Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of the catecholamine neurotransmitters, dopamine, norepinephrine, and epinephrine, which are essential during development. TH 20 anatomically and functionally discrete nuclei in the mammalian CNS and peripheral nervous system, several of which sustain activity-dependent induction of TH. We have identified regulatory sites that direct TH expression in cell culture: principal among these are the cAMP response element (CRE) and the AP1 site. We wi11 mutate the TH CRE and AP I sites in a TH-driven reporter to the consensus DNA binding site for Ga14, a yeast transcription factor absent in mammalian cells. Expression from the mutated enhancers will be assessed in transgenic mice to determine if the CRE or API sites are important in vivo, for cell specific and trans- synaptic induction. Although many transcription factors composed of the transactivation domain of various transcription factors fused to a GaI4 DNA binding domain will be used in cultured cells and in transgenic mice to rescue basal and induced expression from the mutated CRE site. We seek to develop particle-mediated transfection into brain slices as an alternative to promoter mapping in transgemic mice. Reporter constructs will be biolistically transfected into acutely prepared where TH neurons retain their local physiological and anatomical connections. By co-staining for the endogenous TH gene, specificity at the single cell level can be assessed. We have identified a 4.5 KB 5' flanking region that directs expression to all TH CNS and PNS groups in transgenic mice. We hypothesize that discrete elements in this region direct cell-specific expression. Promoter mutations will be used to direct expression of an epitope- tagged TH transgene. Those lines lacking expression in discrete TH cells will be bred to a TH knockout mouse to restore catecholamines to all the but the ablated regions, creating place-specific knockouts.
This aim will not only map putative cell-specific elements in vivo, but provide new animal models for Parkinson's disease, dysautonomia, affected disorders and drug abuse.
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