Introduction

Myelin-associated glycoprotein (MAG, Siglec-4) is a protein of the Siglec family and a functional ligand of Nogo-66 receptors. MAG is a membranous glycoprotein produced by Schwann cells in the peripheral nervous system and by oligodendrocytes in the central nervous system and is selectively localized in myelin near the surface of axons (Quarles,2007; Akbik et al., 2012). The special location of MAG on cell membranes indicates the functional interaction of glia and axons both in the peripheral nervous system and the central nervous system. Many published studies on MAG have focused on its ability to inhibit axonal regeneration(Fujita et al., 2011; Geoffroy and Zheng, 2014; He et al.,2016). Notably, a study by Liu et al. (2002) demonstrated that the inhibitory effect of MAG on neuritogenesis is mediated by its binding to Nogo receptors (NgR).

MAG and two other proteins, oligodendrocyte myelin glycoprotein and neurite outgrowth inhibitor (Nogo), are known as major myelin-associated inhibitors (Filbin, 2006).Although these three inhibitors have distinct structures, all act via binding to NgRs or receptor complexes (Quarles,2009). It has been verified that NgR receptor complexes include NgR, p75, Lingo-1, and trisialoganglioside GT1b (Cao et al., 2010, 2016). Recently, paired immunoglobulin-like receptor B (PirB), another membranous protein located on neuronal membranes, was also found to contain a binding motif of MAG. The signals generated from the binding of MAG and NgR complexes are mainly transduced through p75 by activating RhoA and related downstream effectors,eventually leading to changes in the actin cytoskeleton that underlies growth cone collapse or repulsion (Nieder?st et al., 2002). Therefore, the activation of RhoA and its downstream signaling pathway is critical for the inhibitory action of MAG on the growth of neuronal processes. Commercially available MAG-Fc is a recombinant chimeric protein of MAG and the Fc of IgG. Because of the Fc fragment, it is not clear whether MAG-Fc has the same properties as endogenous MAG. Therefore, we tested the actions of MAGFc in neuro-2a cells, a cell line originating from a mouse neuroblastoma, to verify the availability of MAG-Fc in vitro.We also studied the effect and mechanism of MAG-Fc on neuritogenesis.

Materials and Methods

Cell culture

Neuro-2a cells, purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were plated in 94-well plates at a density of 1 × 10–4cells/well and in 6-well plates at a density of 1 × 10–6cells/well. The cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM)supplemented with 10% fetal bovine serum (ExCell Bio,Auckland, New Zealand), 1% penicillin/streptomycin (Solarbio, Beijing, China), and 2 mM glutamine at 37°C in a humidified 5% CO2incubator (Thermo, Waltham, MA, USA).The medium was refreshed every three days, and cells were passaged at 80% confluency using 0.25% trypsin (TransGen,Beijing, China).

Immuno fluorescence

Immunofluorescence staining of MAG, NgR, PirB, and Rho-associated protein kinase (ROCK) was fly, neuro-2a cells in 96-well plates were harvested after culturing for 1 day. Then, the cells were rinsed twice with 0.01 M chilled phosphate buffered saline (PBS), followed by fixation with 4% paraformaldehyde for 30 minutes. The cells were then incubated for 10 minutes with 0.1% TritonX-100 in PBS for membrane permeability. Afterwards, 10% normal serum was used to block non-specific staining. The cells were incubated with primary antibodies for MAG (rabbit anti-MAG, 1:400; Millipore Corporation, Billerica, MA,USA), NgR (rabbit anti-NgR, 1:500; Millipore Corporation),PirB (goat anti-PirB, 1:200; Santa Cruz Biotechnology, Inc.,Dallas, TX, USA) or ROCK (rabbit anti-ROCKII, 1:500;Abcam Inc., Cambridge, MA, USA) at 4°C overnight. After washing, donkey anti-rabbit or donkey anti-goat IgG conjugated with Alexa Fluorescence-488 or -594 (1:500; Life Technologies, Carlsbad, CA, USA) was applied to the cells and incubated for 1 hour at room temperature in the dark.The cells were mounted with ProLong-Gold antifade reagent containing DAPI (Invitrogen). Images were captured under an inverted fluorescence microscope (IX71, Olympus, Tokyo, Japan). Six wells were used for each primary controls incubated with antibody diluents instead of the primary antibody were also included.

Visualization of MAG and its intervention on neurite outgrowth

Commercially available recombinant rat MAG-Fc chimera (R&D System, Minneapolis, MN, USA), which shows the same bioactivity as natural MAG (Vinson et al., 2001),was used as a source of exogenous MAG. The expression and semi-quantification of MAG-Fc in neuro-2a cells were verified using anti-MAG immunofluorescence (mouse anti-MAG monoclonal antibody, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). MAG-Fc was diluted to different working concentrations (5, 10, 20, 30 and 40 nM) with DMEM. MAG-Fc was added to the medium when most of the neuro-2a cells were attached to the well bottom, which occurred by 4 hours after plating.