Library

Notes: 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.

Notes: 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.

Notes: 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.

Notes: Abstract : The hph-1 mouse is characterized by low levelsof GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitativedouble-lable in situ hybridization technique was used to examine CNS GTPCHmRNA expression within serotonin, dopamine, and norepinephrine neurons of maleand female wild-type and hph-1 mice. In wild-type male and femaleanimals the highest levels of GTPCH mRNA expression were observed withinserotonin neurons, followed by norepinephrine and then dopamine neurons.Wild-type female animals were found to express lower levels of GTPCH mRNA ineach cell type when compared with levels seen in wild-type males. GTPCH mRNAabundance in all three cell types was lower in hph-1 male than inwild-type male mice, with the greatest reduction in serotonin neurons. GTPCHmRNA levels were also lower in hph-1 female than in wild-type femalemice, again with the greatest reduction occurring in serotonin neurons.Comparison of hph-1 male and hph-1 female mice revealed thatthe sex-linked difference in GTPCH mRNA expression observed in wild-typeneurons was only present within female dopamine neurons. Overall, theseresults indicate that not only are basal levels of GTPCH mRNA expressionheterogeneous across wild-type murine monoamine cell types but that geneexpression is also modified in a sex-linked and cell-specific fashion by thehph-1 gene locus. The hph-1 mutation does not lie within theGT-PCH mRNA coding region. The 5′ flanking region of the GTPCH gene wascloned and sequenced and shown to be identical for both wild-type andhph-1 genomic DNA. Transient transfection assays performed in PC12cells demonstrated that this 5′ flanking region was sufficient toinitiate transcription of a luciferase reporter gene. Although thehph-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.

Notes: 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.

Notes: 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.

Notes: 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.