Previous microarray studies in the Miles'laboratory have shown that serum and glucocorticoid- regulated kinase 1, Sgk1, is prominently up regulated by acute ethanol (2 g/kg) in the prefrontal cortex (PFC) of DBA/2J mice. Functionally, Sgk1 is an important focal point of intracellular signaling cross-talk through which the cell surface receptors, nuclear receptors, and cellular stress pathways converge to control many cellular processes including receptor or ion channel trafficking, cell proliferation and/or apoptotic responses. In aforementioned microarray studies, Sgk1 was accompanied by a highly correlated group of genes, many of which are also known to respond to glucocorticoids. This suggests that stress-related signaling events might play an important role in ethanol regulation of the Sgk1 gene network. Recently a brain-specific and more stable isoform of Sgk1, Sgk1.1, which is not regulated by glucocorticoids was also identified. Prior work by others also shows that Sgk1 can play an important role modulating synaptic plasticity occurring in memory. Based on these findings, it is hypothesized that Sgk1 induction might play an important role in modifying behavioral responses to ethanol. This hypothesis can be explored through the following specific aims: 1) characterize ethanol regulation of Sgk1 in the PFC of DBA/2J mice, 2) determine the role of HPA axis signaling in ethanol regulation of Sgk1 in the PFC, and 3) modulate Sgk1 in the PFC and determine the effects on ethanol related behaviors through adeno-associated virus (AAV). Ethanol regulation of Sgk1 in the PFC can be characterized by measuring Sgk1 protein and isoform levels following acute ethanol exposure through western blotting followed by densitometry analysis and the quantitative polymerase chain reaction (Q-rtPCR). There are 5 total isoforms of Sgk1-4 resulting from translational events and 1 (Sgk1.1) resulting from alternative promoter utilization and splicing. At least 2 of the 4 isoforms produced from translational events are present in the brain and can be measured by western blotting and densitometry techniques. Sgk1.1 can be measured through Q-rtPCR. The role of HPA axis signaling in ethanol regulation of Sgk1 can be determined pharmacologically through the use of drugs, aminoglutethimide, metyrapone, and RU-486, which inhibit enzymes involved in glucocorticoid synthesis or block the glucocorticoid receptor. These drugs can be followed by acute ethanol treatment and Q-rtPCR measurement of Sgk1 expression. A gene delivery approach is used to determine the effects of Sgk1 over- expression on ethanol related behaviors. Sgk1 is over-expressed through the delivery of an epitope tagged AAV expressing FLAG-Sgk1 into the PFC of DBA/2J mice using stereotaxic microinjection techniques. Following stereotaxic microinjection, ethanol related behavioral responses including acute locomotor activation and sensitization are recorded. Following behavioral measures, viral expression and placement is determined through the use of immunohistochemistry.
Ethanol dependence remains a persistent and serious problem in the United States as approximately 7.4 percent of Americans meet the criteria for ethanol abuse and more than half of American adults have a family member affected by alcoholism. The neuroadaptations that occur leading to and following ethanol dependence and their mechanisms have yet to be elucidated. The work proposed here could shed light on molecular mechanisms underlying alcoholism and eventually have implications in the development of novel pharmacological agents for alcoholism or alcohol-related neurological disorders.
|Costin, Blair N; Dever, Seth M; Miles, Michael F (2013) Ethanol regulation of serum glucocorticoid kinase 1 expression in DBA2/J mouse prefrontal cortex. PLoS One 8:e72979|
|Costin, Blair N; Wolen, Aaron R; Fitting, Sylvia et al. (2013) Role of adrenal glucocorticoid signaling in prefrontal cortex gene expression and acute behavioral responses to ethanol. Alcohol Clin Exp Res 37:57-66|
|O'Brien, M A; Costin, B N; Miles, M F (2012) Using genome-wide expression profiling to define gene networks relevant to the study of complex traits: from RNA integrity to network topology. Int Rev Neurobiol 104:91-133|