Two Monoclonal Antibodies Recognising aa 634-668 and aa 1026-1055 of NogoA Enhance Axon Extension and Branching in Cultured Neurons

Abstract

In a previous study, we generated two monoclonal antibodies (mAbs) in mice, aNogoA-N and aNogo-66 mAb, which were raised against recombinant N-terminal fragments of rat NogoA and Nogo-66, respectively. When compared with the commercial rabbit anti-rat NogoA polyclonal antibody (pAb), which can specifically recognise NogoA, the two mAbs were also specific for the NogoA antigen in immunofluorescence histochemical (IHC) staining and Western blot (WB) analysis. Serial truncations of NogoA covering the N-terminal region of NogoA (aa 570–691) and Nogo-66 (aa 1026–1091) were expressed in E. coli. The epitopes recognised by aNogoA-N and aNogo-66 are located in the aa 634–668 and aa 1026–1055 regions of NogoA, respectively. Both mAbs remarkably enhanced the axon growth and branching of cultured hippocampal neurons in vitro. These results suggest that the antibodies that bind to aa 634–668 and aa 1026–1055 of NogoA may have stimulatory effects on axon growth and branching. Additionally, the two mAbs that we generated are specific for NogoA and significantly block NogoA function. In conclusion, two sites in NogoA located within aa 634–668 and aa 1026–1055 are recognised by our two antibodies and are novel and potentially promising targets for repair after central nervous system (CNS) injury.

Axonal branching in lateral olfactory tract is promoted by Nogo signaling

“…Deng et al . reported that axonal branches are reduced in rat embryonic hippocampal neurons cultured on exogenous Nogo-A-coated dishes18. Because the methodology in this previous report is different from that used in the current study, this finding is inconsistent with our data.…”

“…Recent studies have shown that Nogo-NgR1 signaling plays a key role in axonal branching161718. Deng et al .…”

“…Recent studies have shown that Nogo-A induces axonal branching in cultured dorsal root ganglion neurons16 and cultured midbrain neurons17. In contrast, exogenous Nogo-A inhibits axonal branching in cultured hippocampal neurons18. Thus, Nogo-NgR1 signaling plays a key role in axonal branching of the developing brain.…”

Electroacupuncture enhances rehabilitation through miR-181b targeting PirB after ischemic stroke

“…Primary cortical neurons were cultured as previously described43. In brief, neurons were isolated from SD rat embryos (E18.5 d), washed with D-Hank’s solution three times under sterile conditions and seeded at a density of 1 × 10 5 cells/cm 2 on plates coated with poly-L-lysine (50 mg/mL) (Sigma, USA).…”

Electroacupuncture Improves Neurobehavioral Function Through Targeting of SOX2-Mediated Axonal Regeneration by MicroRNA-132 After Ischemic Stroke

“…MAP2 was abundantly expressed in dendrites and was used as dendrite marker (Harada et al, 2002), TAU was used as axon marker given its abundant expression in axons (Llorens-Martin et al, 2012), and NF200 was used as nerve fiber marker (Schumacher et al, 1999). The average lengths of the longest neurites from three independent experiments were analyzed as previously described (Deng et al, 2014).…”