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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.

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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.

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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.

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

    Li Chen & Mel B Feany

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Correspondence to Mel B Feany.

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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)

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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

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