Effect of 5-azacytidine on the methylation aspects of NMDA receptor NR2B gene in the cultured cortical neurons of mice.

Our previous study revealed that the exposure of the drug 5-Azacytidine and ethanol to the cultured cortical neurons of mice causes demethylation of cytosine residues in the CpG island of the NMDA receptor NR2B gene (Marutha Ravindran and Ticku, Mol Brain Res 121:19-27, 2004). In the present study, we further analyzed methylation in the CpG island with various concentration frame and time frame of exposure of the cultured cortical neurons with 5-azacytidine to identify whether methylation in the NR2B gene is site specific or region specific. Methylation was studied by digesting the genomic DNA with methylation sensitive HpaII, MspI, AciI or HhaI enzyme following the exposure of cultured cortical neurons of mice with 5-azacytidine by performing PCR and Southern hybridization. We observed demethylation of DNA at 1, 3 and 5 muM concentrations of 5-azacytidine in the regions (5982-6155), (6743-7466) and at 3 and 5 muM concentrations of 5-azacytidine used in the region (6477-6763). Similarly in the time frame study with 5-azacytidine, demethylation of DNA was observed at 24 h and 36 h of incubation with 5-azacytidine in the regions (5982-6155), (6743-7466) and at 36 h of incubation with 5-azacytidine used in the region (6477-6763). Our experimental results demonstrate that the methylation in the CpG islands of the NR2B gene may not be site specific or region specific in the cultured cortical neurons of mice.

Emerging role of epigenetics in the actions of alcohol.

This review deals with the recent developments on the epigenetic effects of ethanol. A large body of data have come from studies in liver and in neuronal systems and involve post-translational modifications in histones and methylations in DNA. Ethanol causes site selective acetylation, methylation, and phosphorylation in histone. With respect to methylations the methyl group donating system involving S-adenosyl methionine appears to play a central role. There is contrasting effect of acetylation versus methylation on the same site of histone, as it relates to the transcriptional activation. Epigenetic memory also appears to correlate with liver pathology and Mallory body formation. Experimental evidence supports transcriptional regulation of genes in the CNS by DNA methylations. These studies are contributing towards a better understanding of a novel epigenetic regulation of gene expression in the context of alcohol. The critical steps and the enzymes (e.g., histone acetyltransferase, histone deacetylase, DNA methyltransferase) responsible for the epigenetic modifications are prime targets for intense investigation. The emerging data are also beginning to offer novel insight towards defining the molecular actions of ethanol and may contribute to potential therapeutic targets at the nucleosomal level. These epigenetic studies have opened up a new avenue of investigation in the alcohol field.

Effect of chronic administration of ethanol on the regulation of the delta-subunit of GABA(A) receptors in the rat brain.

In the present study, we investigated the effect of chronic ethanol (CE) administration on the polypeptide levels of the delta-subunit of GABA(A) receptors and [(3)H]muscimol binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat brain. CE administration resulted a down-regulation of polypeptide levels of the delta-subunit of GABA(A) receptors in the rat cerebellum and hippocampus, whereas there were no changes in the delta-subunit polypeptide levels in the rat cerebral cortex. Further, CE administration caused a down-regulation of native delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum as determined by [(3)H]muscimol binding to the immunoprecipitated receptor assemblies. These results indicate that the delta-subunit-containing GABA(A) receptors may play a role in chronic ethanol-induced tolerance and dependence.

Low concentrations of ethanol do not affect radioligand binding to the delta-subunit-containing GABAA receptors in the rat brain.

In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain. Ethanol (1-50 mM) neither affected [(3)H]muscimol (3 nM) binding nor diazepam-insensitive [(3)H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA(A) receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [(3)H]muscimol (15 nM) binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native delta-subunit-containing GABA(A) receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

Tyrosine kinase phosphorylation of GABAA receptor subunits following chronic ethanol exposure of cultured cortical neurons of mice.

Previous studies from our laboratory revealed that acute ethanol exposure inhibits phosphorylation of mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK) in mice. In the present study, we have further investigated effect of chronic administration of ethanol on tyrosine kinase phosphorylation of GABA(A) receptor subunits in the mouse cultured cortical neurons. We observed that there was an up-regulation in tyrosine kinase phosphorylation of the GABA(A) receptor beta(2) and gamma(2) subunits following chronic ethanol exposure, whereas there was no effect on alpha(1) subunit of the GABA(A) receptor in the cultured cortical neurons of mice as determined by Western blotting. These results suggest a potential role for tyrosine kinase phosphorylation of some of the GABA(A) receptor subunits in chronic ethanol-induced tolerance and dependence.

Comparison of chronic ethanol and chronic intermittent ethanol treatments on the expression of GABA(A) and NMDA receptor subunits.

We examined the mRNA and protein levels of GABA(A) and NMDA receptor (NMDAR) subunits in cultured mouse cortical neurons following exposure to chronic ethanol (CE) or chronic intermittent ethanol (CIE), and after 5 days of withdrawal. With respect to GABA(A) receptor mRNA, both treatments decreased the levels of alpha1 and alpha2 subunits, and increased the level of alpha4. However, only CE treatment caused parallel changes in the protein levels; alpha2 and alpha4 protein levels did not change after CIE. Both treatments did not alter beta2 and beta3 mRNA levels, but they increased beta2/3 protein levels. The gamma2 subunit mRNA levels decreased with both treatments, but protein levels did not change. Most of the changes returned to control levels after withdrawal, except for the gamma2 subunit protein, which was lower than controls. In the case of NMDAR subunit, both treatments greatly increased the levels of NR2B mRNA, but barely altered NR1 mRNA and polypeptide levels. CIE treatment caused a relatively higher increase in NR2B protein, and this was the only sustained increase after long-term withdrawal. Taken together, our results show that CIE regimen has less pronounced effects on GABA(A) receptor expression, but increases NR2B expression more dramatically than CE treatment in cultured cortical neurons. These differential effects on subunit expression may result in altered receptor structure and function as a result of ethanol exposure.

Role of CpG islands in the up-regulation of NMDA receptor NR2B gene expression following chronic ethanol treatment of cultured cortical neurons of mice.

Our earlier studies have indicated that chronic ethanol treatment produces an increase in the rate of NR2B gene expression but acute ethanol treatment doesn't cause any change in the expression in adult cortex and cultured fetal cortical neurons. To determine the molecular mechanism involved in the up-regulation of NR2B gene expression, we studied methylation in CpG islands in the regions (5843-6276) and (6477-6763) in mouse fetal cortical cultured neurons. However, acute administration of ethanol (3.5 g/kg, i.p.) as a 25% solution diluted in 0.9% saline to the adult mice did not cause any change in the methylation status of the NR2B gene. Therefore, these observations indicate that chronic ethanol causes demethylation in CpG islands in the regions (5843-6276) and (6477-6763), thereby probably resulting in increased expression of the NR2B gene. In contrast, acute ethanol treatment did not cause any change in the NR2B gene expression or its demethylation. We also investigated the changes in hypersensitive sites in CpG islands and transcription initiation sites in the cortical cultured neurons following chronic ethanol treatment. These experiments revealed that chronic ethanol treatment does not alter the pattern of hypersensitive sites in adult cortex and cultured cortical neurons.

Effect of chronic administration of ethanol on GABAA receptor assemblies derived from alpha2-, alpha3-, beta2- and gamma2-subunits in the rat cerebral cortex.

Chronic administration of ethanol decreased the immunoprecipitation of the [(3)H]flunitrazepam binding activity for GABA(A) receptor assemblies derived from alpha(2)-, alpha(3)- and gamma(2)-subunits in the rat cerebral cortex. However, the [(3)H]muscimol binding sites derived from these subunits were not affected. Thus, chronic ethanol causes the down-regulation of the benzodiazepine sites derived from the alpha(2)-, alpha(3)- and gamma(2)-subunits without affecting the GABA binding sites.

Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain.

We have previously reported that there is an up-regulation of the NR2B gene expression in the adult cortex and cultured fetal cortical neurons of mice following chronic ethanol treatment due to demethylation of cytosine residues in the NR2B gene CpG island. In the present study, we investigated the methylation pattern of the NR2B CpG island as a function of the mouse age by digesting the cortex genomic DNA with HpaII enzyme, amplifying the interested regions by performing PCR and detecting the methylated regions by Southern hybridization so as to determine whether age affects the methylation process. We observed demethylation of various regions of NR2B gene (5227-5567), (5647-6003), (6091-6445), (6424-7024) of adult mouse cortex. Our results indicate that methylation of NR2B gene in the mouse brain is age-dependent phenomenon.

Comparison of the behavioral effects of gamma-hydroxybutyric acid (GHB) and its 4-methyl-substituted analog, gamma-hydroxyvaleric acid (GHV).

Gamma-hydroxybutyrate (GHB), a metabolite of GABA, is a drug of abuse and a therapeutic. The illicit use of GHB precursors and analogs reportedly has increased worldwide. Gamma-hydroxyvaleric (GHV) is a 4-methyl-substituted analog of GHB that reportedly is abused and is marketed as a dietary supplement and replacement for GHB. The purpose of these studies was to compare the pharmacological and behavioral profiles of GHV and GHB. In radioligand binding studies, GHV completely displaced [(3)H]NCS-382 with approximately 2-fold lower affinity than GHB and did not markedly displace [(3)H]GABA from GABA(B) receptors at a 20-fold larger concentration. In drug discrimination procedures, GHV did not share discriminative stimulus effects with GHB or baclofen. GHV shared other behavioral effects with GHB, such as sedation, catalepsy, and ataxia, although larger doses of GHV were required to produce these effects. Lethality (50%) was observed after the largest dose of GHV (5600mg/kg), a dose that produced less-than-maximal catalepsy and ataxia. To the extent that large doses of GHV might be taken to in an attempt to produce GHB-like effects (e.g., hypnosis) GHV toxicity may pose a greater public health concern than GHB.

Changes in methylation pattern of NMDA receptor NR2B gene in cortical neurons after chronic ethanol treatment in mice.

Chronic ethanol treatment produced an increase in the rate of NR2B gene expression in adult cortex and cultured fetal cortical neurons. To determine the possible molecular mechanism involved in the upregulation of NR2B gene expression, we studied methylation in CpG island in mouse fetal cortical neurons. Our results demonstrate that chronic ethanol administration causes demethylation of cytosine residues in CpG island in cortical neurons which may be responsible for the changes in the expression of NR2B gene.

Alternate splice variants of mouse NR2B gene.

Two alternate spliced transcripts of N-methyl-D-aspartate receptor (NR2B) gene derived from a common promoter have been reported earlier. In the present study, we have examined the expression of alternative splice variants of NR2B gene using 5' rapid amplification of C terminal end (RACE) in the RNA derived from mouse cortex. RT-PCR was performed to confirm the RACE data. Here we report the occurrence of three transcripts containing a novel alternate spliced exon, we refer to as exon 1'. This new exon is located upstream of the transcription start site identified by Klein et al. [Gene 208 (1998) 259] in the 5' untranslated region (UTR) of NR2B, thus making a new alternate 5' end of the NR2B transcript. The molecular nature of the transcripts containing the new exon appears to be highly heterogeneous. These results are interesting in that they are of potential importance in translational regulation of the NR2B gene.

The molecular effects of alcohol: clues to the enigmatic action of alcohol.

Chronic ethanol treatment (50 mM, five days) induces stabilization of NR1 receptor subunit mRNA in cultured fetal cortical neurons. In this paper, we investigate the mechanism(s) by which ethanol mediates its effects on NR1 mRNA. Specifically, we have determined if cellular localization of NR1 mRNA in cortical neurons and/or de novo protein synthesis is essential for ethanol-mediated stabilization of NR1 mRNA. Subcellular fractionation studies show that all detectable NR1 mRNA is associated with rough endoplasmic reticulum, indicating that subcellular distribution of NR1 mRNA in fetal cortical neurons does not play a role in ethanol-mediated NR1 mRNA stabilization. However, inhibition of protein synthesis by cycloheximide abolished the mRNA stabilizing effect of ethanol on NR1 mRNA, thus suggesting de novo protein synthesis is crucial for the action of ethanol on NR1 mRNA stabilization.

Quantitative autoradiographic analysis of the new radioligand [(3)H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([(3)H]NCS-382) at gamma-hydroxybutyric acid (GHB) binding sites in rat brain.

(2E)-(5-Hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid (NCS-382) is an antagonist for gamma-hydroxybutyric acid (GHB) at GHB receptor sites. Advantages of using [(3)H]NCS-382 over [(3)H]GHB in radioligand binding studies are that unlike GHB, NCS-382 does not appear to bind to, activate, or interfere with the functioning of GABA(B) or GABA(A) receptors, either directly or indirectly. Herein we establish a protocol for use of [(3)H]NCS-382 by quantitative autoradiography. GHB was used to define non-specific binding, since it displaced [(3)H]NCS-382 to an extent equivalent to NCS-382. Among many areas of brain examined, two regions in which high specific binding of [(3)H]NCS-382 occurred were the hippocampus and cerebral cortex. Areas such as the striatum and nucleus accumbens exhibited intermediate levels of specific binding. No or very low binding was observed in other areas such as the cerebellum and dorsal raphe nucleus. The distribution of GHB binding sites as defined by [(3)H]NCS-382 suggests that GHB may play a role in neuromodulation or neurotransmission in frontal brain areas.

Ethanol-mediated inhibition of mitogen-activated protein kinase phosphorylation in mouse brain.

Ethanol (1.5-3.5 g/kg body weight) was administered intraperitoneally to mice and the phosphorylation of MAP (mitogen-activated protein) kinase in the cerebral cortex was determined using phospho-specific MAP kinase antibodies. Ethanol inhibited the phosphorylation of MAP kinase in a dose- and time-dependent manner. Developmental studies demonstrated that the levels of phospho-MAP kinase increased from fetal cortex (prenatal) to 16-day-old mice (postnatal) and remained constant up to 4 months of age. However, ethanol (3.5 g/kg) decreased the phospho-MAP kinase staining in all of the age groups studied. Subcellular fractionation studies demonstrated that ethanol inhibited the phosphorylation of MAP kinase in both the cytosol as well as nucleus, but did not alter the levels of MAP kinase. Likewise, MK-801 (0.4 mg/kg) or flurazepam (75 mg/kg) also decreased the phospho-MAP kinase content. These data indicate that ethanol may inhibit the phosphorylation of MAP kinase in vivo by either inhibiting NMDA receptors or activating GABA receptors.

Role of GABA(A) receptors in the ethanol-mediated inhibition of extracellular signal-regulated kinase.

In the present study, we demonstrate the involvement of GABA(A) receptors in the ethanol-mediated modulation of extracellular signal-regulated kinases (ERK). Intraperitoneal (i.p.) administration of ethanol (3.5 g), flurazepam (75 mg) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptane-5,10-iminemaleate (MK-801) (0.4 mg/kg body weight) decreased, while picrotoxin (10 mg/kg body weight) increased, the phosphorylation of ERK following 10 min of their injection. However, the picrotoxin-induced phosphorylation of ERK was inhibited by ethanol, but was not affected by MK-801. These results indicate that ethanol's inhibitory effect on ERK phosphorylation may involve the modulation of GABA(A) receptor function.

Ethanol does not alter the binding of the gamma-hydroxybutyric acid (GHB) receptor ligand [3H]NCS-382 in the rat brain.

We investigated the effect of ethanol on the binding of the gamma-hydroxybutyric acid (GHB) receptor ligand [3H]NCS-382 in the rat cerebral cortex and hippocampus. Ethanol (50-100 mM) did not alter the binding of [3H]NCS-382. Furthermore, acute (3g/kg, p.o.) as well as chronic (9-15 g/kg/day p.o. for 6 days) administration of ethanol also did not have any significant effect on the binding of [3H]NCS-382 in the rat cerebrocortical and hippocampal membranes. These observations suggest that ethanol does not interact directly with the GHB receptor in vitro or in vivo, and GHB receptor may not be involved in the pharmacological effects of ethanol.

Characterization of the picrotoxin site of GABAA receptors.

This unit describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics, and competition assays (IC50, Ki determination) are included.

The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.

BACKGROUND: The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5'-regulatory area following CIE treatment. METHODS: Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5'-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity. RESULTS: Analysis of individual CpG methylation sites within the NR2B 5'regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors. CONCLUSIONS: These results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.

Characterization and pharmacology of the GHB receptor.

Radioligand binding using [(3)H]NCS-382, an antagonist of the GHB receptor, revealed specific binding sites in the rat cerebrocortical and hippocampal membranes. Scatchard analysis of saturation isotherms revealed two different populations of binding sites. NCS-382 was about 10 times more potent than GHB in inhibiting [(3)H]NCS-382 binding. A variety of ligands for other receptors did not affect [(3)H]NCS-382 binding. Quantitative autoradiographic analysis of [(3)H]NCS-382 binding revealed similar characteristics. Thus [(3)H]NCS-382, being more potent and selective, offers advantage over [(3)H]GHB as a radioligand. Unlike GHB, several analogues of GHB such as UMB68 (a tertiary alcohol analogue of GHB), UMB86 (4-hydroxy-4-napthylbutanoic acid, sodium salt), UMB72 [4-(3-phenylpropyloxy)butyric acid, sodium salt], UMB73 (4-benzyloxybutyric acid, sodium salt), UMB66 (3-chloropropanoic acid), gamma-hydroxyvaleric acid (that is, GHV, a 4-methyl-substituted analogue of GHB), 3-HPA (3-hydroxyphenylacetic acid), and ethers of 3-hydroxyphenylacetic acid (UMB108, UMB109, and UMB119) displaced [(3)H]NCS-382 without affecting [(3)H]GABA binding to GABA(B) receptor. Thus these compounds offer an advantage over GHB as an experimental tool. Our study, aimed at exploring the potential involvement of the GHB receptor in the pharmacology of ethanol, indicated that ethanol does not affect [(3)H]NCS-382 binding in the rat brain, thereby suggesting that ethanol does not interact directly with the GHB receptor. Our study, aimed at exploring the involvement of the GHB receptor in the pathology of succinate semialdehyde dehydrogenase deficiency, which is known to cause elevation of GHB levels, revealed no change in the affinity, receptor density or displacement potency as determined by using [(3)H]NCS-382 as a radioligand in Aldh5a1(-/-) vs. Aldh5a1(+/+) mouse brain.