Acta Neuropathologica

, Volume 124, Issue 1, pp 37–50 | Cite as

An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology

  • Gabor G. Kovacs
  • Uta Wagner
  • Benoit Dumont
  • Maria Pikkarainen
  • Awad A. Osman
  • Nathalie Streichenberger
  • Irene Leisser
  • Jérémy Verchère
  • Thierry Baron
  • Irina Alafuzoff
  • Herbert Budka
  • Armand Perret-Liaudet
  • Ingolf Lachmann
Original Paper
First Online:
Received:
Revised:
Accepted:

Abstract

α-Synuclein is the major protein associated with Lewy body dementia, Parkinson’s disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44–57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.

Keywords

α-Synuclein Biomarker Parkinson’s disease Lewy body dementia Multiple system atrophy 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This study was partly performed in the frame of the EU FP6 Project Neuroscreen LSHB-CZ-2006-037719 contract no. 037719. Patent pending for 5G4 antibody.

Supplementary material

401_2012_964_MOESM1_ESM.pdf (7.1 mb)
Supplementary material 1 (PDF 7237 kb)

References

  1. 1.
    Alafuzoff I, Parkkinen L, Al-Sarraj S et al (2008) Assessment of alpha-synuclein pathology: a study of the BrainNet Europe Consortium. J Neuropathol Exp Neurol 67:125–143PubMedCrossRefGoogle Scholar
  2. 2.
    Baba M, Nakajo S, Tu PH et al (1998) Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am J Pathol 152:879–884PubMedGoogle Scholar
  3. 3.
    Beach TG, White CL, Hamilton RL et al (2008) Evaluation of alpha-synuclein immunohistochemical methods used by invited experts. Acta Neuropathol 116:277–288PubMedCrossRefGoogle Scholar
  4. 4.
    Braak H, Sastre M, Del Tredici K (2007) Development of alpha-synuclein immunoreactive astrocytes in the forebrain parallels stages of intraneuronal pathology in sporadic Parkinson’s disease. Acta Neuropathol 114:231–241PubMedCrossRefGoogle Scholar
  5. 5.
    Campbell BC, Li QX, Culvenor JG et al (2000) Accumulation of insoluble alpha-synuclein in dementia with Lewy bodies. Neurobiol Dis 7:192–200PubMedCrossRefGoogle Scholar
  6. 6.
    Campbell BC, McLean CA, Culvenor JG et al (2001) The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson’s disease. J Neurochem 76:87–96PubMedCrossRefGoogle Scholar
  7. 7.
    Chandra S, Chen X, Rizo J, Jahn R, Sudhof TC (2003) A broken alpha-helix in folded alpha-Synuclein. J Biol Chem 278:15313–15318PubMedCrossRefGoogle Scholar
  8. 8.
    Conway KA, Harper JD, Lansbury PT Jr (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39:2552–2563PubMedCrossRefGoogle Scholar
  9. 9.
    Croisier E, MRes DE, Deprez M et al (2006) Comparative study of commercially available anti-alpha-synuclein antibodies. Neuropathol Appl Neurobiol 32:351–356PubMedCrossRefGoogle Scholar
  10. 10.
    Culvenor JG, McLean CA, Cutt S et al (1999) Non-Abeta component of Alzheimer’s disease amyloid (NAC) revisited. NAC and alpha-synuclein are not associated with Abeta amyloid. Am J Pathol 155:1173–1181PubMedCrossRefGoogle Scholar
  11. 11.
    Deramecourt V, Bombois S, Maurage CA (2006) Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies. J Neuropathol Exp Neurol 65:278–288PubMedGoogle Scholar
  12. 12.
    Dickson DW, Liu W, Hardy J et al (1999) Widespread alterations of alpha-synuclein in multiple system atrophy. Am J Pathol 155:1241–1251PubMedCrossRefGoogle Scholar
  13. 13.
    Duda JE, Giasson BI, Mabon ME, Lee VM, Trojanowski JQ (2002) Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases. Ann Neurol 52:205–210PubMedCrossRefGoogle Scholar
  14. 14.
    El-Agnaf OM, Salem SA, Paleologou KE et al (2006) Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J 20:419–425PubMedCrossRefGoogle Scholar
  15. 15.
    Giasson BI, Jakes R, Goedert M et al (2000) A panel of epitope-specific antibodies detects protein domains distributed throughout human alpha-synuclein in Lewy bodies of Parkinson’s disease. J Neurosci Res 59:528–533PubMedCrossRefGoogle Scholar
  16. 16.
    Goedert M (2001) Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci 2:492–501PubMedCrossRefGoogle Scholar
  17. 17.
    Hamodrakas SJ, Liappa C, Iconomidou VA (2007) Consensus prediction of amyloidogenic determinants in amyloid fibril-forming proteins. Int J Biol Macromol 41:295–300PubMedCrossRefGoogle Scholar
  18. 18.
    Hoyer W, Cherny D, Subramaniam V, Jovin TM (2004) Impact of the acidic C-terminal region comprising amino acids 109–140 on alpha-synuclein aggregation in vitro. Biochemistry 43:16233–16242PubMedCrossRefGoogle Scholar
  19. 19.
    Iwai A, Masliah E, Yoshimoto M 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 14:467–475PubMedCrossRefGoogle Scholar
  20. 20.
    Kahle PJ, Neumann M, Ozmen L et al (2000) Subcellular localization of wild-type and Parkinson’s disease-associated mutant alpha -synuclein in human and transgenic mouse brain. J Neurosci 20:6365–6373PubMedGoogle Scholar
  21. 21.
    Kauppinen T, Martikainen P, Alafuzoff I (2006) Human postmortem brain tissue and 2-mm tissue microarrays. Appl Immunohistochem Mol Morphol 14:353–359PubMedCrossRefGoogle Scholar
  22. 22.
    Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174PubMedCrossRefGoogle Scholar
  23. 23.
    Kovacs GG, Botond G, Budka H (2010) Protein coding of neurodegenerative dementias: the neuropathological basis of biomarker diagnostics. Acta Neuropathol 119:389–408PubMedCrossRefGoogle Scholar
  24. 24.
    Langston JW, Sastry S, Chan P, Forno LS, Bolin LM, Di Monte DA (1998) Novel alpha-synuclein-immunoreactive proteins in brain samples from the Contursi kindred, Parkinson’s, and Alzheimer’s disease. Exp Neurol 154:684–690PubMedCrossRefGoogle Scholar
  25. 25.
    Martikainen P, Louhelainen AM, Kauppinen T, Alafuzoff I (2006) Human brain tissue microarrays as a platform to investigate diseases of the nervous system. Brain Res 1089:33–43PubMedGoogle Scholar
  26. 26.
    Martin FL, Williamson SJ, Paleologou KE, Allsop D, El-Agnaf OM (2004) Alpha-synuclein and the pathogenesis of Parkinson’s disease. Protein Pept Lett 11:229–237PubMedCrossRefGoogle Scholar
  27. 27.
    Miake H, Mizusawa H, Iwatsubo T, Hasegawa M (2002) Biochemical characterization of the core structure of alpha-synuclein filaments. J Biol Chem 277:19213–19219PubMedCrossRefGoogle Scholar
  28. 28.
    Mihajlovic M, Lazaridis T (2008) Membrane-bound structure and energetics of alpha-synuclein. Proteins 70:761–778PubMedCrossRefGoogle Scholar
  29. 29.
    Mollenhauer B, Locascio JJ, Schulz-Schaeffer W, Sixel-Doring F, Trenkwalder C, Schlossmacher MG (2011) alpha-Synuclein and tau concentrations in cerebrospinal fluid of patients presenting with parkinsonism: a cohort study. Lancet Neurol 10:230–240PubMedCrossRefGoogle Scholar
  30. 30.
    Mougenot AL, Betemps D, Hogeveen KN et al (2010) Production of a monoclonal antibody, against human alpha-synuclein, in a subpopulation of C57BL/6 J mice, presenting a deletion of the alpha-synuclein locus. J Neurosci Methods 192:268–276PubMedCrossRefGoogle Scholar
  31. 31.
    Osman AA, Uhlig H, Thamm B, Schneider-Mergener J, Mothes T (1998) Use of the phage display technique for detection of epitopes recognized by polyclonal rabbit gliadin antibodies. FEBS Lett 433:103–107PubMedCrossRefGoogle Scholar
  32. 32.
    Paleologou KE, Kragh CL, Mann DM et al (2009) Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. Brain 132:1093–1101PubMedCrossRefGoogle Scholar
  33. 33.
    Pikkarainen M, Martikainen P, Alafuzoff I (2010) The effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers. J Neuropathol Exp Neurol 69:40–52PubMedCrossRefGoogle Scholar
  34. 34.
    Rivers RC, Kumita JR, Tartaglia GG et al (2008) Molecular determinants of the aggregation behavior of alpha- and beta-synuclein. Protein Sci 17:887–898PubMedCrossRefGoogle Scholar
  35. 35.
    Saito Y, Kawashima A, Ruberu NN et al (2003) Accumulation of phosphorylated alpha-synuclein in aging human brain. J Neuropathol Exp Neurol 62:644–654PubMedGoogle Scholar
  36. 36.
    Sampathu DM, Giasson BI, Pawlyk AC, Trojanowski JQ, Lee VM (2003) Ubiquitination of alpha-synuclein is not required for formation of pathological inclusions in alpha-synucleinopathies. Am J Pathol 163:91–100PubMedCrossRefGoogle Scholar
  37. 37.
    Schulz-Schaeffer WJ (2010) The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies, Parkinson’s disease and Parkinson’s disease dementia. Acta Neuropathol 120:131–143PubMedCrossRefGoogle Scholar
  38. 38.
    Sharon R, Bar-Joseph I, Frosch MP, Walsh DM, Hamilton JA, Selkoe DJ (2003) The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson’s disease. Neuron 37:583–595PubMedCrossRefGoogle Scholar
  39. 39.
    Song YJ, Halliday GM, Holton JL et al (2009) Degeneration in different parkinsonian syndromes relates to astrocyte type and astrocyte protein expression. J Neuropathol Exp Neurol 68:1073–1083PubMedCrossRefGoogle Scholar
  40. 40.
    Tong J, Wong H, Guttman M et al (2010) Brain alpha-synuclein accumulation in multiple system atrophy, Parkinson’s disease and progressive supranuclear palsy: a comparative investigation. Brain 133:172–188PubMedCrossRefGoogle Scholar
  41. 41.
    Totterdell S, Meredith GE (2005) Localization of alpha-synuclein to identified fibers and synapses in the normal mouse brain. Neuroscience 135:907–913PubMedCrossRefGoogle Scholar
  42. 42.
    Tu PH, Galvin JE, Baba M et al (1998) Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble alpha-synuclein. Ann Neurol 44:415–422PubMedCrossRefGoogle Scholar
  43. 43.
    Ueda K, Saitoh T, Mori H (1994) Tissue-dependent alternative splicing of mRNA for NACP, the precursor of non-A beta component of Alzheimer’s disease amyloid. Biochem Biophys Res Commun 205:1366–1372PubMedCrossRefGoogle Scholar
  44. 44.
    Vilar M, Chou HT, Luhrs T et al (2008) The fold of alpha-synuclein fibrils. Proc Natl Acad Sci USA 105:8637–8642PubMedCrossRefGoogle Scholar
  45. 45.
    Waxman EA, Duda JE, Giasson BI (2008) Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions. Acta Neuropathol 116:37–46PubMedCrossRefGoogle Scholar
  46. 46.
    Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT Jr (1996) NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 35:13709–13715PubMedCrossRefGoogle Scholar
  47. 47.
    Winner B, Jappelli R, Maji SK et al (2011) In vivo demonstration that {alpha}-synuclein oligomers are toxic. Proc Natl Acad Sci USA 108:4194–4199PubMedCrossRefGoogle Scholar
  48. 48.
    Yu S, Li X, Liu G et al (2007) Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody. Neuroscience 145:539–555PubMedCrossRefGoogle Scholar
  49. 49.
    Zhou J, Broe M, Huang Y et al (2011) Changes in the solubility and phosphorylation of alpha-synuclein over the course of Parkinson’s disease. Acta Neuropathol 121:695–704Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Gabor G. Kovacs
    • 1
  • Uta Wagner
    • 2
  • Benoit Dumont
    • 3
    • 4
  • Maria Pikkarainen
    • 5
  • Awad A. Osman
    • 2
  • Nathalie Streichenberger
    • 6
  • Irene Leisser
    • 1
  • Jérémy Verchère
    • 7
  • Thierry Baron
    • 7
  • Irina Alafuzoff
    • 5
    • 8
  • Herbert Budka
    • 1
  • Armand Perret-Liaudet
    • 3
    • 4
  • Ingolf Lachmann
    • 2
  1. 1.Institute of Neurology, Medical University of ViennaViennaAustria
  2. 2.AJ Roboscreen GmbHLeipzigGermany
  3. 3.Neurobiology Laboratory, Department of BiochemistryCentre de Biologie et Pathologie Est, Hospices Civils de LyonLyonFrance
  4. 4.Centre de Recherche en Neurosciences de Lyon, Equipe BioRan Pr Luc ZimmerCNRS, INSERM, Claude Bernard Lyon1 UniversityLyonFrance
  5. 5.Institute of Clinical Medicine, NeurologyUniversity of Eastern FinlandKuopioFinland
  6. 6.Pathology and BiochemistryGroupement Hospitalier Est, Hospices Civils de Lyon/Claude Bernard UniversityLyonFrance
  7. 7.Agence Nationale de Sécurité Sanitaire (Anses)Unité Maladies Neuro-dégénérativesLyonFrance
  8. 8.Department of Immunology, Genetics and PathologyUppsala University and Uppsala University HospitalUppsalaSweden

Personalised recommendations