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

Animals

Six-week-old male C57BL/6 mice were purchased from SPF (Beijing) Biotechnology Co., LTD (Beijing, China) and raised and maintained for 2 weeks on the ventilated conditions (22 °C, 40–60% humidity, 12 h light/dark) before experiment beginning. Eight-week-old mice were randomization divided into two groups (n = 6 mice/group), the experimental animals were numbered and assigned into the control group and the experimental group through random selection of numbers. The control group fed with normal rodent food, Cuprizone (CPZ) groups were fed with 0.2% CPZ-containing (Sigma, St. Louis, MO, USA) diet for 8 weeks to induce demyelination. Mice were weighed on a daily basis. Animals were sacrificed by dislocation of cervical vertebra for the experiments right after the end of 8 weeks-fed. In this study, six mice were used in each treatment group. Three were sacrificed for histological and IF study, while the other three were sacrificed for LC-MS study. There were no exclusions in each experimental group. All mice were sacrificed by cervical dislocation after the experiment. The animal and the protocol used in this study was approved by the Animal Ethics Committee of Tongren Hospital in China (approval no. 2022-045-01).

Western blot analysis

Western blot was performed as we previously reported (Zhao et al., 2023). Briefly, total protein was extracted from the corpus callosum of brain tissues by radioimmunoprecipitation assay lysis (RIPA) (Beyotime, Jiangsu, China) buffer containing phenylmethyl sulfonylfluoride (PMSF) (Beyotime, Jiangsu, China) on ice for 30 min. Then, the protein was centrifuged at 4 °C at 12,000 g for 10 min and the supernatant was collected. SDS-PAGE protein buffer (Beyotime, Jiangsu, China) added to the total protein and boiling at 100 °C for 5 min. A total of 12% SmartPAGE^TM Precast Protein Gel (Smart Lifescience, Jiangsu, China) were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following the manufacturer’s protocol. About 10 μg protein of each sample were loaded in each lane of western blot. Antibodies of MAG (9043S, 1:1,000; Cell Signaling Technology (CST), Danvers, MA, USA), MOG (45268S, 1:1,000; CST, Danvers, MA, USA), MBP (78896S, 1:1,000; CST, Danvers, MA, USA), GFAP (80788S, 1:1,000; CST, Danvers, MA, USA), β-Actin (81115-1-RR, 1:1,000; Proteintech, Rosemont, IL, USA) were used for detected the relative protein expression. The relative protein expression was normalized to β-Actin.

Immunofluorescence

After the animals were routinely anesthetized and the hearts perfused, mouse brains were harvested and fixed with 4% PFA, followed by the corpus callosum region. IF were performed as reported previously (Ai et al., 2022). For immunofluorescence assays, paraffin section (4 μm) of brain tissues were performed for IF. The slices after baking, dewaxing, and gradient alcohol rehydration are first heated in 1X EDTA antigen retrieval solution to detect the minimum value of the antigen. Then, the section was rinsed with phosphate buffered saline (PBS) at room temperature, soaked in TritonX-100 0.5% liquid and put it in PBS for 15 min for further permeation. After inactivating the endogenous peroxidase with 3% H₂O₂ for 10 min, the sections were washed three times with PBS, blocked with 5% BSA for 30 min, incubated with the primary antibody overnight at 4 °C, washed three times with PBS at room temperature, and the second antibody was added and incubated at 37 °C for 50 min and rinse with PBS three times. Finally, after DAPI was counter-stained for 10 min, rinsed with PBS three times, dropwise anti-fluorescence quencher was added, and the slides were mounted before microscopy. Images were captured by Nikon Eclipse E600 inverted fluorescent microscope.

Antibodies of MAG (9043S, 1:100; Cell Signaling Technology (CST), Danvers, MA, USA), MOG (45268S, 1:200; CST, Danvers, MA, USA), MBP (78896S, 1:100; CST, Danvers, MA, USA), GFAP (80788S, 1:50; CST, Danvers, MA, USA) were used for IF.

Luxol fast blue (LFB) staining

Paraffin sections (4 μm) deparaffinized and incubated in LFB solution (ServiceBio, Wuhan, Hubei, China) for 4 h at 60 °C. After cooling for 15 min, the slides were washed with running water for 1 min, and soak in 70% water soak in ethanol for 10 s. Then, the sections were rinsed again and immersed in lithium carbonate solution for 1 min. This step was repeated three times until the background color fades. Finally, the slices are dehydrated and covered with neutral balsam. These images were observed by an optical microscope (Nikon, Minato City, Tokyo, Japan).

Samples preparation for LC-MS

The corpus callosum of brain tissues samples were ground into powder by adding liquid nitrogen, adding TRIS-HCl according to the volume ratio of 1:3, heating and boiling for 10 min, and then crushed by ultrasound at 100 HZ in ice water bath, over 5 s, at an interval of 5 s, ultrasonic for 2 min. Then, the final concentration of 1 M ice ethyl acid was added into the sample tube, and vortex for 2 min. Add the final concentration of about 50% acetonitrile. Centrifuge at 12,000g at 4 °C for 10 min, remove the supernatant and transfer it to a clean EP tube for freeze-drying. Add 80% acetone solution, vortex, shake, water bath ultrasound for 2 min, then, 20,000g centrifuge for 30 min at 4 °C, take the supernatant, transfer to a clean EP tube, freeze drying. Redissolved with 200 μL 0.1% TFA solution, desalt with 80 μg C18. Freeze-dried for LC-MS.

Liquid chromatography-mass spectrometry (LC-MS) analysis

Peptide analysis was performed with the Q Exactive HF (Thermo Fisher Scientific, Waltham, MA, USA) as previously reported (Lyratzakis et al., 2021). For LC-MS/MS injection, 2.5 μg dried peptide fractions were dissolved in 2% acetonitrile with 0.1% formic acid, and subsequently loaded on reverse phase columns (trapping cartridge 5 µm C18-beads, L = 5 mm, inner diameter = 100 µm; Thermo Fisher Scientific, Waltham, MA, USA), analytical column (C18, 3 µm, 150 mm × 75 µm; Eksigent, Dublin, CA, USA). Eluted peptides were separated over a 78 min gradient of water (buffer A: water with 0.1% formic acid) and acetonitrile (buffer B: acetonitrile with 0.1% formic acid). Typically, gradients were ramped from 5% to 32% B in 70 min at flowrates of 600 nL min⁻¹. Electrospray voltage of 2.0 kV vs. the inlet of the mass spectrometer was used. The mass spectrometer was operated in data-dependent acquisition mode to automatically switch between Orbitrap-MS and ion trap acquisition. Survey of full-scan MS spectra (from m/z 300 to 1,400) were acquired in the Orbitrap with a resolution of R = 120,000. Target ions already selected for MS/MS were dynamically excluded for 18 s and the minimum intensity was 5,000.

Bioinformatics analysis

Bioinformatics analysis were performed as previously reported (Dong et al., 2021). Briefly, UniProt (http://beta.uniprot.org, Release 2021_03) were used for identified the peptide precursor protein. Isoelectric point (pI) and Mw of DEPs were calculated by the ProParm tool (https://web.expasy.org/protparam/). GO and KEGG pathways analysis of DEPs precursor protein was predicted by websites (http://geneontology.org/ and http://www.kegg.jp/). STRING (https://string-db.org) were used for predicted the interaction of DEPs precursor protein. Interactions was set up based on the experiments, databases, co-expression, neighborhood, gene fusion and co-occurrence. The heatmap and volcano plot of DEPs were generated by Version 3.2.2 of R software. The predict bioactive of DEPs were predicted by PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/).

Statistical analysis

Data were analyzed by the GraphPad Prism with unpaired t-test. A p < 0.05 was indicated as a statistically significant difference.

Establishment of cuprizone-induced demyelination of central nervous system (CNS) mouse model

To investigate whether the endogenous peptides participate in the demyelination of CNS, we established a mouse model of demyelination with fed a diet containing 0.2% CPZ with normal rodent chow for 8 weeks (Fig. 1A). The body weight of the mice was measured weekly during the CPZ-fed period. During the first week of CPZ treated, there was no significant difference in body weight between CPZ-fed mice and control (20.5 ± 0.57 g vs. 21.1 ± 1.3, Fig. 1B). However, the weight growth of CPZ-induced mice was significantly lower than that of control group after 2 weeks fed (Fig. 1B). The brain tissue of corpus callosum was selected as a representative region of white matter to perform IF, Luxol fast blue (LFB) staining and western blot because it has been extensively examined in CPZ-induced animal model (Morell et al., 1998; Han et al., 2020a). IF analysis revealed that a strong loss of myelin marker MBP, MOG (Myelin oligodendrocyte glycoprotein) and MAG expression were detected in CPZ treatment compared with control (Fig. 1C), and a strong increase of glial fibrillary acidic protein (GFAP) positive area in the CPZ group compared with control group (Fig. 1C). In addition, LFB staining of brain sections showed that the well characteristic rows of interfascicular oligodendrocytes in CPZ diet group was significantly decreased than that in normal diet group (Fig. 1D). Similarly, the expression of MBP, MOG and MAG were decreased in the CC of MS mice while the expression of GFAP was dramatically increased in the CPZ-induced mice (Figs. 1E and 1F). Taken together, these results suggest that 8 weeks of CPZ-diet induced the demyelination of CNS.

Diverse expression patterns of peptides in cuprizone-induced demyelination

To evaluate the peptide profile of CPZ-induced demyelination, corpus callosum of mouse brain was isolated, and peptides were detected by LC-MS technology. A total of 6,796 peptides were identified and 6,729 peptides were quantified. Furthermore, the expression of 217 peptides was significantly changed (fold change ≥ 2, p ≤ 0.05) in CPZ-induced mice compared with controls, which including 181 up-regulated peptides and 36 down-regulated peptides (Figs. 2A and 2B). The upregulated peptides (fold change ≥ 5) and downregulated peptides (Fold Change ≤ 0.04) in CPZ-treatment mice compared with the control group are shown in Table 1, respectively.

Characteristics of differentially expressed peptide

The physical and biochemistry characteristics of 217 DEPs were analyzed. The results showed that the molecular weight (Mw) of the major up-regulated DEPs in CPZ group was concentrated in the range of 1,000 to 3,000 D (Fig. 3A). However, the molecular weights of the down-regulated peptides were mostly concentrated in the range of 1,400 to 2,600 D (Fig. 3A). The isoelectric point of DEPs was mostly distributed in the range of 3 to 12 (Fig. 3B). Furthermore, the Mw vs. pI of DEPs was distributed within the area of 2 to 6 and 8 to 12 (Fig. 3C). Endogenous peptides were usually produced by protein precursors, we identified 35 proteins that generated more than two peptides. In particular, 11 differentially expression proteins were produced by MBP (Fig. 3D) (Table 2). According to the analysis of N-terminal and C-terminal amino acid of the up-regulated DEPs, it was determined that alanine (A), arginine (R), leucine (L), lysine (K), serine (S) and valine (V) appeared more frequently (Fig. 3E). Similarly, alanine (A), arginine (R), leucine (L), lysine (K) and serine (S) were also the amino acid with high frequently in the N-terminal and C-terminal amino acid of down-regulated DEPs (Fig. 3F).

GO and KEGG pathway analysis

To gain overall insights into the function differences between these differentially expressed peptides, we preformed GO and KEGG pathways analysis using precursor proteins of all 217 discrepancy expressed peptides. Systematic analysis of cellular component, molecular function and biological process showed that myelin sheath, microtubule binding and transport are the most prominent aspects that are correlated with changes in CNS function, respectively (Figs. 4A–4C). KEGG analysis uncovered the distinct expressed peptides related to oxidative phosphorylation, Alzheimer’s disease and Parkinson’s disease (Fig. 4D).

Given that myelin related protein is crucial in maintaining OLs states, and the DEPs function enriched in myelin sheath, we next analyzed myelin-related protein derived peptides expressed levels in CPZ-induced mice compared with control. A total of 11 peptides derived from MAB and one peptide derived from MAG were differently expressed in the CC of CPZ-treated mice (Fig. 5). As shown in Table 2, DEPs of MAB and MAG were localized at Myelin_HBP and IgV_CD33 functional domain, respectively. The sequences of peptide excreted from MBP_157–175, MBP_148–160, MBP_163–177, MBP_110–125, MBP_150–164, MBP_151–175 and MAG_78–90 were homologous with human MBP or MAG protein, respectively. Furthermore, PeptideRanker was used for predicted the bioactivity of these peptides, among 13 DEPs, the predicted bioactivity of MBP_157–175 peptide (RHGFLPRHRDTGILDSIGR) were more than 0.5, indicated that MBP_157–175 peptide may participate in the mechanisms of disease progression.

Interaction network analysis

MS is mainly caused by demyelination injury of CNS. GO analysis of DEPs precursor protein revealed that 34 proteins were involved in myelin sheath process. To uncover the potential interaction between these DEPs which involved in myelin biological processes, STRING was used to conduct association network analysis of the precursor proteins of these DEPs. The results shown that these 34 proteins had interaction with each other (Fig. 6A). In addition, we also found that these 34 proteins paly a core role in the whole DEPs precursor protein interaction (Fig. 6B). Taken together, these results suggest that those DEPs involved in the progress of myelin sheath may play an important role in MS.