Alcohol abuse disorders are highly prevalent particularly in military combat veterans, with yet to be fully elucidated molecular biological underpinnings. Human and animal studies indicate that the stable downregulation of genes protective against the reinforcing effects of ethanol, such as brain derived neurotrophic factor (BDNF), may result in increased alcohol consumption. BDNF is a known regulator of synaptic plasticity that may play a significant role in ethanol's addictiv and neurodegenerative properties. DNA methylation is one component of epigenetic gene regulation. Its ability to endure an animal's lifespan, and even across generations, indicates its potential utility as a means for encoding information. Hypermethylation of the BDNF promoter has been shown to decrease its expression. Studies suggest active DNA demethylation in neurons results from GADD45 proteins (GADD45a, b, g) recruiting cytidine deaminases (CDs) and thymidine glycosylases (TGs) to gene promoters. Recently, we reported decreased BDNF IXabcd mRNA expression in psychotic subjects'postmortem brain samples associated with decreased GADD45b BDNF IXabcd promoter binding and increased 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC). Our principal objective is to determine the role of DNA demethylation in alcohol-drinking behavior utilizing two separate animal models. In the first approach, we will utilize one of the most frequently used murine models in alcohol studies, the C57BL/6 (C57) and DBA/2J (DBA) strains. On average C57 mice voluntarily consume 10- fold more ethanol than DBA mice. Our preliminary data indicate, by comparison to DBA mice, C57 mice have lower BDNF expression in the shell of the nucleus accumbens (NAc), as well as less BDNF IXa mRNA expression in the NAc as a whole. Additionally, prior studies show divergent responses in BDNF expression between C57 and DBA mice in reaction to acute ethanol. Taken together, these data suggest that BDNF dysregulation in the reward circuitry may, in part, contribute to high ethanol consumption in C57 mice. We will validate that BDNF accounts for this difference by determining if the infusion of BDNF protein into the NAc shell of C57 mice reduces voluntary ethanol consumption. We will also determine if deficient BDNF expression in the NAc in C57 mice may be explained by altered expression of DNA methylating (DNMT1, DNMT3a, and DNMT3b) or DNA demethylating genes, such as CDs, TGs, or the GADD45s. We will measure mRNA expression of these transcripts, as well as GADD45b and BDNF protein in the NAc and ventral tegmental area (VTA). We will focus on GADD45 proteins because they appear to be the lynchpins of the demethylation process. We will determine whether there is reduced GADD45b binding and increased 5MC and 5HMC at BDNF promoters in these brain regions in C57 mice. In addition, we will test if the C57 strain's inability to increase BDNF in relation to acute ethanol is due to dysfunctional demethylating machinery. We will attempt to induce the alcohol-drinking phenotype of C57 mice in the DBA strain by in vivo knockdown of GADD45b in the NAc shell via siRNA strategy. In our second approach, we will use a GADD45b knockout (KO) mouse because GADD45b is responsible for activity-dependent demethylation of BDNF. We will evaluate whether acute ethanol increases BDNF expression and promoter demethylation in the NAc and VTA in mice lacking GADD45b. Finally, we will test whether the BDNF-deficient GADD45b KO mice have increased alcohol preference. Ultimately, these studies will identify novel molecular targets in the actions of ethanol and ethanol drinking behavior, and promote the development of pharmacotherapy for alcoholism.
Alcohol use disorders (AUDs) are present in 11-15% of active-duty returning military from Iraq, (compared with approximately 8.5% in the US population as a whole), and have a high comorbidity with mood and anxiety disorders such as Major Depressive Disorder and Post-Traumatic Stress Disorder (PTSD). Despite efforts to develop new medications for AUDs, there is still a lack of more effective treatments. Studies in humans and animal models have indicated altered gene expression mediated in part by epigenetic factors, such as DNA methylation, contribute to alcohol addiction. This research will attempt to characterize those factors responsible for the removal of the DNA methyl mark with the ultimate goals of identifying novel targets for pharmacological development for the treatment of alcoholism.
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| Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P et al. (2015) Gadd45b and N-methyl-D-aspartate induced DNA demethylation in postmitotic neurons. Epigenomics 7:567-79 |
