Mechanisms of Glucocorticoid Resistance in Prenatal Alcohol Exposure

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Project Summary Many of the physiological processes affected by prenatal alcohol exposure (PAE) are regulated by glucocorticoids (GCs). GC resistance (i.e., reduced sensitivity to the actions of GCs), which may result from aberrant in utero glucocorticoid programming, is associated with both a variety of chronic diseases, many of which are immune function related, and PAE. Our goal is to identify molecular mechanisms of alcohol-mediated alterations in the programming of glucocorticoid sensitivity in the developing fetus, and to track these changes into adulthood. We will use this information to develop targeted interventions that reverse or reduce the effects of PAE on GC sensitivity and consequent responses of immune signaling molecules. This proposal will test the hypothesis that PAE modifies the long noncoding RNA, Growth arrest-specific 5 (Gas5), which acts as a glucocorticoid receptor (GR) decoy to regulate GR-mediated gene expression. Our hypotheses are two-fold: 1.) PAE produces glucocorticoid resistance that is expressed as: a.) dysregulation of the hypothalamic pituitary (HPA) axis and an increase in pro-inflammatory to anti-inflammatory cytokine ratio under stressful conditions, b.) a critical developmental shift in the normal stress and immune hyporesponsive early postnatal periods and c.) a decrease in the GRα/GRβ ratio. 2.) This maladaptive glucocorticoid resistance is programmed in the fetal brain as a result of an elevation in fetal brain Gas5 and maintained in adulthood by increases in FK506-bindinig protein-51 (FKBP51) levels. We will test these hypotheses using our established mouse model of PAE and three specific study designs: Aim 1.) PAE produces a measurable increase in glucocorticoid resistance: We will confirm physiologically relevant GC resistance in adult male and female PAE and saccharin (SAC) control mice by assessing HPA responding, pro- and anti-inflammatory cytokine/chemokine protein levels and levels of specific GR-regulated gene transcripts (including cytokines/chemokines) in response to stress activation. GC resistance will also be assessed by the ratio of GRα/GRβ, as well as measures of the levels of FKPB5 methylation. Aim 2.) PAE affects the developmental programming of GC responding: We will determine the impact of PAE on both the immune and stress hyporesponsive periods using maternal separation. HPA axis responsiveness, levels of frontal cortical cytokines/chemokines, FKBP51 protein and Gas5 RNA expression and their associations with GR will be determined. We will also measure the relative expression of GR isoforms GRα and GRβ. Aim 3.) Inhibition or reduction of prenatal Gas5 should restore normal GC sensitivity in the PAE mice. Delivery of an LNA-oligonucleotide target site blocker directed at the GRE binding region of Gas-5 or shRNA mediated gas5 knockdown during the embryonic period will restore normal GC responding and sensitivity (inclusive of stress responses, immune signaling molecules and nuclear GR-regulated genes) in adult PAE mice.