Quantitative proteomics in A30P*A53T α-synuclein transgenic mice reveals upregulation of Sel1l

Abstract

α-Synuclein is an abundantly expressed neuronal protein that is at the center of focus in understanding a group of neurodegenerative disorders called synucleinopathies, which are characterized by the intracellular presence of aggregated α-synuclein. However, the mechanism of α-synuclein biology in synucleinopathies pathogenesis is not fully understood. In this study, mice overexpressing human A30P*A53T α-synuclein were evaluated by a motor behavior test and count of TH-positive neurons, and then two-dimensional liquid chromatography-tandem mass spectrometry coupled with tandem mass tags (TMTs) labeling was employed to quantitatively identify the differentially expressed proteins of substantia nigra pars compacta (SNpc) tissue samples that were obtained from the α-synuclein transgenic mice and wild type controls. The number of SNpc dopaminergic neurons and the motor behavior were unchanged in A30P*A53T transgenic mice at the age of 6 months. Of the 4,715 proteins identified by proteomic techniques, 271 were differentially expressed, including 249 upregulated and 22 downregulated proteins. These alterations were primarily associated with mitochondrial dysfunction, oxidative stress, ubiquitin-proteasome system impairment, and endoplasmic reticulum (ER) stress. Some obviously changed proteins, which were validated by western blotting and immunofluorescence staining, including Sel1l and Sdhc, may be involved in the α-synuclein pathologies of synucleinopathies. A biological pathway analysis of common related proteins showed that the proteins were linked to a total of 31 KEGG pathways. Our findings suggest that these identified proteins may serve as novel therapeutic targets for synucleinopathies.

Fig 1. Behavioral and immunohistochemical analysis of transgenic mice and their wild-type littermates.

(A) RT-PCR was used to verify the mRNA expression of α-synuclein-A30P*A53T transgenic mice (TG) and wild-type littermates (WT). (B) The rotarod test was used to measure TG mice and WT controls at the age of 6 months (n = 15). (C) Grip strength tests were performed in TG mice and WT controls (n = 15). (D and E) Open field tests were performed in TG mice and WT controls (n = 15). (F) Immunostaining of tyrosine hydroxylase (TH)-positive neurons of SNpc in TG mice and WT controls. (G) The number of TH-immunoreactive positive neurons in the SNpc was counted stereologically (n = 3).

Fig 2. Proteomic process flow chart and standardized sample evaluation.

(A) Depiction of the experimental design workflow. (B) Data reproducibility reflected by Pearson correlation coefficients. (C) Mass error distribution of all identified peptides. (D) Peptide length distribution.

Fig 3. Heatmap and the subcellular locations of proteins.

(A) Heatmap of proteins identified as significantly differentially regulated (p < 0.05). (B) Subcellular locations of all identified proteins. (C) Subcellular locations of upregulated proteins. (D) Subcellular locations of downregulated proteins.

Fig 4. GO-based enrichment analysis.

(A) GO-based enrichment analysis of upregulated proteins. (B) GO-based enrichment analysis of down-regulated proteins.

Fig 5. KEGG pathway-based enrichment analysis.

(A) Pathway-based enrichment of upregulated proteins. (B) Pathway-based enrichment of downregulated proteins.

Fig 6. Validation of altered proteins in the SNpc tissue of TG mice and WT controls by western blotting and immunofluorescence staining.

(A) The protein levels of Sel1l were detected by western blot in the SNpc tissue of TG mice and WT controls. (B) Quantification of the protein levels of Sel1l from (A) (n = 3). (C) The protein levels of Sdhc were measured by western blot in the SNpc tissue of TG mice and WT controls. (D) Quantification of the protein levels of Sdhc from (C) (n = 3). (E) Sel1l was monitored by dual immunolabeling of TH (red) and Sel1l (green) in the SNpc regions of TG mice and WT controls. (F) Sdhc was assessed by dual immunolabeling of TH (red) and Sdhc (green) in the SNpc regions of TG mice and WT controls.