The catecholamine (CA) neurotransmitters, dopamine (DA), norepinephrine (NE), and epinephrine, play a profound role in diverse brain functions. Tyrosine hydroxylase (TH) catalyzes the conversion of tyrosine to L-dopa, the first and rate-limiting step in CA biosynthesis, and dopamine beta-hydroxylase (DBH) converts DA to NE. Thus, the proper expression of TH and DBH is essential for specification and maintenance of the neurotransmitter phenotypes of CA neurons. Additionally, their abnormal regulation is implicated in several neurological and psychiatric disorders. A typical example is NE deficiency disease, in which NE is undetectable while DA is remarkably upregulated. Our previous experiments have delineated important aspects of the control mechanisms of TH and DBH gene expression and identified seven novel polymorphisms from two NE deficient patients. These data led to the following hypotheses in regard to the regulation of CA neurotransmitter identities in normal as well as in diseased brain. First, we hypothesize that the TH gene is induced and maintained by significantly distinct mechanisms in different subsets of CA neurons and that several key transcription factors may be involved in these processes. To address this hypothesis, we will test how candidate transcription factors, e.g., Nurrl, Phox2a/2b, and GATA3, may regulate TH gene transcription using cell culture systems and in vitro differentiation paradigm of embryonic stem cells. To investigate in vivo regulation of TH gene expression, transgenic mice will be generated using mutant TH promoter(s) containing altered binding sites of candidate factors. Secondly, our in vitro promoter analysis suggests that the DBH gene is a direct target of Phox2a/2b. To address this hypothesis using an in vivo system, we will generate and characterize transgenic mice using wild type and mutant reporter constructs. We will also test if in vivo promoter strength and cell-specificity can be improved by engineering the Phox2a/2b binding sites to consensus motif in the DBH promoter. Finally, we will initiate studies to examine how mutations identified in NE deficient patients may affect DBH gene expression, by analyzing their effect on the transcriptional activity.The proposed studies will further advance our understanding of molecular mechanisms underlying the regulation of neurotransmitter phenotypes in CA neurons at the molecular and systemic levels. This information may translate into early diagnosis and/or novel therapeutic approaches for the myriad of human diseases associated with CA dysregulation.
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