2000 WHRG Grant Program

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A Nuclear Receptor Co-Activator and Estrogen Action in the Brain

Anthony P. Auger, Ph.D.
School of Medicine, University of Maryland, Baltimore

Normal steroid hormone action in the brain is critical for the development and maintenance of a wide variety of physiological and cognitive functions. Estrogen replacement or treatment paradigms are being highly investigated and implemented for the treatment or alleviation of disorders such as bone loss associated with osteoporosis, improving memory loss associated with Alzheimer's, or preventing neuronal loss associated with stroke. However, little is known about the mechanisms controlling steroid hormone action in the brain.

Estrogen acts in the brain by binding to intracellular receptors located predominantly in neurons. Upon ligand binding, the steroid-receptor complex binds to a hormone response element (HRE) located on DNA where it regulates gene transcription and ultimately neuronal function. Recent studies reveal that steroid receptors interact with other proteins, referred to as nuclear receptor co-activators, that are critical for steroid receptor action at the HRE. The majority of studies to date have utilized transfection assays in cell culture systems in which large quantities of steroid receptors are expressed in order to elucidate interactions with co-activators and co-repressors. While a valid approach, this technique does not allow for assessment of the in vivo role of nuclear receptor co-activators and their physiological significance in developing brain, which remains largely unknown at this time. Therefore, we propose to examine the physiological importance of a nuclear receptor co-activator, originally identified as a binding protein to the cAMP response element binding protein (CBP), in mediating estrogen action in developing brain.

We will use the rat as an animal model for investigating the functional role of CBP in mediating estrogen action in the brain. To test the hypothesis that the nuclear receptor co-activator, CBP, is critical to estrogen action in the brain, we will use antisense oligodeoxynucleotides (ODNs) to reduce CBP protein expression. Antisense ODNs are synthetic stretches of DNA that work by binding to targeted mRNAs through complimentary Watson-Crick base pairing and inhibit translation of the mRNA by hybridization arrest or ablate it by increasing the activity of RNAse H, an enzyme that cleaves the RNA strand of DNA/RNA hybrids. Antisense ODNs provide a unique way to transiently reduce the expression of targeted proteins at particular time points during brain development. Briefly, we will infuse CBP antisense or control ODNs into the neonatal rat brain (i.e. hypothalamus) on the day of birth, and again on the 2 subsequent days, resulting in 3 infusions per rat. One day after birth, rats will be injected with steroid hormone (i.e. estrogen or androgen), and then allowed to develop normally. Neonatal treatment with estrogen following birth results in dramatic physiological and behavioral changes in adulthood. Some outcomes of early abnormal steroid hormone action are infertility, precocious puberty, and altered sexual orientation. Early steroid action also effects neuronal survival, migration, and morphology. Therefore, we can use these steroid-induced outcomes as an assay to determine the functional roles of CBP in mediating steroid action in the brain. Elucidating the functional significance of nuclear receptor co-activators will contribute to the design of therapeutic compounds such as "designer estrogens or anti-estrogens" and further our understanding of endocrine anomalies such as precocious puberty, infertility and early menopause. Nuclear receptor co-activators may also prove informative in understanding the complex and as yet poorly understood actions of so-called "environmental estrogens".

Many aspects of adult female reproductive physiology are governed by steroid hormone action within the brain. Disruptions in normal steroid hormone action in the developing brain can result in generalized endocrine disorders, such as adrenal hyperplasia, infertility, or early/precocious puberty; therefore understanding how steroid hormone action is regulated within the brain is critical in understanding how endocrine disorders occur. Steroid hormones act in the brain by binding to intracellular receptors located predominantly in neurons. Upon ligand binding, the steroid-receptor complex binds to a hormone response element (HRE) located on DNA where it regulates gene transcription and cell function. Recent studies reveal that steroid receptors interact with other proteins, referred to as nuclear receptor co-activators, that are critical for steroid receptor action on the genome. Although many studies have investigated the importance of nuclear receptor co-activators in using cell culture systems, little is known about the functional roles of these proteins in developing brain. We propose to investigate the importance of a nuclear receptor co-activator, identified as binding protein to the cAMP response element binding protein (CBP), in mediating steroid hormone action in developing brain.