Catecholamine neurotransmitter systems have been implicated in the patho-physiology of some central nervous system diseases including schizophrenia, manic depression, Parkinson's and Alzheimer's syndromes. The understanding of the expression and regulation of this important metabolic pathway is not only of biological interest but critical to exploring the fundamental basis of these disorders and more rationally approaching their diagnosis and therapy. Our strategy requires the structural analysis of the genes for all four catecholamine synthesizing enzymes in order to investigate the coordinate and differential regulation in vivo. We have isolated and characterized cDNAs for tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine B-hydroxylase and phenylethanolamine N-methyltransferase. These are now being used to study changes in mRNA expression under various physiological conditions. In addition, genomic clones for each of the enzymes are being identified and characterized with these molecular probes. The following experiments are proposed to accomplish these goals: 1. Definition of the basic structural elements of the catecholamine enzyme genes by isolation of genomic clones, mapping and sequence analysis; 2. Determination of those physiological regulators which influence the transcription of these genes in vivo using primary bovine chromaffin cells as a model; 3. Identification and characterization of the genetic elements responsible for cell-type specific and regulated expression of each enzyme gene by transfection assay and mutagenesis of putative regulatory elements; 4. Examination of the interaction of nuclear trans-acting factors with regulatory regions in the gene using footprinting and in vitro transcription assays; 5. Investigation of possible common elements and/or factors shared among the genes which underly coordinate regulation of the pathway using competition assays. These experiments will not only further our understanding of this important biosynthetic pathway but will provide the techniques and molecular tools to approach the purification of trans-acting factors regulating the tissue specific and regulated expression of the catecholamine enzyme genes. Their identification and characterization will be key to understanding this regulation at the molecular level.
Showing the most recent 10 out of 56 publications
