NMDA receptor up-regulation is a major neuroadaptive process that causes excitatory syndrome upon withdrawal from chronic ethanol exposure. The mechanisms of neuroadaptation of NMDA receptors during withdrawal remain unclear, but several lines of evidence suggest that changed gene expression plays important roles. MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs which are involved in the fine tuning of gene expression in various biological processes. We have recently shown a distinct expression pattern of miRNA in response to chronic intermittent ethanol exposure, including the up- regulation of miR-152, -150, and -126 in mice VTA DA neuron. This proposed study is designed to study the role of miRNA in the DNA methylation underlying neuroadaptive up-regulation of NR2B gene during alcohol withdrawal. Therefore, we hypothesize that alcohol withdrawal-induced up-regulation of miR-152, -150 and - 126 modulates DNA methylation by down-regulating MeCP2 and are subsequently involved in neuroadaptive up-regulation of the NR2B gene.
Specific Aim 1 : Identify and validate that miR-152, -150 and -126 target MeCP2 and regulate DNA methylation of the NR2B gene in vitro. We will confirm these miRNAs (1) biologically interact with the 3'UTR of MeCP2 mRNA using a lentiviral delivery system to introduce these miRNAs into cultured neurons;(2) functionally inhibit the target effort level (repression) using an in vitro reporter gene assay;and (3) determie the regulatory role of these miRNAs on DNA methylation of the NR2B gene using bisulfate pyrosequencing.
Specific Aim 2 : To investigate whether the miRNAs are involved in the regulation of ethanol withdrawal-induced adaptation in ethanol dependence mice. TH- and GAD67-GFP transgenic mice will be exposed to alcohol with a CIE regimen followed by 5 days withdrawal. The DAergic or GABAergic neurons in the VTA of midbrain dopamine neurons in the mice will be obtained using laser capture microdissection and used for the analysis of the differential expression of these miRNAs, mRNA of MeCP2 and NR2B genes as well as DNA methylation of the NR2B promoter. The proposed study will determine a novel mechanism that miRNAs contribute to the epigenetic regulation of the NR2B gene during ethanol withdrawal of mice. For the first time, single neuronal populations obtained by laser capture microdissection will be used to study epigenetic mechanism on alcohol withdrawal-related neuroadaptation.
Alcoholism is a major health problem in the US, consuming 15% of total health costs. Withdrawal syndrome is the major causal factor for the onset and development of alcohol dependence. miRNAs are new regulatory elements for gene expression and represent new therapeutic targets for many diseases. The results from this study will provide new and valuable insight into the epigenetic regulation of alcoholism. The long-term goal of this study is to delineate the epigenetic mechanisms underlying alcohol dependence that will enable development of novel epigenetic therapeutic strategies for the prevention or relief of withdrawal. The proposed study is aimed at understanding the role of microRNA in epigenetic mechanisms underlying alcohol withdrawal adaptation. The information generated will provide new and valuable insight into the mechanisms regulating alcohol drinking related problems and may serve as a basis of designing epigenetic therapy of alcohol dependence and abuse.