Corticosteroid actions in the brain are critical for blood pressure regulation, neural plasticity, and responses to stress. The two major classes of corticosteroids, mineralocorticoids and glucocorticoids, exert their effects primarily through intracellular receptors- the mineralocorticoid (MR) and glucocorticoid (GR) receptors- that elicit changes in cellular behavior through direct effects on the transcriptional activity of specific target genes. While in some brain sites, MR acts as a receptor for the mineralocorticoid, aldosterone, in other brain structures, including hippocampus, MR acts as a high affinity glucocorticoid receptor, mediating the stimulatory effects on neuronal excitability of low levels of corticosterone (the principal glucocorticoid in rats). High corticosterone levels, as seen in stress, depression, or Cushing's syndrome activate both MR and GR and suppress neuronal activity. While intermittent elevation of corticosterone is important for behavioral plasticity, chronic elevations can damage neurons, cause cognitive dysfunction and may be implicated in the pathogenesis of hypertension. Our long-term aims are to gain mechanistic insight into how MR and GR function as transcriptional regulators with distinct specificities in brain, initially focusing on hippocampal neurons. Towards this end we will: 1)Localize the N-terminal activation domain of rat MR and identify specific amino acids required for MR activity using a random mutagenesis screen in yeast. Once activation mutants have been identified they will be tested in transfection experiments using a hippocampal neuronal cell line. We will then go on to identify, clone and characterize hippocampal factors that interact specifically with MR (and/or GR) N-terminal regions; 2)Determine the mechanistic basis of hippocampal-specific expression and corticosteroid repression of the 5-HT1A receptor gene, a key steroid target gene in hippocampus. Using cultured hippocampal neurons and cloned 5-HT1A genomic DNA, we will identify hippocampal-specific and steroid-regulated response elements;3) Identify and characterize target genes in hippocampus. We will being by characterizing the expression pattern and steroid response of Synaptotagmin I and Tmp21, genes recently found by us to be steroid regulated in hippocampus. We will then to go on to study the transcriptional regulation of these genes using approaches similar to those described above for 5-HT1A. Finally, we will use a PCR-based cDNA subtraction method to identify genes differentially regulated by MR and GR.
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