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Molecular Neurobiology

, Volume 53, Issue 3, pp 1925–1934 | Cite as

α-Synuclein Over-Expression Induces Increased Iron Accumulation and Redistribution in Iron-Exposed Neurons

  • Richard OrtegaEmail author
  • Asuncion Carmona
  • Stéphane Roudeau
  • Laura Perrin
  • Tanja Dučić
  • Eleonora Carboni
  • Sylvain Bohic
  • Peter Cloetens
  • Paul LingorEmail author
Article

Abstract

Parkinson’s disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson’s disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson’s disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

Keywords

α-Synuclein Iron Parkinson’s disease Synchrotron Neuroimaging 

Notes

Acknowledgments

Authors sincerely acknowledge Prof. Seung-Jae Lee from Konkuk University in Seoul, Korea, for kindly providing the α-synuclein adenovirus vector. The AAV vectors expressing wild-type α-synuclein/EGFP and EGFP were a kind gift from Dr. Sebastian Kügler (Göttingen). The authors are thankful to the European Synchrotron Radiation Facility for beamtime allocation and to R. Tucoulou and G. Devès for their assistance during the experiment. We also thank CENBG staff from AIFIRA platform. This work was financially supported in part by the French Agence Nationale pour la Recherche (ANR) program PIRIBIO (ANR-09-PIRI-0029-01) and by the PROCOPE program from the French Ministry of European and Foreign Affairs (PHC Procope 25148UC).

References

  1. 1.
    Wirdefeldt K, Adami HO, Cole P, Trichopoulos D, Mandel J (2011) Epidemiology and etiology of Parkinson’s disease: a review of the evidence. Eur J Epidemiol 26(Suppl 1):S1–S58CrossRefPubMedGoogle Scholar
  2. 2.
    Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, et al (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276:2045–2047Google Scholar
  3. 3.
    Neystat M, Lynch T, Przedborski S, Kholodilov N, Rzhetskaya M, Burke RE (1999) Alpha-synuclein expression in substantia nigra and cortex in Parkinson’s disease. Mov Disord 14:417–422CrossRefPubMedGoogle Scholar
  4. 4.
    Devine MJ, Gwinn K, Singleton A, Hardy J (2011) Parkinson’s disease and alpha-synuclein expression. Mov Disord 26:2160–2168CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M (1997) Alpha-synuclein in Lewy bodies. Nature 388:839–840CrossRefPubMedGoogle Scholar
  6. 6.
    Miller DW, Hague SM, Clarimon J, Baptista M, Gwinn-Hardy K, Cookson MR, Singleton AB (2004) Alpha-synuclein in blood and brain from familial Parkinson disease with SNCA locus triplication. Neurology 62:1835–1838CrossRefPubMedGoogle Scholar
  7. 7.
    Wong BX, Duce JA (2014) The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders. Front Pharmacol 5:81CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kozlowski H, Luczkowski M, Remelli M, Valensin D (2012) Copper, zinc and iron in neurodegenerative diseases (Alzheimer’s, Parkinson’s and prion diseases). Coord Chem Rev 256:2129–2141CrossRefGoogle Scholar
  9. 9.
    Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farer M, Wolozin B (2000) The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J Neurosci 20:6048–6054PubMedGoogle Scholar
  10. 10.
    Kostka M, Högen T, Danzer KM, Levin J, Habeck M, Wirth A, Wagner R, Glabe CG, et al (2008) Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers. J Biol Chem 283:10992–11003Google Scholar
  11. 11.
    Li WJ, Jiang H, Song N, Xie JX (2010) Dose- and time-dependent alpha-synuclein aggregation induced by ferric iron in SK-N-SH cells. Neurosci Bull 26:205–210CrossRefPubMedGoogle Scholar
  12. 12.
    Hillmer AS, Putcha P, Levin J, Högen T, Hyman BT, Kretzschmar H, McLean PJ, Giese A (2010) Converse modulation of toxic alpha-synuclein oligomers in living cells by N’-benzylidene-benzohydrazide derivates and ferric iron. Biochem Biophys Res Commun 391:461–466CrossRefPubMedGoogle Scholar
  13. 13.
    Peng Y, Wang C, Xu HH, Liu YN, Zhou F (2010) Binding of alpha-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes. J Inorg Biochem 104:365–370CrossRefPubMedGoogle Scholar
  14. 14.
    He Q, Song N, Xu H, Wang R, Xie J, Jiang H (2011) Alpha-synuclein aggregation is involved in the toxicity induced by ferric iron to SK-N-SH neuroblastoma cells. J Neural Transm 118:397–406CrossRefPubMedGoogle Scholar
  15. 15.
    Jinsmaa Y, Sullivan P, Gross D, Cooney A, Sharabi Y, Goldstein DS (2014) Divalent metal ions enhance DOPAL-induced oligomerization of alpha-synuclein. Neurosci Lett 569:27–32CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Friedlich AL, Tanzi RE, Rogers JT (2007) The 5′-untranslated region of Parkinson’s disease alpha-synuclein messengerRNA contains a predicted iron responsive element. Mol Psychiatry 12:222–223CrossRefPubMedGoogle Scholar
  17. 17.
    Febbraro F, Giorgi M, Caldarola S, Loreni F, Romero-Ramos M (2012) α-Synuclein expression is modulated at the translational level by iron. Neuroreport 23:576–580CrossRefPubMedGoogle Scholar
  18. 18.
    Scherzer CR, Grass JA, Liao Z, Pepivani I, Zheng B, Eklund AC, Ney PA, Ng J et al (2008) GATA transcription factors directly regulate the Parkinson’s disease-linked gene alpha-synuclein. Proc Natl Acad Sci U S A 105:10907–10912CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hirsch EC, Brandel JP, Galle P, Javoy-Agid F, Agid Y (1991) Iron and aluminum increase in the substantia nigra of patients with Parkinson’s disease: an X-ray microanalysis. J Neurochem 56:446–451CrossRefPubMedGoogle Scholar
  20. 20.
    Castellani RJ, Siedlak SL, Perry G, Smith MA (2000) Sequestration of iron by Lewy bodies in Parkinson’s disease. Acta Neuropathol 100:111–114CrossRefPubMedGoogle Scholar
  21. 21.
    Golts N, Snyder H, Frasier M, Theisler C, Choi P, Wolozin B (2002) Magnesium inhibits spontaneous and iron-induced aggregation of alpha-synuclein. J Biol Chem 277:16116–16123CrossRefPubMedGoogle Scholar
  22. 22.
    Binolfi A, Rasia RM, Bertoncini CW, Ceolin M, Zweckstetter M, Griesinger C, Jovin TM, Fernandez CO (2006) Interaction of alpha-synuclein with divalent metal ions reveals key differences: a link between structure, binding specificity and fibrillation enhancement. J Am Chem Soc 128:9893–9901CrossRefPubMedGoogle Scholar
  23. 23.
    Bharathi Rao KS (2007) Thermodynamics imprinting reveals differential binding of metals to alpha-synuclein: relevance to Parkinson’s disease. Biochem Biophys Res Commun 359:115–120CrossRefGoogle Scholar
  24. 24.
    Davies P, Moualla D, Brown DR (2011) Alpha-synuclein is a cellular ferrireductase. PLoS ONE 6:e15814CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Carmona A, Devès G, Ortega R (2008) Quantitative micro-analysis of metal ions in subcellular compartments of cultured dopaminergic cells by combination of three ion beam techniques. Anal Bioanal Chem 390:1585–1594CrossRefPubMedGoogle Scholar
  26. 26.
    Ortega R, Devès G, Carmona A (2009) Bio-metals imaging and speciation in cells using proton and synchrotron radiation X-ray microspectroscopy. J R Soc Interface 6:S649–S658CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Carmona A, Cloetens P, Devès G, Bohic S, Ortega R (2008) Nano-imaging of trace metals by synchrotron X-ray fluorescence into dopaminergic single cells and neurite-like processes. J Anal At Spectrom 23:1083–1088CrossRefGoogle Scholar
  28. 28.
    Dučić T, Barski E, Salome M, Koch JC, Bähr M, Lingor P (2013) X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons. J Neurochem 124:250–261CrossRefPubMedGoogle Scholar
  29. 29.
    Zhu M, Li W, Lu C (2012) Role of alpha-synuclein protein levels in mitochondrial morphology and cell survival in cell lines. PLoS One 7:e36377CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Rendón WO, Martínez-Alonso E, Tomás M, Martínez-Martínez N, Martínez-Menárguez JA (2013) Golgi fragmentation is Rab and SNARE dependent in cellular models of Parkinson’s disease. Histochem Cell Biol 139:671–684CrossRefPubMedGoogle Scholar
  31. 31.
    Lee HJ, Shin SY, Choi C, Lee YH, Lee SJ (2002) Formation and removal of alpha-synuclein aggregates in cells exposed to mitochondrial inhibitors. J Biol Chem 277:5411–5417CrossRefPubMedGoogle Scholar
  32. 32.
    Parihar MS, Parihar A, Fujita M, Hashimoto M, Ghafourifar P (2009) Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells. Int J Biochem Cell Biol 41:2015–2024CrossRefPubMedGoogle Scholar
  33. 33.
    McLean PJ, Kawamata H, Hyman BT (2001) Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons. Neuroscience 104:901–912CrossRefPubMedGoogle Scholar
  34. 34.
    Rasia RM, Bertoncini CW, Marsh D, Hoyer W, Cherny D, Zweckstetter M, Griesinger C, Jovin TM et al (2005) Structural characterization of copper(II) binding to alpha-synuclein: insights into the bioinorganic chemistry of Parkinson’s disease. Proc Natl Acad Sci U S A 102:4294–4299CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Brown DR (2009) Metal binding to alpha-synuclein peptides and its contribution to toxicity. Biochem Biophys Res Commun 380:377–381CrossRefPubMedGoogle Scholar
  36. 36.
    Büttner S, Faes L, Reichelt WN, Broeskamp F, Habernig L, Benke S, Kourtis N, Ruli D, et al (2013) The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to α-synuclein toxicity in Parkinson’s disease models. Cell Death Differ 20:465–477Google Scholar
  37. 37.
    Mosharov EV, Larsen KE, Kanter E, Phillips KA, Wilson K, Schmitz Y, Krantz DE, Kobayashi K et al (2009) Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons. Neuron 62:218–229CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Ying Z, Lin F, Gu W, Su Y, Arshad A, Qing H, Deng Y (2011) α-Synuclein increases U251 cells vulnerability to hydrogen peroxide by disrupting calcium homeostasis. J Neural Transm 118:1165–1172CrossRefPubMedGoogle Scholar
  39. 39.
    Knöferle J, Ramljak S, Koch JC, Tönges L, Asif AR, Michel U, Wouters FS, Heermann S et al (2010) TGF-beta 1 enhances neurite outgrowth via regulation of proteasome function and EFABP. Neurobiol Dis 38:395–404CrossRefPubMedGoogle Scholar
  40. 40.
    Lingor P, Unsicker K, Krieglstein K (2000) GDNF and NT-4 protect midbrain dopaminergic neurons from toxic damage by iron and nitric oxide. Exp Neurol 163:55–62CrossRefPubMedGoogle Scholar
  41. 41.
    Saal KA, Koch JC, Tatenhorst L, Szegő EM, Ribas VT, Michel U, Bähr M, Tönges L et al (2015) AAV.shRNA-mediated downregulation of ROCK2 attenuates degeneration of dopaminergic neurons in toxin-induced models of Parkinson’s disease in vitro and in vivo. Neurobiol Dis 73:150–162CrossRefPubMedGoogle Scholar
  42. 42.
    Taschenberger G, Garrido M, Tereshchenko Y, Bähr M, Zweckstetter M, Kügler S (2011) Aggregation of αSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons. Acta Neuropathol 123:671–683CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Koch JC, Barski E, Lingor P, Bähr M, Michel U (2011) Plasmids containing NRSE/RE1 sites enhance neurite outgrowth of retinal ganglion cells via sequestration of REST independent of NRSE dsRNA expression. FEBS J 278:3472–3483CrossRefPubMedGoogle Scholar
  44. 44.
    Galvani P, Colleoni M, Origgi M, Santagostino A (1995) Mitochondrial toxicity of iron and the protective role of ferritin on dopaminergic PC12 cell line. Toxicol in Vitro 9:365–368CrossRefPubMedGoogle Scholar
  45. 45.
    Koch JC, Tönges L, Barski E, Michel U, Bähr M, Lingor P (2014) ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS. Cell Death Dis 5:e1225CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedGoogle Scholar
  47. 47.
    Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T, Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci U S A 107:12564–12569CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Roudeau S, Carmona A, Perrin L, Ortega R (2014) Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells. Anal Bioanal Chem 406:6979–6991CrossRefPubMedGoogle Scholar
  49. 49.
    Campbell JL, Boyd NI, Grassi N, Bonnick P, Maxwell JA (2010) The Guelph PIXE software package IV. Nucl Inst Methods B 268:3356–3363CrossRefGoogle Scholar
  50. 50.
    Mayer M (1997) SIMNRA user’s guide. Report IPP 9/113, Max-Planck-Inst. für Plasmaphysik, Garching, Germany. www2.if.usp.br/~lamfi/guia-simnra.pdf
  51. 51.
    Solé VA, Papillon E, Cotte M, Walter P, Susini J (2007) A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra. Spectrochim Acta B 62:63–68CrossRefGoogle Scholar
  52. 52.
    Fox J (2005) The R commander: a basic statistics graphical user interface to R. J Stat Softw 14:1–42Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Richard Ortega
    • 1
    • 2
    Email author
  • Asuncion Carmona
    • 1
    • 2
  • Stéphane Roudeau
    • 1
    • 2
  • Laura Perrin
    • 1
    • 2
  • Tanja Dučić
    • 3
  • Eleonora Carboni
    • 4
    • 5
  • Sylvain Bohic
    • 6
    • 7
  • Peter Cloetens
    • 7
  • Paul Lingor
    • 4
    • 5
    Email author
  1. 1.University of Bordeaux, CENBG, UMR 5797GradignanFrance
  2. 2.CNRS, IN2P3, CENBG, UMR 5797GradignanFrance
  3. 3.CELLS—ALBACerdanyola del VallèsSpain
  4. 4.Department of NeurologyUniversity of MedicineGöttingenGermany
  5. 5.DFG-Research Center for Nanoscale Microscopy and Molecular Physiology of the BrainGöttingenGermany
  6. 6.INSERM U-836 Team 6, Rayonnement Synchrotron et Recherche MédicaleGrenobleFrance
  7. 7.European Synchrotron Radiation Facility (ESRF), X-ray Imaging GroupGrenobleFrance

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