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.

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

Fig. 1

Overview of the phosphoproteomics analysis in transgenic and wildtype mice. a Schematic outline of the workflow for the quantitative phosphoproteomics analysis. PD Parkinson’s disease group, CON Control group. b Representative image of reverse transcriptase PCR genotyping results. The left-most lane represents the DNA ladder. c Latency of falling in the rotarod test for the CON and PD groups. d Coomassie Blue staining of polyacrylamide gels, showing protein samples extracted from substantia nigra pars compacta tissue. The protein content in CON1, CON2, PD1 and PD2 group is almost similar

Fig. 2

Quality control indices for the quantitative phosphoproteomics dataset. a Image scatter plot showing the distribution of mass error (ppm) and the peptide scores for the phosphorylation proteomics. b Histogram of the distribution of the lengths of the tryptic peptides. c Numbers of phosphorylated sites per protein for the proteins analyzed. d Data reproducibility reflected by red–green heatmap obtained from Pearson correlation analysis

Fig. 3

Phosphorylation sites differentially quantitated between the alpha-synuclein transgenic and wildtype mice. a Diagram showing the numbers of differentially quantitated phosphorylation sites. b Distribution of the differentially quantitated phosphorylation proteins. c A 10 × 10 dot plot depicting the subcellular localization analysis of the differentially quantitated phosphorylation proteins. d Heatmap visualizing the relative abundances of the differentially quantitated phosphorylation sites in all groups

Fig. 4

Bioinformatics analysis of the upregulated phosphoproteins. a Histogram showing the top ten Gene Ontology (GO) items with respect to the upregulated phosphorylation proteins, obtained from the STRING online database. The molecular function, cellular component, and biological process are shown in red, green, and blue, respectively. b The top ten pathways in the KEGG pathway enrichment analysis for the upregulated phosphorylation proteins, obtained from the STRING online database. c The weblog motif for an alignment of 356 input sequences identified in the phosphoproteomics analysis. The modification site was centrally located for the statistical analysis and the pre-aligned sequences were submitted as 13 residues

Fig. 5

Functional classification of the downregulated phosphoproteins. a Diagram showing the detailed rankings of three Gene Ontology (GO) vocabularies. b KEGG pathway enrichment analysis of the downregulated phosphorylation proteins. c Amino acid sequence pattern for the downregulated phosphorylation sites with modified serine/threonine at position 0

Fig. 6

Scansite-based database search of potential upstream kinases that might act on the differentially quantitated phosphopeptides. a Heatmap depicting the numbers of potential upstream kinase profiles with upregulated and downregulated phosphorylation sites. The numeric values in white represent the amount of predicted times with the corresponding upstream kinase. b Cdk5–substrate interaction network for upregulated (red) and downregulated (green) phosphorylation sites, analyzed with Cytoscape

Fig. 7

Cdk5 was activated in PD model and it phosphorylates Ser1889 of Ank2. a Expression level of p25 protein was detected by western blots in MPP⁺-induced primary cortical neurons at 0, 0.5 h and 1 h. The β-actin as loading control. b The intensity of western blots with p25 was statistically quantified (n = 3; one-way ANOVA, F = 20.44, P = 0.0021; post hoc Tukey’s multiple comparisons test, 0 vs. 0.5 h, P = 0.0390, 0 vs. 1 h, P = 0.0017). c The p35 protein levels were statistically analyzed (n = 3; one-way ANOVA, F = 75.70, P < 0.0001; post hoc Tukey’s multiple comparisons test, 0 vs. 0.5 h, P = 0.0109, 0 vs. 1 h, P < 0.0001). d The p25 and tyrosine hydroxylase (TH) protein levels were quantified by immunoblotting in MPTP-induced mice model. NS, normal saline. e Quantitative analysis of immunoblotting with p25 was statistically analyzed (n = 3; unpaired t test, t = 8.281, P = 0.0012). f The immunoblotting of p35 was quantified and statistically analyzed (n = 3; unpaired t test, t = 5.543, P = 0.0052). g Mass spectrum from the synthetic peptide HPPVSPSSKTEK. The green color ‘S’ represented the phosphorylated amino acid site. All the data was showed as mean ± SD