永津 俊治

Previously we reported that the synthesis of catecholamines, dopamine, and noradrenaline was enhanced by overexpression of V-1 protein, a neuronal protein active in the initial stage of development of the rat cerebellum, in the neuronal cell line PC12D, a model of dopamine cells (Yamakuni et al. [1998] J. Biol. Chem. 273:27051-27054). To investigate the physiological role of this protein, we examined the effect of V-1 overexpression on cell toxicity induced by nitric oxide (NO) used at low concentrations. Two clones of PC12D cells overexpressing V-1, transfectants termed V1-46 and V1-69, were significantly more resistant to NOR3 (an NO donor) but not to etoposide (an inhibitor of topoisomerase II)-induced apoptotic cell death than the control cells (termed C-7 and C-9) that had been transfected with the vector alone. The addition of L-DOPA, dopamine, or noradrenaline to the medium did not abolish NOR3-induced cell death in PC12D cells. Moreover, pretreatment of V1-46 and V1-69 cells with L-alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase, to inhibit catecholamine biosynthesis did not affect the resistance to NO toxicity. These results indicate that the catecholamine levels increased by V-1 overexpression did not produce the protection against NOR3-induced toxicity. We further showed that overexpression of V-1 enhanced the synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). In addition, pretreatment with BH4 or with sepiapterin, which is converted to BH4 intracellularly, significantly protected PC12D cells in a dose-dependent manner. The increased BH4 synthesis by V-1 overexpression was dose dependently inhibited by pretreatment with diaminohydroxypyrimidine (DAHP), an inhibitor of GTP-cyclohydrolase 1, which is the rate-limiting enzyme for the biosynthesis of BH4, concomitantly with the loss of protective effect afforded by V-1 overexpression. Furthermore, the addition of BH4 or sepiapterin to DAHP-pretreated V146 and V1-69 cells restored cell vi-ability. Taken together, these results indicate that V1 protein plays an important role in protection against cell death induced by NO at low levels by promoting the synthesis of BH4. Moreover, these findings suggest the up-regulation of V1 expression as a possible therapeutic target for protection against the insult of NO-induced oxidative stress. (C) 2003 Wiley-Liss, Inc.

The causative gene for dopa-responsive dystonia (DRD) is discovered to be that for guanosine triphosphate cyclohydrolase I (GCH), an enzyme involved in tetrahydrobiopterin biosynthesis. DRD patients are heterozygous in terms of the mutations. More than 70 mutations are found in this gene in DRD patients. A defective GCH gene results in a decreased biopterin content and thus in a decreased dopamine production in the brain. Analysis of the molecular etiology of DRD should help understand the pathophysiology of basal ganglia disorders, including Parkinson's disease. This chapter emphasizes that the nigro-striatal dopaminergic neurons are highly susceptible to a deficiency of tetrahydrobiopterin and the resulting defect in dopamine production. There are several types of dopa-responsive basal ganglia diseases. Parkinson's disease is the most well-known disease in that category. DRD is a disorder characterized by childhood or adolescent-onset of dystonia and by a dramatic response to low-dose L-dopa. DRD is also caused by dysfunction of nigro-striatal dopaminergic neurons, although its main symptom is dystonia, not parkinsonism. It is noted that blocking of dopamine receptors by neuroleptics produces a dystonic reaction in childhood, whereas in adults it results in parkinsonism. Parkinsonian symptoms sometimes appears later in DRD patients in adolescence. © 2003 Elsevier Inc. All rights reserved.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AA V) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AA V vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ), into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AA V-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AA V vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

The structure of the major tetrahydropterin in Escherichia coli Was determined as (6R)-5,6,7,8-tetrahydro-L-monapterin, i.e. (6R)-2-amino-5,6,7,8-tetrahydro-6-[(1S,2S)-1,2,3-trihydroxypropyl]pteridin-4(3H)-one. Although the stereochemical structure of the trihydroxypropyl side chain has been determined previously by fluorescence detected circular dichroism analysis on its aromatic derivative, the most important configuration at C(6) has not been clarified. The major difficulties for the determination of the chirality were instability toward air oxidation and very low concentration of the tetrahydropterin derivative. In the present study, the C(6)-configuration was determined as R by comparing its stable hexaacetyl derivative with authentic (6R)- and (6S)-hexaacetyl-5,6,7,8-tetrahydro-L-monapterins by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry (LC-MS). (6R)-5,6,7,8-Tetrahydro-L-monapterin is a new unconjugated tetrahydropterin from natural sources.

Neuropilin I is the specific receptor for Sema3A and plays a role in nerve fiber guidance. We report that neuropilin I and Sema3A mutant mouse embryos, generated by targeted gene disruption, showed displacement of sympathetic neurons and their precursors and abnormal morphogenesis in the sympathetic trunk. We also show that Sema3A suppressed the cell migration activity of sympathetic neurons from wild-type but not neuropilin I mutant embryos in vitro and instead promoted their accumulation into compact cell masses and fasciculation of their neurites. These findings suggest that the neuropilin 1-mediated Sema3A signals regulate arrest and aggregation of sympathetic neuron precursors and sympathetic neurons themselves at defined target sites and axon fasciculation to produce the stereotyped sympathetic nerve pattern.

Absolute configurations of 6-(1-hydroxyalkyl)pterin derivatives are determined by fluorescence detected circular dichroism (FDCD) spectroscopy. This method is at least 10 times more sensitive than CD analysis and specific even existence of 10 times excess amounts of chiral sugars or nucleic acids.

Specific degeneration of the nigrostriatal 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, i.e., muscle rigidity, akinesia, and resting tremor, observed in Parkinson's disease (PD). We and other workers have found changes in the levels of cytokines, neurotrophins, and other apoptosis-related factors in the nigro-striatal region of postmortem brain and/or in the cerebrospinal fluid (CSF) from PD patients, or from animal models of PD such as 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP)-induced PD in mice or 6-hydroxydopamine (6-OHDA)-induced PD in rats. The most remarkable changes observed specifically in the nigrostriatal region were decreased levels of neurotrophins supporting DA neurons. These results indicate that the process of cell death in the nigrostriatal DA neurons in PD may be the so-called programmed cell death, i.e., apoptosis. Thus gene therapy for PD should aim both at supplementing the decreased striatal DA level by introducing the genes of DA-synthesizing enzymes into non-DA cells in the striatum and at supporting or restoring DA neurons by preventing apoptosis by introducing genes that block the process of apoptosis.

ジーンターゲッティング法を用いて6-ピルボイルテトラヒドロプテリン合成酵素(PTPS)遺伝子ノックアウトマウスの作成を行った.ホモ接合型マウスはほぼメンデルの法則にしたがって生まれたが,48時間以内に死亡した.ホモ接合型新生仔では脳に含まれるビオプテリン,カテコールアミン,セロトニンの量が極めて少なかった.変異マウスの生化学的測定ではホモ接合型マウスのチロシン水酸化酵素活性が著しく低下していた

We examined the modifying effect of whole-leaf Aloe arborescens Miller var. natalensis Berger (designated as 'ALOE') on azoxymethane (AOM)-induced aberrant crypt foci (ACF), putative preneoplastic lesions, in the rat colorectum. Male F344 rats (4 weeks old) were fed the basal diet, or experimental diets containing 1% or 5% ALOE for 5 weeks. One week later, all rats except those in the vehicle-treated groups were injected s.c. with AOM (15 mg/kg, once weekly for 3 weeks). At 9 weeks of age, all the rats were killed, and the colorectum and liver were evaluated for ACF and cytosolic quinone reductase (QR; a phase 2 enzyme), respectively. In rats given AOM and ALOE (1% or 5% in diet) the numbers of ACF/ colorectum, aberrant crypts/colorectum, aberrant crypts/focus and large ACF/colorectum were significantly decreased compared with those of rats given AOM alone (all p < 0.01). No ACF were found in rats treated without AOM. In addition, ALOE significantly increased cytosolic QR activity in the liver (p < 0.01). These results indicated that ALOE inhibited the development of AOM-induced ACF in the rat colorectum, with increased QR activity in the liver, and therefore suggested that ALOE might have a chemopreventive effect against colon carcinogenesis at least in the initiation stage. Copyright (C) 2001 John Wiley & Sons, Ltd.

Manganese and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are known to induce neurological pathologies similar to that of parkinsonism. Previous studies performed in rat striatal slices have shown that MPTP and related compounds inhibit tyrosine hydroxylation, a rate-limiting step of dopamine biosynthesis. Here, we reported that manganese inhibited tyrosine hydroxylation in rat striatal slices. In addition, manganese caused increase in the levels of lactate indicating that aerobic glycolysis was inhibited in striatal slices. This inhibition was unique to manganese since other divalent cations, such as magnesium and zinc, did not increase lactate concentrations. These results suggest that the mechanisms by which manganese produces dysfunction of the nervous system are similar to those of MPTP. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

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, cine of which is phenytalanine 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-CW 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. (C) 2000 Elsevier Science B.V. All rights reserved.

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 (BH(4)), 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.