Quantitative Phosphoproteomic Analysis in Alpha-Synuclein Transgenic Mice Reveals the Involvement of Aberrant p25/Cdk5 Signaling in Early-stage Parkinson’s Disease

Abstract

A30P and A53T mutations in the gene encoding alpha-synuclein—a presynaptic protein—are the most frequently identified genetic causes of Parkinson’s disease (PD). Aberrant alpha-synuclein likely plays central roles in dopaminergic neuronal death and motor symptoms in PD. This study investigated the protein phosphorylation profile in early-stage PD through phosphoproteomic analyses of tissue samples from the substantia nigra pars compacta (SNpc) of 6-month-old alpha-synuclein transgenic mice (A30P/A53T double-mutant human alpha-synuclein; hm2α-SYN-39 strain). We identified 5351 phosphorylation sites in 2136 phosphoproteins. Of these, 357 upregulated sites in 245 proteins and 50 downregulated sites in 46 proteins were differentially phosphorylated between alpha-synuclein transgenic and wildtype mice. Bioinformatic analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, and motif analyses, were used to elucidate the molecular and cellular mechanisms underlying double-mutant human alpha-synuclein overexpression. Scansite-based computational analysis and prediction of differentially quantitated phosphoproteins identified the neuronal protein cyclin-dependent kinase 5 (Cdk5) as the most significantly enriched kinase. Biochemical experiments suggested that the p25/Cdk5 pathway was activated in an MPP+-induced cell culture model and MPTP-induced mouse model. Moreover, Cdk5 could directly phosphorylate the Ank2 protein at Ser1889 in vitro. Therefore, quantitative phosphoproteomic using an alpha-synuclein transgenic mouse model offers a powerful approach for elucidating the protein phosphorylation mechanism underlying SNpc dopaminergic neuronal death in an animal model of PD.

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Data Availability

The MS proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PXD013965.

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Acknowledgements

We also thank the members of the PTM BioLabs for technical support and helpful discussion and we would like to thank Xiaojun Peng (Department of Proteomics, PTM BioLabs, Inc.) for the guidance about bioinformatics analyses.

Funding

This work was supported financially by grants from National Natural Science Foundation of China (No. 31571044,31871073 to B.T., and No. 31600821 to P.Z.), Program for New Century Excellent Talents in University (No. NCET-10–0415 to B.T.), China Postdoctoral Scientific Foundation (No. 2018T110774 to P.Z.), Natural Science Foundation of Hubei Province (No. 2017CFB465 to P.Z.), Educational Commission of Hubei Province of China (No. D20182102 to P.Z.) and the Fundamental Research Funds for the Central Universities (HUST: 2019kfyXJJS081 to P.Z.).

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FH, GQ, QZ and PZ conducted the breeding and reproduction of wild-type and transgenic mice. FH and QZ performed the experiments of mice genotyping and behavior. GQ, QZ, JM, BT and PZ analyzed the data by using bioinformatics and statistical method. HC, TL, ML, JC and JM contribute to the generation of all figures and tables. JM, BT and PZ designed the study and wrote the manuscript.

Corresponding authors

Correspondence to Bo Tian or Pei Zhang.

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The authors declare that they have no conflict of interest.

Research Involving Animal Participants

All the procedures were in line with institutional guidelines and were approved by the Animal Care and Use Committee of the core animal facility (Tongji Medical College, Huazhong University of Science and Technology, China).

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He, F., Qi, G., Zhang, Q. et al. Quantitative Phosphoproteomic Analysis in Alpha-Synuclein Transgenic Mice Reveals the Involvement of Aberrant p25/Cdk5 Signaling in Early-stage Parkinson’s Disease. Cell Mol Neurobiol 40, 897–909 (2020). https://doi.org/10.1007/s10571-019-00780-7

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Keywords

  • Phosphoproteomics
  • Alpha-synuclein
  • Parkinson's disease
  • Substantia nigra pars compacta
  • Cyclin-dependent kinase 5