Rosalie M. Uht - US grants

Long term objectives - To elucidate neuronal mechanisms of estrogen and glucocorticoid receptor (ER & GR) transcriptional regulation by combining molecular genetic and physiologic approaches. SPECIFIC AIMS 1- To investigate the molecular mechanisms which underlie an observed functional interaction between the ER & GR at the AP-1 cis element (IRE). Specific Aim 1 : To establish the ER/GR/IRE interaction. Specific Aim 2: To determine the effect of different Jun and Fos family members on the interaction by transfection studies. Specific Aim 3: To investigate molecular mechanisms of the interaction in mammalian cells and yeast. SPECIFIC AIMS 4&5: To analyze mechanisms of ER and GR transcriptional regulation through estrogen and glucocorticoid response elements (ERE & GRE) and the IRE in vitro and in vivo neuronal systems. Specific Aim 4: To analyze ER and GR transcriptional regulation at ERE, GRE, and IRE in a neuronal cell line. Specific Aim 5: To generate mice transgenic for ERE, GRE and TRE activated reporter transgenes. Accomplishing these specific aims will elucidate molecular mechanisms of the stress response and effects of stress on reproductive function. Specific Aims 1-3 will be accomplished by transfection studies in mammalian cells and transformation of yeast. Yeast will provide a highly efficient means for identifying protein surfaces required for the ER/GR/TRE interaction. Specific Aims 4&5 will be accomplished by constructing response element driven reporter transgenes, testing them in the GT1 neuronal cell line, and making transgenic mice. Steroid manipulations of mice will permit identification of neurons and neuronal networks which contain transcriptionally functional ER and GR.

1) The Applicant is in the fourth year of her Clinical Investigator Award (K08 DK02335-04). She is an Assistant Research Biochemist in the Metabolic Research Unit at UCSF currently spending 100 percent time in research activities. 2) Research Accomplishments: Under the K08 award, Dr. Uht has published a first authored paper and has co-authored several others. She was also selected to participate in the NSF sponsored Young Investigator Symposium in the 1996 Workshop on Steroid Hormones and Brain Function and invited to present at the 1998 American Neuroendocrine Society Workshop. She has authored several abstracts, presenting her work in poster form at national meetings. 3) Research Plan: Long Term Objectives: to elucidate the molecular mechanisms by which estrogens and glucocorticoids regulate physiologic and pathophysiologic states in the nervous system by combining molecular and in vivo approaches. SPECIFIC AIMS 1, 4, and to a large extent 2, of the original K08 have been accomplished. The current proposal is designed to expedite progress on the remaining Specific Aims. They are: Specific Aim 1: Generate mice transgenic for steroid receptor activated reporter genes. Specific Aim 2: Analyze mechanisms by which ER and GR mediate transcriptional regulation at certain AP-1 sites. Specific Aim 3: Establish a yeast system which will permit analysis of factors required for the ER/GR/AP-1 site interaction. 4) Environment/Career Development: The environment at UCSF remains rich and supportive. If granted, R03 will greatly facilitate the development of the reporter transgenic mice and the yeast system. The preliminary data obtained will be used to support an application for an R01. The R03 will also facilitate publishing manuscripts to report work currently in progress.

DESCRIPTION (provided by applicant): Nineteen million people a year in the United States experience depression. A potentially lethal disease, depression puts a strain on family members, leads to lost hours at school and work, is costly to treat, and places a substantial burden on society. In light of this, it is surprising that the pathogenesis of depression is still poorly understood. One feature of the illness that is clear - depression is highly correlated with an abnormal response to stress. The stress response is mediated by the hypothalamic-pituitary-adrenal (HPA) axis. Activity of the HPA axis is normally tightly regulated, in large part because the end products, glucocorticoids, are potent down- regulators of the system. In depression, neurons in the hypothalamus that synthesize and secrete the peptide that triggers the stress response, corticotropin-releasing hormone (CRH), are insensitive to glucocorticoid down-regulation. Because these neurons are the final common integrators of inputs from the central nervous system and the hormonal milieu, if they are abnormally responsive, the entire functioning of the axis is negatively affected. The experiments described in this proposal are designed to elucidate mechanisms by which glucocorticoids down-regulate expression of the CRH gene (crh). Aims 1 and 2 take advantage of a relatively new and powerful technique, chromatin immunoprecipitation (ChIP), and a relatively new neuronal cell line (IVB), which has many features of CRH cells in the hypothalamus. Aim 1 is designed to determine which arrays of co-regulatory proteins are required for down-regulation of crh expression. Aim 2 is designed to determine the role that chromatin modification enzymes and their epigenetic marks play in regulating crh. In Aim 3, the work will be taken into an in vivo setting. Data gained from Aims 1 and 2 will guide the in vivo work by narrowing the array of possible candidates to be chosen for analysis. Two in vivo settings will be studied. First, patterns of coregulators and histone modification enzymes will be assessed at the circadian peak and trough of corticosterone (Cort). These experiments will permit analysis of the effect physiological changes in Cort levels exert on coregulators and histone modification enzymes. Second, rats will be administered metyrapone at short time points, and the same parameters assessed as in the circadian studies. Taken together, the studies will permit molecular analysis of epigenetic mechanisms that regulate the stress response. This unique combination of approaches to the study of depression will produce a novel array of data, leading to a greater understanding of mechanisms of HPA regulation, and increasing our ability to identify novel drug targets for the treatment of depression. PUBLIC HEALTH RELEVANCE: Depression is a remarkably common illness that carries a lifetime risk of 20-25% - one in every five people will experience an episode of depression in the United States. A potentially lethal disease, depression puts strain on family members, leads to lost hours at school and work, is costly to treat, and places a substantial burden on society. Many cases are treatable with drugs such as fluoxetine (Prozac), but rarely is any single drug continually efficacious for a given individual. The current trend is to use a combination of drugs;however, many of these drugs'mechanisms of action are poorly understood. Optimizing this poly-pharmaceutical approach can be better accomplished by defining various steps in the pathogenesis of depression, which will lead to the identification of novel drug targets. One aspect of depression is clear - it is inexorably linked to a dysfunctional response to stress. The physiological system that mounts the stress response is the hypothalamic-pituitary-axis. Stressors are integrated in the hypothalamus, which sits at the base of the brain. These cells synthesize and secrete corticotropin-releasing hormone, or CRH. It is this peptide that triggers the stress response, and it is the cells that synthesize CRH that are abnormal. The proposed studies are designed to elucidate mechanisms by which the stress steroids, glucocorticoids, exert their usual, and profound, inhibitory effect on the CRH neurons. The studies take advantage of a powerful technique, chromatin immunoprecipitation, which is currently in its infancy as an investigatory tool of the nervous system. Additionally, the studies incorporate a relatively new cell line derived from the hypothalamus that has many features of CRH neurons. Data from the cell line will be used to develop mechanistic models of the stress response, which will be tested in vivo in the last set of proposed experiments.