Tetrahydrobiopterin (BH4) is the cofactor for tyrosine hydroxylase and tryptophan hydroxylase, which are the initial and rate-limiting enzymes in catecholamine and serotonin synthesis. BH4 is an important regulator of catecholamine synthesis, since BH4 administration increases brain dopamine synthesis, whereas inhibition of endogenous BH4 synthesis in brain leads to catecholamine deficits. Of all the genes encoding proteins involved in pre- and postsynaptic catecholamine metabolism, only the altered expression of genes encoding BH4 biosynthetic enzymes is known to cause human illness. Specifically, genetic defects in the expression of any one of several BH4 biosynthetic enzymes cause BH4 deficiency in liver and brain at birth in atypical phenylketonuria (PKU); the BH4 deficit causes brain biogenic amine deficiency and neurological impairment. Since many other systems requiring biogenic amine synthesis function normally in these patients, atypical PKU may be caused by tissue-specific regulatory mutations of BH4 metabolism. Thus, altered expression of the """"""""candidate genes"""""""" encoding BH4 biosynthetic enzymes and tyrosine hydroxylase may be responsible for the BH4 and catecholamine deficits observed in aging, Alzheimer's disease, and other neuropsychiatric disorders, which warrants the study of BH4-related genes. The BH4 biosynthetic enzymes to be studied are GTP cyclohydrolase and sepiapterin reductase, which are the initial and final enzymes. In rodents, GTP cyclohydrolase may be the main rate-controlling enzyme in BH4 synthesis. In humans, sepiapterin reductase may also contribute to regulating BH4 production, though further study is required. The first goal of this proposal is to clone the human cDNAs for GTP cyclohydrolase and sepiapterin reductase to examine altered genetic expression of these enzymes and tyrosine hydroxylase in human brain in aging and Alzheimer's disease. Expression of these enzyme will be examined more completely by measuring multiple parameters including tissue mRNAs, amounts of enzymes, enzyme activities, and end-products of biosynthesis (BH4 and catecholamines). Part of the second goal using young and old rats is to test the combined effects of aging and neuro-degeneration on tyrosine hydroxylase, GTP cyclohydrolase, and sepiapterin reductase gene expression in surviving nigrostriatal dopamine neurons following striatal kainic acid lesions of non-dopamine neurons. Other rats will be treated with drugs affecting BH4 and catecholamine metabolism to investigate regulatory interactions between BH4 and catecholamine biosynthesis. The hypothesis will be tested in brain and adrenal catecholamine-producing cells that coordinate regulation causes a concomitant enhancement of GTP cyclohydrolase and sepiapterin reductase gene expression when tyrosine hydroxylase expression is enhanced. Coordinate regulation will be studied in rat brain and adrenal after treatments with: 1) inhibitors of GTP cyclohydrolase; 2) activators of BH4 and catecholamine synthesis, and 3) kainic acid in striatum, which the PI has shown to increase striatal tyrosine hydroxylase and GTP cyclohydrolase activities. These studies in human and rat tissues will advance the knowledge of regulation of BH4 and catecholamine biosynthesis and its involvement in aging and Alzheimer's disease. The cloning of human cDNAs for BH4 biosynthetic enzymes will allow related studies on: 1) characterizing the genes and promoter regulation; 2) chromosomal assignments, and 3) genetic linkage analyses in neuropsychiatric illnesses. Thus, the PI's long-term objective to utilize molecular biological approaches for identifying altered BH4 and catecholamine metabolism in human neuropsychiatric illness will be achieved.
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