Toshiharu Nagatsu

A novel member of the epidermal growth factor (EGF) family, the neural- and thymus-derived activator for ErbB kinases (NTAK), has been purified and cloned. Four alternative spliced isoforms have been detected in the rat adrenal pheochromocytoma cell line, PC-12 cells. In vivo, NTAK is only expressed in the brain of rat E11.5 embryos, and in the brain and thymus of adult rats. The soluble 46kDa form binds directly to ErbB3 and B4, but not to ErbB1 or B2. NTAK, however, trans-activates ErbB1 and B2 via heterodimerization with ErbB3 or B4. NTAK stimulates the differentiation of MDA-MB-453 cells and competitively inhibits the binding of ¹²⁵I-neuregulin to these cells. In addition to these neuregulin-like properties, NTAK exhibits limited structural homology to neuregulins in the immunoglobulin (Ig)-like, EGF-like and hydrophobic domains. Thus, NTAK appears to be a new member of the EGF family displaying neuregulin properties.

Parkinson's disease (PD) is an aging-related neurodegenerative disorder with motor disturbances and is characterized by selective neuronal cell death of the nigrostriatal dopamine (DA) neurons by unknown causes. Sporadic PD is thought to be caused by the combined effects of the genes related to the risk factors and some environmental factors such as neurotoxins. We found that a number of cytokines, neurotrophins (growth factors), and apoptosis-related modifiers are increased in parkinsonian brain. These results suggest that up-regulation of cytokines, neurotrophins, and apoptosis-related modifiers in the striatum may be involved in the pathogenesis of neurodegeneration in PD.

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)-1 beta, 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-1 beta level was increased and NGF one decreased in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP)-induced parkinsonian mice, and TNF-a 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-cr 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 neuroprotective effect of nicotine via nicotinic acetylcholine receptor (nAChR) has been reported in differentiated PC12 cells. We examined the effects of nerve growth factor (NGF) and nicotine on the expressions of the nAChR subunits alpha 3, alpha 5, alpha 7, beta 2, beta 3, and beta 4, in PC12 cells using Northern blot analysis. NGF increased the transcriptions of alpha 5 (1.4- to 2.3-fold), alpha 7 (1.7- to 2.5-fold), and beta 4 (1.9- to 3.0-fold) subunits, but changes in alpha 3 and beta 2 subunit levels were smaller than alpha 5, alpha 7, or beta 4. Nicotine also increased the levels of mRNA encoding alpha 5 and beta 2 subunits. The pattern of subunit mRNA changes was different between NGF and nicotine treatment. No signal for beta 3 subunit was detected. The results suggest that NGF changes the expression of nAChR in a subtype-specific manner over the course of the differentiation, and the disproportionate subunit expressions might be related to the neuroprotective effect exerted by nicotine. (C) 1999 Elsevier Science ireland Ltd. All rights reserved.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH) is the first and rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase (TH). Our previous study reported the presence of GCH in several neuronal groups in animal brains using a newly raised anti-GCH antibody. The present study aims at elucidating whether GCH and TH coexist in the same neurons of the human brain with the aid of immunohistochemical dual labeling. GCH-immunoreactivity was observed in the cell bodies and fibers of monoaminergic neurons of the human brain. Neurons which contain both enzymes are seen in the human substantia nigra, ventral tegmental area, locus coeruleus, dorsal raphe, and zona incerta. In these regions, almost all the cells also show immunoreactivity for aromatic L-amino acid decarboxylase (AADC), the second step enzyme for catecholamine synthesis, indicating that these neurons are catecholaminergic. However, some neurons in the dorsal and dorsomedial hypothalamic nuclei are stained only for GCH or TH. They appear to constitute an independent cell group in the human brain. The present observation suggests that L-dopa is not produced in the cells immunoreactive for TH but not for GCH, and that TH in these cells which lack GCH may have an unidentified role other than dopa synthesis.

Objective To examine whether tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) genes can be cotransduced into the same target striatal cells using adeno-associated virus (AAV) vectors, and to determine whether the cotransduction would result in better biochemical change than the TH gene alone. Methods TH and AADC genes were cotransduced into cultured striatal cells with separate AAV vectors. Expressions of TH and AADC were detected by immunocytochemistry; intracellular catecholamine levels were assayed by high-performance liquid chromatography (HPLC). Results TH and AADC genes were efficiently cotransduced into the striatal cells. Specifically, the coexpression of TH and AADC resulted in more effective dopamine production compared with the TH gene alone. Conclusion Using AAV vectors, coexpression of TH and AADC in the striatal cells might be a useful approach to gene therapy for Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum, A promising approach to the gene therapy of PD is intrastriatal expression of enzymes in the biosynthetic pathway for dopamine, Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC), Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC, Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and lit vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.

The postsynaptic density (PSD) fraction prepared from the rat forebrain contained a transcription factor, cAMP response element-binding protein (CREB). The occurrence of CREB in the PSD was confirmed by immunoelectron microscopic examination. CREB in the PSD fraction was phosphorylated both by protein kinase A and Ca2+/calmodulin-dependent protein kinase II (CaMKII) endogenous to the fraction, and dissociated from the PSD after phosphorylation, especially under CaMKII-activated conditions. The fraction containing CREB that was released from PSD after phosphorylation possessed cAMP response element (CRE)-binding activity. Thus, PSD anchors functionally active CREB. These results suggest that CREB anchored to the PSD is liberated by phosphorylation upon specific synaptic stimulation, translocates into the nucleus, and then triggers synaptic activity-dependent changes in gene expression. (C) 1998 Elsevier Science B.V. All rights reserved.

The development of autosomal dominant DOPA-responsive dystonia (AD-DRD) is stipulated by mutations in GTP-cyclohydrolase I gene. GTP-cyclohydrolase I is the first and key enzyme of tetrahydrobiopterin biosynthesis. Its deficiency in nirgostriatal dopaminergic neurons cause a decrease in tyrosine hydroxylase activity and therefore dopamine deficiency. However, administration of low doses of dopamine can control the development of AD-DRD. Determination of GTP-cyclohydrolase I activity in mononuclear blood cells is convenient diagnostic method.

The effect of cytokines on apolipoprotein E (apo E) production and secretion was investigated in a human hepatoma cell line HepG2. Incubation of HepG2 cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF-alpha) for 48 h resulted in a significant dose-related decrease of apo E concentration in the culture medium, white intracellular apo E content increased without change in mRNA level. In contrast, IL-1 beta and TNF-alpha decreased both intracellular and medium apo A-I. Elution profiles of cholesterol and apolipoproteins revealed that apo E was present in apo E-rich high density lipoprotein (HDL) fraction and apo A-I was in apo E-rich HDL and small HDL fractions. IL-1 beta and TNF-alpha decreased both apo E and apo A-I in these fractions. The present results suggest that IL-1 beta and TNF-alpha suppress hepatic apo A-I expression and secretion but not expression of apo E, which could contribute to the abnormal Lipid metabolism in certain cytokine-mediated inflammatory diseases. (C) 1998 Academic Press Limited.

The causative genes of hereditary dystonia (hereditary progressive dystonia, HPD dopa-responsive dystonia, DRD) were discovered in 1994-1995. HPD/DRD is causesd by the deficiency of dopamine to less than 20% of the normal level in the nigro-striatum of the brain owing to the mutations of the dopamine synthesizing enzymes. Autosomal dominant dystonia (Segawa's disease) was found to be caused by mutations of GTP cyclohydrolase I which synthesizes tetrahydrobiopterin, the cofactor of tyrosine hydroxylase, by Ichinose et al. (Nature Genetics, 1994) in Japan. Autosomal recessive dystonia was reported to be caused by mutations of tyrosine hydroxylase by Ludecke et al. (Human Genetics, 1995) in Germany. Hereditary dystonia, especially autosomal dominant Segawa's disease can be completely controlled by L-dopa administration. Measurement of the activity of GTP cyclohydrolase I in mononuclear blood cells is useful for the diagnosis of Segawa's disease.

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N2-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 μmol/mol creatinine. The present results indicate that the peak found with elution from the C18 column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N-2-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 mu mol/mol creatinine. The present results indicate that the peak found with elution from the C-18 column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.

5-Hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) were injected intraperitoneally (i.p.) into the laboratory shrew (Suncus murinus). Immunocytochemical and immunofluorescence studies were carried out on serial or same sections of the brain, which were reacted with specific antisera to dopamine (DA) or serotonin (5-HT) produced in our laboratory. We observed that cell bodies and nerve terminals of many catecholaminergic (CAnergic) neurons exhibited 5-HTP uptake and conversion of the precursor into 5-HT. However, the locus ceruleus showed scarcely any 5-HT immunoreactivity. This suggests that the precursor uptake mechanism may be different among various CAnergic groups. In contrast to these findings on CAnergic neurons, all serotoninergic (5-HTnergic) neurons after L-DOPA administration showed DA immunopositive reaction in their cell bodies and nerve terminals, suggesting that 5-HTnergic neurons may have the same capacity for precursor uptake. On the other hand, we observed that all aromatic L-amino acid decarboxylase (AADC)-only-positive neuron groups showed both DA and 5-HT immunoreactions after L-DOPA and 5-HTP administration, respectively, in the double-staining immunofluorescence method. From these results, AADC-only-positive neurons may be considered to belong to the amine precursor uptake and decarboxylation (APUD) system.

Transforming growth factor (TGF)-beta 1 content was measured for the first time in the brain (caudate nucleus, putamen, and cerebral cortex) and in ventricular cerebrospinal fluid (VCSF) from control and parkinsonian patients by a sandwich enzyme immunoassay. The concentrations of TGF-beta 1 were significantly higher in the dopaminergic striatal regions in parkinsonian patients than those in controls, but were not significantly different in the cerebral cortex between parkinsonian and control patients. Furthermore, the concentrations of TGF-beta 1 in VCSF were significantly higher in parkinsonian patients than those in non-parkinsonian control patients. Since TGF-beta 1 has potent regulatory activity on cell growth, these results suggest that TGF-beta 1 may have some significant modulatory role in the process of neurodegeneration in Parkinson's disease.