5812 base pairs of rat GTP cyclohydrolase I (GTPCH) 5-flanking region were cloned and sequenced, and the transcription start site was determined for the gene in rat liver. Progressive deletion analysis using transient transfection assays of luciferase reporter constructs defined the core promoter as a highly conserved 142-base pair GC-rich sequence upstream from the cap site. DNase I footprint analysis of this region revealed (5 3 3) a Sp1/GC box, a noncanonical cAMP-response element (CRE), a CCAAT-box, and an E-box. Transcription from the core promoter in PC12 but not C6 or Rat2 cells was enhanced by incubation with 8-bromo-cyclic AMP. Mutagenesis showed that both the CRE and CCAAT-box independently contribute to basal and cAMP-dependent activity. The combined CRE and CCAAT-box cassette was also found to enhance basal transcription and confer cAMP sensitivity on a heterologous minimal promoter. The addition of the Sp1/GC box sequence to this minimal promoter construct inhibited basal transcription without affecting the cAMP response. EMSA showed that nuclear proteins from PC12 but not C6 or Rat2 cells bind the CRE as a complex containing activating transcription factor (ATF)-4 and CCAAT enhancer-binding protein ␤, while both PC12 and C6 cell nuclear extracts were recruited by the CCAAT-box as a complex containing nuclear factor Y. Overexpression of ATF-4 in PC12 cells was found to transactivate the GT-PCH promoter response to cAMP. These studies suggest that the elements required for cell type-specific cAMPdependent enhancement of gene transcription are located along the GTPCH core promoter and include the CRE and adjacent CCAAT-box and the proteins ATF-4, CCAAT enhancer-binding protein ␤, and nuclear factor Y.

Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by ϳ20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GT-PCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L-phenylalanineinduced increase in tetrahydrobiopterin levels. 7,8-Dihydroneopterin, a reduced pteridine capable of inhibiting GTPCH in a GFRP-dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was reversed by Lphenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhibitory tone mediated by GFRP and that tetrahydrobiopterin levels are maintained by the balance of intracellular concentrations of tetrahydrobiopterin and L-phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback-regulated, L-phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPCH activity. Key Words: 5,6,7,8-Tetrahydrobiopterin-GTP cyclohydrolase I-GTP cyclohydrolase I feedback regulatory protein-Dopamine-Serotonin.

Abstract:The hph-1 mouse is characterized by low levels of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative double-label in situ hybridization technique was used to examine CNS GTPCH mRNA expression within serotonin, dopamine, and norepinephrine neurons of male and female wild-type and hph-1 mice. In wild-type male and female animals the highest levels of GTPCH mRNA expression were observed within serotonin neurons, followed by norepinephrine and then dopamine neurons. Wild-type female animals were found to express lower levels of GTPCH mRNA in each cell type when compared with levels seen in wild-type males. GTPCH mRNA abundance in all three cell types was lower in hph-1 male than in wild-type male mice, with the greatest reduction in serotonin neurons. GTPCH mRNA levels were also lower in hph-1 female than in wild-type female mice, again with the greatest reduction occurring in serotonin neurons. Comparison of hph-1 male and hph-1 female mice revealed that the sex-linked difference in GTPCH mRNA expression observed in wildtype neurons was only present within female dopamine neurons. Overall, these results indicate that not only are basal levels of GTPCH mRNA expression heterogeneous across wild-type murine monoamine cell types but that gene expression is also modified in a sex-linked and cell-specific fashion by the hph-1 gene locus. The hph-1 mutation does not lie within the GT-PCH mRNA coding region. The 5Ј flanking region of the GTPCH gene was cloned and sequenced and shown to be identical for both wild-type and hph-1 genomic DNA. Transient transfection assays performed in PC12 cells demonstrated that this 5Ј flanking region was sufficient to initiate transcription of a luciferase reporter gene. Although the hph-1 mutation does not lie within the 5Ј flanking region of the GTPCH gene, this region of the gene can function as a core promoter and is thus crucial to the control of GTPCH gene expression. Key Words: GTP cyclohydrolase I-Tetrahydrobiopterin-hph-1 mouse-Hereditary progressive dystonia-Monooxygenases.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridization. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1.2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2- and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.

Fig. 4 Human GTPCH promoter activity is stimulated by 8Br-cAMP treatment. (a) GTPCH 5¢-flanking sequence showing restriction sites used for generating the luciferase reporter gene deletion constructs 1171GTPCHluc (DraI), 613GTPCHluc (HindIII), and 313GTPCHluc (AccIII). Also shown are putative Sp1, CRE, CCAAT-box and TATAbox like sequences located within the 313GTPCHluc construct. (b) SK-N-BE(2)M17 cells were transfected with GTPCHluc constructs and pCMV-b-gal DNA. Then, 5 mM 8Br-cAMP was added 18 h later and the cultures continued for another 8 h, when cells were lysed and assayed for luciferase and b-galactosidase activities. Luciferase activity was divided by b-galactosidase activity to correct for transfection efficiency and was expressed as relative luciferase activity. Data are the mean ± SE of three independent experiments each determined in triplicate.