Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

α-Synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease

Nature Neuroscience volume 8, pages 657–663 (2005)Cite this article

Abstract

α-Synuclein is phosphorylated at serine 129 (Ser129) in intracellular protein aggregates called Lewy bodies. These inclusion bodies are the characteristic pathologic lesions of Parkinson disease. Here we define the role of phosphorylation of Ser129 in α-synuclein toxicity and inclusion formation using a Drosophila model of Parkinson disease. Mutation of Ser129 to alanine to prevent phosphorylation completely suppresses dopaminergic neuronal loss produced by expression of human α-synuclein. In contrast, altering Ser129 to the negatively charged residue aspartate, to mimic phosphorylation, significantly enhances α-synuclein toxicity. The G protein–coupled receptor kinase 2 (Gprk2) phosphorylates Ser129 in vivo and enhances α-synuclein toxicity. Blocking phosphorylation at Ser129 substantially increases aggregate formation. Thus Ser129 phosphorylation status is crucial in mediating α-synuclein neurotoxicity and inclusion formation. Because increased number of inclusion bodies correlates with reduced toxicity, inclusion bodies may protect neurons from α-synuclein toxicity.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: α-Synuclein is phosphorylated at Ser129.
Figure 2: Phosphorylation of Ser129 is critical for α-synuclein induced degeneration of dopaminergic neurons.
Figure 3: Ser129 phosphorylation enhances retinal degeneration.
Figure 4: Over-expressing Gprk2 increases α-synuclein phosphorylation and neurotoxicity.
Figure 5: Blocking Ser129 phosphorylation increases inclusion formation.
Figure 6: Increased inclusion formation correlates with increased proteinase K resistance.

Similar content being viewed by others

References

  1. Iwai, A. et al. The precursor protein of non-Aβ component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system. Neuron 14, 467–475 (1995).

    Article  CAS  Google Scholar 

  2. Chandra, S. et al. Double-knockout mice for α- and β-synucleins: effect on synaptic functions. Proc. Natl. Acad. Sci. USA 101, 14966–14971 (2004).

    Article  CAS  Google Scholar 

  3. Polymeropoulos, M.H. et al. Mutation in the α-synuclein gene identified in families with Parkinson's disease. Science 276, 2045–2047 (1997).

    Article  CAS  Google Scholar 

  4. Kruger, R. et al. Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease. Nat. Genet. 18, 106–108 (1998).

    Article  CAS  Google Scholar 

  5. Zarranz, J.J. et al. The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164–173 (2004).

    Article  CAS  Google Scholar 

  6. Singleton, A.B. et al. α-Synuclein locus triplication causes Parkinson's disease. Science 302, 841 (2003).

    Article  CAS  Google Scholar 

  7. Spillantini, M.G. et al. α-Synuclein in Lewy bodies. Nature 388, 839–840 (1997).

    Article  CAS  Google Scholar 

  8. Fujiwara, H. α-Synuclein is phosphorylated in synucleinopathy lesions. Nat. Cell Biol. 4, 160–164 (2002).

    Article  CAS  Google Scholar 

  9. Giasson, B.I. et al. Oxidative damage linked to neurodegeneration by selective α-synuclein nitration in synucleinopathy lesions. Science 290, 985–989 (2000).

    Article  CAS  Google Scholar 

  10. Shimura, H. et al. Ubiquitination of a new form of α-synuclein by parkin from human brain: implications for Parkinson's disease. Science 293, 263–269 (2001).

    Article  CAS  Google Scholar 

  11. Okochi, M. et al. Constitutive phosphorylation of the Parkinson's disease associated α-synuclein. J. Biol. Chem. 275, 390–397 (2000).

    Article  CAS  Google Scholar 

  12. Pronin, A.N., Morris, A.J., Surguchov, A. & Benovic, J.L. Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J. Biol. Chem. 275, 26515–26522 (2000).

    Article  CAS  Google Scholar 

  13. Ellis, C.E., Schwartzberg, P.L., Grider, T.L., Fink, D.W. & Nussbaum, R.L. α-Synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases. J. Biol. Chem. 276, 3879–3884 (2001).

    Article  CAS  Google Scholar 

  14. Saito, Y. et al. Accumulation of phosphorylated α-synuclein in aging human brain. J. Neuropathol. Exp. Neurol. 62, 644–654 (2003).

    Article  CAS  Google Scholar 

  15. Ross, C.A. & Poirier, M.A. Protein aggregation and neurodegenerative disease. Nat. Med. 10 (Suppl.), S10–S17 (2004).

    Article  Google Scholar 

  16. Feany, M.B. & Bender, W.W. A Drosophila model of Parkinson's disease. Nature 404, 394–398 (2000).

    Article  CAS  Google Scholar 

  17. Takahashi, M. et al. Phosphorylation of α-synuclein characteristic of synucleinopathy lesions is recapitulated in α-synuclein transgenic Drosophila. Neurosci. Lett. 336, 155–158 (2003).

    Article  CAS  Google Scholar 

  18. Leger, J., Kempf, M., Lee, G. & Brandt, R. Conversion of serine to aspartate imitates phosphorylation-induced changes in the structure and function of microtubule-associated protein tau. J. Biol. Chem. 272, 8441–8446 (1997).

    Article  CAS  Google Scholar 

  19. Auluck, P.K., Meulener, M.C. & Bonini, N.M. Mechanisms of suppression of α-synuclein neurotoxicity by geldanamycin in Drosophila. J. Biol. Chem. 280, 2873–2878 (2005).

    Article  CAS  Google Scholar 

  20. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M. & Bonini, N.M. Chaperone suppression of α-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295, 865–868 (2002).

    Article  CAS  Google Scholar 

  21. Yang, Y., Nishimura, I., Imai, Y., Takahashi, R. & Lu, B. Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila. Neuron 37, 911–924 (2003).

    Article  CAS  Google Scholar 

  22. Cassill, J.A., Whitney, M., Joazeiro, C.A., Becker, A. & Zuker, C.S. Isolation of Drosophila genes encoding G protein-coupled receptor kinases. Proc. Natl. Acad. Sci. USA 88, 11067–11070 (1991).

    Article  CAS  Google Scholar 

  23. Li, H., Chaney, S., Roberts, I.J., Forte, M. & Hirsh, J. Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster. Curr. Biol. 10, 211–214 (2000).

    Article  CAS  Google Scholar 

  24. Neumann, M. et al. Misfolded proteinase K-resistant hyperphosphorylated α-synuclein in aged transgenic mice with locomotor deterioration and in human α-synucleinopathies. J. Clin. Invest. 110, 1429–1439 (2002).

    Article  CAS  Google Scholar 

  25. Conway, K.A. et al. Acceleration of oligomerization, not fibrillization, is a shared property of both α-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. USA 97, 571–576 (2000).

    Article  CAS  Google Scholar 

  26. Giasson, B.I., Uryu, K., Trojanowski, J.Q. & Lee, V.M. Mutant and wild type human α-synucleins assemble into elongated filaments with distinct morphologies in vitro. J. Biol. Chem. 274, 7619–7622 (1999).

    Article  CAS  Google Scholar 

  27. Giasson, B.I., Murray, I.V., Trojanowski, J.Q. & Lee, V.M. A hydrophobic stretch of 12 amino acid residues in the middle of α-synuclein is essential for filament assembly. J. Biol. Chem. 276, 2380–2386 (2001).

    Article  CAS  Google Scholar 

  28. Forloni, G. Neurotoxicity of β-amyloid and prion peptides. Curr. Opin. Neurol. 9, 492–500 (1996).

    Article  CAS  Google Scholar 

  29. Pals, P. et al. α-Synuclein promoter confers susceptibility to Parkinson's disease. Ann. Neurol. 56, 591–595 (2004).

    Article  CAS  Google Scholar 

  30. Valente, E.M. et al. Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304, 1158–1160 (2004).

    Article  CAS  Google Scholar 

  31. Paisan-Ruiz, C. et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron 44, 595–600 (2004).

    Article  CAS  Google Scholar 

  32. Zimprich, A. et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44, 601–607 (2004).

    Article  CAS  Google Scholar 

  33. Goldberg, M.S. & Lansbury, P.T., Jr. Is there a cause-and-effect relationship between α-synuclein fibrillization and Parkinson's disease? Nat. Cell Biol. 2, E115–E119 (2000).

    Article  CAS  Google Scholar 

  34. Collins, S.R., Douglass, A., Vale, R.D. & Weissman, J.S. Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol. 2, e321 (2004).

    Article  Google Scholar 

  35. Saudou, F., Finkbeiner, S., Devys, D. & Greenberg, M.E. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95, 55–66 (1998).

    Article  CAS  Google Scholar 

  36. Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R. & Finkbeiner, S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810 (2004).

    Article  CAS  Google Scholar 

  37. Matsuo, E.S. et al. Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau. Neuron 13, 989–1002 (1994).

    Article  CAS  Google Scholar 

  38. Lee, M.K. et al. Human α-synuclein-harboring familial Parkinson's disease-linked Ala-53 → Thr mutation causes neurodegenerative disease with α-synuclein aggregation in transgenic mice. Proc. Natl. Acad. Sci. USA 99, 8968–8973 (2002).

    Article  CAS  Google Scholar 

  39. Cruz, J.C., Tseng, H.C., Goldman, J.A., Shih, H. & Tsai, L.H. Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles. Neuron 40, 471–483 (2003).

    Article  CAS  Google Scholar 

  40. Shulman, J.M. & Feany, M.B. Genetic modifiers of tauopathy in Drosophila. Genetics 165, 1233–1242 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Jackson, G.R. et al. Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 34, 509–519 (2002).

    Article  CAS  Google Scholar 

  42. Chen, H.K. et al. Interaction of Akt-phosphorylated ataxin-1 with 14–3-3 mediates neurodegeneration in spinocerebellar ataxia type 1. Cell 113, 457–468 (2003).

    Article  CAS  Google Scholar 

  43. Choi, J.Y. et al. Rapid purification and analysis of α-synuclein proteins: C-terminal truncation promotes the conversion of α-synuclein into a protease-sensitive form in Escherichia coli. Biotechnol. Appl. Biochem. 36, 33–40 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Teplow for advice on protein fibrillization experiments, T. Iwatsubo for providing psyn#64 antibody and C.S. Zuker for the Gprk2 antibody. This study was funded by grants from the US National Institutes of Health (AG88001, NS41536) to M.B.F.

Author information

Authors and Affiliations

  1. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, 02115, Massachusetts, USA

    Li Chen & Mel B Feany

Authors
  1. Li Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mel B Feany
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mel B Feany.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Quantitative comparison of α-synuclein expression levels in transgenic flies. (a) Fly heads were homogenized and serial loadings (1, 1/2, 1/4 head equivalents from left to right for each genotype) were analysed with antibody against total α-synuclein. WT, gmr-GAL4; UAS-α-synuclein; S129A, gmr-GAL4; UAS-S129A; S129D, gmr-GAL4; UAS-S129D. (b) Intensities of the corresponding bands were quantitated by a densitometer using NIH ImageJ program. Readings from wild-type α-synuclein group were normalized to 1 and relative intensity ratios were calculated. Values represent mean ± SEM. Flies were 1 day old. (JPG 48 kb)

Supplementary Fig. 2

Inclusion formation and phospho-synuclein immunoreactivity in dopaminergic neurons. (a) psyn#64 detected phosphorylated α-synuclein deposition in thread and grain-like structures in the dorsomedial neuropil in flies expressing wild-type α-synuclein (elav-GAL4/+; UAS-α-synuclein/+). (b) Inclusion bodies (arrow heads) in a dorsomedial dopamine neuron in a transgenic fly expressing S129A α-synuclein (Ddc-GAL4/+; UAS-S129A/+). Flies were 20 days old. (JPG 25 kb)

Supplementary Fig. 3

Fibrillization of α-synuclein. (a) The turbidity due to protein aggregation of either wild-type or mutant α-synuclein was monitored by measuring absorbance at 405 nm. A53T aggregates most rapidly whereas differences in aggregation among wild-type, S129A, and S129D α-synuclein were not seen. Values represent mean α SEM of four independent experiments. (b-d) Negatively stained electron micrographs of filaments assembled in vitro from wild-type (b), S129A (c), or S129D α-synuclein (d). (e) Sarkosyl insoluble filaments extracted from fly brains expressing wild-type α-synuclein. The 10-nm gold particles attached to the secondary antibody appear as black dots. The scale bar equals 100 nm. (JPG 89 kb)

Supplementary Fig. 4

Western blot analysis of α-synuclein differentially extracted from fly heads expressing wild-type α-synuclein. The same membrane was sequentially labelled with the psyn#64 and clone 42. Flies were 30 days old. (JPG 29 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, L., Feany, M. α-Synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease. Nat Neurosci 8, 657–663 (2005). https://doi.org/10.1038/nn1443

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn1443

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing