Alcohol protects the CNS by activating HSF1 and inducing the heat shock proteins.

Although alcohol abuse and dependence have profound negative health consequences, emerging evidence suggests that exposure to low/moderate concentrations of ethanol protects multiple organs and systems. In the CNS, moderate drinking decreases the risk of dementia and Alzheimer's disease. This neuroprotection correlates with an increased expression of the heat shock proteins (HSPs). Multiple epidemiological studies revealed an inverse association between ethanol intoxication and traumatic brain injury mortality. In this case, ethanol-induced HSPs limit the inflammatory immune response diminishing cell death and improving the neurobehavioural outcome. Ethanol also protects the brain against ischemic injuries via the HSPs. In our laboratory, we demonstrated that ethanol increased the expression of several HSP genes in neurons and astrocytes by activating the transcription factor, heat shock factor 1 (HSF1). HSF1 induces HSPs that target misfolded proteins for refolding or degradation, increasing the survival chances of the cells. These data indicate that ethanol neuroprotection is mediated by the activation HSF1 and the induction of HSPs.

Alcohol regulates gene expression in neurons via activation of heat shock factor 1.

Drinking alcohol causes widespread alterations in gene expression that can result in long-term physiological changes. Although many alcohol-responsive genes (ARGs) have been identified, the mechanisms by which alcohol alters transcription are not well understood. To elucidate these mechanisms, we investigated Gabra4, a neuron-specific gene that is rapidly and robustly activated by alcohol (10-60 mM), both in vitro and in vivo. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs. The activation of Gabra4 by alcohol or high temperature is dependent on the binding of heat shock factor 1 (HSF1) to a short downstream DNA sequence, the alcohol response element (ARE). Alcohol and heat stimulate the translocation of HSF1 from the cytoplasm to the nucleus and the induction of HSF1-dependent genes, Hsp70 and Hsp90, in cultured neurons and in the mouse cerebral cortex in vivo. The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol.

GABAA receptors in the thalamus: alpha4 subunit expression and alcohol sensitivity.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) has long been implicated in the anxiolytic, amnesic, and sedative behavioral effects of alcohol. A large number of studies have investigated the interactions of alcohol with GABA receptors. Many investigators have reported effects of "high concentrations" (50-100 mM) of alcohol on GABA-mediated synaptic inhibition, but effects of the "low concentrations" (1-30 mM) of alcohol normally associated with mild intoxication have been elusive until recently. A novel form of "tonic inhibition" has been described in the central nervous system (CNS) that is generated by the persistent activation of extrasynaptic gamma-aminobutyric acid type A receptors (GABAA-Rs). These receptors are specific GABAA-R subtypes and distinct from the synaptic subtypes. Tonic inhibition regulates the excitability of individual neurons and the activity and rhythmicity of neural networks. Interestingly, several reports show that tonic inhibition is sensitive to low concentrations of alcohol. The thalamus is a structure that is critically important in the control of sleep and wakefulness. GABAergic inhibition in the thalamus plays a crucial role in the generation of sleep waves. Among the various GABAA-R subunits, the alpha1, alpha4, beta2, and delta subunits are heavily expressed in thalamic relay nuclei. Tonic inhibition has been demonstrated in thalamocortical relay neurons, where it is mediated by alpha4beta2delta GABAA-Rs. These extrasynaptic receptors are highly sensitive to gaboxadol, a novel hypnotic, but insensitive to benzodiazepines. Tonic inhibition is absent in thalamic relay neurons from alpha4 knockout mice, as are the sedative and analgesic effects of gaboxadol. The sedative effects of alcohol can promote sleep. However, alcohol also disrupts the normal sleep pattern and reduces sleep quality. As a result, sleep disturbance caused by alcohol can play a role in the progression of alcoholism. As an important regulator of sleep cycles, inhibition in the thalamus may therefore be involved in both the sedative effects of alcohol and the development of alcoholism. Investigating the effects of alcohol on both synaptic and extrasynaptic GABAA-Rs in the thalamus should help us to understand the mechanisms underlying the interaction between alcohol and sleep.

Contribution of a glial glutamate transporter to GABA synthesis in the retina.

Neuronal glutamate transporters have been shown to play a role in GABA synthesis by enhancing glutamate uptake. In the present study, we have examined whether a glial glutamate transporter, GLAST, has a role in GABA synthesis in the mammalian retina. We found that the retinal GABA level was about two-fold higher in the GLAST-/- mouse retina compared to that in the wild type. Endogenous glutamate level was also increased about 2-fold in the mutant. Therefore, loss of GLAST results in a higher retinal GABA level, probably due to increased availability of its precursor, glutamate. An increase in GABAergic activity can be expected to affect trigger features such as directional selective response of neurons in the GLAST-/- mouse retina.

Brief alcohol exposure alters transcription in astrocytes via the heat shock pathway.

Astrocytes are critical for maintaining homeostasis in the central nervous system (CNS), and also participate in the genomic response of the brain to drugs of abuse, including alcohol. In this study, we investigated ethanol regulation of gene expression in astrocytes. A microarray screen revealed that a brief exposure of cortical astrocytes to ethanol increased the expression of a large number of genes. Among the alcohol-responsive genes (ARGs) are glial-specific immune response genes, as well as genes involved in the regulation of transcription, cell proliferation, and differentiation, and genes of the cytoskeleton and extracellular matrix. Genes involved in metabolism were also upregulated by alcohol exposure, including genes associated with oxidoreductase activity, insulin-like growth factor signaling, acetyl-CoA, and lipid metabolism. Previous microarray studies performed on ethanol-treated hepatocyte cultures and mouse liver tissue revealed the induction of almost identical classes of genes to those identified in our microarray experiments, suggesting that alcohol induces similar signaling mechanisms in the brain and liver. We found that acute ethanol exposure activated heat shock factor 1 (HSF1) in astrocytes, as demonstrated by the translocation of this transcription factor to the nucleus and the induction of a family of known HSF1-dependent genes, the heat shock proteins (Hsps). Transfection of a constitutively transcriptionally active Hsf1 construct into astrocytes induced many of the ARGs identified in our microarray study supporting the hypothesis that HSF1 transcriptional activity, as part of the heat shock cascade, may mediate the ethanol induction of these genes. These data indicate that acute ethanol exposure alters gene expression in astrocytes, in part via the activation of HSF1 and the heat shock cascade.

The regulation of neuronal gene expression by alcohol.

In recent years there has been an explosion of interest in how genes regulate alcohol drinking and contribute to alcoholism. This work has been stimulated by the completion of the human and mouse genome projects and the resulting availability of gene microarrays. Most of this work has been performed in drinking animals, and has utilized the extensive genetic variation among different mouse strains. At the same time, a much smaller amount of effort has gone into the in vitro study of the mechanisms underlying the regulation of individual genes by alcohol. These studies at the cellular and sub-cellular level are beginning to reveal the ways in which alcohol can interact with the transcriptional, translational and post-translational events inside the cell. Detailed studies of the promoter regions within several individual alcohol-responsive genes (ARGs) have been performed and this work has uncovered intricate signaling pathways that may be generalized to larger groups of ARGs. In the last few years several distinct ARGs have been identified from 35,000 mouse genes, by both the "top-down" approach (ex vivo gene arrays) and the "bottom-up" methods (in vitro promoter analysis). These divergent methodologies have converged on a surprisingly small number of genes encoding ion channels, receptors, transcription factors and proteins involved in synaptic function and remodeling. In this review we will describe some of the most interesting cellular and microarray work in the field, and will outline specific examples of genes for which the mechanisms of regulation by alcohol are now somewhat understood.

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors.

Excitatory amino acid transporters (EAATs) are involved in regulating extracellular glutamate levels at synaptic regions in the CNS. EAAT1, 2, 3, and 5 have been found in the mammalian retina, but the presence of EAAT4 has remained controversial. Recently, we found a high level of EAAT4 mRNA in the human retina, and this observation lead us to examine whether EAAT4 was expressed in the mammalian retina. Immunoblotting studies showed the presence of EAAT4-immunoreactive proteins in human and mouse retinas, corresponding to EAAT4 monomers and dimers. Immunohistochemistry revealed that EAAT4 was localized in rod and cone photoreceptor outer segments in the human retina, and in the outer and inner segments of mouse and ground squirrel retinas. In no case was EAAT4 found in the outer plexiform layer or in any other layer in the retina. EAAT4 expression by photoreceptors was confirmed by immunoblotting a purified rod outer segment preparation, which showed the presence of a 50-kDa EAAT4-immunoreactive protein. In addition, the EAAT4-associated protein, GTRAP41, was found in the human, mouse, and squirrel retinas as well as in the rod outer segment preparation. Further immunocytochemical and co-immunoprecipitation experiments demonstrated that GTRAP41 was colocalized and interacted in vivo with EAAT4. Importantly, glutamate uptake and drug inhibition experiments showed that an EAAT4-like glutamate uptake system is present in the rod outer segments. Finally, we examined whether glutamate signaling mediated by EAAT4 can modulate rod outer segment phagocytosis by the retinal pigment epithelium. Results of the present study show that EAAT4 is present in the outer segments, a nonsynaptic region of photoreceptors, where it might provide a feedback mechanism for sensing extracellular glutamate or serve as an outer barrier to prevent glutamate from escaping from the retina.

An extrasynaptic GABAA receptor mediates tonic inhibition in thalamic VB neurons.

Whole cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) and reticular (RTN) neurons in mouse brain slices. A bicuculline-sensitive tonic current was observed in VB, but not in RTN, neurons; this current was increased by the GABA(A) receptor agonist 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol (THIP; 0.1 microM) and decreased by Zn(2+) (50 microM) but was unaffected by zolpidem (0.3 microM) or midazolam (0.2 microM). The pharmacological profile of the tonic current is consistent with its generation by activation of GABA(A) receptors that do not contain the alpha(1) or gamma(2) subunits. GABA(A) receptors expressed in HEK 293 cells that contained alpha(4)beta(2)delta subunits showed higher sensitivity to THIP (gaboxadol) and GABA than did receptors made up from alpha(1)beta(2)delta, alpha(4)beta(2)gamma(2s,) or alpha(1)beta(2)gamma(2s) subunits. Western blot analysis revealed that there is little, if any, alpha(3) or alpha(5) subunit protein in VB. In addition, co-immunoprecipitation studies showed that antibodies to the delta subunit could precipitate alpha(4), but not alpha(1) subunit protein. Confocal microscopy of thalamic neurons grown in culture confirmed that alpha(4) and delta subunits are extensively co-localized with one another and are found predominantly, but not exclusively, at extrasynaptic sites. We conclude that thalamic VB neurons express extrasynaptic GABA(A) receptors that are highly sensitive to GABA and THIP and that these receptors are most likely made up of alpha(4)beta(2)delta subunits. In view of the critical role of thalamic neurons in the generation of oscillatory activity associated with sleep, these receptors may represent a principal site of action for the novel hypnotic agent gaboxadol.

Glutamate transport by retinal Muller cells in glutamate/aspartate transporter-knockout mice.

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Muller cells in GLAST-/- mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild-type and GLAST-/- mouse retinas, but the Vmax was approximately 50% lower in the mutant. In Na+-free medium, the Vmax was further reduced by 40%. In patch-clamp recordings of dissociated Muller cells from GLAST-/- mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Muller cells. [3H]D-Aspartate uptake autoradiography, however, showed that Na+-dependent, high-affinity transporters account for most of the glutamate uptake by Muller cells, and that Na+-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Muller cells in the GLAST-/- mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Muller cells, new Na+-dependent, high-affinity glutamate transporters remain to be identified.