パーキンソン病におけるN-メチル転移酵素遺伝子異常の解明

• Project/Area Number: 09877033
• Research Category: Grant-in-Aid for Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Pathological medical chemistry
• Research Institution: Nagoya Institute of Technology
• Principal Investigator: 直井 信 名古屋工業大学, 工学部, 教授 (50022786)
• Project Period (FY): 1997
• Project Status: Completed (Fiscal Year 1997)
• Budget Amount: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: パーキンソン病 / N-methyl(R)salso1inol / (R)salso1inol N-methyltransferase / リンパ球 / 黒質線条体 / 1,,2-dimethyl-6, 7-dihydroxisoquinoliniunion / 神経毒 / 内在性病因物質

Research Abstract

申請者は最近,ヒト脳内で神経毒N-methyl(R)salsolinol[NM(R)Sal〕が生成されることがパーキンソン病(PD)の病因に関与する可能性が高いとの結果を得ている。今回この神経毒を生合成する酵素系をPD患者のリンパ球サンプルで測定し,PDの病因に関与する酵素とそれをコードする遺伝子を明らかとするため研究を行った。PD患者,正常のヒトリンパ球を採取分離し,NM(R)Salの生成に関与している(R)sa1solinol synthase,(R)salso1inol N-methyltransferaseとN-methyl(R)salsolino1 oxidaseの酵素活性を測定した。その中で中性に至適pHを有する(R)salso1inol N-methyltransferase 活性がPD患者のみで有意に増加していた。PD患者で酵素活性は100.2±81.8pmol/min/mg proteinと対照正常群より有意に18.9±15.0pmo1/min/mg protein増加していた(Anal.Neurol.in press)。一方アルカリ側に至適pHを持つ(R)salso1inol N-methyltransferase とN-methyl(R)salso1inol oxidase活性には差がなかった。この線条体での中性(R)salso1inol N-methyltransferase 活性と,黒質でのNM(R)Sa1の酸化生成物1,2-dimethyl-6,7-dihydroxyisoquino-linium ionの濃度との間に明らかな相関が認められた(Neurosci,Lett,235,81-84,1997)。同一患者からのリンパ球での酵素活性と脳脊髄液でのNM(R)Sal濃度に相関が認められ,本酵素が脳内の神経毒濃度を決定していることが示唆された。
ヒト脳から中性(R)salso1inol N-methyltransferase の精製を行い,分子量35KDのタンパクとして精製された。現在本酵素のタンパク化学的性状を検討し,分子生物学研究を進める用意をしている。

Research Products

• [Publications] Wakako Maruyama: "(-)-Deprenyl protects human dopaminergic neuroblastoma SH-SY5Y cells from apoptosis induced by peroxynitrite and nitric oxide." Journal of Neurochemsitry in press.(1998)
• [Publications] Makoto Naoi: "(R)Salso1inol N-methyltransferase activity increases in parkinsonian lymphocytes." Annals of Neurology in press. (1998)
• [Publications] Tetsuo Ohkuwa: "A novel assay for glutathione reductase activity by use of high performance liquid chromatography with electrochemical detection." Journal of Chromatography B in press.(1998)
• [Publications] Chiyoko Minami: "Preparation of (R)-N-methylsalsolinol via reversed-phase high-performance liquid chromatography using β-cyclodextrin as a mobile-phase additive." Journal of Chromatography B. 702. 245-248 (1997)
• [Publications] Makoto Naoi: "A neutral N-methyltransferase activity in the striatum determines the level of an endogenous MPP^+-like neurotoxin, 1, 2-dimethyl-6, 7-dihydroxyisoquinolnium ion, in the substantia nigra of human brains." Neuroscience Letters. 235. 81-84 (1997)
• [Publications] Wakako Maruyama: "A neurotoxin N-methyl(R)salsolinol induces apoptotic cell death in differentiated human dopaminergic neuroblastoma SH-SY5Y cells." Neuroscience Letters. 232. 147-150 (1997)
• [Publications] Wakako Maruyama: "An endogenous dopaminergic neurotoxin, N-methyl-(R)-salsolinol, induces DNA damage in human dopaminergic neuroblastoma SH-Sy5Y cells." Journal of Neurochemsitry. 69. 322-329 (1997)
• [Publications] Masao Kawai: "Convenient enantioselective preparation of salsolinol-1-carboxylic acid" Tetrahydron : Asymmetry. 8. 1487-1490 (1997)
• [Publications] Wakako Maruyama: "A dopaminergic neurotoxin, 1(R), 2(N)-dimethyl-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquino-line, N-methyl(R)salsolnol, and its oxidation product, 1, 2(N)-diemthyl-6, 7-dihydroxyliso-quinolinium ion, accumulate in the nigro-striatal system-of the human brain." Neuroscience Letters. 223. 61-64 (1997)
• [Publications] Yulin Deng: "Assay for the (R)- and (S)-enantiomers of salsolinols in biological samples and foods with ion-pair high-performance liquid chromatography using β-cyclodextrin as a chiral mobile phase additive." Journal of Chromatography B. 689. 313-320 (1997)
• [Publications] Tsutomu Takahashi: "Cytotoxicity of endogenous isoquinolines to human dopaminergic neuroblastoma SH-SY5Y cells." Journal of Neural Transmission. 104. 59-66 (1997)
• [Publications] Tetsuo Ohkuwa: "Glutathione status and reactive oxygen generation in tissue of young and old exercised rats" Acta Physiological Scandinavica. 159. 237-244 (1997)
• [Publications] Makoto Naoi: "N-Methyl-(R)salsolinol as a dopaminergic neurotoxin ; from an animal model to an early marker of parkinson's disease." "Advances in Research on Neurodegeneration 5 "Journal of Neural Transmission, Supplement. 50. 89-105 (1997)
• [Publications] Makoto Naoi: "Oxidation of N-methyl(R)salsolinol : Involvement to neurotoxicity and neuroprotection by endogenous catechol isoquinolines." Journal of Neural Transmission. Supplement. 52. 131-144 (1997)
• [Publications] 直井信: "パーキンソン病発症候補物質" 脳と神経. 49. 793-803 (1997)
• [Publications] 直井信: "パーキンソン病の成因-最近の進歩-" 薬局. 48. 6-19 (1997)
• [Publications] 直井信: "酸化的ストレス Key Word 1998-'99" 神経変性疾患. 32-33 (1997)
• [Publications] Makoto Naoi: "Animal model of parkinson's disease induced by naturally-occurring 1(R ), 2(N), dimethyl-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline" Biogenic Amines. 12. 135-147 (1996)
• [Publications] Hideki Miura: "Effects of moclobemide on forced-swimming stress and brain monoamine levels in mice" Pharmacology Biochemistry and Behavior. 53. 469-475 (1996)
• [Publications] Makoto Naoi: "Dopamine-derived endogenous 1(R), 2(N)-dimethyl-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline, N-methyl-(R)-salsolinol, induced parkinsonism in rat : biochemical, pathological and behavioral studies" Brain Research. 709. 285-295 (1996)
• [Publications] Wakako Maruyama: "Identification of 3-nigro-L-tyrosine, a product of nitric oxide and superoxide, as an indicative stress in the human brain" Journal of Chromatography B. 676. 153-158 (1996)
• [Publications] Wakako Maruyama: "A dopaminergic neurotoxin, (R)-N-methylsalsolinol, increases in parkinsonian cerebrospinal fluid" Analus of Neurology. 40. 199-122 (1996)
• [Publications] Yulin Deng: "Mechanism of enantioseparation of salsolinols, endogenous neurotoxins in human brain, with ion-pair chromatography using γ-cyclodextrin as a mobile phase additive" Analytical Chemistry. 68. 2826-2831 (1996)
• [Publications] Wakako Maruyama: "The metabolism of L-DOPA and L-threo-3, 4-dihydroxyphenylserine and their effects on monoamines in the human brain:analysis of the intraventricular fluid from parkinsonian patients" Journal of the Neurological Sciences. 139. 141-148 (1996)
• [Publications] Makoto Naoi: "A novel enzyme enantio-selectively synthesizes (R)salsolinol, a precursor of a dopaminergic neurotoxin, N-methyl(R)salsolinol" Neurosciences Letters. 212. 183-186 (1996)
• [Publications] Kazuo Matubara: "Modulation of tryptophan hydroxylase activity in vitro by ethanol depends on biopterin cofactor concentration" Alcohol. 13. 455-459 (1996)
• [Publications] Wakako Maruyama: "Stereospecific occurrence of a parkinsonism-inducing catechol isoquinoline, N-methyl(R)salsolinol, in the human intraventricular fluid" Journal of Neural Transmission. 103. 1069-1076 (1996)
• [Publications] Philippe Dostert: "Effects of dopamine-derived isoquinolines on hydroxyl radical formation by dopamine autoxidation." Biogenic Amine. 12. 149-160 (1996)
• [Publications] 丸山和佳子: "Parkinson病" 最新医学. 51. 68-77 (1996)
• [Publications] 直井 信: "パーキンソン病と内因性・外因性神経毒(パーキンソン病-病態と治療)" 日医雑誌. 91. 996 (1996)
• [Publications] 直井 信: "1.神経毒(パーキンソン病の今日の課題)" Progress in Medicine. 16. 8-13 (1996)
• [Publications] Wakako Maruyama: "Dopamine-derived 1-methyl-6,7-dihydroxyisoquinolines as hydroxyl radical promoters and scavengers in the rat brain : in vivo and in vitro studies." Journal of Neurochemistry. 64. 2635-2643 (1995)
• [Publications] Wakako Maruyama: "Non-enzymatic oxidation of 1, 2(N)-dimethyl-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinolines into 1, 2(N)-dimethyl-6, 7-dihydroxyisoquinolinium ion, produces hydroxyl radicals." Free Radical Biology and Medicine. 19. 67-75 (1995)
• [Publications] Yulin Deng: "Determination of (R)-and (S)-enantiomers of salsolinol and N-methylsalsolinol by use of a chiral HPLC column" Journal of Chromatography B. 670. 47-54 (1995)
• [Publications] Makoto Naoi: "Enzymatic oxidation of dopaminergic neurotoxin, l(R), 2(N)-dimethyl-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline into 1, 2(N)-dimethyl-6, 7-dihydroxyisoquinolinium ion." Life Sciences. 57. 1061-1066 (1995)
• [Publications] Kazuo Matsubra: "N-Methyl-4-phenylpyridinium and an endogenously formed analog, N-methylated β-carbolinium, inhibit striatal tyrosine hydroxylation in freely moving rats." Neuroscience Letters. 199. 199-202 (1995)
• [Publications] Makoto Naoi: "Molecular Toxiology" VSP. Utrecht-Tokyo (in press), (1998)
• [Publications] Wakako Maruyama: "Molecular Toxiology" VSP. Utrecht-Tokyo (in press), (1998)
• [Publications] Makoto Naoi: "Progress in Alzheimer's and Parkinson's disease" Plenum Publisher (in press), (1998)
• [Publications] Wakako Maruyama: "Progress in Alzheimer's and Parkinson's disease" Plenum Publisher (in press), (1998)
• [Publications] Makoto Naoi: "Progress in HPLC-HPCE, Volume 7 "Neurochemical Markers of Degenerative Nervous Diseases and Drug Addiction"" VSP. Utrecht-Tokyo (in press), (1998)
• [Publications] Wakako Maruyama: "Progress in HPLC-HPCE, Volume 7 "Neurochemical Markers of Degenerative Nervous Diseases and Drug Addiction"" VSP. Utrecht-Tokyo (in press), (1998)
• [Publications] Makoto Naoi: "Advences in Neurolgy. "Parkinson's disease"" Rappincott-Raven, Philadelphia (in press), (1998)
• [Publications] Makoto Naoi: "Progress in HPLC "Multielectrode EC Array Applications to Trace Analysis of Neurotransmitters, Pharmaceuticals and Environmental Markers"" VSP. Utrecht-Tokyo, 20 (1997)
• [Publications] Wakako Maruyama: " Progress in HPLC "Multielectrode EC Array Applications to Trace Analysis of Neurotransmitters, Pharmaceuticals and Environmental Markers"" VSP. Utrecht-Tokyo, 13 (1997)
• [Publications] Yulin Deng: "Progress in HPLC "Multielectrode EC Array Applications to Trace Analysis of Neurotransmitters,Pharmaceuticals and Environmental Markers"" VSP. Utrecht-Tokyo, 38 (1997)
• [Publications] Wakako Maruyama: "Progress in HPLC "Multielectrode EC Array Applications to Trace Analysis of Neurotransmitters, Pharmaceuticals and Environmental Markers"" VSP. Utrecht-Tokyo, 12 (1997)
• [Publications] Makoto Naoi: "Handbook of Endogenous Neurotoxins" Birkhaeser, Boston, 20 (1997)