Stabilization of α-synuclein oligomers using formaldehyde

Autoři: Harm Ruesink aff001;  Lasse Reimer aff001;  Emil Gregersen aff001;  Arne Moeller aff001;  Cristine Betzer aff001;  Poul Henning Jensen aff001
Působiště autorů: Danish Research Institute of Translational Neuroscience—DANDRITE, Aarhus University, Aarhus, Denmark aff001;  Department of Biomedicine, Aarhus University, Aarhus, Denmark aff002;  Department of Structural Biology, The Max Planck Institute of Biophysics, Frankfurt, Germany aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0216764


The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.

Klíčová slova:

Amyloid proteins – Antigens – Cross-linking – Enzyme-linked immunoassays – Monomers – Oligomers – Parkinson disease – Formaldehyde


1. Goedert M., Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci, 2001. 2(7): p. 492–501. doi: 10.1038/35081564 11433374

2. Eliezer D., Kutluay E., Bussell R. Jr., and Browne G., Conformational properties of alpha-synuclein in its free and lipid-associated states. J Mol Biol, 2001. 307(4): p. 1061–73. doi: 10.1006/jmbi.2001.4538 11286556

3. Iwai A., Masliah E., Yoshimoto M., Ge N., Flanagan L., de Silva H.A., et al., The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system. Neuron, 1995. 14(2): p. 467–75. doi: 10.1016/0896-6273(95)90302-x 7857654

4. Burre J., Sharma M., Tsetsenis T., Buchman V., Etherton M.R., and Sudhof T.C., Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science, 2010. 329(5999): p. 1663–7. doi: 10.1126/science.1195227 20798282

5. Chartier-Harlin M.C., Kachergus J., Roumier C., Mouroux V., Douay X., Lincoln S., et al., Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. Lancet, 2004. 364(9440): p. 1167–9. doi: 10.1016/S0140-6736(04)17103-1 15451224

6. Kruger R., Kuhn W., Muller T., Woitalla D., Graeber M., Kosel S., et al., Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet, 1998. 18(2): p. 106–8. doi: 10.1038/ng0298-106 9462735

7. Polymeropoulos M.H., Lavedan C., Leroy E., Ide S.E., Dehejia A., Dutra A., et al., Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science, 1997. 276(5321): p. 2045–7. doi: 10.1126/science.276.5321.2045 9197268

8. Zarranz J.J., Alegre J., Gomez-Esteban J.C., Lezcano E., Ros R., Ampuero I., et al., The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol, 2004. 55(2): p. 164–73. doi: 10.1002/ana.10795 14755719

9. Spillantini M.G., Schmidt M.L., Lee V.M., Trojanowski J.Q., Jakes R., and Goedert M., Alpha-synuclein in Lewy bodies. Nature, 1997. 388(6645): p. 839–40. doi: 10.1038/42166 9278044

10. Paleologou K.E., Kragh C.L., Mann D.M., Salem S.A., Al-Shami R., Allsop D., et al., Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. Brain, 2009. 132(Pt 4): p. 1093–101. doi: 10.1093/brain/awn349 19155272

11. Roberts R.F., Wade-Martins R., and Alegre-Abarrategui J., Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson’s disease brain. Brain, 2015. 138(6): p. 1642–1657.

12. Sekiya H., Kowa H., Koga H., Takata M., Satake W., Futamura N., et al., Wide distribution of alpha-synuclein oligomers in multiple system atrophy brain detected by proximity ligation. Acta Neuropathol, 2019. 137(3): p. 455–466. doi: 10.1007/s00401-019-01961-w 30721406

13. Winner B., Jappelli R., Maji S.K., Desplats P.A., Boyer L., Aigner S., et al., In vivo demonstration that alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A, 2011. 108(10): p. 4194–9. doi: 10.1073/pnas.1100976108 21325059

14. Karpinar D.P., Balija M.B.G., Kügler S., Opazo F., Rezaei-Ghaleh N., Wender N., et al., Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models. The EMBO Journal, 2009. 28(20): p. 3256–3268. doi: 10.1038/emboj.2009.257 19745811

15. Ludtmann M.H.R., Angelova P.R., Horrocks M.H., Choi M.L., Rodrigues M., Baev A.Y., et al., alpha-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease. Nat Commun, 2018. 9(1): p. 2293. doi: 10.1038/s41467-018-04422-2 29895861

16. Danzer K.M., Krebs S.K., Wolff M., Birk G., and Hengerer B., Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha-synuclein pathology. J Neurochem, 2009. 111(1): p. 192–203. doi: 10.1111/j.1471-4159.2009.06324.x 19686384

17. Danzer K.M., Haasen D., Karow A.R., Moussaud S., Habeck M., Giese A., et al., Different species of alpha-synuclein oligomers induce calcium influx and seeding. J Neurosci, 2007. 27(34): p. 9220–32. doi: 10.1523/JNEUROSCI.2617-07.2007 17715357

18. Cappai R., Leck S.L., Tew D.J., Williamson N.A., Smith D.P., Galatis D., et al., Dopamine promotes alpha-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway. Faseb j, 2005. 19(10): p. 1377–9. doi: 10.1096/fj.04-3437fje 15946991

19. Lashuel H.A., Petre B.M., Wall J., Simon M., Nowak R.J., Walz T., et al., Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils. J Mol Biol, 2002. 322(5): p. 1089–102. doi: 10.1016/s0022-2836(02)00735-0 12367530

20. Mysling S., Betzer C., Jensen P.H., and Jorgensen T.J.D., Characterizing the Dynamics of α-Synuclein Oligomers Using Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry. Biochemistry, 2013. 52(51): p. 9097–9103. doi: 10.1021/bi4009193 24191706

21. Betzer C., Movius A.J., Shi M., Gai W.P., Zhang J., and Jensen P.H., Identification of synaptosomal proteins binding to monomeric and oligomeric alpha-synuclein. PLoS One, 2015. 10(2): p. e0116473. doi: 10.1371/journal.pone.0116473 25659148

22. Betzer C., Lassen L.B., Olsen A., Kofoed R.H., Reimer L., Gregersen E., et al., Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation. EMBO Rep, 2018. 19(5).

23. Lassen L.B., Gregersen E., Isager A.K., Betzer C., Kofoed R.H., and Jensen P.H., ELISA method to detect α-synuclein oligomers in cell and animal models. PLoS ONE, 2018. 13(4): p. e0196056. doi: 10.1371/journal.pone.0196056 29698510

24. Ono K., The Oligomer Hypothesis in α-Synucleinopathy. Neurochemical Research, 2017. 42(12): p. 3362–3371. doi: 10.1007/s11064-017-2382-x 28828740

25. El-Agnaf O.M., Salem S.A., Paleologou K.E., Curran M.D., Gibson M.J., Court J.A., et al., Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson's disease. Faseb j, 2006. 20(3): p. 419–25. doi: 10.1096/fj.03-1449com 16507759

26. Park M.J., Cheon S.-M., Bae H.-R., Kim S.-H., and Kim J.W., Elevated levels of α-synuclein oligomer in the cerebrospinal fluid of drug-naïve patients with Parkinson's disease. Journal of clinical neurology (Seoul, Korea), 2011. 7(4): p. 215–222.

27. Tokuda T., Qureshi M.M., Ardah M.T., Varghese S., Shehab S.A., Kasai T., et al., Detection of elevated levels of alpha-synuclein oligomers in CSF from patients with Parkinson disease. Neurology, 2010. 75(20): p. 1766–72. doi: 10.1212/WNL.0b013e3181fd613b 20962290

28. Cremades N., Chen S.W., and Dobson C.M., Chapter Three—Structural Characteristics of α-Synuclein Oligomers, in International Review of Cell and Molecular Biology, Sandal M., Editor. 2017, Academic Press. p. 79–143.

29. Bousset L., Pieri L., Ruiz-Arlandis G., Gath J., Jensen P.H., Habenstein B., et al., Structural and functional characterization of two alpha-synuclein strains. Nat Commun, 2013. 4: p. 2575. doi: 10.1038/ncomms3575 24108358

30. Jensen P.H., Islam K., Kenney J., Nielsen M.S., Power J., and Gai W.P., Microtubule-associated protein 1B is a component of cortical Lewy bodies and binds alpha-synuclein filaments. J Biol Chem, 2000. 275(28): p. 21500–7. doi: 10.1074/jbc.M000099200 10764738

31. Lindersson E., Beedholm R., Hojrup P., Moos T., Gai W., Hendil K.B., et al., Proteasomal inhibition by alpha-synuclein filaments and oligomers. J Biol Chem, 2004. 279(13): p. 12924–34. doi: 10.1074/jbc.M306390200 14711827

32. Huang C., Ren G., Zhou H., and Wang C.C., A new method for purification of recombinant human alpha-synuclein in Escherichia coli. Protein Expr Purif, 2005. 42(1): p. 173–7. doi: 10.1016/j.pep.2005.02.014 15939304

33. Gewering T., Januliene D., Ries A.B., and Moeller A., Know your detergents: A case study on detergent background in negative stain electron microscopy. J Struct Biol, 2018. 203(3): p. 242–246. doi: 10.1016/j.jsb.2018.05.008 29852220

34. Tao H., Lee S.C., Moeller A., Roy R.S., Siu F.Y., Zimmermann J., et al., Engineered nanostructured β-sheet peptides protect membrane proteins. Nature methods, 2013. 10(8): p. 759–761. doi: 10.1038/nmeth.2533 23817067

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden