1997 WHRG Grant Program

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Estrogen-Induced Gene Expression in the Brain
Margaret M. McCarthy, Ph.D.
School of Medicine, University of Maryland

Premarin, a synthetic estrogen, is one of the most widely used prescription drugs in the United States. Hormone replacement therapy in post-menopausal women has been found to have significant health benefits by preventing bone loss associated with osteoporosis and lowering the risk of cardiovascular disease. A relatively recently discovered and somewhat unexpected health benefit of estrogen replacement is a significant reduction in the risk of development of Alzheimer's disease, as well as improved cognitive function in women suffering from Alzheimer's or other forms of dementia. The mechanism(s) by which estrogen exerts positive influences on the brain are essentially unknown. Research in animal models suggests that estrogen may have neurotrophic and/or neuroprotective effects on the brain functioning, but the cellular basis for these actions remain unclear.

Estrogen exerts its biological action by binding to its cogent receptor. The estrogen receptor is a member of a superfamily of steroid receptors, all of which when activated by ligand binding function as transcription factors. An activated steroid receptor is transported to the cell nucleus where it binds to selective sites on the DNA and modulates gene expression. The coordinated activation and suppression of a series of proteins will then result in what is identified as "steroid hormone action". An initial series of gene products may also activate a second wave of gene expression, adding to the variety of biological responses. For this reason the mechanism of steroid hormone action is often complex and variable.

Using the rat as an animal model, the primary goal of the current proposal is to attempt to identify the major gene products induced by estrogen exposure in the brain. However, the distribution of estrogen receptors in the brain is highly heterogenous and steroid hormone action can be regionally specific. Therefore, a second major goal is to determine regionally specific effects of estrogen on gene expression in the brain. These goals will be accomplished with the use of a cloning strategy referred to as "gene expression screen". Briefly, cDNA (reverse transcribed mRNA) libraries will be generated from specific brain regions (cortex, hippocampus, and hypothalamus) of a female rat that has been treated with estrogen for 24 hours and a female rat that has been deprived of estrogen for at least two weeks. In a process known as subtractive hybridization, the cDNAs from the estrogen-deprived condition will be amplified by PCR and then allowed to hybridize with the cDNA from the estrogen-treated condition. The cDNA sequences that are common to the two conditions will hybridize with each other and will be removed from the mix by precipitation. The cDNAs that are unique to the estrogen-treated condition will remain and will then be used to screen the original library to pull out the full length clone. These clones will be sequenced and compared to the sequences already available in the Genbank database. Any previously unidentified sequences will be computer analyzed for possible hints regarding function (i.e., amino acid content, regulatory sequences, etc.). The methodology used is based on that previously established for determining the profile of gene expression after activation of the thyroid receptor (also a member of the superfamily of steroid receptors) in the process of tadpole tail reabsorption. In this case, less than 30 genes were found to be activated by the thyroid receptor within 24 hours. A similar level of activation of gene expression is anticipated in the brain, although there may be substantial regional variation in both the number and identity of gene products induced by estrogen. These data will lay important ground work for increasing our understanding of estrogen action in the brain and provide insite into the neuroportective effects of this steroid.