The acetylome of adult mouse sciatic nerve.

Lysine acetylation is a reversible post-translational modification (PTM) involved in multiple physiological functions. Recent studies have demonstrated the involvement of protein acetylation in modulating the biology of Schwann cells (SCs) and regeneration of the peripheral nervous system (PNS). However, the mechanisms underlying these processes remain partially understood. Here, we characterized the acetylome of the mouse sciatic nerve (SN) and investigated the cellular distribution of acetylated proteins. We identified 483 acetylated proteins containing 1442 acetylation modification sites in the SN of adult C57BL/6 mice. Bioinformatics suggested that these acetylated SN proteins were mainly located in the myelin sheath, mitochondrial inner membrane, and cytoskeleton, and highlighted the significant differences between the mouse SN and brain acetylome. Manual annotation further indicated that most acetylated proteins (> 45%) were associated with mitochondria, energy metabolism, and cytoskeleton and cell adhesion. We verified three newly discovered acetylation-modified proteins, including neurofilament light polypeptide (NEFL), neurofilament medium/high polypeptide (NFM/H), and periaxin (PRX). Immunofluorescence illustrated that the acetylated proteins, including acetylated alpha-tubulin, were mainly co-localized with S100-positive SCs. Herein, we provided a comprehensive acetylome for the mouse SN and demonstrated that acetylated proteins in the SN were predominantly located in SCs. These results will extend our understanding and promote further study of the role and mechanism of protein acetylation in SC development and PNS regeneration.

Cyr61 promotes Schwann cell proliferation and migration via αvß3 integrin.

BACKGROUND: Schwann cells (SCs) play a crucial role in the repair of peripheral nerves. This is due to their ability to proliferate, migrate, and provide trophic support to axon regrowth. During peripheral nerve injury, SCs de-differentiate and reprogram to gain the ability to repair nerves. Cysteine-rich 61 (Cyr61/CCN1) is a member of the CCN family of matrix cell proteins and have been reported to be abundant in the secretome of repair mediating SCs. In this study we investigate the function of Cyr61 in SCs. RESULTS: We observed Cyr61 was expressed both in vivo and in vitro. The promoting effect of Cyr61 on SC proliferation and migration was through autocrine and paracrine mechanisms. SCs expressed αvß3 integrin and the effect of Cyr61 on SC proliferation and migration could be blocked via αvß3 integrin. Cyr61 could influence c-Jun protein expression in cultured SCs. CONCLUSIONS: In this study, we found that Cyr61 promotes SC proliferation and migration via αvß3 integrin and regulates c-Jun expression. Our study contributes to the understanding of cellular and molecular mechanisms underlying SC's function during nerve injury, and thus, may facilitate the regeneration of peripheral nerves after injury.

A proteomic view on the differential phenotype of Schwann cells derived from mouse sensory and motor nerves.

Schwann cells (SCs) are myelin-forming glial cells of the peripheral nervous system. Recent studies suggested that SCs comprise two phenotypes: sensory SCs and motor SCs, which are associated with the modality-specific promotion of sensory and motor axon growth during peripheral neuronal regeneration. However, the molecular basis of the two phenotypic SCs is unclear. We established a workflow to obtain highly purified SCs derived from sensory nerve (SNdSCs) and motor nerve (MNdSCs) from B6; D2-Tg(s100B-EGFP)1Wjt/J mice. Subsequently, a quantitative proteomic analysis based on iTRAQ labeling was performed to compare the proteome of SNdSCs and MNdSCs. A total of 6,567 proteins were identified, of which 63 and 11 proteins were overexpressed in SNdSCs and MNdSCs, respectively. Three of the overexpressed proteins were further validated by western blot and immunocytochemistry: GMFB and CNPase, which were overexpressed in sensory SNdSCs, and histone H4, which was overexpressed in MNdSCs. The expression pattern of the three proteins was also validated in the dorsal roots and ventral roots. Bioinformatics analysis indicated that proteins highly expressed in SNdSCs are mainly involved in RNA processing and protein synthesis, while those overexpressed in MNdSCs are related to cell proliferation. Real-time cell analysis confirmed that the proliferation activity of MNdSCs is higher than that of SNdSCs. This study is the first to provide a proteomic view of the differential phenotype of mouse SNdSCs and MNdSCs. The data may serve as a valuable source for the study of the biological characteristics of these two SC phenotypes and their roles in nerve-specific regeneration.

Achyranthes bidentata polypeptide k suppresses neuroinflammation in BV2 microglia through Nrf2-dependent mechanism.

BACKGROUND: Activated microglia play a critical role in regulating neuroinflammatory responses in central nervous system. Previous studies have shown that Achyranthes bidentata polypeptide k's (ABPPk's) neuroprotective effects are partly due to its anti-inflammatory effect, but the mechanism remains unknown. This study is aimed to investigate the anti-inflammatory effect of ABPPk on lipopolysaccharide (LPS)-activated neuroinflammation in BV2 microglia. METHODS: We pretreated BV2 microglia with different concentrations of ABPPk (0.04-5 µg/mL) for 30 minutes, and then stimulated microglia with LPS for 24 hours. Pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO) and prostaglandin E2 (PGE2) production were measured by enzyme-linked immunosorbent assay (ELISA) kits. Inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), phosphorylated nuclear factor kappa B (NF-κB), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) expression levels were detected by western blot. Glutathione (GSH) level was measured by GSH-Glo™ Glutathione assay. Immunofluorescent staining was used to detect the nuclear translocation of NF-κB and Nrf2. BV2 microglia transfected with Nrf2 siRNA were used to investigate the effect of Nrf2 on the anti-inflammatory activity of ABPPk. RESULTS: ABPPk (0.2-5 µg/mL) reduced the iNOS mediated NO and COX-2 mediated PGE2 production significantly in LPS-activated BV2 microglia. ABPPk (1 and 5 µg/mL) also suppressed the production of TNF-α and IL-6 significantly. NF-κB is phosphorylated and translocated into nuclear in LPS-activated BV2 microglia, but ABPPk is shown to inhibit the phosphorylation and translocation of NF-κB in a concentration-dependent way. ABPPk increased the protein expression levels of HO-1 and Nrf2, as well as the GSH content in BV2 microglia. Immunofluorescent staining showed that ABPPk also promoted nuclear translocation of Nrf2. After knocking down Nrf2 in BV2 cells with siRNA interference, ABPPk's inhibitory effect on pro-inflammatory mediators also disappeared. CONCLUSIONS: The present study suggests that ABPPk inhibits neuroinflammation in BV2 microglia through Nrf2-dependent mechanism. This provides some strong evidence for the potential of this neuroprotective natural compound to treat neurodegenerative diseases such as ischemic stroke and Parkinson's disease.