click on name to contact
Principal Investigators: Susan E. Bergeson and Peter J. Syapin
SPECIFIC AIMS
Numerous studies have shown that alcoholism and many specific alcohol-related responses are affected by both environmental and genetic components (Schuckit et al. 1972; Goodwin et al. 1974; Cloninger et al.1981; Crabbe et al. 1994; Bergeson et al. 2003 and numerous others; and see Schuckit, 2009 for a recent review). Historically, alcoholism research has predominantly focused on the actions of neurons although pproximately 50-60% of the brain consists of glial cells. Little is known about the role various glial cells play in mediating either damage or protection from the effects of alcohol in brain. Microglia are the immunoresponsive cells that either act to protect brain or, through chronic activation, promote oxidative damage. They make up approximately 20% of total glial cells, a substantive portion of brain tissue. Therefore, our overarching hypothesis is that some changes, which we define as "molecular memory" occur in microglia at the molecular level and drive, in part, a new homeostasis during alcohol intoxication that resets the transcriptome, and persists for some time. A growing number of reports point either directly or indirectly to epigenetic involvement in alcoholism and alcohol-induced consequences (Kim and Shukla 2005; Park et al., 2005; Wang et al., 2007; Pandey et al., 2008, Pietrzykowski et al., 2008, Shukla et al, 2008; Haycock and Ramsay, 2009, Wang et al., 2009; for a review see: Shukla et al., 2008). Most of the early work began with a focus on liver and FASD-affected brain. However, there is increasing excitement toward understanding the epigenetics of alcohol drinking in brain. The development of a global picture of alcohol-induced "molecular memory", including through epigenetic mechanisms, specifically in microglia is proposed in this exploratory R21 application through two primary aims:
The specific aims of this proposal are:
Aim 1. To test the hypothesis that alcohol and LPS have direct and specific effects on the microglial transcriptome.
Rationale: To our knowledge, alcohol-mediated changes in microglia gene expression have not been determined despite the obvious roles these pluripotent cells play in mediating homeostasis of brain tissue and regulating a neuroinflammatory response in brain tissue.
Approach: Cultured N9 microglial cells will be treated with and without LPS (lipopolysaccharide) and 44 mM ethanol for 24 hrs and will be harvested for array analyses of mRNA and miRNA expression and DNA methylation. In addition, ChIP-on-chip will be used to identify genomic level transcription factor usage and chromatin structural changes. Transcript content is not only regulated by up- and down-stream promoter region sequence, but microRNAs, histone modifications, DNA-methlyation and chromatin remodeling play a regulatory role for some specific gene expression levels. We wish to systematically identify the role of each.
Aim 2. To test the hypothesis that alcohol concentration and the brain environment interact to effect "molecular memory" regulation of the global microglial transcriptome.
Rationale: Although we test the direct affects of alcohol on microglia in culture in Aim 1, we feel that it is especially important to understand how microglia respond differently in the brain environment. A contrast of the two approaches should allow us to make greater inroads into understanding the importance of the complex responses of microglia in brain.
Approach: Male and Female C57BL/6J mice will be given alcohol in two ways. First, to control dose equivalency across mice, 2 and 4 g/kg of 20% ethanol will be administered by gavage. In order to discover differences elicited by free choice consumption over a longer time period, the mice will be given alcohol through a drinking-in-the-dark paradigm. Again, the aforementioned molecular analyses will be completed on microglia freshly isolated from cortex at the end of the intoxication period and over a time course following a return to the sober state. A linear process of bioinformatics analyses we have developed will be utilized to compare and contrast direct alcohol effects in culture to the complex response in brain. A validation of a limited number of significant candidate responses will be completed and will form the preliminary data for a future proposal focused on elucidating the detailed molecular mechanisms of microglial "molecular memory" and connection to function.