Novel mutations in the guanosine triphosphate cyclohydrolase 1 gene associated with DYT5 dystonia.

OBJECTIVES: To better understand the relationship between mutation of the guanosine triphosphate cyclohydrolase I (GCH1) gene and the etiology of DYT5 dystonia and to accumulate data on the mutation in the Japanese population for genetic diagnosis of the disease. SETTING: Japanese population. Patients Eight Japanese patients with suspected DYT5 dystonia were analyzed. Intervention Direct genomic sequencing of 6 exons of GCH1 was performed. MAIN OUTCOME MEASURES: For patients who did not exhibit any abnormality in the sequence analysis, the possibility of exon deletions was examined. In cases for which cerebrospinal fluid was available, the concentrations of neopterin and biopterin were measured as an index of GCH1 enzyme activity. RESULTS: In 2 patients, we found a new T106I mutation in exon 1 of GCH1, a position involved in the helix-turn-helix structure of the enzyme. In the third patient, we found a new mutation (a 15-base pair nucleotide deletion) in exon 5 that may cause a frameshift involving the active site. In the fourth patient, we detected a known nucleotide G>A substitution in the splice site of intron 5, which has been reported to produce exon 5-skipped messenger RNA. The concentrations of both neopterin and biopterin in the cerebrospinal fluid of the third and fourth patients were markedly lower than the normal range, indicating that the GCH1 enzyme was functionally abnormal in these mutations. Gene dosage analysis showed that the fifth patient had a deletion of both exon 3 and exon 4, whereas the sixth patient had a deletion of exon 3. CONCLUSIONS: We found several novel, as well as known, GCH1 mutations in Japanese patients with DYT5 dystonia. In some of them, the GCH1 enzyme activity was proved to be impaired.

Molecular mechanism for pterin-mediated inactivation of tyrosine hydroxylase: formation of insoluble aggregates of tyrosine hydroxylase.

Tyrosine hydroxylase (TH), an iron-containing enzyme, catalyzes the first and rate-limiting step of catecholamine biosynthesis, and requires tetrahydrobiopterin (BH4) as a cofactor. We found that preincubation of recombinant human TH with BH4 results in the irreversible inactivation of the enzyme at a concentration far less than the Km value toward BH4 in spite of its cofactor role, whereas oxidized biopterin, which has no cofactor activity, does not affect the enzyme activity. We show that TH is inactivated by BH4 in competition with the binding of dopamine. The sequential addition of BH4 to TH results in a gradual decrease in the intensity of the fluorescence and CD spectra without changing their overall profiles. Sedimentation velocity analysis demonstrated an association of TH molecules with each other in the presence of BH4, and studies using gel-permeation chromatography, turbidity measurements, and transmission electron microscopy demonstrated the formation of amorphous aggregates with large molecular weights following the association of the TH proteins. These results suggest that BH4 not only acts as a cofactor, but also accelerates the aggregation of TH. We propose a novel mechanism for regulating the amount of TH protein, and discuss its physiological significance.

Viral-mediated temporally controlled dopamine production in a rat model of Parkinson disease.

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.

A case of late-onset Segawa syndrome (autosomal dominant dopa-responsive dystonia) with a novel mutation of the GTP-cyclohydrase I (GCH1) gene.

We report a case of a 46-year-old Japanese woman with hereditary progressive dystonia with marked diurnal fluctuations and dopa-responsive dystonia (HPD/DRD). She developed difficulty in walking at the age of 44 years due to bradykinesia as well as hand tremors, muscle rigidity, increased tendon reflexes and mild dystonia in the lower extremities, all of which responded remarkably to low doses of levodopa (150 mg/day). Biopterin and neopterin concentrations in the cerebrospinal fluid (CSF) were decreased. Analysis of the guanosine 5'-triphosphate cyclohydrolase I (GCH1) gene revealed a novel mutation (W53X) in one allele. The GCH1 activity that was expressed in mononuclear blood cells was almost half the normal value (usually 2-20% of the normal value (39.0+/-9.2 pmol/ml) in patients with HPD/DRD). The relatively conserved GCH1 activity that is expressed in stimulated peripheral blood mononuclear cells may be related to the late clinical symptoms in this patient.

Genetically rescued tetrahydrobiopterin-depleted mice survive with hyperphenylalaninemia and region-specific monoaminergic abnormalities.

One of the possibly mutated genes in DOPA-responsive dystonia (DRD, Segawa's disease) is the gene encoding GTP cyclohydrolase I, which is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. Based on our findings on 6-pyruvoyltetrahydropterin synthase (PTS) gene-disrupted (Pts(-/-)) mice, we suggested that the amount of tyrosine hydroxylase (TH) protein in dopaminergic nerve terminals is regulated by the intracellular concentration of BH4. In this present work, we rescued Pts(-/-) mice by transgenic introduction of human PTS cDNA under the control of the dopamine beta-hydroxylase promoter to examine regional differences in the sensitivity of dopaminergic neurons to BH4-insufficiency. The DPS-rescued (Pts(-/-), DPS) mice showed severe hyperphenylalaninemia. Human PTS was efficiently expressed in noradrenergic regions but only in a small number of dopaminergic neurons. Biopterin and dopamine contents, and TH activity in the striatum were poorly restored compared with those in the midbrain. TH-immunoreactivity in the lateral region of the striatum was far weaker than that in the medial region or in the nucleus accumbens. We concluded that dopaminergic nerve terminals projecting to the lateral region of the striatum are the most sensitive to BH4-insufficiency. Biochemical and pathological changes in DPS-rescued mice were similar to those in human malignant hyperphenylalaninemia and DRD.

Arg(184)His mutant GTP cyclohydrolase I, causing recessive hyperphenylalaninemia, is responsible for dopa-responsive dystonia with parkinsonism: a case report.

We describe a 54-year-old man with dominant adult-onset dopa-responsive dystonia (DRD) with parkinsonism caused by an Arg184His mutation in guanosine 5'-triphosphate cyclohydrolase I (GCH-I). This is the first mutation in the GCH-I gene that has been proven to be responsible for both recessive and dominant phenotypes.

Effects of chronic treatment with L-arginine on atherosclerosis in apoE knockout and apoE/inducible NO synthase double-knockout mice.

OBJECTIVE: L-arginine serves as a substrate for the formation of NO by the NO synthase (NOS) enzymes. In some studies, dietary supplementation of L-arginine reduces atherosclerosis through the restoration of NO release and improvement in endothelial function. In the present study, we investigate the effect of L-arginine supplementation on the development of atherosclerosis in a mouse model. METHODS AND RESULTS: Apolipoprotein E (apoE) knockout (ko) and apoE/inducible NOS (iNOS) double-ko mice were fed a western-type diet with or without L-arginine supplementation in the drinking water (25 g/L). L-Arginine did not affect the lesion area after 16 weeks or 24 weeks in apoE ko mice. However, L-arginine negates the protective effect of iNOS gene deficiency. In contrast to apoE/iNOS dko mice without arginine supplementation, lesion areas were increased in apoE/iNOS double-ko mice with arginine supplementation at 24 weeks. This was associated with an increase in thiobarbituric acid-reactive malondialdehyde adducts, nitrotyrosine staining within lesions, and a decrease in the ratio of reduced tetrahydrobiopterin to total biopterins. CONCLUSIONS: Although L-arginine supplementation does not affect lesion formation in the western-type diet-fed apoE ko mice, it negates the protective effect of iNOS gene deficiency in this model. This raises the possibility that L-arginine supplementation may paradoxically contribute to, rather than reduce, lesion formation by mechanisms that involve lipid oxidation, peroxynitrite formation, and NOS uncoupling.

Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes.

One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.

Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.

We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH(+) neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH(+) neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (approximately 5 weeks). In addition, 8% of the colonies contain large patches of Pax6(+)-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa.