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Post-translational modifications of soluble α-synuclein regulate the amplification of pathological α-synuclein

Abstract

Cell-to-cell transmission and subsequent amplification of pathological proteins promote neurodegenerative disease progression. Most research on this has focused on pathological protein seeds, but how their normal counterparts, which are converted to pathological forms during transmission, regulate transmission is less understood. Here we show in cultured cells that phosphorylation of soluble, nonpathological α-synuclein (α-Syn) at previously identified sites dramatically affects the amplification of pathological α-Syn, which underlies Parkinsonʼs disease and other α-synucleinopathies, in a conformation- and phosphorylation site-specific manner. We performed LC–MS/MS analyses on soluble α-Syn purified from Parkinsonʼs disease and other α-synucleinopathies, identifying many new α-Syn post-translational modifications (PTMs). In addition to phosphorylation, acetylation of soluble α-Syn also modified pathological α-Syn transmission in a site- and conformation-specific manner. Moreover, phosphorylation of soluble α-Syn could modulate the seeding properties of pathological α-Syn. Our study represents the first systematic analysis how of soluble α-Syn PTMs affect the spreading and amplification of pathological α-Syn, which may affect disease progression.

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Fig. 1: Soluble α-Syn phosphorylation modulates pathological α-Syn amplification.
Fig. 2: Soluble α-Syn phosphorylation modulates GCI-α-Syn and LB-α-Syn amplification.
Fig. 3: Systematically identify PTMs on soluble α-Syn from different α-synucleinopathies.
Fig. 4: Soluble α-Syn acetylation modulates pathological α-Syn amplification.
Fig. 5: The effect of soluble α-Syn PTMs on pathological α-Syn transmission is conformation dependent.
Fig. 6: PTMs of soluble α-Syn modify the spreading of pathological α-Syn in primary neurons.
Fig. 7: PTMs on soluble α-Syn modify the seeding properties of pathological α-Syn.

Data availability

Peptide identification was performed in MaxQuant (1.6.1.0) using human reference database from Uniprot (reviewed canonical and isoforms; downloaded on 1 January 2018) and the search results were exported into Scaffold 4 (Proteome Software). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE37 partner repository with the dataset identifier PXD037994. Figure 3 and Supplementary Tables 48 are associated with LC–MS/MS data. Source data are provided with this paper.

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Acknowledgements

We thank Quantitative Proteomics Resource Core of School of Medicine at the University of Pennsylvania and Proteomics Core at Children’s Hospital of Philadelphia (CHOP) for performing the LC–MS/MS analysis. We thank S. Seeholzer, L. Spruce, H. Ding, H. Fazelinia and H. Lee (from CHOP) for helping with LC–MS/MS experiments. We thank D. Riddle for providing primary neurons, L. Romero for helping with quantification and all the other members of the Center for Neurodegenerative Disease Research for their support. We thank Lucy Ruoxi Shi for helping with the cartoon illustration of this work. This work was supported by NIH/NINDS Udall Center under grant NS53488 (to V.M.-Y.L.), NIA under grant U19 AG062418 (Center on Alpha-Synuclein Strains in Alzheimerʼs Disease and Related Dementias) (to V.M.-Y.L.), NIH/NINDS R01-NS103873 to E.J.P. (to V.M.-Y.L.), NIH/NINDS R01-NS128964 (to C.P.), CurePSP grant 672-2020-12 (to C.P.), the Ofer Nimerovsky Family Fund (to V.M.-Y.L.), the Jeff and Anne Keefer Fund (to V.M.-Y.L.) and the MSA Coalition Seed Grant (to C.P. and V.M.-Y.L.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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C.P., V.L., S.Z. and R.Z. conceived experiments. C.P., S.Z. and R.Z. performed most of the experiments. B.P. and E.J.P. generate pY39 and pS87 α-synuclein monomer. H.X., M.F.O., R.J.G., J.Z., W.X. and E.M.K. performed cell culture experiments. L.Z. and H.A.L. performed biochemical experiments. S.-J.K. generated PFF. C.K.W., S.M. and H.V. identified and prepared human brain tissue for the preparation of pathological α-synuclein. X.C., C.Y. and B.A.G. performed LC–MS/MS experiments. Y.L. analyzed the data. C.P., J.Q.T., V.M.-Y.L., E.J.P. and H.A.L. wrote the manuscript.

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Correspondence to Virginia M.-Y. Lee or Chao Peng.

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Zhang, S., Zhu, R., Pan, B. et al. Post-translational modifications of soluble α-synuclein regulate the amplification of pathological α-synuclein. Nat Neurosci (2023). https://doi.org/10.1038/s41593-022-01239-7

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