Neurotoxicity Research

, Volume 35, Issue 2, pp 432–440 | Cite as

Novel Alpha-Synuclein Oligomers Formed with the Aminochrome-Glutathione Conjugate Are Not Neurotoxic

  • Sandro Huenchuguala
  • Birgitta Sjödin
  • Bengt Mannervik
  • Juan Segura-AguilarEmail author


Aminochrome induces neurotoxic alpha-synuclein oligomer formation relevant to the etiology of Parkinson’s disease. Oxidative stress produces aminochrome from dopamine, but conjugation with glutathione catalyzed by glutathione transferase M2-2 significantly decreases aminochrome-induced toxicity and alpha-synuclein oligomer formation. Notably, in the presence of the aminochrome-glutathione conjugate, previously unknown species of alpha-synuclein oligomers are formed. These aminochrome-glutathione oligomers of alpha-synuclein differ from formerly characterized oligomers and (i) have high molecular weight, and are stable and SDS-resistant, as determined by the Western blot method, (ii) show positive NBT-quinone-protein staining, which indicates the formation of alpha-synuclein adducts containing aminochrome. Furthermore, aminochrome-glutathione alpha-synuclein oligomers (iii) have distinctive shape and size, as determined by transmission electron microscopy, and (iv) are not toxic in U373MG cells. In conclusion, glutathione conjugated with aminochrome induces a new type of alpha-synuclein oligomers of a different size and shape, which have no demonstrable toxicity.


Alpha-synuclein Parkinson’s disease Glutathione Glutathione transferase Dopamine Aminochrome Oligomers 


Funding information

This study was supported through funding from FONDECYT (1100165, 1061083, 1120337, 7040028, 1170033), and a CONICYT doctoral scholarship and a doctoral thesis support scholarship no. 24121454, as well as by the Swedish Research Council.


  1. Aguirre P, Urrutia P, Tapia V, Villa M, Paris I, Segura-Aguilar J, Nuñez MT (2012) The dopamine metabolite aminochrome inhibits mitochondrial complex I and modifies the expression of iron transporters DMT1 and FPN1. Biometals 25:795–803. CrossRefPubMedGoogle Scholar
  2. Arriagada C, Paris I, Sanchez de las Matas MJ, Martinez-Alvarado P, Cárdenas S, Castañeda P, Graumann R, Sanchez de las Matas MJ, Martinez-Alvarado P, Cardenas S, Castañeda P, Graumann R, Perez-Pastene C, Olea-Azar C, Couve E, Herrero MT, Caviedes P, Segura-Aguilar J (2004) On the neurotoxicity mechanism of leukoaminochrome o-semiquinone radical derived from dopamine oxidation: mitochondria damage, necrosis, and hydroxyl radical formation. Neurobiol Dis 16:468–477. CrossRefPubMedGoogle Scholar
  3. Bisaglia M, Tosatto L, Munari F, Tessari I, de Laureto PP, Mammi S, Bubacco L (2010) Dopamine quinone interact with alpha-synuclein to form unstructured adducts. Biochem Biophys Res Commun 394:424–428. CrossRefPubMedGoogle Scholar
  4. Breydo L, Wu JW, Uversky VN (2012) α-Synuclein misfolding and Parkinson’s disease. Biochim Biophys Acta 1822:261–285. CrossRefPubMedGoogle Scholar
  5. Briceño A, Muñoz P, Brito P, Huenchuguala S, Segura-Aguilar J, Paris IB (2016) Aminochrome toxicity is mediated by inhibition of microtubules polymerization through the formation of adducts with tubulin. Neurotox Res 29:381–393. CrossRefPubMedGoogle Scholar
  6. Cappai R, Leck SL, Yew DJ, Williamson NA, Smith DP, Galatis D, Sharples RA, Curtain CC, Ali FE, Cherny RA, Culvenor JG, Bottomley SP, Masters CL, Barnham KJ, Hill AF (2005) Dopamine promotes alpha-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway. FASEB J 19:1377–13779. CrossRefPubMedGoogle Scholar
  7. Conway KA, Rochet JC, Bieganski RM, Lansbury PT Jr (2001) Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct. Science 294:1346–1349. CrossRefPubMedGoogle Scholar
  8. Cuevas C, Huenchuguala S, Muñoz P, Villa M, Paris I, Mannervik B, Segura-Aguilar J (2015) Glutathione transferase-M2-2 secreted from glioblastoma cell protects SH-SY5Y cells from aminochrome neurotoxicity. Neurotox Res 27:217–228. CrossRefPubMedGoogle Scholar
  9. Dagnino-Subiabre A, Cassels BK, Baez S, Johansson A-S, Mannervik B, Segura-Aguilar J (2000) Glutathione transferase M2-2 catalyzes conjugation of dopamine and Dopa o-quinones. Biochem Biophys Res Commun 274:32–36. CrossRefPubMedGoogle Scholar
  10. Danzer KM, Haasen D, Karow AR, Moussaud S, Habeck M, Giese A, Kretzschmar H, Hengerer B, Kostka M (2007) Different species of α-synuclein oligomers induce calcium influx and seeding. J Neurosci 27:9220–9232. CrossRefPubMedGoogle Scholar
  11. de Araújo FM, Ferreira RS, Souza CS, Dos Santos CC, Rodrigues TLRS, E Silva JHC, Gasparotto J, Gelain DP, El-Bachá RS, D Costa MF, Fonseca JCM, Segura-Aguilar J, Costa SL, Silva VDA (2018) Aminochrome decreases NGF, GDNF and induces neuroinflammation in organotypic midbrain slice cultures. Neurotoxicology 66:98–106. CrossRefPubMedGoogle Scholar
  12. Dimant H, Kalia SK, Kalia LV, Zhu LN, Kibuuka L, Ebrahimi-Fakhari D, McFarland NR, Fanz Z, Hyman BT, McLean PJ (2013) Direct detection of alpha-synuclein oligomers in vivo. Acta Neuropathol Commun 1:6CrossRefGoogle Scholar
  13. Herrera A, Muñoz P, Steinbusch HWM, Segura-Aguilar J (2017) Are dopamine oxidation metabolites involved in the loss of dopaminergic neurons in the nigrostriatal system in Parkinson’s disease? ACS Chem Neurosci 8:702–711. CrossRefPubMedGoogle Scholar
  14. Huenchuguala S, Muñoz P, Zavala P, Villa M, Cuevas C, Ahumada U, Graumann R, Nore BF, Couve E, Mannervik B, Paris I, Segura-Aguilar J (2014) Gluthathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction. Autophagy 10:618–630. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Huenchuguala S, Muñoz P, Segura-Aguilar J (2017) The importance of mitophagy in maintaining mitochondrial function in U373MG cells. bafilomycin A1 restores aminochrome-induced mitochondrial damage. ACS Chem Neurosci 8(10):2247–2253. CrossRefPubMedGoogle Scholar
  16. Khurana R, Coleman C, Ionescu-Zanetti C, Carter SA, Krishna V, Grover RK, Roy R, Singh S (2005) Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol 151:229–238. CrossRefPubMedGoogle Scholar
  17. Lashuel HA, Overk CR, Oueslati A, Masliah E (2013) The many faces of α-synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci 14:38–48. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Meléndez C, Muñoz P, Segura-Aguilar J (2018, 2018) DT-diaphorase prevents aminochrome-induced lysosome dysfunction in SH-SY5Y cells. Neurotox Res.
  19. Muñoz PS, Segura-Aguilar J (2017) DT-diaphorase protects against autophagy induced by aminochrome-dependent alpha-synuclein oligomers. Neurotox Res 32:362–367. CrossRefPubMedGoogle Scholar
  20. Muñoz P, Paris I, Sanders LH, Greenamyre JT, Segura-Aguilar J. (2012) Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity. Biochim Biophys Acta 1822:1125–1136.
  21. Muñoz P, Cardenas S, Huenchuguala S, Briceño A, Couve E, Paris I, Segura-Aguilar J (2015) DT-Diaphorase prevents aminochrome-induced alpha-synuclein oligomer formation and neurotoxicity. Toxicol Sci 145:37–47. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Norris EH, Giasson BI, Hodara R, Xu S, Trojanowski JQ, Ischiropoulos H, Lee VMY (2005) Reversible inhibition of alpha-synuclein fibrillization by dopaminochrome-mediated conformational alterations. J Biol Chem 280:21212–21219. CrossRefPubMedGoogle Scholar
  23. Ochs SD, Westfall TC, Macarthur H (2005) The separation and quantification of aminochromes using high-pressure liquid chromatography with electrochemical detection. J Neurosci Methods 142:201–208. CrossRefPubMedGoogle Scholar
  24. Paris I, Perez-Pastene C, Cárdenas S, Iturra P, Muñoz P, Couve E, Caviedes P, Segura-Aguilar J (2010) Aminochrome induces disruption of actin, alpha, and beta-tubulin cytoskeleton networks in substantia-nigra-derived cell line. Neurotox Res 18:82–92. CrossRefPubMedGoogle Scholar
  25. Polymeropoulos MH, Laveda C, Leroy E, Ide SE, Dejehia A, Dutra A et al (1997) Mutation of alpha-synuclein gene identified in families with Parkinson’s disease. Science 276:2045–2047. CrossRefPubMedGoogle Scholar
  26. Rockenstein E, Nuber S, Overk CR, Ubhi K, Mante M, Patrick C et al (2014) Accumulation of oligomers-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo. Brain 137:1496–1513. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Santos CC, Araújo FM, Ferreira RS, Silva VB, Silva JHC, Grangeiro MS, Soares ÉN, Pereira ÉPL, Souza CS, Costa SL, Segura-Aguilar J, Silva VDA (2017) Aminochrome induces microglia and astrocyte activation. Toxicol in Vitro 42:54–60. CrossRefPubMedGoogle Scholar
  28. Segura-Aguilar J (2015) A new mechanism for protection of dopaminergic neurons mediated by astrocytes. Neural Regen Res 10:1225–1227. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Segura-Aguilar J (2017) On the role of endogenous neurotoxins and neuroprotection in Parkinson's disease. Neural Regen Res 12:897–901. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Segura-Aguilar J, Baez S, Widersten M, Welch CJ, Mannervik B (1997) Human class Mu glutathione transferases, in particular isoenzyme M2-2, catalyze detoxication of the dopamine metabolite aminochrome. J Biol Chem 272:5727–5731. CrossRefPubMedGoogle Scholar
  31. Segura-Aguilar J, Muñoz P, Paris I (2016) Aminochrome as new preclinical model to find new pharmacological treatment that stop the development of Parkinson’s disease. Curr Med Chem 23:346–359. CrossRefPubMedGoogle Scholar
  32. Shimotakahara S, Matsui M, Sakuma C, Takahashi T, Fujimoto T, Furihata K, Kojima M, Seino S, Machinami T, Shibusawa Y, Uéda K, Tashiro M (2013) Dopamine cannot promote oligomerization of unoxidized α-synuclein. J Biophys Chem 4:110–114. CrossRefGoogle Scholar
  33. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J, Gwinn-Hardy K (2003) α-synuclein locus triplication causes Parkinson’s disease. Science 302:841. CrossRefPubMedGoogle Scholar
  34. Tokuda T, Qureshi MM, Ardah MT, Varghese S, Shehab SA, Kasai T, Ishigami N, Tamaoka A, Nakagawa M, El-Agnaf OM (2010) Detection of elevated levels of α-synuclein oligomers in CSF from patients with Parkinson disease. Neurology 75:1766–1772. CrossRefGoogle Scholar
  35. Van Rooijen BD, Claessens MM, Subramaniam V (2010) Membrane interactions of oligomeric alpha-synuclein: potential role in Parkinson’s disease. Curr Protein Pept Sci 11:334–342. CrossRefPubMedGoogle Scholar
  36. Xiong R, Siegel D, Ross D (2014) Quinone-induced protein handling changes: implications for major protein handling systems in quinone-mediated toxicity. Toxicol Appl Pharmacol 28:285–295. CrossRefGoogle Scholar
  37. Zafar KS, Siegel D, Ross D (2006) A potencial role for cyclized quinones derived from dopamine, DOPA, and 3,4-dihydroxyphenylacetic acid in proteasomal inhibition. Mol Pharmacol 70:1079–1086. CrossRefPubMedGoogle Scholar
  38. Zhou ZD, Lim TM (2009) Dopamine (DA) induced irreversible proteasome inhibition via DA derived quinones. Free Radic Res 43:417–430. CrossRefPubMedGoogle Scholar
  39. Zhou ZD, Lim TM (2010) Glutathione conjugates with dopamine-derived quinones to form reactive or non-reactive glutathione-conjugates. Neurochem Res 35:1805–1818. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sandro Huenchuguala
    • 1
    • 2
  • Birgitta Sjödin
    • 3
  • Bengt Mannervik
    • 3
  • Juan Segura-Aguilar
    • 1
    Email author
  1. 1.Molecular and Clinical Pharmacology, ICBM, Faculty of MedicineUniversity of ChileSantiago-7Chile
  2. 2.Escuela de Tecnología Médica, Facultad de SaludUniversidad Santo TomásOsornoChile
  3. 3.Department of Biochemistry and BiophysicsStockholm UniversityStockholmSweden

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