Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection. Investigative ophthalmology & visual science 49

PURPOSE. LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes cell death after neuronal injury and spinal cord injury. The authors sought to examine whether blocking LINGO-1 function with LINGO-1 antagonists promotes retinal ganglion cell (RGC) survival after ocular hypertension and optic nerve transection. METHODS. An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. LINGO-1 expression in the retinas was investigated using immunohistochemistry and Western blotting. Soluble LINGO-1 protein (LINGO-1-Fc) and anti-LINGO-1 mAb 1A7 were injected into the vitreous body to examine their effects on RGC survival after ocular hypertension and optic nerve transection. Signal transduction pathways mediating neuroprotective LINGO-1-Fc effects were characterized using Western blotting and specific kinase inhibitors. RESULTS. LINGO-1 was expressed in RGCs and up-regulated after intraocular pressure elevation. Blocking LINGO-1 function with LINGO-1 antagonists, LINGO-1-Fc and 1A7 significantly reduced RGC loss 2 and 4 weeks after ocular hypertension and also promoted RGC survival after optic nerve transection. LINGO-1-Fc treatment blocked the RhoA, JNK pathway and promoted Akt activation. LINGO-1-Fc induced Akt phosphorylation, and the survival effect of LINGO-1 antagonists was abolished by Akt phosphorylation inhibitor. CONCLUSIONS. The authors demonstrated that blocking LINGO-1 function with LINGO-1 antagonists rescues RGCs from cell death after ocular hypertension and optic nerve transection. They also delineated the RhoA and PI-3K/Akt pathways as the predominant mediator of LINGO-1-Fc neuroprotection in this paradigm of RGC death. (Invest Ophthalmol Vis Sci. 2008;49: 975-985) DOI:10.1167/iovs.07-1199 T he inability of injured central nerve system (CNS) neurons to spontaneously regenerate has been ascribed to the presence of Nogo66, 1,2 myelin-associated glycoprotein (MAG), 11,12 LINGO-1 is expressed in CNS neurons and is upregulated after injury or cell death, suggesting that LINGO-1 may be involved in the cell injury response. 14,15 A soluble LINGO-1 protein (LINGO-1-Fc) significantly reduced the apoptotic death of neurons and oligodendrocytes after spinal cord transection. 14 The presence of LINGO-1 in the adult retina 9 prompted us to investigate the effect of LINGO-1 antagonists on protecting the neurons after retinal injury. Glaucoma, a leading cause of blindness, is a neurodegenerative disease characterized by slow, progressive degeneration of retinal ganglion cells (RGCs) and their axons. 16 Elevated intraocular pressure (IOP) is an important trigger for the progression of glaucomatous optic neuropathy. Current standard therapy for glaucoma is to lower the IOP by medication or surgery, which may delay disease progression but does not alter RGC loss and axon degeneration. Therefore, more attention is being focused on new therapeutic strategies with the aim of preserving, protecting, and rescuing RGCs and their axons. In this study, we hypothesized that LINGO-1 antagonists may protect RGCs in glaucoma. To test this hypothesis, we induced rat ocular hypertension by laser photocoagulation and measured RGC survival with the treatment of LINGO-1 antagonists. Optic nerve transection, as an acute model of secondary degeneration, leads to significant optic neuropathy. We further confirmed the effect of LINGO-1 antagonists on the RGC survival after optic nerve transection. A common crucial signaling event for three myelin inhibitors after binding with the NgR1 complex is the activation of RhoA, a member of small guanosine triphosphatase (GTPases), which are known regulators of the actin cytoskeleton. 11,17 The inactivation of RhoA promotes axon regeneration after spinal cord injury in rats and mice. 19 Application of C3-07, another Rho antagonist, promoted the regeneration of RGC axons and completely prevented RGC cell death for 1 week after optic nerve crush

Determination of the Disulfide Structure of Murine Meteorin, a Neurotrophic Factor, by LC–MS and Electron Transfer Dissociation-High-Energy Collisional Dissociation Analysis of Proteolytic Fragments

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC–MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure–function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials