Journal of Molecular Neuroscience

, Volume 18, Issue 3, pp 229–238 | Cite as

Mutant and wild-type α-synuclein interact with mitochondrial cytochrome C oxidase

  • Hanock Elkon
  • Jermy Don
  • Eldad Melamed
  • Ilan Ziv
  • Anat Shirvan
  • Daniel Offen


α-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson’s disease (PD). Furthermore, rare mutations of α-synuclein cause autosomal-dominant PD. However, it is unknown how α-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) α-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant α-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant α-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant α-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of α-synuclein, a major LB protein, to COX, a key enzyme of the mitochondrial respiratory system. This interaction suggests that α-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

Index Entries

α-synuclein coimmunoprecipitation cytochrome c oxidase in vitro translation Parkinson’s disease potassium cyanide two-hybrid system 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abeliovich A., Schmitz Y., Farinas I., Choi-Lundberg D., Ho W. H., Castillo P. E., et al. (2000) Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25, 239–252.PubMedCrossRefGoogle Scholar
  2. Alves-Rodrigues A., Gregori L., and Figueiredo-Pereira M. E. (1998) Ubiquitin, cellular inclusions and their role in neurodegeneration. Trends Neurosci. 21, 516–520.PubMedCrossRefGoogle Scholar
  3. Baba M., Nakajo S., Tu P. H., Tomita T., Nakaya K., Lee V. M., et al. (1998) Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am. J. Pathol. 152, 879–884.PubMedGoogle Scholar
  4. Barzilai A., Zilkha-Falb R., Daily D., Stern N., Offen D., Ziv I., et al. (2000) The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity. J. Neural Transm. Suppl. 60, 59–76.PubMedGoogle Scholar
  5. Bennet C. M., Bishop J. F., Leng Y., Chock B., Chase T. N., and Mouradian M. (1999) Degradation of α-synuclein by proteasome. J. Biol. Chem. 274, 33855–33858.CrossRefGoogle Scholar
  6. Burke P. A. and Poyton R. O. (1998) Structure/function of oxygen-regulated isoforms in cytochrome c oxidase. J. Exp. Biol. 201, 1163–1175.PubMedGoogle Scholar
  7. Casali C., Bonifati V., Santorelli F. M., Casari G., Fortini D., Patrignani A., et al. (2001) Mitochondrial myopathy, parkinsonism, and multiple mtDNA deletions in a Sephardic Jewish family. Neurology 56, 802–805.PubMedGoogle Scholar
  8. Cheng G., Cleary A. M., Ye Z. S., Hong D. I., Lederman S., and Baltimore D. (1995) Involvement of CRAF1, a relative of TRAF, in CD40 signaling. Science 267, 1494–1498.PubMedCrossRefGoogle Scholar
  9. Cohen G., Farooqui R., and Kesler N. (1997) Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow. Proc. Natl. Acad. Sci. USA 94, 4890–4894.PubMedCrossRefGoogle Scholar
  10. Conway K. A., Harper J. D., and Lansbury P. T. (1998) Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat. Med. 4, 1318–1320.PubMedCrossRefGoogle Scholar
  11. Davidson W. S., Jonas A., Clayton D. F., and George J. M. (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273, 9443–9449.PubMedCrossRefGoogle Scholar
  12. El-Agnaf O. M., Jakes R., Curran M. D., Middleton D., Ingenito R., Bianchi E., et al. (1998) Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments. FEBS Lett. 440, 71–75.PubMedCrossRefGoogle Scholar
  13. Engelender S., Kaminsky Z., Guo X., Sharp A. H., Amaravi R. K., Kleiderlein J. J., et al. (1999) Synphilin-1 associates with α-synuclein and promotes the formation of cytosolic inclusions. Nat. Genet. 22, 110–114.PubMedCrossRefGoogle Scholar
  14. Feldman J. M. and Feldman M. D. (1990) Sequelae of attempted suicide by cyanide ingestion: a case report. Int. J. Psychiatry Med. 20, 173–179.PubMedCrossRefGoogle Scholar
  15. Filloux F. and Townsend J. J. (1993) Pre- and postsynaptic neurotoxic effects of dopamine demonstrated by intrastriatal injection. Exp. Neurol. 119, 79–88.PubMedCrossRefGoogle Scholar
  16. Forno L. S. (1996) Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55, 259–272.PubMedCrossRefGoogle Scholar
  17. Ghee M., Fournier A., and Mallet J. (2000) Rat alpha-synuclein interact with tat binding protein 1, a component of the 26S proteasomal complex. J. Neurochem. 75, 2221–2224.PubMedCrossRefGoogle Scholar
  18. Grandas F., Artieda J., and Obeso J. A. (1989) Clinical and CT scan findings in a case of cyanide intoxication. Mov. Disord. 4, 188–193.PubMedCrossRefGoogle Scholar
  19. Gu M., Cooper J. M., Taanman J. W., and Schapira A. H. (1998) Mitochondrial DNA transmission of the mitochondrial defect in Parkinson’s disease. Ann. Neurol. 44, 177–186.PubMedCrossRefGoogle Scholar
  20. Hashimoto M., Takeda A., Hsu L. J., Takenouchi T., and Masliah E. (1999) Role of cytochrome c as a stimulator of α-synuclein aggregation in Lewy body disease. J. Biol. Chem. 274, 28849–28852.PubMedCrossRefGoogle Scholar
  21. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., and Pease L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.PubMedCrossRefGoogle Scholar
  22. Hsu L. J., Sagara Y., Arroyo A., Rockenstein E., Sisk A., Mallory M., et al. (2000) alpha-synuclein promote mitochondrial deficit and oxidative stress. Am. J. Pathol. 157, 401–410.PubMedGoogle Scholar
  23. Iwai A., Masliah E., Yoshimoto M., Ge N., Flanagan L., de Silva H. A., et al. (1995) The precursor protein of non-A beta component of Alzheimer’s disease amyloid is a presynaptic protein of the central nervous system. Neuron. 14, 467–475.PubMedCrossRefGoogle Scholar
  24. Jenco J. M., Rawlingson A., Daniels B., and Morris A. J. (1998) Regulation of phospholipase D2: selective inhibition of mammalian phospholipase Disoenzymes by alpha- and beta-synuclein. Biochemistry 37, 4901–4909.PubMedCrossRefGoogle Scholar
  25. Kahle P. J., Neumann M., Ozmen L., Muller V., Jacobsen H., Schindzielorz A., et al. (2000) Subcellular localization of wild-type and Parkinson’s disease-associated mutant alpha-synuclein in human and transgenic mouse brain. J. Neurosci. 20, 6365–6373.PubMedGoogle Scholar
  26. Kanda S., Bishop J. F., Eglitis M. A., Yang Y., and Mouradian M. M. (2000) Enhanced vulnerability to oxidative stress by alpha-synuclein mutations and c-terminal truncation. Neuroscience 97, 279–284.PubMedCrossRefGoogle Scholar
  27. Ko L., Mehta N. D., Farrer M., Easson C., Hussey J., Yen S., et al. (2000) Sensitization of neuronal cells to oxidative stress with mutated human alpha-synuclein. J. Neurochem. 75, 2546–2554.PubMedCrossRefGoogle Scholar
  28. Kruger R., Kuhn W., Muller T., Woitalla D., Graeber M., Kosel S., et al. (1998) Ala30Pro mutation in the gene encoding α-synuclein in Parkinson’s disease. Nat. Genet. 18, 106–108.PubMedCrossRefGoogle Scholar
  29. Lantos P. L. and Papp M. I. (1994) Cellular pathology of multiple system atrophy. J. Neurol. Neurosurg. Psychiatry 57, 129–133.PubMedCrossRefGoogle Scholar
  30. Lee F. J., Liu F., Pristupa Z. B., and Niznik H. B. (2001) Direct binding and functional coupling of alpha-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis. FASEB J. 15, 916–926.PubMedCrossRefGoogle Scholar
  31. Masliah E., Rockenstein E., Veinbergs I., Mallory M., Hashimoto M., Takeda A., et al. (2000) Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science 287, 1265–1269.PubMedCrossRefGoogle Scholar
  32. Mezey E., Dehejia A., Harta G., Papp M. I., Polymeropoulos M. H., and Brownstein M. J. (1998) Alpha synuclein in neurodegenerative disorders: murderer or accomplice? Nature Med. 4, 755–757.PubMedCrossRefGoogle Scholar
  33. Mizuno Y., Ikebe S., Hattori N., Nakagawa-Hattori Y., Mochizuki H., Tanaka M., and Ozawa T. (1995) Role of mitochondria in the etiology and pathogenesis of Parkinson’s disease. Biochim. Biophys. Acta. 1271, 265–274.PubMedGoogle Scholar
  34. Narhi L., Wood S. J., Steavenson S., Jiang Y., Wu G. M., Anafi D., et al. (1999) Both familial PD mutations accelerate α-synuclein aggregation. J. Biol. Chem. 274, 9843–9846.PubMedCrossRefGoogle Scholar
  35. Offen D., Strenin H., Ziv I., Melamed E., and Hochman A. (1996) Prevention of dopamine-induced cell death by thiol antioxidants: possible implications for treatment of Parkinson’s disease. Exp. Neurol. 141, 32–39.PubMedCrossRefGoogle Scholar
  36. Offen D., Ziv I., Gorodin S., Glater E., Hochman A., and Melamed E. (1997) Dopamine-melanin induces apoptosis in PC12 cells: Possible implications for the etiology of Parkinson’s disease. Neurochem. Int. 141, 32–39.Google Scholar
  37. Ostrerova N., Petrucelli L., Farrer M., Mehta N., Choi P., Hardy J., and Wolozin B. (1999) alpha-Synuclein shares physical and functional homology with 14-3-3 proteins. J. Neurosci. 19, 5782–5791.PubMedGoogle Scholar
  38. Polymeropoulos M. H., Lavedan C., Leory E., Ide S. E., Dehejia A., Dutra A., Pike B., et al. (1997) Mutation in the α-Synuclein gene identified in families with Parkinson’s Disease. Science 276, 2045–2047.PubMedCrossRefGoogle Scholar
  39. Rosenberg N. L., Myers J. A., and Martin W. R. (1989) Cyanide-induced parkinsonism: clinical, MRI, and 6-fluorodopa PET studies. Neurology 39, 142–144.PubMedGoogle Scholar
  40. Sato T., Irie S., and Reed J. C. (1995) A novel member of the TRAF family of putative signal transducing proteins binds to the cytosolic domain of CD40. FEBS Lett. 358, 113–118.PubMedCrossRefGoogle Scholar
  41. Schapira A. H., Cooper J. M., Dexter D., Clark J. B., Jenner P., and Marsden C. D. (1990) Mitochondrial complex I deficiency in Parkinson’s disease. J. Neurochem. 54, 823–827.PubMedCrossRefGoogle Scholar
  42. Schapira A. H., Gu M., Taanman J. W., Tabrizi S. J., Seaton T., Cleeter M., and Cooper J. M. (1998) Mitochondria in the etiology and pathogenesis of Parkinson’s disease. Ann. Neurol. 44, S89-S98.PubMedGoogle Scholar
  43. Sian J., Dexter D. T., Lees A. J., Daniel S., Jenner P., and Marsden C. D. (1994) Glutathione-related enzymes in brain in Parkinson’s disease. Ann. Neurol. 36, 356–361.PubMedCrossRefGoogle Scholar
  44. Simantov R., Blinder E., Ratovitski T., Tauber M., Gabbay M., and Porat S. (1996) Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acids antisense to the dopamine transporter. Neuroscience 74, 39–50.PubMedCrossRefGoogle Scholar
  45. Spillantini M. G., Crowther R. A., Jakes R., Hasegawa M., and Goedert M. (1998) alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. USA 95, 6469–6473.PubMedCrossRefGoogle Scholar
  46. Spillantini M. G., Schmidt M. L., Lee V. M., Trojanowski J. Q., Jakes R., and Goedert M. (1997) Alpha-synuclein in Lewy bodies. Nature 388, 839–840.PubMedCrossRefGoogle Scholar
  47. Tabrizi S. J., Orth M., Wilkinson J. M., Taanman J. W., Warner T. T., Cooper J. M., and Schapira A. H. (2000) Expression of mutant alpha-synuclein causes increased susceptibility to dopamine toxicity. Hum. Mol. Genet. 9, 2683–2689.PubMedCrossRefGoogle Scholar
  48. Tanaka Y., Engelender S., Igarashi S., Rao R. K., Wanner T., Tanzi R. E., et al. (2001) Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis. Hum. Mol. Genet. 10, 919–926.PubMedCrossRefGoogle Scholar
  49. Trimmer P. A., Swerdlow R. H., Parks J. K., Keeney P., Bennett J. P. Jr, Miller S. W., et al. (2000) Abnormal mitochondrial morphology in sporadic Parkinson’s and Alzheimer’s disease cybrid cell lines. Exp. Neurol. 162, 37–50.PubMedCrossRefGoogle Scholar
  50. Tu P. H., Galvin J. E., Baba M., Giasson B., Tomita T., Leight S., et al. (1998) Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble alpha-synuclein. Ann. Neurol. 44, 415–422.PubMedCrossRefGoogle Scholar
  51. Turnbull S., Tabner B. J., El-Agnaf O. M., Moore S., Davies Y., and Allsop D. (2001) alpha-synuclein implicated in Parkinson’s disease catalyses the formation of hydrogen peroxide in vitro. Free Radic. Biol. Med. 30, 1163–1170.PubMedCrossRefGoogle Scholar
  52. Ueda K., Fukushima H., Masliah E., Xia Y., Iwai A., Yoshimoto M., et al. (1993) Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90, 11282–11286.PubMedCrossRefGoogle Scholar
  53. Uitti R. J., Rajput A. H., Ashenhurst E. M., and Rozdilsky B. (1985) Cyanide-induced parkinsonism: a clinicopathologic report. Neurology 35, 921–925.PubMedGoogle Scholar
  54. van der Putten H., Wiederhold K. H., Probst A., Barbieri S., Mistl C., Danner S., et al. (2000) Neuropathology in mice expressing human alpha-synuclein. J. Neurosci. 20, 6021–6029.PubMedGoogle Scholar
  55. Weinreb P. H., Zhen W., Poon A. W., Conway K. A., and Lansbury P. T. Jr. (1996) NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 35, 13709–13715.PubMedCrossRefGoogle Scholar
  56. Zhou W., Hurlbert M. S., Schaack J., Prasad K. N., and Freed C. R. (2000) Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells. Brain Res. 866, 33–43.PubMedCrossRefGoogle Scholar
  57. Ziv I., Barzilai A., Offen D., Stein R., Achiron A., and Melamed E. (1996) Dopamine-induced, genotoxic activation of programmed cell death. A role in nigrostriatal neuronal degeneration in Parkinson’s disease? Adv. Neurol. 69, 229–233.PubMedGoogle Scholar
  58. Ziv I., Melamed E., Nardi N., Luria D., Achiron A., Offen D., and Barzilai A. (1994) Dopamine induces apoptosis-like cell death in cultured chick sympathetic neurons—a possible novel pathogenetic mechanism in Parkinson’s disease. Neurosci. Lett. 170, 136–140.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2002

Authors and Affiliations

  • Hanock Elkon
    • 1
    • 2
  • Jermy Don
    • 3
  • Eldad Melamed
    • 1
    • 2
  • Ilan Ziv
    • 1
    • 2
  • Anat Shirvan
    • 1
    • 2
  • Daniel Offen
    • 1
    • 2
  1. 1.Felsenstein Medical Research Center, Department of Clinical Biochemistry, Sackler School of MedicineTel-Aviv UniversityIsrael
  2. 2.Department of NeurologyRabin Medical CenterPetah-TikvaIsrael
  3. 3.Faculty of Life Sciences Bar-Ilan UniversityRamat-GanIsrael

Personalised recommendations