DNA vaccination efficiently induces antibodies to Nogo-A and does not exacerbate experimental autoimmune encephalomyelitis.

Antibodies against the neurite outgrowth inhibitor Nogo-A enhance axonal regeneration following spinal cord injury. However, antibodies directed against myelin components can also enhance CNS inflammation. The present study was designed to assess the efficacy of DNA vaccination for generating antibodies against Nogo-A and to study their pathogenic potential in a mouse model for multiple sclerosis. Mice were immunized by a single i.m. injection of a plasmid expression vector encoding either full length membrane-integral Nogo-A equipped with a signal peptide or two versions of its large N-terminal extramembrane region. The presence of serum antibodies to Nogo-A was measured 4 weeks after injection by ELISA, Western blotting and immunohistochemistry. DNA vaccination efficiently induced production of Nogo-A-specific antibodies that recognized recombinant, intracellular Nogo-A in cell culture but also stained native Nogo-A on the oligodendrocyte surface. Experimental autoimmune encephalomyelitis was induced in DNA-vaccinated mice by immunization with proteolipid peptide (a.a. 139-154). In contrast to vaccination with DNA encoding myelin oligodendrocyte glycoprotein that exacerbates this disease, Nogo-A DNA vaccination did not enhance clinical severity of disease. In summary, DNA vaccination is a simple and efficient method for generating an antibody response to Nogo-A. No pathogenicity was observed even during a full-blown inflammatory response of the central nervous system.

Intrinsic resistance of oligodendrocytes to prion infection.

Within the CNS, the normal form of cellular prion protein (PrP(C)) is expressed on neurons, oligodendrocytes, and astrocytes. The contribution of these cell types to prion replication and pathogenesis is unclear. To assess the role of oligodendrocytes, we expressed PrP(C) under the control of the myelin basic protein (MBP) promoter in mice lacking endogenous PrP(C). PrP(C) was detected in oligodendrocytes and Schwann cells but not in neurons and astrocytes. MBP-PrP mice never developed scrapie after intracerebral, intraperitoneal, or intraocular challenge with scrapie prions. Transgenic brains did not contain protease-resistant prion protein and did not transmit scrapie when inoculated into PrP(C)-overexpressing indicator mice. To investigate whether prion spread within the CNS depends on oligodendrocytic PrP(C), we implanted PrP(C)-overexpressing neuroectodermal grafts into MBP-PrP brains. After intraocular prion inoculation, none of the grafts showed spongiform encephalopathy or prion infectivity. Hence oligodendrocytes do not support cell-autonomous prion replication, establishment of subclinical disease, and neural spread of prions. Prion resistance sets oligodendrocytes aside from both neurons and astrocytes.

Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions.

Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.