Role of Aromatic l-Amino Acid Decarboxylase for Dopamine Replacement by Genetically Modified Fibroblasts in a Rat Model of Parkinson's Disease

Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l -amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l -amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l -amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l -DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l -amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l -amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic l -amino acid decarboxylase produced less l -DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l -amino acid decarboxylase near striatal grafts producing l -DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

Biopterin cofactor and monoamine-synthesizing monooxygenases

The activities of three pterin-requiring monooxygenases, phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase, are regulated by the level of the pterin cofactor, $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$, which is synthesized from guanosine triphosphate (GTP). Since tyrosine hydroxylase or tryptophan hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines (dopamine, norepinephrine and epinephrine) or serotonin in monoaminergic neurons, biosynthesis of tetrahydrobiopterin from GTP may also regulate the tissue level of monoamine transmitters. Recent evidences indicate that biosynthesis of tetrahydrobiopterin and that of biogenic monoamines may be regulated each other.     

Neurochemical Effects Following Peripheral Administration of Tetrahydropterin Derivatives to the hph-1 Mouse

Abstract: The hph-1 mouse, which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 µmol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6-methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N -benzyl-1,4-dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1–4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 µmol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.     

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.     

IMP dehydrogenase inhibitors reduce intracellular tetrahydrobiopterin levels through reduction of intracellular GTP levels. Indications of the regulation of GTP cyclohydrolase I activity by restriction of GTP availability in the cells.

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.     

Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

Molecular Biology of Catecholamine-Related Enzymes in Relation to Parkinson's Disease

1. Catecholamine (dopamine, norepinephrine, and epinephrine) biosynthesis is regulated by tyrosine hydroxylase (TH). TH activity is regulated by the concentration of the cofactor tetrahydrobiopterin (BH4), whose level is regulated by GTP cyclohydrolase I (GCH) activity. Thus, GCH activity indirectly regulates TH activity and catecholamine levels.2. TH activity in the nigrostriatal dopaminergic neurons is most sensitive to the decrease in BH4.3. Mutations of GCH result in reductions in GCH activity, BH4, TH activity, and dopamine, causing either recessively inherited GCH deficiency or dominantly inherited hereditary progressive dystonia [HPD; Segawa's disease; also called dopa-responsive dystonia (DRD)].4. In juvenile parkinsonism and Parkinson's disease, which have dopamine deficiency in the basal ganglia as HPD/DRD, the GCH gene may be normal, and the molecular mechanism of the dopamine deficiency in the basal ganglia is different from that in HPD/DRD.     

Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.     

GTP cyclohydrolase I is coinduced in hepatocytes stimulated to produce nitric oxide

GTP cyclohydrolase I is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide (NO) synthase. Here we show that GTP cyclohydrolase I mRNA was present in unstimulated hepatocytes and was up-regulated 2- to 3-fold concurrently with iNOS induction induced in vivo by LPS injection and in vitro by stimulation with LPS and inflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta, and interferon-gamma. Hepatocyte GTP cyclohydrolase I enzyme activity increased 2-fold in vivo after LPS. This coinduction of GTP cyclohydrolase I resulted in increased total intracellular biopterin which supported induced NO synthesis. The addition of a GTP cyclohydrolase I inhibitor to the stimulated hepatocytes decreased intracellular biopterin levels and resulted in a decrease in NO production. The results show that GTP cyclohydrolase I is up-regulated by certain acute inflammatory conditions. Further, the results indicate that biopterin is essential as a cofactor for induced NO synthase activity in hepatocytes.     

Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice

J. Neurochem. (2012) 122, 1211-1218. ABSTRACT: In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4) ) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-) ) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4) . In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4) , increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.     

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.     

GTP cyclohydrolase I gene,tetrahydrobiopterin, and tysorine hydroxylase gene: Their relations to dystonia and parkinsonism

Catecholamine biosynthesis is regulated by tyrosine hydroxylase (TH) requiring tetrahydrobiopterin (BH4) as the cofactor. We found four (human TH type 1–4) and two isoforms (TH type 1 and 2) in humans and monkeys, while non-primate animals have a single TH corresponding to human TH type 1. BH4 is synthesized from GTP, and GTP cyclohydrolase I (GCH) is the first and regulatory enzyme. Mutations in GCH gene were found to cause both GCH deficiency with autosomal recessive trait and hereditary progressive dystonia with marked diurnal fluctuation (HPD) (Segawa's disease)/or DOPA-responsive dystonia (DRD) with autosomal dominant trait. When GCH activity is decreased to less than 20% of the normal value, the activity of TH in the nigrostriatal dopaminergic neurons may be first decreased resulting in decreases in TH activity and dopamine level, and in the symptoms of HPD/DRD. In contrast to HPD/DRD, juvenile parkinsonism (JP) have normal GCH activity. In Parkinson's disease (PD), GCH, TH, and dopamine in the striatum may decrease in parallel, as the secondary effects caused by cell death. Special issue dedicated to Dr. Kinya Kuriyama.     

Isolation and characterization of GTP cyclohydrolase I from mouse liver. Comparison of normal and the hph-1 mutant

GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of pterin compounds, was purified from of C3H mouse liver by 192-fold to apparent homogeneity, using Ultrogel AcA34, DEAE-Trisacryl, and GTP-agarose gels. Its native molecular weight was estimated at 362,000. When the enzyme was subjected to electrophoresis on a denaturing polyacrylamide gel, only one protein band was evident, and its molecular weight was estimated at 55,700. The NH2-terminal amino acid of this enzyme was serine. These results indicate the enzyme consists of six to eight subunits. No coenzyme or metal ion was required for activity. This enzyme activity was inhibited by most of divalent cations and was slightly activated by potassium ion. The Km value for GTP was determined to be 17.3 microM. The temperature and pH optima for the activity were 60 degrees C and pH 8.0-8.5, respectively. The expected products, a dihydroneopterin compound and formic acid, were found in a molar ratio of 1.01. A polyclonal antiserum generated against the purified enzyme was used to compare GTP cyclohydrolase I from the hph-1 mutant and normal mouse. The hph-1 mutant liver contained only 8% of normal specific activity, but a normal amount of GTP cyclohydrolase I antigen as compared with the C3H mouse. Subunit molecular weight and electrophoretic behavior of GTP cyclohydrolase I from hph-1 mutant were not different from those of the enzyme from C3H mouse. These results suggest that the hph-1 mutation may involve alteration of the catalytic site but does not detectably alter the whole enzyme structure.     

Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GCH), 6-pyruvoyltetrahydropterin synthase (PTS), and sepiapterin reductase (SPD). GCH is the rate-limiting enzyme. BH4 is a cofactor for three pteridine-requiring monooxygenases that hydroxylate aromatic L-amino acids, i.e., tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and phenylalanine hydroxylase (PAH), as well as for nitric oxide synthase (NOS). The intracellular concentrations of BH4, which are mainly determined by GCH activity, may regulate the activity of TH (an enzyme-synthesizing catecholamines from tyrosine), TPH (an enzyme-synthesizing serotonin and melatonin from tryptophan), PAH (an enzyme required for complete degradation of phenylalanine to tyrosine, finally to CO2 + H2O), and also the activity of NOS (an enzyme forming NO from arginine), Dominantly inherited hereditary progressive dystonia (HPD), also termed DOPA-responsive dystonia (DRD) or Segawa's disease, is a dopamine deficiency in the nigrostriatal dopamine neurons, and is caused by mutations of one allele of the GCH gene. GCH activity and BH4 concentrations in HPD/DRD are estimated to be 2-20% of the normal value. By contrast, recessively inherited GCH deficiency is caused by mutations of both alleles of the GCH gene, and the GCH activity and BH4 concentrations are undetectable. The phenotypes of recessive GCH deficiency are severe and complex, such as hyperphenylalaninemia, muscle hypotonia, epilepsy, and fever episode, and may be caused by deficiencies of various neurotransmitters, including dopamine, norepinephrine, serotonin, and NO. The biosynthesis of dopamine, norepinephrine, epinephrine, serotonin, melatonin, and probably NO by individual pteridine-requiring enzymes may be differentially regulated by the intracellular concentration of BH4, which is mainly determined by GCH activity. Dopamine biosynthesis in different groups of dopamine neurons may be differentially regulated by TH activity, depending on intracellular BH4 concentrations and GCH activity. The nigrostriatal dopamine neurons may be most susceptible to a partial decrease in BH4, causing dopamine deficiency in the striatum and the HPD/DRD phenotype.