Kei Hirayama

GTP cyclohydrolase I gene expression in the brains of male and female hph-1 mice.

Abstract : The hph‐1 mouse is characterized by low levels of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative double‐lable 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 wild‐type 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 5′ 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 5′ flanking region was sufficient to initiate transcription of a luciferase reporter gene. Although the hph‐1 mutation does not lie within the 5′ 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.

Tetrahydrobiopterin Cofactor Biosynthesis: GTP Cyclohydrolase I mRNA Expression in Rat Brain and Superior Cervical Ganglia

Abstract: 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 hybridisation. 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.

Nigrostriatal dopamine neurons express low levels of GTP cyclohydrolase I protein

Abstract: A previous study using the in situ hybridization technique showed that serotonin neurons contain substantially more GTP cyclohydrolase I mRNA than do either dopamine or norepinephrine neurons. The objective of the current study was to determine whether these differences in mRNA abundance are predictive of the amount of GTP cyclohydrolase I protein available for tetrahydrobiopterin biosynthesis. The double‐label immunofluorescence technique was used to localize GTP cyclohydrolase I protein to the tyrosine hydroxylase‐positive A9 dopamine neurons of the substantia nigra and the A6 norepinephrine neurons of the locus ceruleus or the tryptophan hydroxylase‐positive B6/B7 serotonin neurons of the dorsal raphe nucleus. Although GTP cyclohydrolase I immunofluorescence within serotonin and norepinephrine neurons was relatively intense, the fluorescence signal within dopamine neurons was faint to nondetectable. An immunoautoradiographic technique was developed to quantify these apparent differences in GTP cyclohydrolase I protein expression at the cellular level. Significant differences between all three neurochemical subdivisions were found and comparisons showed that on average serotonin neurons contain between 2.3‐ and 7.3‐fold more GTP cyclohydrolase I protein than do either norepinephrine or dopamine neurons, respectively. Nigrostriatal dopamine neurons thus appear to synthesize and maintain tetrahydrobiopterin at low levels. Because dopamine and norepinephrine neurons express essentially equal amounts of GTP cyclohydrolase I mRNA, posttranscriptional events may serve to maintain low levels of GTP cyclohydrolase I protein within dopamine neurons. Phenotypic differences in GTP cyclohydrolase I protein expression across populations of monoamine neurons may be an important control point in neurotransmitter biosynthesis.

Tetrahydrobiopterin biosynthesis in C6 glioma cells: induction of GTP cyclohydrolase I gene expression by lipopolysaccharide and cytokine treatment

The possibility that 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis is stimulated in glial cells by treatment with lipopolysaccharide (LPS) and tumor necrosis factor (TNF-alpha) was examined in the astrocyte-derived C6 glioma cell line. Under basal culture conditions BH4 levels were found to be at the limit of detection. Concurrent treatment with 10 micrograms/ml LPS and 50 ng/ml TNF-alpha caused a time-dependent 13-fold increase in the levels of BH4. This treatment paradigm also induced nitric oxide synthase activity, as evidenced by increased levels of nitrite, an oxidized metabolite of NO, in the culture medium. LPS and TNF-alpha treatment led to a 25-fold increase in GTPCH enzyme activity, the first and rate-limiting enzyme in BH4 synthesis, and a corresponding 23-fold increase in GTPCH protein levels. Northern blot analysis showed that increased levels of GTPCH mRNA preceded changes in GTPCH protein, GTPCH enzyme activity and BH4 levels and reached a maximal of 44-fold that was sustained for at least 48 h. These results demonstrate that LPS and TNF-alpha stimulate de-novo BH4 biosynthesis and suggest that C6 cells offer a model system for studying the molecular events that control the induction of GTPCH gene expression and BH4 synthesis in glial cells.

GTP Cyclohydrolase I Feedback Regulatory Protein Is Expressed in Serotonin Neurons and Regulates Tetrahydrobiopterin Biosynthesis

Abstract : 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 ~20 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 GTPCH 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‐phenylalanine‐induced 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 L‐phenylalanine 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.

Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN-BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter.

GTP cyclohydrolase I (GTPCH) gene expression was investigated in the human monoamine‐containing neuroblastoma cell line SK‐N‐BE(2)M17. Northern blot analysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced forms of GTPCH mRNA were detected with this assay. Incubation with 8Br‐cAMP, but not nerve growth factor or leukemia inhibitory factor, produced a rapid increase in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly between 10 and 24 h. Treatment with 8Br‐cAMP did not significantly modify the stability of GTPCH mRNA but did increase GTPCH transcription as determined by transient transfection assays of a luciferase reporter construct containing 1171 bp of human GTPCH 5′‐flanking sequence. Cis‐acting elements required for maximal basal and cAMP‐dependent transcription were localized by deletion analysis to the 146 bp proximal promoter. DNase I footprint analysis of the proximal promoter using SK‐N‐BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Sp1‐like site and the other a combined CRE‐Sp1‐CCAAT‐box element. EMSA and supershift assays demonstrated that the combined CRE‐Sp1‐CCAAT‐box element recruits ATF‐2 and NF‐Y but not Sp1–4 or Egr‐1–3. NF‐Y binding was confirmed using pure recombinant human NF‐Y protein. Transcription of the human GTPCH gene in human SK‐N‐BE(2)M17 cells is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF‐Y and ATF‐2.

Tetrahydrobiopterin Turnover in Cultured Rat Sympathetic Neurons: Developmental Profile, Pharmacologic Sensitivity, and Relationship to Norepinephrine Synthesis

Abstract: We have examined the turnover of 5,6,7,8‐tetrahydrobiopterin (BH4) and the effect of decreasing BH4 levels on in situ tyrosine hydroxylase (TH) activity and norepinephrine (NE) content in a homogeneous population of NE‐containing neurons derived from the superior cervical ganglion (SCG) of the neonatal rat and maintained in tissue culture. Initial studies indicated that the level of BH4 within SCG cultures increased fourfold between 5 and 37 days in vitro (DIV). This increase in BH4 levels was determined to result from an increase in the rate of BH4 biosynthesis without a change in the rate of degradation. Regardless of culture age, the BH4 content of SCG neurons was observed to turn over with a half‐life of ∼2.5 h. BH4 synthesis by SCG neurons was found to be five times more sensitive to inhibition by 2,4‐diamino‐6‐hydroxypyrimidine (DAHP) and 25 times less sensitive to inhibition by N‐acetylserotonin than was previously reported for CNS neurons in culture. Under basal conditions, the rates of in situ TH activity and BH4 biosynthesis were similar. In response to inhibition of BH4 biosynthesis by DAHP and a 90–95% decrease in BH4 levels, in situ TH activity declined by 75%. NE levels declined by 30% following a 24‐h period of inhibition of BH4 synthesis. After 2 days of BH4 synthesis inhibition, the level of NE was decreased by 47%. On treatment days 3 and 4, the decline in NE content plateaued at 24% of control levels. In contrast, treatment of cultures for 24 h with the direct‐acting inhibitor of TH, α‐methyl‐p‐tyrosine, produced an 84% decline in NE content that was maintained over the 4‐day treatment period, indicating that the slow decline in NE content following inhibition of BH4 synthesis was not the result of the slow turnover rate of NE. These results demonstrate that despite an almost complete loss of BH4, sympathetic neurons were able to maintain neurotransmitter content, albeit at reduced levels, by retaining a level of TH activity above the value that might have been predicted based on the reduced level of BH4.

Regulation of GTP Cyclohydrolase I Gene Expression and Tetrahydrobiopterin Content in Cultured Sympathetic Neurons by Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor

Abstract: Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8‐tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration‐dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3–7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF‐dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78–96% reduction in GTPCH mRNA content relative to β‐actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4‐dependent enzyme and the rate‐limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.

Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content by nerve growth factor in cultures of superior cervical ganglia.

Monolayer cultures of superior cervical ganglia free of support cells were maintained in the presence of 100 ng/ml 7S-NGF for 4 days. The concentration of NGF was then changed to between 50 and 400 ng/ml and cultures continued for an additional 7 days. Tetrahydrobiopterin (BH4) content, GTP cyclohydrolase (GTPCH) enzyme activity and mRNA levels were then determined. All three of these measures were found to be elevated between 2- to 4-fold by treatment with increasing concentrations of NGF. Tyrosine hydroxylase (TH) enzyme activity and mRNA levels were increased from 8 to 13-fold by these same treatments. These results indicate that the content of BH4 within sympathetic neurons can be regulated by NGF receptor-mediated changes in GTPCH gene expression. Moreover, concomitant increases in TH enzyme activity and BH4 content demonstrate a coordinated regulation by NGF of this enzyme and its essential cofactor.

Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA

6-Pyruvoyl tetrahydropterin synthase catalyzes the second step in the biosynthesis of tetrahydrobiopterin. In the present study, the reverse transcription-polymerase chain reaction technique was used to clone a portion of 6-pyruvoyl tetrahydropterin synthase cDNA from rat pineal gland RNA. The sequence of this cDNA was found to be essentially identical to that previously reported for the rat liver. 6-Pyruvoyl tetrahydropterin synthase mRNA levels in various rat tissues, including the brain, were then analyzed by Northern blot and nuclease protection assay. A single 1.35 kb transcript of 6-pyruvoyl tetrahydropterin synthase mRNA was detected by Northern blot analysis in the rat adrenal gland, brain-stem, and liver. Quantitation by nuclease protection assay demonstrated that 6-pyruvoyl tetrahydropterin synthase mRNA was most abundant in the adrenal gland, kidney, and pineal gland (19.5-25.5 amol/microgram RNA). Relatively homogeneous levels of 6-pyruvoyl tetrahydropterin synthase mRNA were found in various brain regions including the cerebellum, substantia nigra and locus coeruleus (4.12-12 amol/microgram RNA). In the adrenal gland, 6-pyruvoyl tetrahydropterin synthase and tyrosine hydroxylase mRNAs were elevated between 3 and 4-fold 24 h after a single dose of reserpine (10 mg/kg), a treatment known to increase tetrahydrobiopterin levels in this tissue. This result suggests that although 6-pyruvoyl tetrahydropterin synthase is not believed to be rate-limiting in the tetrahydrobiopterin biosynthetic pathway, control of gene expression for this enzyme may play an essential role in regulating the synthesis of this important cofactor.