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GTP cyclohydrolase I (GCH) catalyzes the first and rate limiting step reaction for the de novo synthesis of 5,6,7,8-tetrahydrobiopterin (BH4). The expression of GCH is dramatically elevated by immune activation, while the mechanism remains to be elucidated. In this study, we investigated the transcription mechanism of the GCH gene using lipopolysaccharide (LPS) to stimulate mouse macrophage RAW264.7 cells. With luciferase assay, we found a highly conserved enhancer region spanning approximately 300 bp in intron 1 of GCH gene as a response element to LPS stimulation. The same enhancer region was also responsible for the induction of the GCH gene by IFN-γ and TNF-α in HUVECs. With electrophoresis mobility shift assay (EMSA) and site directed mutation analysis, we identified two key fragments containing C/EBP and Ets binding motifs within the enhancer. Furthermore, C/EBP-β was involved in LPS activated GCH transcription through direct binding to the enhancer shown by supershift, chromatin immunoprecipitation, and RNA interference experiments. In conclusion, our findings uncovered a novel mechanism of GCH transcriptional regulation by immune activation.

SummaryAlthough the mechanisms underlying the spatial pattern formation of sensory maps have been extensively investigated, those triggering sensory map formation during development are largely unknown. Here we show that the birth of pups instructively and selectively regulates the initiation of barrel formation in the somatosensory cortex by reducing serotonin concentration. We found that preterm birth accelerated barrel formation, whereas it did not affect either barreloid formation or barrel structural plasticity. We also found that serotonin was selectively reduced soon after birth and that the reduction of serotonin was triggered by birth. The reduction of serotonin was necessary and sufficient for the effect of birth on barrel formation. Interestingly, the regulatory mechanisms described here were also found to regulate eye-specific segregation in the visual system, suggesting that they are utilized in various brain regions. Our results shed light on roles of birth and serotonin in sensory map formation.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamine but its transcriptional regulation is not fully understood. Using a reporter assay with cultured rat cortical neurons, we demonstrated that the TH gene promoter was activated by brain-derived neurotrophic factor (BDNF), through its specific receptor TrkB and the ERK/MAP kinase pathway. Using a series of mutant TH gene promoters, we found that the cAMP-response element (CRE) plays a crucial role in the TH promoter activity and the Egr-1-responsive element (ERE), at least in part, is responsible for the BDNF-induced activation. Notably, the influx of Ca(2+) evoked via the N-methyl-D-aspartate receptor (NMDA-R) but not via the L-type voltage-dependent Ca(2+) channel (L-VDCC) selectively antagonized the activation of the gene promoter, suggesting a new link between the catecholaminergic and glutamatergic systems. The Ca(2+) signals evoked via NMDA-R did not affect the phosphorylation of ERK1/2 induced by BDNF. These results suggest that the TH gene's transcription is positively regulated by BDNF, through the CRE and ERE of the promoter, but selectively antagonized by the Ca(2+) signals evoked via NMDA-R without disturbing the ERK/MAP kinase pathway, the regulation by which may underlie the development of the catecholaminergic system in the brain.

Sepiapterin reductase (SPR) is an enzyme that acts in the third and final step of tetrahydrobiopterin (BH4) biosynthesis. The human Spr gene locates within the region of 2.5MB mapped to PARK3, an autosomal dominant form of familial Parkinson's diseases. In order to explore the role of SPR in the metabolism of BH4, we produced and analyzed Spr-deficient mice. Most of Spr-null mice survived beyond two weeks. Whereas the BH4 contents in the homozygous mutant mice were greatly decreased than those in wild-type and heterozygous mice, the substantial amounts of BH4 were remained even 17 days after delivery. Spr-null mice exhibited severe monoamine deficiencies and a tremor-like phenotype after weaning. The amount of TH protein in the brain of Spr-null mice was less than 10% of wild-type, while TH protein in the adrenal, phenylalanine hydroxylase protein in the liver, and nNOS in the brain were not altered. These data suggest an essential role of SPR in the biosynthesis of BH4, and that the SPR gene could be a candidate gene for PARK3.

International audience; Regulation of gene expression is necessary to avoid possible adverse effects of gene therapy due to excess synthesis of transgene products. To reduce transgene expression, we developed a viral vector-mediated somatic regulation system using inducible Cre recombinase. A recombinant adeno-associated virus (AAV) vector expressing Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor alpha (CreER(T2)) was delivered along with AAV vectors expressing dopamine-synthesizing enzymes to rats of a Parkinson disease model. Treatment with 4-hydroxytamoxifen, a synthetic estrogen receptor modulator, activated Cre recombinase within the transduced neurons and induced selective excision of the tyrosine hydroxylase (TH) coding sequence flanked by loxP sites, leading to a reduction in transgene-mediated dopamine synthesis. Using this strategy, aromatic L-amino acid decarboxylase (AADC) activity was retained so that l-3,4-dihydroxyphenylalanine (L-dopa), a substrate for AADC, could be converted to dopamine in the striatum and the therapeutic effects of L-dopa preserved, even after reduction of TH expression in the case of dopamine overproduction. Our data demonstrate that viral vector-mediated inducible Cre recombinase can serve as an in vivo molecular switch, allowing spatial and temporal control of transgene expression, thereby potentially increasing the safety of gene therapy.

Sepiapterin reductase (SPR) is the enzyme that catalyzes the final step of the synthesis of tetrahydrobiopterin (BH4), the cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase (NOS). Although SPR is essential for synthesizing BH4, the distribution of SPR in the human brain has not yet been clarified. In the present study, we purified recombinant human SPR from cDNA, raised an antibody against human SPR (hSPR), and examined the localization of SPR protein and SPR activity. Human brain homogenates from the substantia nigra (SN), caudate nucleus (CN), gray and white matters of the cerebral cortex (CTX), and dorsal and ventral parts of the medulla oblongata (MO) were subjected to Western blot analysis with anti-hSPR antibody or with anti-TH antibody. Whereas TH protein showed a restricted localization, being mainly detected in the SN and CN, SPR protein was detected in all brain regions examined. SPR activity was relatively high compared with the activity of GTP cyclohydrolase I (GCH), the rate-limiting biosynthetic enzyme of BH4, and was more widely distributed than GCH activity. Immunohistochemistry revealed SPR immunoreactivity in pyramidal neurons in the cerebral CTX, in a small number of striatal neurons, and in neurons of the hypothalamic and brain stem monoaminergic fields and olivary nucleus. Double-staining immunohistochemistry showed that TH and SPR were colocalized in the SN dopamine neurons. Localization of SPR immunoreactive neurons corresponded to monoamine or NOS neuronal fields, and also to the areas where no monoamine or NOS neurons were located. The results indicate that there might be a BH4 biosynthetic pathway where GCH is not involved and that SPR might have some yet unidentified function(s) in addition to BH4 biosynthesis.

Three of the catecholamine-synthesizing enzymes, i.e., tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase, and dopamine b-hydroxylase, were earlier shown to be up-regulated in cloned PC12D cells overexpressing V-1, a cdc10/SWI6 motifcontaining protein. GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), known as an essentialcofactor for TH; and here we found the increased expression of GCH in V-1-overexpressing cl ones. Both GCH activity and total biopterin content were highly increased in the V-1 clones; whereas the activity of sepiapterin reductase, enzyme in the finalstep of the BH 4 biosynthesis, was not altered. Biochemical analyses revealed increased levels of GCH protein, mRNA, and transcription in the V-1 clones. Promoter analysis showed increased reporter activity in the construct with 150 bp of the promoter region of the human GCH gene, suggesting the involvement of cAMP-responsive element-mediated transcriptional regulation. 2002 Elsevier Science (USA). All rights reserved.

It has been reported that several mRNA isoforms of tyrosine 3-monooxygenase (tyrosine hydroxylase; TH) occur only in primates. New TH isoforms produced by skipping of exon 3 in the adrenal medulla of patients with progressive supranuclear palsy (PSP) have recently been reported, J. Neurochem. 67 (1996) 19. Here, we looked for the presence of new TH isoforms in control brains and adrenal medulla and in brains from patients with PSP. We found a novel type of TH mRNA in the adrenal medulla from one of the control subjects. The mRNA lacked exon 4, resulting in a premature stop codon at amino acid 147. This result suggests the importance of alternative splicing in the regulation of TH activity.

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum , Saccharomyces cerevisiae , Drosophila melanogaster , mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis .

We compared in rats with 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonism the content of tumor necrosis factor (TNF)-alpha in the nigrostriatal dopaminergic region of the control side with that of the 6-OHDA-injected experimental side, and explored the effects of 6-OHDA injection combined with the immunosuppressant FK506 treatment (0.5 or 4 mg/kg per day for 2 weeks). The ratios of the concentration of TNF-alpha in the striatum and substantia nigra on the 6-OHDA injection side to that on the control side in the 6-OHDA hemiparkinsonism rats were significantly higher than those in the control rats without 6-OHDA treatment, whereas those in the rats treated with 6-OHDA and FK506 were not significantly different from those in the control rats. Thus FK506 attenuated increased TNF-alpha level in the nigrostriatal dopaminergic region injured by 6-OHDA treatment.

The active site of sepiapterin reductase (SPR), which is a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family and acts as the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin cofactor (BH4), was investigated by truncation and site-directed mutagenesis. The truncation mutants showed that N-terminal and C-terminal residues contribute to bind coenzyme and substrate, respectively. The mutant rSPRA29V showed decreased activity; however, the A-X-L-L-S sequence, which has been reported as a putative pterin binding site, was estimated to preferably work as a component in the region for binding coenzyme rather than substrate. Site-directed mutants of rSPRS158D, rSPRY171V, and rSPRK175I showed low, but significant, activity having similar Km values and kcat/Km values less than 25%, for both sepiapterin and NADPH. Both amino acids Tyr-171 and Ser-158 are located within a similar distance to the carbonyl group of the substrate in the crystal structure of mouse SPR, and the double point mutant rSPRY171V+S158D was indicated to be inactive. These results showed that Ser-158, Tyr-171, and Lys-175 contributed to the catalytic activity of SPR, and both Tyr-171 and Ser-158 are simultaneously necessary on proton transfer to the carbonyl functional groups of substrate.

The human SNF2alpha (or hbrm) and SNF2beta (or BRG1) proteins have previously been shown to enhance transcriptional activation by nuclear receptors (NRs) in cultured human cells, and to be present in SWI/SNF complexes which are thought to be involved in control of transcription by facilitating remodelling of chromatin templates. Using the yeast two-hybrid system, we now demonstrate that the N-terminal regions of hSNF2alpha and hSNF2beta, preceding the DNA-dependent ATPase domain, specifically interact with the region of the estrogen receptor (ER) which includes the ligand binding domain and the ligand-dependent activation function AF-2. These interactions are increased by estrogen, but not by the ER AF-2 antagonist hydroxytamoxifen. Furthermore, mutants of ER that lack AF-2 activity are unable to interact with hSNF2alpha and -beta. These results suggest that the human homologues of the yeast SWI2/SNF2 protein may participate in the enhancement of transcription by the ER in vivo through interactions with the AF-2 activating domain, thus leading to ligand-dependent remodelling of chromatin templates.

Monkeys are known to be highly susceptible to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) which produces parkinsonism, as in humans. We have previously reported that only monkeys and humans have multiple isoforms of tyrosine hydroxylase (TH), the first enzyme for dopamine biosynthesis, with only two TH mRNA isoforms, type 1 and type 2, being present inMacaca fascicularis. In the present study we have measured TH mRNA type 1 and 2 content in the substantia nigra, locus coeruleus, and adrenal gland of normal control monkeys and in MPTP-produced parkinsonian monkeys (Macaca fascicularis) using a newly developed, sensitive and quantitative assay based on the reverse transcription-polymerase chain reaction. Marked decreases in TH mRNA type 1 and 2 content were observed, specifically in the substantia nigra of the monkeys with MPTP-parkinsonism compared to control monkeys. These results are similar to our recent data showing marked decreases in TH mRNA type 1, 2, 3 and 4 content in the substantia nigra of patients with Parkinson's disease, and suggest that MPTP-treated monkeys closely replicate changes in TH isoforms in human Parkinson's disease.

We investigated the effect of subcutaneous injection of nicotine on in vitro tyrosine hydroxylase (TH) activity in adrenal gland and brain of the transgenic mice carrying an 11-kb fragment containing the entire human TH gene. Injection of 5 mg nicotine/kg (as free base) for 3 days caused a statistically significant increase in vitro TH activity in the adrenal gland, whereas brain TH activity was not affected at all. The adrenal gland of non-transgenic C57BL/6J mice treated in the same way as for transgenic mice tended to enhance TH activity, although not to a significant level. This observation might indicate the possibility that the machinery used by nicotine in regulating the properties or expression of TH in the adrenal gland should be similar between transgenic and non-transgenic mice.

Glial cell line-derived neurotrophic factor (GDNF) was measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme-linked immunosorbent assay. In both groups, the levels of GDNF in the various brain regions were lower (pg/mg protein) than those of brain-derived growth factor (ng/mg order), and were significantly higher in the nigro-striatal dopaminergic regions (substantia nigra, caudate nucleus, putamen) than in the cerebellum and frontal cortex (P < 0.05). However, the content of GDNF in the dopaminergic regions showed no significant difference between parkinsonian and control patients.

1. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. 2. The structures of TH from various species have been elucidated. 3. We have cloned and determined the sequences of four types of human TH cDNA and human TH genomic DNA. 4. We have compared the amino acid sequences of TH from various species. 5. The results indicate that the amino acid sequences of TH are highly conserved among various species, and that TH consists of the regulatory domain containing serine residues which are phosphorylated by protein kinases and of the catalytic domain where the substrates, tyrosine and oxygen, and the cofactor, tetrahydrobiopterin, are bound. 6. Comparison of amino acid sequences among TH from various species can give us useful information on the functional importance of each amino acid residue.

The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole. The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus. The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al. (Proc. Natl. Acad. Sci. USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.