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. 2020 Aug;40(6):897-909. doi: 10.1007/s10571-019-00780-7. Epub 2020 Feb 3.

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.

Keywords: Alpha-synuclein; Cyclin-dependent kinase 5; Parkinson's disease; Phosphoproteomics; Substantia nigra pars compacta.

MeSH terms

  • Animals
  • Cyclin-Dependent Kinase 5 / metabolism*
  • Databases, Protein
  • Disease Models, Animal
  • Down-Regulation
  • Gene Ontology
  • Humans
  • Male
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Parkinson Disease
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proteomics*
  • Signal Transduction*
  • Substrate Specificity
  • Up-Regulation
  • alpha-Synuclein / metabolism*

Substances

  • Phosphoproteins
  • alpha-Synuclein
  • Cyclin-Dependent Kinase 5