Journal of Neural Transmission

, Volume 115, Issue 6, pp 831–842 | Cite as

Role of reactive nitrogen and reactive oxygen species against MPTP neurotoxicity in mice

  • Hironori Yokoyama
  • Sho Takagi
  • Yu Watanabe
  • Hiroyuki Kato
  • Tsutomu ArakiEmail author
Parkinson's Disease and Allied Conditions - Original Article


There is growing evidence indicating that reactive nitrogen species (RNS) and reactive oxygen species (ROS) are a major contributor to the pathogenesis and progression of Parkinson’s disease. Here we investigated whether edaravone (free radical scavenger), minocycline (inducible nitric oxide synthase, iNOS inhibitor), 7-nitroindazole (neuronal NOS, nNOS inhibitor), fluvastatin (endothelial NOS, eNOS activator) and pitavastatin (eNOS activator) can protect against MPTP neurotoxicity in mice under the same condition. The present study showed that 7-nitroindazole could protect dose-dependently against the striatal dopamine depletions in mice 5 days after MPTP treatment. In contrast, edaravone, minocycline, fluvastatin and pitavastatin did not show the neuroprotective effect on MPTP-induced striatal dopamine depletion. Our immunohistochemical study showed that TH (tyrosine hydroxylase) and DAT (dopamine transporter) immunoreactivity was decreased significantly in the striatum and substantia nigra 5 days after MPTP treatment. The administration of 7-nitroindazole showed a protective effect against the severe reductions in levels of TH and DAT immunoreactivity in the striatum and substantia nigra 5 days after MPTP treatment. Furthermore, our Western blot analyses study showed the remarkable loss of TH protein levels in the striatum 5 days after MPTP treatment. In contrast, 7-nitroindazole prevented a significant loss in TH protein levels in the striatum 5 days after MPTP treatment. On the other hand, GFAP (glial fibrillary acidic protein) immunoreactivity increased significantly in the striatum and substantia nigra, 5 days after MPTP treatment. 7-Nitroindazole ameliorated severe increases in number of GFAP immunoreactive astrocytes in the striatum and substantia nigra 5 days after MPTP treatment. Furthermore, our Western blot analyses study showed the increase of GFAP protein levels in the striatum 5 days after MPTP treatment. 7-Nitroindazole prevented a significant increase in the GFAP protein levels in the striatum 5 days after MPTP treatment. The present results indicate that 7-nitroindazole can protect dose-dependently against the striatal dopamine depletions in mice 5 days after MPTP treatment. In contrast, edaravone, minocycline, fluvastatin and pitavastatin did not show the neuroprotective effect on MPTP-induced striatal dopamine depletions. These findings demonstrate that the overexpression of nNOS may play a major role in the neurotoxic processes of MPTP, as compared to the production of ROS, the overexpression of iNOS and the modulation of eNOS. Thus, our findings provide strong evidence for neuroprotective properties of nNOS inhibitor in this animal model of Parkinson’s disease.


Parkinson’s disease Oxidative stress Immunohistochemistry Western blot analysis Dopaminergic system Mice 



This study was supported in part by the Grant-in-Aid for Scientific Research (12877163, 13671095 and 13670627) from the Ministry of Science and Education in Japan. We thank Yuko Kamiyama for technical assistance with HPLC.


  1. Agid Y (1991) Parkinson’s disease: pathophysiology. Lancet 337:1321–1324PubMedCrossRefGoogle Scholar
  2. Alam ZI, Jenner A, Daniel SE, Lees AJ, Gairns N, Marsden CD, Jenner P, Halliwell B (1997) Oxidative DNA damage in the parkisonian brain: an apparent selective increase 8-hydroxyguanine levels in substantia nigra. J Neurochem 69:1196–1203PubMedCrossRefGoogle Scholar
  3. Amin AR, Thakker GD, Patel PD, Vyas PR, Patel RN, Patel IR, Abramson SB (1996) A novel mechanism of action of tetracyclines: effects on nitric oxide synthase. Proc Natl Acad Sci USA 93:14014–14019PubMedCrossRefGoogle Scholar
  4. Araki T, Tanji H, Fujihara K, Kato H, Itoyama Y (1999) Increases in [3H]FK-506 and [3H]L-NG-nitro-arginine binding in the rat brain after nigrostriatal dopaminergic denervation. Metab Brain Dis 14:21–31PubMedCrossRefGoogle Scholar
  5. Araki T, Mikami T, Tanji H, Matsubara M, Imai Y, Mizugaki M, Itoyama Y (2001) Biochemical and immunohistological changes in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse. Eur J Pharm Sci 12:231–238PubMedCrossRefGoogle Scholar
  6. Beal MF (2003) Mitochondria, oxidative damage, and inflammation in Parkinson’s disease. Ann NY Acad Sci 991:120–131PubMedCrossRefGoogle Scholar
  7. Beal MF, Hymn BT, Koroshetz W (1993) Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative disease? Trends Neurosci 16:125–131PubMedCrossRefGoogle Scholar
  8. Beckman GC, Koppenol WH (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271:C1424–C1437PubMedGoogle Scholar
  9. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87:1620–1624PubMedCrossRefGoogle Scholar
  10. Beckman JS, Ischiropoulos H, Zhu L, van der Woerd M, Smith C, Chen J, Harrison J, Martin JC, Tsai M (1992) Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite. Arch Biochem Biophys 298:438–445PubMedCrossRefGoogle Scholar
  11. Bernheimer H, Birkmayer W, Hornykeiwicz O, Jellinger K, Seitelberger F (1973) Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci 20:415–455PubMedCrossRefGoogle Scholar
  12. Betarbet R, Scerer TB, Greenamyre JT (2002) Animal models of Parkinson’s disease. Bioessays 24:308–318PubMedCrossRefGoogle Scholar
  13. Bredt DS, Snyder SH (1990) Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87:682–685PubMedCrossRefGoogle Scholar
  14. Bredt DS, Snyder SH (1994) Nitric oxide: a physiologic messenger molecule. Ann Rev Biochem 63:175–195PubMedCrossRefGoogle Scholar
  15. Chen LW, Zhang JP, Shum DKY, Chan YS (2006) Localization of nerve growth factor, neurotropin-3, and glial cell line-derived neurotrophic factor in nestin-expressing reactive astrocytes in the caudate-putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/Bl mice. J Comp Neurol 497:898–909PubMedCrossRefGoogle Scholar
  16. Choi J, Levery AI, Weintraub ST, Rees HD, Gearing M, Chin LS, Li L (2004) Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson’s and Alzheimer’s diseases. J Biol Chem 279:13256–13264PubMedCrossRefGoogle Scholar
  17. D’Amato RJ, Lipman ZP, Snyder H (1986) Selectivity of the parkinsonian neurotoxin MPTP: toxic metabolite MPP+ binds to neuromelanin. Science 231:987–989PubMedCrossRefGoogle Scholar
  18. Dehmer T, Lindenau J, Haid S, Dichgans J, Schultz JB (2000) Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo. J Neurochem 74:2213–2216PubMedCrossRefGoogle Scholar
  19. Dauer W, Przedborski S (2003) Parkinson7 disease: mechanisms and models. Neuron 39:889–909PubMedCrossRefGoogle Scholar
  20. Dawson TM, Snyder SH (1994) Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 14:5147–5159PubMedGoogle Scholar
  21. Dawson VL, Dawson TM, London ED, Bredit DS, Snyder SH (1991) Nitric oxide mediates glutamate neurotoxicity in primary cultures. Proc Natl Acad Sci USA 88:6268–6371Google Scholar
  22. Di Monte DA, Royland JE, Anderson A, Castagnoli K, Castagoli Jr N, Langston JW (1997) Inhibition of monoamine oxidase contributes to the protective effect of 7-nitroindazole against MPTP neurotoxicity. J Neurochem 69:1771–1773PubMedCrossRefGoogle Scholar
  23. Du Y, Ma Z, Lin S, Dodel RC, Gao F, Bales KR, Thiarhou LC, Chernet E, Perry KW, Nelson DL, Luecke S, Phebus LA, Bymaster FP, Paul SM (2001) Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson’s disease. Proc Natl Acad Sci USA 98:14669–14674PubMedCrossRefGoogle Scholar
  24. Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, Liao JK (1998) Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA 95:8880–8885PubMedCrossRefGoogle Scholar
  25. Gerlach M, Ben-Schachar D, Riederer P, Youdim MB (1994) Altered brain metabolism of iron as a cause of neurodegenerative diseases? J Neurochem 63:793–807PubMedCrossRefGoogle Scholar
  26. Hasegawa E, Takeshige K, Oishi T, Murai Y, Minakami S (1990) 1-Methylphenylpyridinium (MPP+) induces NADH-dependent superoxide formation and enhances NADH-dependent lipid peroxidation in bovine heart submitochondrial particles. Biochem Biophys Res Commun 170:1049–1055PubMedCrossRefGoogle Scholar
  27. Hantraye P, Brouillet E, Ferrante R, Pafi S, Dolan R, Matthews RT, Beal MF (1996) Inhibition of neuronal nitric oxide synthase prevents MPTP-induced Parkinsonism in baboons. Nat Med 2:1017–1021PubMedCrossRefGoogle Scholar
  28. Hastings TG (1995) Enzymatic oxidation of dopamine: the role of prostaglandin H synthase. J Neurochem 64:919–924PubMedCrossRefGoogle Scholar
  29. Himeda T, Mizuno K, Kato H, Araki T (2005) Effects of age on immunohistochemical changes in the mouse hippocampus. Mech Ageing Dev 126:673–677PubMedCrossRefGoogle Scholar
  30. Hirsh E, Graybiel AM, Agid YA (1988) Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease. Nature 334:345–348CrossRefGoogle Scholar
  31. Ignarro LJ (1990) Biosynthesis and metabolism of endothelium-derived nitric oxide. Ann Rev Pharmacol Toxicol 30:535–560CrossRefGoogle Scholar
  32. Ischiropoulos H, Zhu L, Chen J, Tsai M, Martin JC, Smith CD, Beckman JS (1992) Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys 298:431–437PubMedCrossRefGoogle Scholar
  33. Jellinger KA (1999) The role of iron in neurodegeneration: prospects for pharmacotherapy of Parkinson’s disease. Drugs Aging 14:115–140PubMedCrossRefGoogle Scholar
  34. Jenner P, Olanow CW (1996) Oxidative stress and the pathogenesis of Parkinson’s disease. Neurology 47:S161–S170PubMedGoogle Scholar
  35. Khan FH, Sen T, Chakrabarti S (2003) Dopamine oxidation products inhibit Na+, K+-ATPase activity in crude synaptosomal-mitochondrial fraction from rat brain. Free Radic Res 37:597–601PubMedCrossRefGoogle Scholar
  36. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392:605–608PubMedCrossRefGoogle Scholar
  37. Kurosaki R, Akasak M, Michimata M, Matsubara M, Ima Y, Araki T (2003) Effects of Ca2+ antagonists on motor activity and the dopaminergic system in aged mice. Neurobiol Aging 24:315–319PubMedCrossRefGoogle Scholar
  38. LaVoice MJ, Hastings TG (1999) Peroxynitrite- and nitrite-induced oxidation of dopamine: implications for nitric oxide in dopaminergic cell loss. J Neurochem 73:2546–2554CrossRefGoogle Scholar
  39. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ, Jonnalagada S, Chernova T, Dehejia A, Lavedan C, Gasser T, Steinbach PJ, Wilkinson KD, Polymeropoulos MH (1998) The ubiquitin pathway in Parkinson’s disease. Nature 395:451–452PubMedCrossRefGoogle Scholar
  40. Liberatore GT, Jackson-Lewis V, Vukosavic S, Mandir AS, vila M, McAuliffe WG, Dawson VL, Dawson TM, Przedborski S (1999) Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of PD. Nat Med 5:1403–1409PubMedCrossRefGoogle Scholar
  41. Lonart G, Johanson KM (1992) Inhibitory effects of nitric oxide on the uptake of [3H]dopamine and [3H]glutamate by striatal synaptosomes. J Neurochem 63:2108–2117CrossRefGoogle Scholar
  42. Marletta MA (1994) Nitric oxide synthase: aspects concerning structure and catalysis. Cell 78:927–930PubMedCrossRefGoogle Scholar
  43. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109–142PubMedGoogle Scholar
  44. Muramatsu Y, Kurosaki R, Watanabe H, Michimata M, Matsubara M, Imai Y, Araki T (2003) Cerebral alterations in a MPTP-mouse model of Parkinson’s disease-an immunocytochemical study. J Neural Transm 110:1129–1144PubMedCrossRefGoogle Scholar
  45. Murphy S, Simmons ML, Agullo L, Garcia A, Feinstein DL, Galea E, Reis DJ, Minc-Colomb D, Schwartz JP (1993) Synthesis of nitric oxide in CNS glial cells. Trends Neurosci 16:323–328PubMedCrossRefGoogle Scholar
  46. Nathan C, Xie QW (1994) Nitric oxide synthases: roles, tolls, and controls. Cell 78:915–918PubMedCrossRefGoogle Scholar
  47. Numagami Y, Zubrow AB, Mishra OP, Delivoria-Papadopoulos M (1997) Lipid free radical generation and brain cell membrane alteration following nitric oxide synthase inhibition during cerebral hypoxia in the newborn piglet. J Neurochem 69:1542–1547PubMedCrossRefGoogle Scholar
  48. Olanow CW, Tatton WG (1999) Etiology and pathogenesis of Parkinson’s disease. Annu Rev Nurosci 22:123–144CrossRefGoogle Scholar
  49. Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, van der Brug M, de Munain AL, Aparico S, Gil AM, Khan N, Johnson J, Martinez JR, Nicholl D, Carrera IM, Pena AS, de Silva R, lees A, Marti-Masso JF, Perez-Tur J, Wood NW, Singleton AB (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 44:595–600PubMedCrossRefGoogle Scholar
  50. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropous T, Johnson WG, Lazzarini AM, Duvoison RC, Di Iorio G, Golbe LI, Nussbaum RL (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276:2045–2047PubMedCrossRefGoogle Scholar
  51. Radi R (2004) Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci USA 101:4003–4008PubMedCrossRefGoogle Scholar
  52. Radi R, Cassina A, Hodara R, Quijano C, Castro L (2002) Peroxynitrite reactions and formation in mitochondria. Free Radic Biol Med 33:1451–1464PubMedCrossRefGoogle Scholar
  53. Reilly DK, Hershey L, Rivera-Calimlim L, Shoulson I (1983) On-off effects in Parkinson’s disease: a controlled investigation of ascorbic acid therapy. Adv Neurol 37:51–60PubMedGoogle Scholar
  54. Rice-Evans CA (1994) Formation of free radicals and mechanisms of action in normal biochemical processes and pathological states. In: Rice-Evans CA, Burdon RH (eds) Free radical damage and its control. Elsevier, Amsterdam, pp 131–153CrossRefGoogle Scholar
  55. Schulz JB, Matthews RT, Muqit MMK, Browne SE, Beal MF (1995) Inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in mice. J Neurochem 64:936–939PubMedCrossRefGoogle Scholar
  56. Schulz JB, Matthews RT, Klockgether T, Dichgans J, Beal MF (1997) The role of mitochondrial dysfunction and neuronal nitric oxide in animal models of neurodegenerative diseases. Mol Cell Biochem 174:193–197PubMedCrossRefGoogle Scholar
  57. Selley ML (2005) Simvastatin prevents 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine- induced striatal dopamine depletion and protein tyrosine nitration in mice. Brain Res 1037:1–6PubMedCrossRefGoogle Scholar
  58. Slivka A, Cohen G (1985) Hydroxyl radical attack on dopamine. J Biol Chem 260:15466–15472PubMedGoogle Scholar
  59. Sriram K, Pai KS, Boyd MR, Ravindranath V (1997) Evidence for generation of oxidative stress in brain by MPTP: in vitro and in vivo studies in mice. Brain Res 749:44–52PubMedCrossRefGoogle Scholar
  60. Sriram K, Miller DB, O’Callaghan P (2006) Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-α. J Neurochem 96:706–718PubMedCrossRefGoogle Scholar
  61. Taira T, Saito Y, Niki T, Iguchi-Ariga SM, Takahashi K, Ariga H (2004) DJ-1 has a role in antioxidative stress to prevent cell death. EMBO Rep 5:213–218PubMedCrossRefGoogle Scholar
  62. The Parkinson Study group (1993) Effects of tocopherol and deprenyl on the progression of disability in early Parkinson’s disease. N Engl J Med 328:176–183CrossRefGoogle Scholar
  63. Tikka T, Fiebich BL, Goldsteins G, keinanen R, Koistinaho J (2001) Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21:2580–2588PubMedGoogle Scholar
  64. Tipton KF, Singer TP (1993) Advances in our understanding of the mechanisms of the neurotoxicity of MPTP and related compounds. J Neurochem 61:1191–1206PubMedCrossRefGoogle Scholar
  65. Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, Ali Z, Del Turco D, Bentivoglio AR, Healy DG, Albanese A, Nussbaum R, González-Maldonado R, Deller T, Salvi S, Cortelli P, Gilks WP, Latchman DS, Harvey RJ, Dallapiccola B, Auburger G, Wood NW (2004) Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304:1158–1160PubMedCrossRefGoogle Scholar
  66. Watanabe T, Yuki S, Egawa M, Nishi H (1994) Protective effects of MCI-186 on cerebral ischemia: possible involvement of free radical scavenging and antioxidant actions. J Pharmacol Exp Ther 268:1597–1604PubMedGoogle Scholar
  67. Wu DC, Jakson-Lewis M, Vila M, Tieu K, Teismann C, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S (2002) Blocakade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 22:1763–1771PubMedGoogle Scholar
  68. Yang L, Sugama S, Chirichigno JW, Gregorio J, Lorenzl S, Shin DH, Browne SE, Shimizu Y, Joh TH, Beal MF, Albers DS (2003) Minocycline enhances MPTP neurotoxicity to dopaminergic neurons. J Neurosci Res 74:278–285PubMedCrossRefGoogle Scholar
  69. Yrjanheikki J, Keinanen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci USA 96:13496–13500PubMedCrossRefGoogle Scholar
  70. Zhang J, Perry G, Smith MA, Robertson D, Olson SJ, Graham DG, Montine TJ (1999) Parkinson’s disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons. Am J Pathol 154:1423–1429PubMedCrossRefGoogle Scholar
  71. Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu du C, Gullans S, Ferrante RJ, Przedborski S, Kristal BS, Friedlander RM (2002) Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 417:74–78PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Hironori Yokoyama
    • 1
  • Sho Takagi
    • 1
  • Yu Watanabe
    • 1
  • Hiroyuki Kato
    • 2
  • Tsutomu Araki
    • 1
    • 3
    Email author
  1. 1.Department of Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical SciencesThe University of TokushimaTokushimaJapan
  2. 2.Department of Neurology, Organized Center of Clinical MedicineInternational University of Health and WelfareTochigiJapan
  3. 3.Department of Drug Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical SciencesThe University of TokushimaTokushimaJapan

Personalised recommendations