Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Abstract

Myelin-associated glycoprotein (MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase (ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Keywords: MAG-Fc; RhoA/ROCK signaling pathways; cell culture; myelin growth inhibitors; myelin-associated glycoprotein; nerve regeneration; neural regeneration; neurite outgrowth; neuro-2a cell line; receptors for myelin-associated glycoprotein.

Figure 1

Immunofluorescence of MAG in neuro-2a cells treated with 20 nM MAG-Fc for 10 minutes to 6 hours. (A) Intracellular level and distribution of MAG detected by immunofluorescence staining (scale bar: 50 μm). Arrowheads: Positive MAG dots in the cytoplasm; arrows: positive MAG dots near the plasma membrane (green: Alexa Fluorescence-488). (B) MAG levels detected by western blotting. (C) MAG immunoreactivity. Data are expressed as the mean ± SE (one-way analysis of variance followed by t-test). **P < 0.01, ***P < 0.001, vs. 0 min (control group). MAG: Myelin-associated glycoprotein; min: minute(s); h: hour(s).

Figure 2

Expression of NgR and PirB in neuro-2a cells with or without MAG treatment. (A) Cultured neuro-2a cells showed NgR-positive immunofluorescence staining, which mainly appeared along the cell membrane. (B) There was increased NgR expression when MAG-Fc was added to the medium at cell plating. NgR staining was clearly observed in the cytoplasm. (C) Double labeling of NgR and MAG was observed in most of the NgR-positive cells. (D) Cultured neuro-2a cells also exhibited positive PirB immunofluorescence staining. In contrast to NgR, PirB immunoreactivity was primarily distributed in the cytoplasm. (E) The level of PirB staining did not increase further with MAD incubation, and was similar to the pattern observed without MAG-Fc treatment. (F) A few cells were double-labeled for PirB and MAG. Scale bars: 50 μm. Green: Anti-NgR or anti-PirB labeled with Alexa Fluorescence-488; red: anti-MAG labeled with Alexa Fluorescence-594; blue: DAPI counterstaining for nuclei; arrowhead: location of double-labeling for NgR/MAG or PirB/MAG. w/o: Without; MAG: myelin-associated glycoprotein; PirB: paired immunoglobulin-like receptor B; NgR: Nogo receptor.

Figure 3

Effects of exogenous MAG treatment on RhoA activity in neuro-2a cells (ELISA assay). Data are expressed as the mean ± SE (n = 6 per group; one-way analysis of variance followed by t-test). **P < 0.01, ***P < 0.001. ELISA: Enzyme-linked immunosorbent assay; MAG: myelin-associated glycoprotein; ns: not significant; min: minutes.

Figure 4

Effects of exogenous MAG-Fc on the phosphorylation of ROCK II in neuro-2a cells. (A, B) Western blot assay showed that after MAG-Fc was administered, increased p-ROCK II levels were observed compared to the control (*P < 0.05, vs. control group), but the increased level dropped after MAG treatment for 40 minutes, although the level remained higher than controls. Data are expressed as the mean ± SE (one-way analysis of variance followed by t-test). (C) Immunofluorescence of cytoplasmic p-ROCK II in the MAG treatment group was generally stronger than that in controls. Some p-ROCK II foci were clearly identified in the cytoplasm, especially in the area adjacent to the membrane (arrows). (a–e) DAPI counterstaining; (f–g) anti-p-ROCK; (k–o) merged images of DAPI (blue) and anti-p-ROCK (green). Scale bar: 50 μm. Green color: Alexa Fluorescence-488; blue color: DAPI counterstaining of nuclei. MAG: myelin-associated glycoprotein; p-ROCK II: phosphorylated Rho-associated protein kinase II; DAPI: 4′,6-diamidino-2-phenylindole; min: minutes.

Figure 5

Linear dose effects of MAG-Fc on neurite outgrowth from neuro-2a cells. (A) Neuro-2a cells treated with a series of MAG-Fc concentrations from 5–40 nM for 24–72 hours showed dose-dependent inhibition of neurite growth based on anti-βIII tubulin (red) immunofluorescence staining (Alexa Fluorescence-594 labeling). Scale bar: 50 μm. (B) The total neurite length of neuro-2a cells was decreased at 24 hours by 5–40 nM MAG treatment; the decrease in neurite length also appeared to be dose-dependent, although there was no significant difference with incubation time. (C) Adding MAG-Fc to neuro-2a cells also decreased the percentage of cells with neurites. The drop in the percentage was clearly observed at 48 and 72 hours of MAG treatment. Data are expressed as the mean ± SE (one-way analysis of variance followed by t-test). *P < 0.05, **P < 0.01, ***P < 0.001, vs. 0 nM group. MAG: Myelin-associated glycoprotein; h: hours.