharide (LPS)‐induced liver injury. Male Wistar rats were pair‐fed either the control or ethanol‐containing (36% of caloric intake as ethanol) form of the Lieber‐DeCarli liquid diet for 6 weeks. Liver injury was induced by the i.v. injection of LPS (1 μg/g bodyweight), with animals being killed at 0, 1, 3, 6, 12 and 24 h after injection. At the later time points, plasma transaminase and transpeptidase activities were significantly elevated in ethanol‐fed LPS‐treated rats compared with control‐fed LPS‐treated animals. At these times after LPS treatment, hepatocytes in ethanol‐fed animals displayed fatty change and necrosis with an associated neutrophil polymorph infiltrate. Time course analysis revealed that plasma TNF‐μ (1–3 h post‐LPS) and IL‐6 (3 h post‐LPS) bioactivity was significantly elevated in ethanol‐fed compared with control‐fed animals. No difference was seen in plasma IL‐1μ concentration (maximal in both groups 6 h post‐LPS). The expression of TNF‐μ, IL‐1μ, IL‐1β and IL‐6 mRNA were elevated between 1 and 6 h post‐LPS in the livers of both control and ethanol‐fed rats. However, ethanol‐fed LPS‐treated animals exhibited significantly higher maximal expression of IL‐1 and IL‐6 mRNA. Comparison of the appearance of cytokine mRNA and plasma bioactivity indicated an effect of ethanol feeding on post‐transcriptional processing and/or the kinetics of the circulating cytokines. Elevated levels of both hepatic cytokine mRNA expression and the preceding plasma cytokines are presumably a necessary prerequisite for hepatic injury seen in this model and, therefore, possibly for the damage seen in human alcoholics. Further studies using this model may lead to significant advances in our understanding of the pathogenic mechanisms of alcoholic liver disease in humans.