DOPA-responsive dystonia. From causative gene to molecular mechanism.
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Dihydroxyphenylalanine↗
Biomedical subjects
T Nagatsu
Publications and source records attributed to T Nagatsu.
[Huntington's disease model mouse and neuronal cell death].
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Animals↗
Characterization of the human NTAK gene structure and distribution of the isoforms for rat NTAK mRNA.
NTAK (neural- and thymus-derived activator for the ErbB kinase, neuregulin-2) is a novel member of the epidermal growth factor (EGF) family. We have isolated and characterized the human NTAK gene, comprising 12 exons spanning in excess of 55 kilobases (kb). The 7. 0kb long mRNA of the human NTAK gene was expressed in the human neuroblastoma SK-N-SH cell line with two alternative isoforms detected. Furthermore, six isoforms have been identified from rat brain and PC-12 cells. Although the alpha isoform of the NTAK gene was found to be expressed in all tissues including brain, the beta isoform was expressed only in rat brain tissues. Potential regulatory regions included consensus binding sites for AP-2, TF-IIIA, Sp-1, and YY-1 located in the 5'-flanking region of the NTAK gene.
Amino Acid Sequence↗
Increase in level of tumor necrosis factor-alpha in 6-hydroxydopamine-lesioned striatum in rats is suppressed by immunosuppressant FK506.
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.
Animals↗
Synaptic integration mediated by striatal cholinergic interneurons in basal ganglia function.
The physiological role of striatal cholinergic interneurons was investigated with immunotoxin-mediated cell targeting (IMCT). Unilateral cholinergic cell ablation caused an acute abnormal turning behavior. These mice showed gradual recovery but displayed abnormal turning by both excess stimulation and inhibition of dopamine actions. In the acute phase, basal ganglia function was shifted to a hyperactive state by stimulation and suppression of striatonigral and striatopallidal neurons, respectively. D1 and D2 dopamine receptors were then down-regulated, relieving dopamine-predominant synaptic perturbation but leaving a defect in controlling dopamine responses. The acetylcholine-dopamine interaction is concertedly and adaptively regulated for basal ganglia synaptic integration.
Acetylcholine↗
Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.
Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.
Animals↗
Normal values and age-dependent changes in GTP cyclohydrolase I activity in stimulated mononuclear blood cells measured by high-performance liquid chromatography.
GTP cyclohydrolase I (GCH1) activity in phytohemaglutinin (PHA)-stimulated mononuclear blood cells (MBCs) is a useful clinical marker for diagnosis of tetrahydrobiopterin (BH4)-related genetic disorders such as recessively inherited GCH1 deficiency and dominantly inherited dopa-responsive dystonia (Segawa's disease). Since the assay is complex, including isolation of MBCs from blood, stimulation of MBCs by PHA under culture, isolation of the protein fraction from the PHA-stimulated MBCs, and the subsequent activity measurement, the reproducibility is problematic in its application to clinical study. We established a sensitive and reproducible method by high-performance liquid chromatography with fluorescence detection for clinical assay of GCH1 in PHA-stimulated MBCs, and measured the normal values of 91 healthy males and females of various ages (1-74 years). The mean normal values were 19.1+/-0.9 pmol/mg protein per h (mean+/-S.E., n=91). There were no significant differences between males and females. The activity tends to be higher in the first decade and to be decreased from the second to third decade and becomes almost stable from the third decade.
Age Factors↗
Modest neuropsychological deficits caused by reduced noradrenaline metabolism in mice heterozygous for a mutated tyrosine hydroxylase gene.
Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme for the biosynthesis of catecholamines that are considered to be involved in a variety of neuropsychiatric functions. Here, we report behavioral and neuropsychological deficits in mice carrying a single mutated allele of the TH gene in which TH activity in tissues is reduced to approximately 40% of the wild-type activity. In the mice heterozygous for the TH mutation, noradrenaline accumulation in brain regions was moderately decreased to 73-80% of the wild-type value. Measurement of extracellular noradrenaline level in the frontal cortex by the microdialysis technique showed a reduction in high K(+)-evoked noradrenaline release in the mutants. The mutant mice displayed impairment in the water-finding task associated with latent learning performance. They also exhibited mild impairment in long-term memory formation in three distinct forms of associative learning, including active avoidance, cued fear conditioning, and conditioned taste aversion. These deficits were restored by the drug-induced stimulation of noradrenergic activity. In contrast, the spatial learning and hippocampal long-term potentiation were normal in the mutants. These results provide genetic evidence that the central noradrenaline system plays an important role in memory formation, particularly in the long-term memory of conditioned learning.
Animals↗
Positive charge intrinsic to Arg(37)-Arg(38) is critical for dopamine inhibition of the catalytic activity of human tyrosine hydroxylase type 1.
Tyrosine hydroxylase (TH), which converts L-tyrosine to L-3, 4-dihydroxyphenylalanine, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N-terminal sequence Arg(37)-Arg(38) of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg(37)-Arg(38) site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum. The prediction of the secondary structure of N-terminal residues 1-60 by computer software specified the location of the Arg(37)-Arg(38) sequence in the turn intervening between the two alpha-helices (residues 16-29 and residues 41-59). These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine.
Amino Acid Sequence↗
Changes in cytokines and neurotrophins in Parkinson's disease.
Degeneration of the dopamine (DA) neurons of the substantia nigra pars compacta and the resulting loss of nerve terminals accompanied by DA deficiency in the striatum are responsible for most of the movement disturbances called parkinsonism, observed in Parkinson's disease (PD). One hypothesis of the cause of degeneration of the nigrostriatal DA neurons is that PD is caused by programmed cell death (apoptosis) due to increased levels of cytokines and/or decreased ones of neurotrophins. We and other workers found markedly increased levels of cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, IL-4, IL-6, transforming growth factor (TFG)-alpha, TGF-beta1, and TGF-beta2, and decreased ones of neurotrophins, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in the nigrostriatal DA regions and ventricular and lumbar cerebrospinal fluid of PD patients. Furthermore, the levels of TNF-alpha receptor R1 (TNF-R1, p55), bcl-2, soluble Fas (sFas), and the activities of caspase-1 and caspase-3 were also elevated in the nigrostriatal DA regions in PD. In experimental animal models of PD, IL-1beta level was increased and NGF one decreased in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian mice, and TNF-alpha level was increased in the substantia nigra and striatum of the 6-hydroxydopamine (6OHDA)-injected side of hemiparkinsonian rats. L-DOPA alone or together with 6OHDA does not increase the level of TNF-alpha in the brain in vivo. Increased levels of proinflammatory cytokines, cytokine receptors and caspase activities, and reduced levels of neurotrophins in the nigrostriatal region in PD patients, and in MPTP- and 6OHDA-produced parkinsonian animals suggest increased immune reactivity and programmed cell death (apoptosis) of neuronal and/or glial cells. These data indicate the presence of such proapoptotic environment in the substantia nigra in PD that may induce increased vulnerability of neuronal or glial cells towards a variety of neurotoxic factors. The probable causative linkage among the increased levels of proinflammatory cytokines and the decreased levels of neurotrophins, candidate parkinsonism-producing neurotoxins such as isoquinoline neurotoxins (Review; Nagatsu, 1997), and the genetic susceptibility to toxic factors, remains for further investigation in the molecular mechanism of PD. The increased cytokine levels, decreased neurotrophin ones, and the possible immune response in the nigrostriatal region in PD indicate new neuroprotective therapy including nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, immunosuppressive or immunophilin-binding drugs such as FK-506, and drugs increasing neurotrophins.
Animals↗
Caspase activities and tumor necrosis factor receptor R1 (p55) level are elevated in the substantia nigra from parkinsonian brain.
The activities of caspase-1 and caspase-3 were measured by use of fluoropeptides as substrates for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients. The activities of caspases in the brain were significantly higher in the substantia nigra from parkinsonian patients than those in the brain from control patients (p < 0.01). However, the activities of caspases in the caudate nucleus, putamen, cerebellum, and frontal cortex showed no significant difference between parkinsonian and control patients. The tumor necrosis factor (TNF) receptor R1 (TNF-R1, p55) level was also elevated in the substantia nigra of the parkinsonian brain in comparison with that of controls (p < 0.05). Since both caspases and TNF-R1 may play important roles in apoptotic cell death through TNF-alpha-induced signaling pathway, our present data suggest the presence of a proapoptotic environment in the substantia nigra of parkinsonian brain, probably inducing vulnerability of neurons and glias towards a variety of noxious factors.
Adult↗
GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression.
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 Tetrahrymena 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.
Amino Acid Sequence↗
Studies on the genotype-phenotype relation in the hph-1 mouse mutant deficient in guanosine triphosphate (GTP) cyclohydrolase I activity.
The guanosine triphosphate (GTP) cyclohydrolase I (GTP-CHI) catalyses the rate-limiting step in the de novo synthesis of tetrahydrobiopterin, a cofactor of three aromatic amino acid hydroxylases, one of which is phenylalanine hydroxylase. The hph-1 mouse mutant deficient in GTP-CHI activity exhibits hyperphenylalaninemia which peculiarly disappears at 3 weeks of age, thus corresponding to the increase in liver GTP-CHI activity. The present gas chromatographic-mass spectrometric analysis of the phenylalanine and catecholamine metabolisms demonstrated the former metabolism to remain disturbed even in adult hph-1, which demonstrated a metabolic basis for sensitivity to the phenylalanine challenge in adult hph-1. A Northern blot analysis showed the hepatic GTP-CHI RNA expression in hph-1 at 2, 3 and 4 weeks of age to parallel the peculiar time course of the enzyme activity previously reported. No mutation was detected in either the coding region or the 5' flanking region (nt.-1 to -746) of the GTP-CHI gene of the hph-1. Further molecular genetic analyses are therefore required to elucidate the mechanism of the peculiar phenotype of hph-1.
Animals↗
Molecular mechanisms of hereditary progressive dystonia with marked diurnal fluctuation, Segawa's disease.
The causative gene for hereditary progressive dystonia with marked diurnal fluctuation/dopa-responsive dystonia (HPD/DRD) was discovered in 1994 to be guanosine triphosphate (GTP) cyclohydrolase I, an enzyme involved in tetrahydrobiopterin biosynthesis. To the present, more than 50 mutations have been found in this gene in HPD/DRD patients. Although it is clear that HPD/DRD is caused by partial deficiency of tetrahydrobiopterin in the brain, several important issues regarding the molecular etiology of HPD/DRD remain to be addressed. We review herein the recent progress in the molecular genetics of HPD/DRD and clarify the points to be answered.
Biopterins↗
Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.
At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.
Aged↗
Direct imaging of phosphorylation-dependent conformational change and DNA binding of CREB by electron microscopy.
BACKGROUND: The second messenger cAMP stimulates the expression of numerous genes through the PKA-dependent phosphorylation of CREB. The cAMP-regulated transcription factor CREB undergoes conformational change in response to phosphorylation by PKA at Ser 133. The phosphorylation enables interaction between the kinase-inducible domain (KID) of CREB and KIX domain of CREB binding protein (CBP). RESULTS: To understand the activation mechanism of CREB-mediated gene expression, we performed the electron-microscope imaging of the transcription machinery. We improved the metal shadowing techniques to achieve higher resolution to detect phosphorylation-induced conformation change of the protein. Homodimer formation of CREB and the complex formation of phosphorylated CREB with CBP were observed under the electron microscope. The binding of the CREB dimer to CREs on the somatostatin and tyrosine hydroxylase promoters were also visualized directly and stereoscopically. CONCLUSIONS: Greatly improved resolution achieved by our modified metal shadowing techniques makes it possible to visualize that the shape of CREB homodimer was changed in phosphorylation-dependent manner and that the promoter DNA strands containing CREs appeared to be bent and twisted slightly by the holding in the crevice of the CREB homodimer. This method may be applicable to visualize transcriptional activation process of nuclear receptors or general transcription machinery.
Animals↗
A branch site mutation leading to aberrant splicing of the human tyrosine hydroxylase gene in a child with a severe extrapyramidal movement disorder.
We report a branch site mutation in the gene of the enzyme tyrosine hydroxylase (TH): a -24t > a substitution two bases upstream of the adenosine in the branchpoint sequence (BPS) of intron 11. As normal lariat formation is therefore prevented, alternative splicing takes place; use of the BPS of intron 12 results in skipping of exon 12, whereas the use of a cryptic branch site in intron 11 leads to partial retention of this intron in the mRNA. This leads in both cases to an aberrant protein product. In the one case, skipping of exon 12 results in the absence of 32 amino acids. In the other, retention of 36 nucleotides of intron 11 in the mRNA results in the incorporation of twelve additional amino acids. The functional consequences of this mutation for the patient, who is also heterozygous for another previously identified mutation, become apparent in a severe clinical phenotype.
Basal Ganglia Diseases↗
NTAKalpha and beta isoforms stimulate breast tumor cell growth by means of different receptor combinations.
Neural- and thymus-derived activator for ErbB kinases (NTAK) is a recently described member of the neuregulin family that binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Rat NTAK has at least five alternative-spliced isoforms: alpha1, alpha2a, alpha2b, beta, and gamma. In order to understand their biological properties, this study focused on the NTAK alpha2a and beta isoforms, which have different EGF-like domains. The effect of these isoforms on cell growth and tyrosine phosphorylation in human breast cancer cells, MDA-MB-453 and T47D, was examined using the recombinant proteins. In terms of cell growth, NTAKalpha2a and NTAKbeta preferentially stimulate T47D cells and MDA-MB-453 cells, respectively, in a dose-dependent manner. Although both NTAKs induce the highest level of tyrosine phosphorylation of ErbB2, NTAKalpha2a and NTAKbeta preferentially induce ErbB3 and ErbB4 phosphorylation, respectively. Thus, NTAKalpha2a and NTAKbeta stimulate cell growth in different ways, by means of different combinations of receptors.
Alternative Splicing↗