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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

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 67:125–143

    PubMed  Article  Google 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 152:879–884

    PubMed  CAS  Google Scholar 

  3. 3.

    Beach TG, White CL, Hamilton RL et al (2008) Evaluation of alpha-synuclein immunohistochemical methods used by invited experts. Acta Neuropathol 116:277–288

    PubMed  Article  CAS  Google 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 114:231–241

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Campbell BC, Li QX, Culvenor JG et al (2000) Accumulation of insoluble alpha-synuclein in dementia with Lewy bodies. Neurobiol Dis 7:192–200

    PubMed  Article  CAS  Google 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 76:87–96

    PubMed  Article  CAS  Google 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 278:15313–15318

    PubMed  Article  CAS  Google 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 39:2552–2563

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Croisier E, MRes DE, Deprez M et al (2006) Comparative study of commercially available anti-alpha-synuclein antibodies. Neuropathol Appl Neurobiol 32:351–356

    PubMed  Article  CAS  Google 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 155:1173–1181

    PubMed  Article  CAS  Google 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 65:278–288

    PubMed  CAS  Google Scholar 

  12. 12.

    Dickson DW, Liu W, Hardy J et al (1999) Widespread alterations of alpha-synuclein in multiple system atrophy. Am J Pathol 155:1241–1251

    PubMed  Article  CAS  Google 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 52:205–210

    PubMed  Article  CAS  Google 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 20:419–425

    PubMed  Article  CAS  Google 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 59:528–533

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Goedert M (2001) Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci 2:492–501

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Hamodrakas SJ, Liappa C, Iconomidou VA (2007) Consensus prediction of amyloidogenic determinants in amyloid fibril-forming proteins. Int J Biol Macromol 41:295–300

    PubMed  Article  CAS  Google 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 43:16233–16242

    PubMed  Article  CAS  Google 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 14:467–475

    PubMed  Article  CAS  Google 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 20:6365–6373

    PubMed  CAS  Google Scholar 

  21. 21.

    Kauppinen T, Martikainen P, Alafuzoff I (2006) Human postmortem brain tissue and 2-mm tissue microarrays. Appl Immunohistochem Mol Morphol 14:353–359

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Kovacs GG, Botond G, Budka H (2010) Protein coding of neurodegenerative dementias: the neuropathological basis of biomarker diagnostics. Acta Neuropathol 119:389–408

    PubMed  Article  CAS  Google 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 154:684–690

    PubMed  Article  CAS  Google 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 1089:33–43

    PubMed  CAS  Google 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 11:229–237

    PubMed  Article  CAS  Google 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 277:19213–19219

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Mihajlovic M, Lazaridis T (2008) Membrane-bound structure and energetics of alpha-synuclein. Proteins 70:761–778

    PubMed  Article  CAS  Google 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 10:230–240

    PubMed  Article  CAS  Google 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 192:268–276

    PubMed  Article  CAS  Google 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 433:103–107

    PubMed  Article  CAS  Google 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 132:1093–1101

    PubMed  Article  Google 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 69:40–52

    PubMed  Article  CAS  Google 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 17:887–898

    PubMed  Article  CAS  Google 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 62:644–654

    PubMed  CAS  Google 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 163:91–100

    PubMed  Article  CAS  Google 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 120:131–143

    PubMed  Article  CAS  Google 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 37:583–595

    PubMed  Article  CAS  Google 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 68:1073–1083

    PubMed  Article  CAS  Google 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 133:172–188

    PubMed  Article  Google Scholar 

  41. 41.

    Totterdell S, Meredith GE (2005) Localization of alpha-synuclein to identified fibers and synapses in the normal mouse brain. Neuroscience 135:907–913

    PubMed  Article  CAS  Google 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 44:415–422

    PubMed  Article  CAS  Google 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 205:1366–1372

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Vilar M, Chou HT, Luhrs T et al (2008) The fold of alpha-synuclein fibrils. Proc Natl Acad Sci USA 105:8637–8642

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Waxman EA, Duda JE, Giasson BI (2008) Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions. Acta Neuropathol 116:37–46

    PubMed  Article  CAS  Google 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 35:13709–13715

    PubMed  Article  CAS  Google 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 108:4194–4199

    PubMed  Article  CAS  Google 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 145:539–555

    PubMed  Article  CAS  Google 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 121:695–704

    Google Scholar 

Download references

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Gabor G. Kovacs.

Additional information

The authors G. G. Kovacs and I. Lachmann contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 7237 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kovacs, G.G., Wagner, U., Dumont, B. et al. An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology. Acta Neuropathol 124, 37–50 (2012). https://doi.org/10.1007/s00401-012-0964-x

Download citation

Keywords

  • α-Synuclein
  • Biomarker
  • Parkinson’s disease
  • Lewy body dementia
  • Multiple system atrophy