Atypical catecholamine function has been implicated in a number of neuropsychiatric disorders. The long term goal of this project is to understand at the DNA sequence level, factors involved in the neuronal expression of catecholamine genes. Specifically, the DNA elements involved in the restricted expression of the pivotal, central nervous system gene, tyrosine hydroxylase (TH) will be defined. Such identified dopaminergic response elements might serve in gene replacement strategies by targeting the expression of effector molecules (such as trophic factors) to dysfunctional dopamine synthesizing neurons. In order to test the hypothesis that elements within the TH promoter contribute to cell type specific expression, three main approaches will be utilized. First, TH promoter/reporter gene constructs will be transfected into a novel, dopamine-synthesizing mesencephalic cell line. DNase footprinting and gel retardation assays will be used to further define general neuronal specific elements as well as sequence motifs associated with a dopaminergic phenotype. Second, a novel amplicon-based Herpes Simplex Virus (HSV-l) system will be utilized to analyze TH promoter/reporter gene constructs. HSV-l constructs containing different TH promoter deletions driving the lacz reporter gene will be packaged into viral particles and used to infect primary cultures derived from TH- expressing peripheral and central nervous system neurons as well as TH- negative tissues such as striatum. Promising constructs will be tested in vivo by stereotactic injection of virus into TH expressing and non- expressing CNS regions. Immuno- and histochemical techniques will be employed to verify cell type specific expression. Third, defined TH promoter/reporter gene constructs encompassing putative dopaminergic response elements will be authenticated by gene transfer into the mouse embryo. Transgenic offspring may serve as valuable models with which to study the development of dopaminergic cell types. The identification of sequence motifs associated with the phenotypic expression of dopamine will lay the ground work for future experiments directed towards the targeted expression of trophic factors or other effector molecules (such as trans- acting factors, kinases, and phosphatases) to specific brain regions thought to be involved in human illnesses.
