| The mechanisms of dopamine regulation and the responsible proteins are very highly conserved in all multi-cellular organisms. Such conservation makes it possible to utilize genetic model systems such as Drosophila melanogaster to identify new components of the cellular network that is responsible for dopamine homeostatic mechanisms and to more clearly define these mechanisms. The research described in this dissertation has resulted in the discovery of previously unknown or uncharacterized components of the dopamine homeostatic network and has provided insights into their roles in normal neuron function and in oxidative stress-induced neurodegeneration.; Catsup is a protein that has novel functions. Loss-of-function Catsup mutants have elevated dopamine levels and abnormally elevated tetrahydrobiopterin, an essential cofactor for dopamine synthesis. The results of this research establish that the enzymes that catalyze the rate limiting steps in dopamine and tetrahydrobiopterin synthesis in the adult central nervous system, tyrosine hydroxylase and GTP-cyclohydrolase, are hyperactivated, confirming that Catsup is a negative regulator of dopamine homeostasis. These studies also demonstrate that the Catsup protein regulates DA transport into synaptic vesicles for storage and release. Catsup protein was found to be present in synaptic vesicles, where it is likely to be in the vesicle membrane, and serves as a negative regulator of Vesicular Monoamine Transporter which is responsible for the transport of dopamine from the cytoplasm into synaptic vesicles.; Previous studies indicate that two proteins, 14-3-3 and alpha-synuclein, have potential functions in dopamine regulation. Drosophila 14-3-3 zeta protein was shown to have multiple roles in maintaining dopamine homeostasis in the brain, including negative regulation of tyrosine hydroxylase and vesicular monoamine transporter; while overexpression of human alpha-synuclein in Drosophila causes inhibition of tyrosine hydroxylase and therefore, dopamine synthesis. In addition, these mutants show differential responses to oxidative stressors, paraquat and hydrogen peroxide, suggesting selective targeting between brain and non-brain tissue.; Dopamine metabolism also was analyzed in a Drosophila model of the human Fragile X syndrome. Drosophila Fragile X mental retardation (dfmr1) mutants show increased production of dopamine and tetrahydrobiopterin. Mutants were found to be hyperactive, a behavioral change that is consistent with the hyperactivity of Fragile X patients. |
