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A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment.However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.NMDA receptor mediates excitatory neurotransmission in the central nervous system and plays a central role in the function of excitatory synapses, which are important in neuronal development, memory formation, and many forms of synaptic plasticity. The level of NMDA receptor function at the synapse critically regulates brain function and cell survival. At synapses, it is believed that the appropriate clustering of this receptor at the postsynaptic membrane is critical for efficient synaptic transmission, and a dynamic balanced process of NMDA receptors is key to remaining constant at the surface level of receptors (Wenthold et al., 2003). Trafficking of NMDA receptor has emerged as a key regulatory mechanism that underlies channel function. Recent experimental evidence supports that NMDA receptors are quite mobile within neurons via endocytosis and lateral diffusion in the membrane (Rao and Craig, 1997;Tovar and Westbrook, 1999;Crump et al., 2001;Snyder et al., 2005). For example, long-term treatment with NMDA receptor antagonists leads to an increase in surface clusters of NMDA receptors and a shift to a more synaptic localization (Rao and Craig, 1997).The NMDA receptor system has emerged as an important site for the action of ethanol. Short-term ethanol exposure depresses NMDA receptor-activated ionic currents and...

BackgroundThe 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.MethodsPrimary 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.ResultsAnalysis 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.ConclusionsThese 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.

BACKGROUND: Elderly individuals react less efficiently to vaccines than do adults, mainly because of T-cell unresponsiveness. In this study, we analysed whether tumour-associated antigen (TAA)-specific CD8 T-cell responses could be induced by vaccination in old mice with metastatic breast cancer. METHODS: The effect of pcDNA-3.1-and Listeria-based vaccines, expressing TAA Mage-b, on Mage-b-specific immune responses was tested in spleens and draining lymph nodes (LNs) of mild (4TO7cg) and aggressive (4T1) syngeneic metastatic mouse breast tumour models at young (3 months) and old (20 months) age. RESULTS: Interferon g and interleukin-2 levels increased significantly in draining LNs and spleens of Mage-b-vaccinated mice compared with those in control groups at young but not old age in both mouse tumour models. A significant increase was observed in the number of IFNg-producing Mage-b-specific CD8 T cells after Mage-b vaccination in the 4T1 model at young but not old age. This correlated with a reduced protective effect of Mage-b vaccination against metastatic breast cancer at old compared with young age. CONCLUSIONS: The absence of CD8 T-cell responses after Mage-b vaccination and the accompanying reduced protection against metastatic breast cancer in old compared with young mice point towards the need for tailoring cancer vaccination to older age.

Background:Repeated alcohol exposure is known to increase subsequent ethanol consumption in mice. However, the underlying mechanisms have not been fully elucidated. One postulated mechanism involves epigenetic modifications, including histone modifications and DNA methylation of relevant genes such as NR2B or BDNF.Methods:To investigate the role of epigenetic mechanisms in the development of alcohol drinking behavior, an established chronic intermittent ethanol exposure reinforced ethanol drinking mouse model with vapor inhalation over two 9-day treatment regimens was used. The DNA methyltransferase inhibitor, 5-azacytidine or the histone deacetylase inhibitor, Trichostatin A was administered (intraperitoneally) to C57BL/6 mice 30min before daily exposure to chronic intermittent ethanol. Changes in ethanol consumption were measured using the 2-bottle choice test.Results:The results indicated that systemic administration of Trichostatin A (2.5 µg/g) facilitated chronic intermittent ethanol-induced ethanol drinking, but systemic administration of 5-azacytidine (2 µg/g) did not cause the same effect. However, when 5-azacytidine was administered by intracerebroventricular injection, it facilitated chronic intermittent ethanol-induced ethanol drinking. Furthermore, the increased drinking caused by chronic intermittent ethanol was prevented by injection of a methyl donor, S-adenosyl-L-methionine. To provide evidence that chronic intermittent ethanol- or Trichostatin A-induced DNA demethylation and histone modifications of the NR2B promoter may underlie the altered ethanol consumption, we examined epigenetic modifications and NR2B expression in the prefrontal cortex of these mice. Chronic intermittent ethanol or Trichostatin A decreased DNA methylation and increased histone acetylation in the NR2B gene promoter, as well as mRNA levels of NR2B in these mice.Conclusions:Taken together, these results indicate that epigenetic modifications are involved in regulating ethanol drinking behavior, partially through altering NR2B expression.

Metastatic breast cancer is an important contributor to morbidity and mortality. Hence, new therapies are needed that target breast cancer metastases. Here, we focus on Mage-b as a possible vaccine target to prevent the development of breast cancer metastases, through activation of Mage-b-specific cytotoxic T lymphocytes (CTL). The syngeneic cell line 4T1, highly expressing Mage-b, was used as a pre-clinical metastatic mouse breast tumor model. BALB/c mice received three preventive intraperitoneal immunizations with Mage-b DNA vaccine mixed with plasmid DNA, secreting granulocyte-macrophage colony stimulating factor (GM-CSF). In addition, antigen-presenting cells were more efficiently recruited to the peritoneal cavity by the injection of thioglycollate broth (TGB), prior to each immunization. Immunization with Mage-b/GM-CSF/TGB significantly reduced the number of metastases by 67% compared to the saline/GM-CSF/TGB and by 69% compared to the vector control/GM-CSF/TGB. Also, tumor growth was significantly reduced by 45% in mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/ GM-CSF/TGB and by 47% compared to the control vector/ GM-CSF/TGB group. In vivo, the number of CD8 T cells significantly increased in the primary tumors and metastases of mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/GM-CSF/TGB and the control vector/ GM-CSF/TGB group, while the number of CD4 T cells significantly decreased. The combination of Mage-b, GM-CSF and TGB did not only induce significantly higher levels of IFNgamma in the lymph nodes of vaccinated compared to control mice, but also induced significantly higher expression levels of Fas-ligand (FasL) in the primary tumors (expressing Fas protein constitutively), compared to the control mice. Whether the interaction between Fas and FasL may have contributed to the smaller tumors needs to be further analyzed.

Altered activity of the serotonin transporter (SERT) has been hypothesized to contribute to the etiology of major depression, anxiety, obsessive‐compulsive disorder, and autism. SERTs are thought to be the primary target for selective serotonin reuptake inhibitor (SSRI)‐class antidepressants, as well as many tricyclic antidepressants (TCA's) used to treat these disorders. Previously, we generated mice expressing a SERT variant (SERT I172M) that displays a dramatic loss of SERT affinity for cocaine, several SSRI's and TCA's, without impacting serotonin (5‐HT) transport (Thompson B.J., et al., 2011). Ex vivo forebrain synaptosomal 5‐HT transport assays with mice carrying the I172M substitution reveal an ~1,000 fold shift in citalopram potency, ~150 fold shift in venlafaxine potency, ~ 60 fold shift in sertraline potency and an ~10 fold shift in fluoxetine potency. Here we demonstrate that citalopram's metabolite, desmethylcitalopram, maintains an ~1000 fold shift in potency for SERT I172M, while the metabolites of venlafaxine, sertraline and fluoxetine display much smaller shifts in potency (~40 fold shift for desvenlafaxine, <2 fold shift for desmethysertraline and <2 fold shift for norfluoxetine). These results highlight the importance of considering the actions of drug metabolites. Ongoing studies are examining the effects chronic SSRIs have on gene expression and behavior.Grant Funding Source : UTHSCSA Faculty Startup Funds