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NgR1 (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgR1 have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgR1 are flanked by N- and C-terminal disulfide-containing ‘cap’ domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgR1(310)–Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgR1 truncation site or the spacing between the NgR1 and Fc domains, or replacing cysteines within the NgR1 or IgG hinge regions. One variant, which incorporates replacements of Cys266 and Cys309 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgR1–Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins. [ABSTRACT FROM AUTHOR]

AbstractFunctional recovery is markedly restricted following traumatic brain injury (TBI), partly due to myelin-associated inhibitors including Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), that all bind to the Nogo-66 receptor-1 (NgR1). In previous studies, pharmacological neutralization of both Nogo-A and MAG improved outcome following TBI in the rat, and neutralization of NgR1 improved outcome following spinal cord injury and stroke in rodent models. However, the behavioral and histological effects of NgR1 inhibition have not previously been evaluated in TBI. We hypothesized that NgR1 negatively influences behavioral recovery following TBI, and evaluated NgR1−/−mice (NgR1−/−study) and, in a separate study, soluble NgR1 infused intracerebroventricularly immediately post-injury to neutralize NgR1 (sNgR1 study) following TBI in mice using a controlled cortical impact (CCI) injury model. In both studies, motor function, TBI-induced loss of tissue, and hippocampal β-amyloid immunohistochemistry were not altered up to 5 weeks post-injury. Surprisingly, cognitive function (as evaluated with the Morris water maze at 4 weeks post-injury) was significantly impaired both in NgR1−/−mice and in mice treated with soluble NgR1. In the sNgR1 study, we evaluated hippocampal mossy fiber sprouting using the Timm stain and found it to be increased at 5 weeks following TBI. Neutralization of NgR1 significantly increased mossy fiber sprouting in sham-injured animals, but not in brain-injured animals. Our data suggest a complex role for myelin-associated inhibitors in the behavioral recovery process following TBI, and urge caution when inhibiting NgR1 in the early post-injury period. [ABSTRACT FROM AUTHOR]