Identification of potential functional peptides involved in demyelinating injury in the central nervous system

Abstract

Multiple sclerosis (MS) is a chronic inflammatory neurologic disease characterized by the demyelinating injury of the central nervous system (CNS). It was reported that the mutant peptide came from myelin proteolipid protein (PLP) and myelin basic protein (MBP) might play a critical role in immunotherapy function of MS. However, endogenous peptides in demyelinating brain tissue of MS and their role in the pathologic process of MS have not been revealed. Here, we performed peptidomic analysis of freshly isolated corpus callosum (CC) from the brains of CPZ-treated mice and normal diet controls of male C57BL/6 mice by LC-MS/MS. Identified a total of 217 peptides were expressed at different levels in MS mice model compared with controls. By performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, we found that the precursor protein of these differently expressed peptides (DEPs) were associated with myelin sheath and oxidative phosphorylation. Our study is the first brain peptidomic of MS mice model, revealing the distinct features of DEPs in demyelination brain tissue. These DPEs may provide further insight into the pathogenesis and complexity of MS, which would facilitate the discovery of the potential novel and effective strategy for the treatment of MS.

Keywords: Central nervous system; Demyelinating injury; MBP; Multiple sclerosis; Peptides.

Figure 1. Characterize the cuprizone induced demyelination of central nervous system in mice.

Schematic illustration of the experimental protocols, the mice fed with normal diet and 0.2% cuprizone administration diet in control group and cpz-induced group for 8 weeks, respectively (A). The body weight of the mice was measured weekly according to the experimental requirement (B). IF staining of MBP, MAG, MOG and GFAP in the corpus callosum of normal fed mice compared with cuprizone fed group (C). Luxol fast blue (LFB) staining of the CPZ-fed and control groups in the corpus callosum region (D). Western blot analysis of the protein expression of MBP, MAG, MOG and GFAP in the control group and cpz-induced group of corpus callosum (E). Statistical analysis of MBP, MAG, MOG and GFAP protein expression level by grey value analysis (F). *p < 0.05, **p < 0.01, scale bar 50 μm.

Figure 2. Hierarchical clustering and volcano plot of differentially expressed peptides.

Each row represents a differentially changed polypeptide, and the polypeptide sequence is listed on the right side of each row (A). Volcano plot of differentially expressed peptides (B).

Figure 3. Characteristics and cleavage patterns of DEPs.

Distribution of the molecular weights of DEPs (A). Distribution of the isoelectric points of DEPs (B). Distribution of isoelectric point vs. molecular weight in DEPs (C). Peptide numbers for each protein precursor of DEPs (D). Features of the cleavage sites in upregulated DEPs (E). Features of the cleavage sites in downregulated DEPs (F).

Figure 4. GO and KEGG pathway analyses of precursor proteins of DEPs.

The top 10 enrichment biological process (BP) categories (A). The top ten enrichment cellular component (CC) categories (B). The top 10 enrichment molecular function (MF) categories (C). The top 10 enrichment pathway analysis (D).

Figure 5. Relative intensity of 11 peptides derived from MBP and one peptide derived from MAG in the CC of CPZ-treated mice.

*p < 0.01, **p < 0.05.

Figure 6. Interaction network analysis of precursor proteins of differentially expressed peptides according to STRING.

The interaction network analysis of precursor proteins of DEPs involved in myelin sheath progress (A). The interaction network analysis of precursor proteins of all DEPs (B).