Axo-glial units are highly organised microstructures propagating saltatory conduction and are disrupted during multiple sclerosis (MS). Nogo receptor 1 (NgR1) has been suggested to govern axonal damage during the progression of disease in the MS-like mouse model, experimental autoimmune encephalomyelitis (EAE). Here we have identified that adult ngr1 -/- mice, previously used in EAE and spinal cord injury experiments, display elongated paranodes, and nodes of Ranvier. Unstructured paranodal regions in ngr1 -/- mice are matched with more distributed expression pattern of Caspr. Compound action potentials of optic nerves and spinal cords from naïve ngr1 -/- mice are delayed and reduced. Molecular interaction studies revealed enhanced Caspr cleavage. Our data suggest that NgR1 may regulate axo-myelin ultrastructure through Caspr-mediated adhesion, regulating the electrophysiological signature of myelinated axons of central nervous system (CNS).

The potential role of Nogo-66 Receptor 1 (NgR1) on immune cell phenotypes and their activation during neuroinflammatory diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is unclear. To further understand the function of this receptor on haematopoietically-derived cells, phenotypic and functional analyses were performed using NgR1-deficient (ngr1-/-) animals. Flow cytometry-based phenotypic analyses performed on blood, spleen, thymus, lymph nodes, bone marrow and central nervous-system (CNS)-infiltrating blood cells revealed no immunological defects in naïve ngr1-/- animals versus wild-type littermate (WTLM) controls. EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or by MOG35-55 peptide, a B cell-independent model. We have demonstrated that in ngr1-/- mice injected with MOG35-55, a significant reduction in the severity of EAE correlated with reduced axonal damage present in the spinal cord when compared to their WTLM controls. However, despite a reduction in axonal damage observed in the CNS of ngr1-/- mice at the chronic stage of disease, no clinical differences could be attributed to a specific genotype when rMOG was used as the encephalitogen. Following MOG35-55-induction of EAE, we could not derive any major changes to the immune cell populations analyzed between ngr1-/- and WTLM mice. Collectively, these data demonstrate that NgR1 has little if any effects on the repertoire of immune cells, their activation and trafficking to the CNS.

Myelin-associated inhibitory factors within the central nervous system (CNS) are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1 (NgR1) has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis (MS). In this study, the animal model of MS, experimental autoimmune encephalomyelitis (EAE) was induced in ngr1+/+ and ngr1-/- female mice following injection with the myelin oligodendrocyte glycoprotein (MOG35-55) peptide. A fate-map analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively (increasing locomotor disability) from both genotypes, using the CD11b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1+/+ and ngr1-/- mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1+/+ and ngr1-/- mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1+/+ and ngr1-/- mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of NgR on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1-/- mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1-/- mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1-/- mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.

Nogo receptor 1 is the high affinity receptor for the potent myelin-associated inhibitory factors that make up part of the inflammatory extracellular milieu during experimental autoimmune encephalomyelitis. Signalling through the Nogo receptor 1 complex has been shown to be associated with axonal degeneration in an animal model of multiple sclerosis, and neuronal deletion of this receptor homologue, in a disease specific manner, is associated with preserving axons even in the context of neuroinflammation. The local delivery of Nogo receptor(1-310)-Fc, a therapeutic fusion protein, has been successfully applied as a treatment in animal models of spinal cord injury and glaucoma. As multiple sclerosis and experimental autoimmune encephalomyelitis exhibit large numbers of inflammatory cell infiltrates within the CNS lesions, we utilized transplantable haematopoietic stem cells as a cellular delivery method of the Nogo receptor(1-310)-Fc fusion protein. We identified CNS-infiltrating macrophages as the predominant immune-positive cell type that overexpressed myc-tagged Nogo receptor(1-310)-Fc fusion protein at the peak stage of experimental autoimmune encephalomyelitis. These differentiated phagocytes were predominant during the extensive demyelination and axonal damage, which are associated with the engulfment of the protein complex of Nogo receptor(1-310)-Fc binding to myelin ligands. Importantly, mice transplanted with haematopoietic stem cells transduced with the lentiviral vector carrying Nogo receptor(1-310)-Fc and recovered from the peak of neurological decline during experimental autoimmune encephalomyelitis, exhibiting axonal regeneration and eventual remyelination in the white matter tracts. There were no immunomodulatory effects of the transplanted, genetically modified haematopoietic stem cells on immune cell lineages of recipient female mice induced with experimental autoimmune encephalomyelitis. We propose that cellular delivery of Nogo receptor(1-310)-Fc fusion protein through genetically modified haematopoietic stem cells can modulate multifocal experimental autoimmune encephalomyelitis lesions and potentiate neurological recovery.

We have previously reported evidence that Nogo-A activation of Nogo-receptor 1 (NgR1) can drive axonal dystrophy during the neurological progression of experimental autoimmune encephalomyelitis (EAE). However, the B-cell activating factor (BAFF/BlyS) may also be an important ligand of NgR during neuroinflammation. In the current study we define that NgR1 and its homologs may contribute to immune cell signaling during EAE. Meningeal B-cells expressing NgR1 and NgR3 were identified within the lumbosacral spinal cords of ngr1+/+ EAE-induced mice at clinical score 1. Furthermore, increased secretion of immunoglobulins that bound to central nervous system myelin were shown to be generated from isolated NgR1- and NgR3-expressing B-cells of ngr1+/+ EAE-induced mice. In vitro BAFF stimulation of NgR1- and NgR3-expressing B cells, directed them into the cell cycle DNA synthesis phase. However, when we antagonized BAFF signaling by co-incubation with recombinant BAFF-R, NgR1-Fc, or NgR3 peptides, the B cells remained in the G0/G1 phase. The data suggest that B cells express NgR1 and NgR3 during EAE, being localized to infiltrates of the meninges and that their regulation is governed by BAFF signaling.