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Purpose Acrylamide is known to induce disorders in the central nervous system in humans and experimental animals. The present study investigated effects of exposure to acrylamide on adult neurogenesis, noradrenergic axons and the level of norepinephrine in the brain of male rats. Method Four groups of 12 male Wistar rats each were exposed to acrylamide at 0, 0.2, 2 and 20 mg/kg body weight by gavage for 5 weeks. Six rats of each groups were injected with 5-bromo-2′-deoxy-uridine (BrdU) after five-week exposure to acrylamide to examine proliferative cells in the dentate gyrus using immunostaining. Density of noradrenergic and serotonergic axons in the prefrontal cortex, hippocampus and cortex behind the bregma was quantified. Remaining 6 rats were decapitated after the last exposure and brains were dissected out to measure monoamine level in the hippocampus and prefrontal cortex using high performance liquid chromatography. Result Exposure to acrylamide dose-dependently decreased the density of noradrenergic axons in the prefrontal cortex with a significant change at 20 mg/kg. Norepinephrine level decreased in the hippocampus at 20 mg/kg. Exposure to acrylamide at 20 mg/kg or less did not change the number of BrdU positive cells, but the result should be considered preliminary. Conclusion The results show that oral exposure to acrylamide induces decrease in noradrenergic axons and norepinephrine level in the brain of rats. Given the similar effects are observed in 1-bromopropane-exposed rats, there may be the common mechanism in the toxicity of soft electrophiles to the central nervous system.

Parkinson's disease (PD) is the second common neurodegenerative disorder. Deficit of the nigro-striatal dopaminergic neurons causes the motor symptoms of PD. While the oxidative stress is thought to be deeply involved in the etiology of PD, molecular targets for the oxidative insults has not been fully elucidated. 6R-5,6,7,8-Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase (TH), the rate-limiting enzyme for production of dopamine, and easily oxidized to its dihydro-form. In this study, we examined the alteration in the metabolism of BH4 caused by a parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP reduced the dopamine content and the in vivo activity of TH in the striatum prior to degeneration of the dopaminergic neurons. We found that administration of BH4 could restore the dopamine content and in vivo TH activity in the striatum of MPTP-treated mice. Unexpectedly, when BH4 was administered with MPTP, BH4 contents in the brain were far higher than those injected without MPTP even at 23 h after the last injection. Because MPTP has been shown to increase ROS production in the dopaminergic neurons, we assumed that the increased ROS oxidizes BH4 into its dihydro-form, excreted from the dopaminergic neurons, taken-up by the neighboring cells, reduced back to BH4, and then accumulated in the brain. We also investigated the action of MPTP in mice lacking quinonoid-dihydropteridine reductase (Qdpr), an enzyme catalyzing regeneration of BH4 from quinonoid dihydrobiopterin. The dopamine depletion induced by MPTP was severer in Qdpr-deficient mice than in wild-type mice. The present data suggest that perturbation of the BH4 metabolism would be the cause of early and persistent dopamine depletion in the striatum.

6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI–TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2. S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.

Some bipolar cells in the human retina are known to express voltage-gated Na+ channels. However, it is unclear which types of channels are expressed, and whether Na+ channel expression is limited to specific types of bipolar cells. In the present study, we examined the types of voltage-gated Na+ channels expressed in human bipolar cells and the morphology of bipolar cells with voltage-gated Na+ currents. To investigate the expression of voltage-gated Na+ channels in human bipolar cells, we examined whether Na+ channel transcripts could be detected in single bipolar cells using the reverse transcription polymerase chain reaction (RT-PCR) technique. The voltage-gated Na+ current was recorded from isolated bipolar cells using the patch-clamp recording technique. Types of bipolar cells that have the Na+ currents were investigated by analyzing their morphology after staining with Lucifer yellow. Using RT-PCR, the SCN2A Na+ channel was detected in 5 of 6 isolated bipolar cells. This suggests that a subset of human bipolar cells expresses the SCN2A Na+ channel. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current in cone bipolar cells with axon terminal boutons that stratified at the ON layer, which includes the stratum 3, 4, and 5 of the inner plexiform layer (IPL, n = 2/11 cells). The fast transient inward current of isolated bipolar cells was blocked by 1 μM of tetrodotoxin (TTX), a voltage-gated Na+ channel blocker. No fast transient inward current was recorded with axon terminals that stratify at the OFF layer, which includes stratum 1 and 2 of the IPL (n = 4). Thus, a subset of ON cone bipolar cells at least expresses the putative voltage-gated Na+ channel SCN2A in the human retina. The Na+ channels in the bipolar cells may serve to amplify the release of neurotransmitter, glutamate, when membrane potential is rapidly depolarized and thereby selectively accelerating light responses.

Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier.

Parkinson's disease (PD) is an aging-related movement disorder mainly caused by a deficiency of neurotransmitter dopamine (DA) in the striatum of the brain and is considered to be due to progressive degeneration of nigro-striatal DA neurons. Most PD is sporadic without family history (sPD), and there are only a few percent of cases of young-onset familial PD (fPD, PARKs) with the chromosomal locations and the genes identified. Tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4)-dependent and iron-containing monooxygenase, catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthesis of catecholamines (DA, noradrenaline, and adrenaline). PD affects specifically TH-containing catecholamine neurons. The most marked neurodegeneration in patients with DA deficiency is observed in the nigro-striatal DA neurons, which contain abundant TH. Accordingly, TH has been speculated to play some important roles in the pathophysiology in PD. However, this decrease in TH is thought to be secondary due to neurodegeneration of DA neurons caused by some as yet unidentified genetic and environmental factors, and thus, TH deficiency may not play a direct role in PD. This manuscript provides an overview of the role of human TH in the pathophysiology of PD, covering the following aspects: (1) structures of the gene and protein of human TH in relation to PD; (2) similarity and dissimilarity between the phenotypes of aging-related sPD and those of young-onset fPD or DOPA-responsive dystonia due to DA deficiency in the striatum with decreased TH activity caused by mutations in either the TH gene or GTP cyclohydrolase I (GCH1) gene; and (3) genetic variants of the TH gene (polymorphisms, rare variants, and mutations) in PD, as discovered recently by advanced genome analysis.

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase that is essential for the biosynthesis of dopamine. Parkinson's disease (PD) is characterized by a progressive degeneration of nigrostriatal dopaminergic neurons, and biomarkers reflecting the degree of neurodegeneration are important not only for basic research but also for clinical diagnosis and the treatment of the disease. Although the total neopterin and biopterin levels in the cerebrospinal fluids (CSF) of the patients with PD were reported, alterations in the composition of reduced and oxidized forms of pteridine compounds have not been examined. In this study, we first examined the time-dependent alterations in BH4 and other reduced pteridine compounds in the CSF of an MPTP-treated monkey as a primate PD model. We found that the CSF levels of BH4 and dihydroneopterin, an intermittent metabolite of BH4-biosynthesis, altered inversely with progression of neurodegeneration, whereas those of dihydrobiopterin and neopterin were relatively low and constant. Next, we assayed the amounts of reduced pteridine compounds in the CSF of 36 pre-symptomatic LRRK2-mutation (N1437H or G2019S) carriers (LRRK2-carrier), 13 patients with PD symptoms (LRRK2-PD), 46 patients with sporadic PD (sPD), and 26 non-PD individuals. The BH4 levels were significantly lower in both the LRRK2-PD and sPD patients, and the LRRK2-carriers exhibited higher BH4 levels compared with the sPD patients. The total neopterin levels in the CSF of the LRRK2-PD were significantly higher than those in the sPD and non-PD individuals, which indicated greater inflammatory responses in the brains of LRRK2-PD patients. The present results suggest that detailed analyses of pteridine levels in the CSF might be useful for understanding the pathophysiology of familial PD and for monitoring PD progression.

(6 R )‐l‐ erythro ‐5,6,7,8‐Tetrahydrobiopterin (BH4) is an essential cofactor for monoamine and nitric oxide (NO) production. Sepiapterin reductase (SPR) catalyzes the final step in BH4 biosynthesis. We analyzed the cardiovascular function of adult Spr gene‐disrupted ( Spr −/− ) mice for the first time. After weaning, Spr −/− mice suffered from hypertension with fluctuation and bradycardia, while the monoamine contents in these mice were less than 10% of those in the wild‐type mice as a result of BH4 depletion. Heart rate variability analysis indicated the sympathetic dominant state in Spr −/− mice. The endothelium‐dependent vascular relaxation in response to acetylcholine was significantly impaired in Spr −/− mice after sexual maturation (above 4 months old). Protein amounts of α 1 adrenergic receptor and eNOS in the aorta were not altered. Spr −/− mice exhibited hypoglycemia and elevation of plasma renin activity. Our results suggest that the hypertension with fluctuation and bradycardia of Spr −/− mice would be caused by an imbalance of sympathetic and parasympathetic input and impaired nitric oxide production in endothelial cells. We suggest an important role of BH4 and SPR in age‐related hypertension and a possible relationship with the cardiovascular instabilities in autonomic diseases, including Parkinson9s disease and spinal cord injury.

Guanosine triphosphate cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin, a cofactor for aromatic amino acid hydroxylases and nitric oxide synthases. As monoamine neurotransmitters are synthesized by the reactions catalyzed by tyrosine hydroxylase and tryptophan hydroxylase, alterations in the content of tetrahydrobiopterin affect the monoamine levels in the brain. Here, we examined the possible association of a functional single-nucleotide polymorphism (SNP) of the GCH1 gene, rs841 (C+243T), with attentional function as assessed by the Continuous Performance Test-Identical Pairs version (CPT-IP) in healthy individuals. We found that homozygous T/T genotype carriers of rs841 scored lower performance on the CPT-IP test. Our data suggest that alterations in GCH1 activity affect attentional function, especially sustained attention and vigilance.

Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders., 患者由来iPS細胞とゲノム編集技術を用いて, BH4代謝病のドパミン合成異常の疾患モデル系構築に成功 : iPS細胞利用による代謝改善生理活性物質の同定と疾患再定義. 京都大学プレスリリース. 2016-10-28.

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson's disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin-proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-(40)Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin-proteasome system in PC12D cells.

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.

Background Human artificial chromosomes (HACs) are generated from the precursor DNA constructs containing α-satellite DNA with CENP-B boxes, and the process could be used for the incorporation of large genes in the HACs. Guanosine triphosphate cyclohydrolase I (GCH1) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthase. Results: We constructed HACs carrying a 180 kb genome segment encoding the human GCH1 gene and its control region from the bacterial artificial chromosome (BAC) with the GCH1 segment by co-transfection with the α-satellite DNA-containing BAC to a human fibroblast cell line. Two cell lines carrying a HAC with GCH1 genes were obtained. Both HACs were composed of multiple copies of precursor BACs and were maintained stably in human and mouse cell lines. The GCH1 activities of the HAC-carrying human fibroblast cell lines were elevated but still highly sensitive to IFN-γ induction, mimicking the response of the gene expression from the authentic chromosomal genes. Conclusion: These HACs will provide a useful system for analysis of the complex regulatory circuit of the GCH1 gene in vivo and also function as a tool for gene delivery in animal models or in therapeutic trials.

Blood pressure is regulated by extrinsic factors including noradrenaline, the sympathetic neurotransmitter that controls cardiovascular functions through adrenergic receptors. However, the fine-tuning system of noradrenaline signaling is relatively unknown. We here show that l-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of catecholamines, sensitizes the vascular adrenergic receptor alpha1 (ADRA1) through activation of L-DOPA receptor GPR143. In WT mice, intravenous infusion of the ADRA1 agonist phenylephrine induced a transient elevation of blood pressure. This response was attenuated in Gpr143 gene-deficient (Gpr143-/y) mice. Specific knockout of Gpr143 in vascular smooth muscle cells (VSMCs) also showed a similar phenotype, indicating that L-DOPA directly modulates ADRA1 signaling in the VSMCs. L-DOPA at nanomolar concentrations alone produced no effect on the VSMCs, but it enhanced phenylephrine-induced vasoconstriction and intracellular Ca2+ responses. Phenylephrine also augmented the phosphorylation of extracellular signal-regulated kinases in cultured VSMCs from WT but not Gpr143-/y mice. In WT mice, blood pressure increased during the transition from light-rest to dark-active phases. This elevation was not observed in Gpr143-/y mice. Taken together, our findings provide evidence for L-DOPA/GPR143 signaling that exerts precursor control of sympathetic neurotransmission through sensitizing vascular ADRA1.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

β Both neopterin and biopterin concentrations in cerebrospinal fluid from patients with Parkinson's disease, in which the nigrostriatal dopamine neurons degenerate, were lower than those from age-matched older control subjects. However, the decrease in biopterin was more marked than that in neopterin, resulting in the increase in the neopterin/ biopterin ratio in Parkinson's disease. These results suggests that neopterin in cerebrospinal fluid in Parkinson's disease may partly be derived from immunoactivated glial cells, besides catecholamine or serotonin n eurons including nigrostriatal dopamine neurons. In accordance to this hypothesis, cytokines (TNF-α, IL-1, IL-2 , IL-6, EGF, TGF-α, TGF-β1) were found to be increased in the striatum and/or in cerebrospinal fluid. The increment of cytokines in the brain in Parkinson's disease may be related to the mechanism of neurodegeneration of dopaminergic neurons in Parkinson's disease . In contrast to Parkinson's disease, in hereditary progressive dystonia/ dopa-responsive dystonia, which is a dopamine deficiency caused by mutations in GTP cyclohydrolase I without neuronal cell death (Segawa's disease), neopterin and biopterin in cerebrospinal fluid decreases in parallel owing to the decreased activity in GTP cyclohydrolase I .

Aromatic l-amino acid decarboxylase (AADC) is a homodimeric pyridoxal phosphate-dependent enzyme responsible for the syntheses of dopamine and serotonin. Defects in the AADC gene result in neurotransmitter deficiencies. Patients with AADC deficiency have severe motor and autonomic dysfunctions. A mouse model of AADC deficiency was recently established. These mice grow poorly and move awkwardly during infancy. They also show high anxiety when they grow up. Because drug therapy provides little or no benefit for many patients with AADC deficiency, a gene therapy has been attempted. The gene therapy employed an adeno-associated virus viral vector that can express the human AADC protein. The vector was injected to the brain of several children with AADC deficiency. The therapy was well tolerated, and all treated patients showed improvement. In the future, the mouse model will also help the development of treatments for AADC deficiency.

Human tyrosine hydroxylase (hTH) exists in four isoforms. The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor. In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4. Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4. Two different Km values for pterin cofactor were observed at a high concentration (200 microM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4. The present results suggest that in the presence of relatively high concentrations (approximately 100 microM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo. We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes.

PARK8 is the most common form of familial Parkinson's disease (PD). We measured biopterin and monoamine metabolite levels in the cerebrospinal fluids of 7 PARK8 patients (I2020T mutation in leucine-rich repeat kinase 2), 2 asymptomatic mutation carriers, and 21 sporadic PD patients. The biopterin levels in PARK8 patients were significantly higher than those in sporadic PD patients, although the symptoms were comparable in both groups, suggesting that PARK8 patients exhibit parkinsonian symptoms with higher biopterin levels than sporadic PD patients.

Rhabdomyolysisの患者に種々の血液浄化法を試みた. 基礎疾患を有するRhabdomyolysisは容易に重症化し, その予防には, ミオグロビンの早期除去が重要となる. 近年, 透析膜の進歩によりハイパフォーマンス膜が開発され, 比較的大きな分子量の物質まで除去できるようになってきた.今回, PMMA膜 (BK2.1P) を用い, QB200ml/minで濾液を測定しミオグロビンの除去率を求めたところ23.1%と高値を示した. さらにこの膜を用いてHDFを行えば, 実際にはもっと高いミオグロビンの除去が可能と思われ, 有効な治療方法と考えられた.

Recently we showed that phosphorylated tyrosine hydroxylase (TH) at Ser40 (p40-TH) was degradated by ubiquitin proteasome system (UPS) and easily insolubilized, and that TH level was decreased in the striatum of tetrahydrobiopterin (BH4) deficient mice. Here we studied the effect of depletion of dopamine (DA) or BH4 to the regulation of the amount of p40-TH. We treated PC12D cells with quinpirole or haloperidol, NSD-1015; an inhibitor of aromatic aminoacid decarboxylase (AADC), or DAHP; an inhibitor of GTP cyclohydrolase I (GCH). The amount of p40-TH was measured by western blotting. Treatment with haloperidol induced the short and transient elevation of p40-TH level (30 min), and exposure to NSD-1015 or DAHP induced the long-lasting elevation of p40-TH after 48 h. We are also examining the effects using primary mesencephalic neurons. The molecular mechanism of long-lasting elevation of p40-TH by DA deficiency is now under investigation.

Some naturally occurring pteridine derivatives carry out various important metabolic transformations which produce and metabolize essential materials for life as cofactors in all kinds of living organisms. For example, the pteridine derivatives work in biosyntheses of amino acids, nucleic acids, neurotransmitters and nitrogen monoxides and metabolisms of purine and aromatic amino acids in the human body. Based on the mechanisms of the metabolisms, such compounds have long been interested in biological chemistry and medicinal chemistry. Some pteridine derivatives are practically used in chemotherapy or diagnosis for various diseases. In Sect. 1, we briefly review the history of pteridine and the biological significances of naturally occurring pteridine derivatives other than tetrahydrobiopterin, e.g., folic acid, molybdenum cofactor. Recent progress on the analysis and synthesis of tetrahydrobiopterin and its analogues is summarized in Sects 2 and 3. In Sects. 4 and 5, The biosynthesis of tetrahydrobiopterin and its functions in catecholamine and NO biosynthesis are described and Sect. 6 describes diseases caused by the deficiency of tetrahydrobiopterin.

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 K m 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.

The main biochemical characteristic of Parkinson's disease (PD) is reduction of the neurotransmitter dopamine and the dopamine-synthesizing enzyme system, including tyrosine hydroxylase (TH, tyrosine 3-monooxygenase, EC 1.14.16.2) and tetrahydrobiopterin (BH(4) co-factor, in nigrostriatal neurons (1). The deficiency in dopamine-synthesizing enzymes is accompanied by cell loss, which is thought to be caused by unknown exogenous environmental factors as well as endogenous genetic factors.

Unique pterins were identified as a major pterin in several microorganisms. D-Monapterin was identified as major pterin in Tetrahymena pyriformis (1 ), which is a protozoa living in fresh water, and L-monapterin was identified in E. coli (2), respectively. However, their biological functions have not been revealed yet. We recently reported presence of reduced forms of those monapterins in each microorganism, and the reduced form was estimated 5,6,7,8-tetrahydro form (3, 4). In this study, we would describe structural determination of unstable natural 5,6,7,8-tetrahydromonapterins in the two microorganisms. First, the configuration of 1′,2′,3′-trihydroxypropyl side chain was determined by fluorescence detected circular dichroism (FDCD) spectra using stable aromatic derivatives, which has strong fluorescence nature, and then configurations of 6-position was determined by LC-MS analysis using stable completely acetyl derivatives.

We found that in Parkinson’s disease (PD) the levels of various cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-2, IL-4, IL-6, epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β1] were significantly increased in the striatum (caudate and putamen) of the postmortem brain and in ventricular or spinal cerebrospinal fluid (VCSF, LCSF). Furthermore, the levels of the apoptosis-related proteins such as bcl-2 and soluble Fas (sFas) in the striatum were also elevated in PD. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonism mice, the levels of IL-1β in the striatum were significantly increased, but those of nerve growth factor (NGF) were significantly decreased, compared with control mice. In hemiparkinsonism rats produced by injection of 6-hydroxydopamine (6-OHDA) into one side of the median forebrain bundle, the levels of TNF-α in the 6-OHDA-treated side were increased in the striatum and substantia nigra, but not in the cerebral cortex, compared with those in the control side. Repeated administration of L-DOPA in the 6-OHDA-treated rats did not change the TNF-α levels in the control side and in the 6-OHDA-treated side in the substantia nigra, striatum, and cerebral cortex.

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.

The present study was carried out to examine the efficacy of plasma exchange in patients with rapidly progressive glomerulonephritis (RPGN). Seventeen patients with RPGN were treated with plasmapheresis as adjunct to immunosuppressive therapy. Of these, 4 had antiglomerular basement membrane (GBM) antibody-mediated glomerulonephritis (GN), 8 had immune-complex GN (5 SLE, 2 HSP, 1 cryoblobulinemia), 5 had pauci-immune GN (3 peripheral antineutrophil cytoplasmic antibody [P-ANCA], 1 cytoplasmic antineutrophil cytoplasmic antibody [C-ANCA], 1 other). Treatment of 10 of these patients with plasmapheresis within the first month of disease onset resulted in a stable renal function for a period extending from 1 to 3 years, except in 2 patients who had high baseline levels of serum creatinine. In the remaining patients, 2 were treated with hemodialysis 6 years later at the end of follow-up. We conclude that plasmapheresis, when used in combination with immunosuppressive drugs, is beneficial, leading to improved renal function.

The main biochemical characteristics of Parkinson’s disease are the reduction of dopamine the neurotransmitter, and of tyrosine hydroxylase (TH) and the biopterin cofactor, the dopamine-synthesizing enzyme system, in the nigrostriatal dopamine neurons. A deficiency in the dopamine-synthesizing enzymes is accompanied by cell loss of the nigrostriatal dopamine neurons, which is assumed to be caused by unknown exogenous, environmental factors and endogenous, genetic factors. Not only the nigrostriatal dopamine neurons but also other catecholamine neurons may be impaired, as suggested by decreases in dopamine β-hydroxylase (DBH) in norepinephrine neurons and phenylethanolamine Nmethyltransferase (PNMT) in epinephrine neurons (Nagatsu et al., 1977; Nagatsu et al., 1984).

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 specificeven existence of 10 times excess amounts of chiral sugars or nucleic acids.

We investigated the effects of 14 heterocyclic amines in food on nigro-striatal dopaminergic neurons. Among 14 compounds tested, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) caused substantial decreases in 3,4-dihydroxy-phenylalanine (DOPA) formation in striatal tissue slice system. When Trp-P-1 or Trp-P-2 was unilaterally infused in the rat striatum by an in vivo micro-dialysis technique, both compounds produced a transient increase of dopamine (DA) and continuous decreases in the metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the perfusate. This suggests that the two compounds inhibit monoamine oxidase (MAO) in vivo. Indeed they were found to be very potent inhibitors of MAO in vitro. Systemic administration of Trp-P-1 to C57 Black mice caused a marked decrease of DOPAC content and a significant increase of DA in the striatum, indicating inhibition of MAO in vivo. These results suggest that Trp-P-1 and Trp-P-2 contained in food could alter the metabolism of DA in the brain.

Administration of carbon tetrachloride to rats by intraperitoneal injection caused significant increases in serum aromatic L-amino acid decarboxylase activity and serum glutamate oxaloacetate and glutamate pyruvate transaminase levels. Moreover, a significant positive correlation coefficient was obtained between the decarboxylase activity and glutamate oxaloacetate transaminase activity.The increment of the decarboxylase activity in the serum caused by carbon tetrachloride was found to be accompanied by a decrease in the decarboxylase activity of the liver, and a significant negative correlation was found between the two enzyme activities. In contrast, no change was found in the decarboxylase activity of the kidney.These observations suggest that aromatic L-amino acid decarboxylase occurs in the serum at least in part as a result of the leakage of the enzyme from the liver into the blood.

Reduction of aldehydes and ketones with 2-propanol was found to proceed efficiently in the presence of hydrous zirconium oxide. The reaction is performed simply and the products are easily isolated in the pure state by filtering off the hydrous zirconium oxide, followed by evaporation of solvents.

Quinonoid dihydropteridine reductase (QDPR) catalyzes the regeneration of tetrahydrobiopterin (BH4), a cofactor for monoamine synthesis, phenylalanine hydroxylation and nitric oxide production. Here, we produced and analyzed a transgenic Qdpr−/− mouse model. Unexpectedly, the BH4 contents in the Qdpr−/− mice were not decreased and even increased in some tissues, whereas those of the oxidized form dihydrobiopterin (BH2) were significantly increased. We demonstrated that unlike the wild-type mice, dihydrofolate reductase regenerated BH4 from BH2 in the mutants. Furthermore, we revealed wide alterations in folate-associated metabolism in the Qdpr−/− mice, which suggests an interconnection between folate and biopterin metabolism in the transgenic mouse model.

Reduction of aldehydes and ketones with 2-propanol was found to proceed efficiently in the presence of hydrous zirconium oxide. The reaction is performed simply and the products are easily isolated in the pure state by filtering off the hydrous zirconium oxide, followed by evaporation of solvents.

Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5′-phosphate as a cofactor, both enzymes were similar.

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 .

Fixed Mo catalysts (supported molecular catalysts), synthesised using the facile reaction between Mo (π-C3H5)4 and the surface acidic OH groups of alumina, silica and silica–alumina, were found to be very active for propene and but-1-ene metathesis in the temperature range 268–308 K. The isolated and coordinatively unsaturated Mo4+ species which are chemically bonded to the support surface are the active species. The nature of the ligands and supports coordinated to the Mo4+ ions produce different catalytic activities and selectivities. The support also works as a reservoir/supplier of reactants. Metathesis on fixed Mo catalysts is explained by a one-site chain mechanism involving carbene intermediates.