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.

Nogo-A is a neurite growth inhibitor involved in regenerative failure and restriction of structural plasticity in the adult CNS. Three major protein products (Nogo-A, -B, and -C) are derived from the nogo gene. Here we describe the embryonic and postnatal expression of the three Nogo isoforms in the rat by in situ hybridization and immunohistochemistry. Northern and Western blot analysis indicated that Nogo-A is predominantly expressed in the nervous system with lower levels also present in testis and heart. In CNS myelin, confocal and immunoelectron microscopy revealed that Nogo-A is expressed in oligodendrocyte cell bodies and processes and localized in the innermost adaxonal and outermost myelin membranes. Additionally, we find Nogo-A to be expressed by projection neurons, in particular during development, and by postmitotic cells in the developing cortex, spinal cord, and cerebellum. The expression levels of Nogo-A/B were not changed significantly after traumatic lesions to the cortex or spinal cord. Nogo-B showed widespread expression in the central and peripheral nervous systems and other peripheral tissues. Nogo-C was mainly found in skeletal muscle, but brain and heart were also found to express this isoform. The localization of Nogo-A in oligodendrocytes fits well with its role as a myelin-associated inhibitor of regenerative fiber growth and structural plasticity. However, expression of Nogo-A in other tissues and, in particular, in neurons and the widespread expression of the two shorter isoforms, Nogo-B and -C, suggest that the Nogo family of proteins might have function(s) additional to the neurite growth-inhibitory activity.

The zebrafish epithalamus, consisting of the pineal complex and flanking dorsal habenular nuclei, provides a valuable model for exploring how left-right differences could arise in the vertebrate brain. The parapineal lies to the left of the pineal and the left habenula is larger, has expanded dense neuropil, and distinct patterns of gene expression from the right habenula. Under the influence of Nodal signaling, positioning of the parapineal sets the direction of habenular asymmetry and thereby determines the left-right origin of habenular projections onto the midbrain target, the interpeduncular nucleus (IPN). In zebrafish with parapineal reversal, neurons from the left habenula project to a more limited ventral IPN region where right habenular axons would normally project. Conversely, efferents from the right habenula adopt a more extensive dorsoventral IPN projection pattern typical of left habenular neurons. Three members of the leftover-related KCTD (potassium channel tetramerization domain containing) gene family are expressed differently by the left and right habenula, in patterns that define asymmetric subnuclei. Molecular asymmetry extends to protein levels in habenular efferents, providing additional evidence that left and right axons terminate within different dorsoventral regions of the midbrain target. Laser-mediated ablation of the parapineal disrupts habenular asymmetry and consequently alters the dorsoventral distribution of innervating axons. The results demonstrate that laterality of the dorsal forebrain influences the formation of midbrain connections and their molecular properties.