Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin

Abstract

CNS myelin contains axon outgrowth inhibitors, such as Nogo, that restrict regenerative growth after injury. An understanding of the mechanism of Nogo signaling through its receptor (NgR) is critical to developing strategies for overcoming Nogo-mediated inhibition. Here we analyze the function of NgR domains in outgrowth inhibition. Analysis of alkaline phosphatase (AP)-Nogo binding in COS-7 cells reveals that the leucine-rich repeat domain is necessary and sufficient for Nogo binding and NgR multimerization. Viral infection of embryonic day 7 chick retinal ganglion cells with mutated NgR demonstrates that the NgR C-terminal domain is required for inhibitory signaling but not ligand binding. The NgR glycosylphosphatidylinositol domain is not essential for inhibitory signaling but may facilitate Nogo responses. From this analysis, we have developed a soluble, truncated version of the Nogo receptor that antagonizes outgrowth inhibition on both myelin and Nogo substrates. These data suggest that NgR mediates a significant fraction of myelin inhibition of axon outgrowth.

Fig. 1.

Nogo binding to NgR deletion mutants.a, Schematic of WTNgR (WT) and the NgR deletion mutants used in this study. NgR mutants include deletions of the N-terminus (ΔNT), LRR domains 1 and 2 (Δ1–2), 3 and 4 (Δ3–4), 5 and 6 (Δ5–6), 7 and 8 (Δ7–8), the LRR C-terminus (ΔLRRCT), the C-terminus (ΔCT), and the complete LRR domain (LRR−). b, COS-7 cells transfected with NgR deletion mutant plasmids were stained for Myc immunoreactivity or tested for 20 nm AP or AP-Nogo binding. All NgR mutant proteins were expressed in COS-7 cells as shown by Myc immunoreactivity. Only WTNgR- and NgRΔCT-transfected COS-7 cells bound to AP-Nogo. Scale bar, 100 μm. c, Quantification of AP-Nogo or AP binding to COS-7 cells transfected with NgR deletion mutants.

Fig. 2.

The CT region of NgR is necessary but not sufficient for Nogo inhibition. a, E7 retinal explants were infected with a recombinant viral preparation of the NgRΔCT. NgRΔCT-infected RGCs were insensitive to a 40 min treatment with 500 nm GSTNogo-66. Expression of NgRΔCT in RGC neurites and growth cones was verified by Myc immunostaining. Scale bar, 50 μm. b, Quantification of the growth cone collapse response of RGCs to GSTNogo-66 after viral infection with NgRΔCT, WTNgR, or a control plexinA1 virus (Plexin). Means ± SEM from 4–10 experiments are reported. Student'st tests comparing WTNgR or NgRΔCT to control plexinA1 values at the indicated Nogo concentration are reported. *p < 0.001. c, E7 RGC explants were infected with recombinant viral preparations of control HSV particles (HSV) or HSVNgRCT. NgRCT alone does not cause growth cone collapse. Scale bar, 100 μm. d, Quantification of E7 RGC growth cone collapse after control HSV or HSVNgRCT infection. Means ± SEM for three experiments are reported. e, Neurite outgrowth of dissociated E13 DRGs plated on control or Nogo substrates and treated with 500 nm soluble GST or GSTNgRCT. GSTNgRCT does not inhibit neurite outgrowth on control spots or modify the response of E13 DRGs to Nogo inhibition. Scale bar, 200 μm. f, Nogo dose–response of E13 DRG neurite outgrowth in the presence of GST or GSTNgRCT. Neurite outgrowth is calculated as micrometers of growth per cell. Means ± SEM from three experiments are reported.

Fig. 3.

Characterization of NgRL1. a, Cell lysates from HEK293T cells transfected with HSVWTNgR or HSVNgRL1 plasmids were fractionated on flotation gradients. WTNgR is found almost exclusively in the caveolin-positive detergent-insoluble fraction. b, NgRL1 is in multiple membrane fractions, with a small proportion in the caveolin-positive detergent-insoluble fraction. c, COS-7 cells were transfected with WTNgR and tested for 10 nm AP-Nogo binding. d, COS-7 cells were transfected with NgRL1 and tested for 10 nm AP-Nogo binding. Cells expressing WTNgR or NgRL1 bind similar amounts of AP-Nogo. Scale bar, 100 μm.

Fig. 4.

The GPI linkage region of NgR is not required for Nogo-mediated inhibition. a, E7 retinal explants were infected with recombinant viral preparations of PlexA1, WTNgR, or NgRL1. Explants were treated with 500 nm GSTNogo-66 for 40 min, fixed, and stained with rhodamine–phalloidin. RGCs infected with PlexA1 control virus are insensitive to Nogo, whereas those infected with WTNgR or NgRL1 collapse in response to Nogo. Scale bar, 50 μm.b, Dose–response of RGCs to GSTNogo-66 after infection with NgR viral preparations. Student's t tests comparing WTNgR or NgRL1 to PlexA1 at the indicated Nogo concentration are reported. *p = 0.01; **p < 0.01. Significance indicators (*) are coded with the appropriate infection. Means ± SEM for 6–10 experiments are reported.

Fig. 5.

NgR interacts with itself. a, COS-7 cells were transfected with WTNgR or NgR deletion mutant plasmids and tested for AP or AP-NgR binding. WTNgR- and NgRΔCT-transfected COS-7 cells bind to AP-NgR. b, Quantification of AP or AP-NgR binding to COS-7 cells transfected with NgR deletion mutants.c, AP-NgR binding to COS-7 cells transfected with NgR as a function of AP-NgR concentration. Mean ± SEM for three experiments. d, COS-7 cells were transfected with WTNgR and treated with AP-NgR in the presence of 25 nm GST or GSTNogo-66 (GSTNg66). AP-NgR interaction with WTNgR is not modified by the presence of GST or GSTNogo-66. Scale bar, 100 μm. e, Quantification of AP-NgR binding to WTNgR-transfected COS-7 cells in the presence of 25 nm GST or GSTNogo-66.

Fig. 6.

Soluble NgREcto antagonizes neurite outgrowth inhibition on Nogo and myelin substrates. a, AP-Nogo stain of COS-7 cells transfected with WTNgR. Cells were stained with 7 nm AP-Nogo in the presence or absence of 70 nmNgREcto. NgREcto blocks AP-Nogo binding to WTNgR. b, Quantification of AP-Nogo (7 nm) binding to WTNgR-transfected COS-7 cells in the presence or absence of 70 nm NgREcto. c, Dissociated E13 DRGs were plated on spots of PBS or 500 ng of NgREcto mixed with Nogo, myelin, or aggrecan. Nogo and myelin inhibition is partially reversed by the addition of NgREcto, whereas aggrecan inhibition is not.d, e, Dose–response of E13 DRG outgrowth on spots of PBS or 500 ng of NgREcto mixed with Nogo, myelin, or aggrecan. f, E13 DRG neurite outgrowth on Nogo or myelin substrates in the presence of 2 μm soluble purified NgREcto (Soluble NgREcto) or PBS. Neurite outgrowth is expressed as micrometers of growth per cell. Means ± SEM for four to six experiments are reported. Student's t tests comparing PBS to NgREcto at the indicated Nogo concentration are reported. *p < 0.01. Scale bar, 200 μm.

Fig. 7.

Model of Nogo receptor-mediated signaling. This schematic illustrates the proposed role each Nogo receptor domain plays in Nogo-signal transduction. See Discussion.