Blocking LINGO-1 in vivo reduces degeneration and enhances regeneration of the optic nerve

Abstract

Background: Two ongoing phase II clinical trials (RENEW and SYNERGY) have been developed to test the efficacy of anti-LINGO-1 antibodies in acute optic neuritis and relapsing forms of multiple sclerosis, respectively. Across a range of experimental models, LINGO-1 has been found to inhibit neuron and oligodendrocyte survival, axon regeneration, and (re)myelination. The therapeutic effects of anti-LINGO-1 antibodies on optic nerve axonal loss and regeneration have not yet been investigated.

Objective: In this series of studies we investigate if LINGO-1 antibodies can prevent acute inflammatory axonal loss, and promote axonal regeneration after injury in rodent optic nerves.

Methods: The effects of anti-LINGO-1 antibody on optic nerve axonal damage were assessed using rodent myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (EAE), and its effects on axonal regeneration were assessed in optic nerve crush injury models.

Results: In the optic nerve, anti-LINGO-1 antibody therapy was associated with improved optic nerve parallel diffusivity measures on MRI in mice with EAE and reduced axonal loss in rat EAE. Both anti-LINGO-1 antibody therapy and the genetic deletion of LINGO-1 reduced nerve crush-induced axonal degeneration and enhanced axonal regeneration.

Conclusion: These data demonstrate that LINGO-1 blockade is associated with axonal protection and regeneration in the injured optic nerve.

Keywords: LINGO-1; Optic neuritis; anti-LINGO-1 antibody; axons and retinal ganglion cells; multiple sclerosis; optic nerve degeneration and regeneration.

Figure 1.

LINGO-1 blockade reduced morbidity and mortality of myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (MOG-EAE) mice. (a) The percentage of MOG-EAE mice reaching complete hindlimb paralysis (grade 3 disease severity) exposed to anti-LINGO-1 monoclonal antibody (mAb) 3B5 or control antibody (ctrl Ab). (b) The percentage survival of MOG-EAE mice exposed to mAb 3B5 or ctrl Ab. Data shown as mean ± standard deviation (SD). Day 16 p values were determined using a log-rank test. LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1.

Figure 2.

LINGO-1 blockade protected optic nerve axonal integrity of myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (MOG-EAE) mice. Analysis of optic nerve axonal integrity by diffusion-weighted imaging in mouse MOG-EAE exposed to anti-LINGO-1 monoclonal antibody (mAb) 3B5 or control antibody (ctrl Ab). Imaging was performed on day 16 or 17 after induction of EAE. Data shown as mean ± SD. p value was determined using a t test. LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1.

Figure 3.

Histological analysis conducted on the 0.5-µm transverse sections of the pre-chiasmal optic nerve in five regions of interest (a). Analysis indicated no significant differences in axon numbers between anti-LINGO-1- and control-treated mice in either the peripheral (b–e) or the central regions (f) of the optic nerve. Axon numbers between the two treatment conditions remained unchanged (Table 2). However, average axon area of the central portion of the optic nerve showed a strong trend toward greater reduction in the placebo-treated mice than the anti-LINGO-1-treated mice when compared with healthy control tissues (Table 2). LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1.

Figure 4.

LINGO-1 blockade protected the optic nerve from axonal degeneration in MOG-EAE rats. (a) Immunohistochemical staining to visualize optic nerve axons in normal or MOG-EAE rats 28 days after the start of the indicated antibody treatments. Scale bar = 25 µm. (b) Quantification of axons from (a). Data in (b) shown as mean ± standard error of the mean (SEM). p values were determined using a one-way analysis of variance followed by Tukey post test. Ctrl Ab: control antibody; DAPI: 4',6-diamidino-2-phenylindole; LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1; mAb: monoclonal antibody; MOG-EAE: myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis; MP: methylprednisolone; Veh: vehicle.

Figure 5.

LINGO-1 is expressed in retinal ganglion cells (RGCs). Immunohistochemical staining of LINGO-1 in the retina 14 days after optic nerve crush. Arrows indicate RGCs. Scale bar = 100 µm. FG: Fluoro-Gold; GCL: ganglion cell layer; INL: inner nuclear layer; LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1; ONL: outer nuclear layer.

Figure 6.

Anti-LINGO-1 monoclonal antibody (mAb) 1A7 prevented optic nerve axonal degeneration and facilitated regeneration in a rat optic nerve crush model. (a) Images of optic nerves showing fluorescein isothiocyanate-conjugated cholera toxin subunit B (FITC-CTB)-labeled axons proximal to the crush site in normal or injured rats (14 days after optic nerve crush) exposed to mAb 1A7 or vehicle (veh). Arrows show the crush site, asterisks show axonal areas. Scale bar = 100 µm. (b) Images of optic nerves showing FITC-CTB-labeled axons beyond the crush site in injured rats exposed to mAb 1A7 or veh. Arrows show axonal outgrowth. Scale bar = 100 µm. (c) Quantification of regenerating axons per section at different distances beyond the crush sites in injured rats exposed to veh or mAb 1A7 through local or intravitreal delivery. Data in (c) shown as mean ± SEM. p values were determined using an unpaired t test. LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1.

Figure 7.

LINGO-1 deletion enhanced optic nerve axon regeneration in a mouse optic nerve crush model. (a) Images of optic nerves showing Alexa Fluor 488-conjugated cholera toxin subunit B-labeled axons around the crush sites at 28 days after injury from wild-type and LINGO-1-null mice. Asterisk shows crush site. Scale bar = 500 µm. (b) Quantification of regenerating axons per section at different distances beyond the crush site. Data in (b) shown as mean ± SEM. p values were determined using an unpaired t test. LINGO-1: leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1.