Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions

Abstract

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.

Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS

“…For example, amino-Nogo can block fibroblast spreading, whereas Nogo-66 cannot 32,34 , indicating that the amino-Nogo receptor is likely to be more widely expressed than Ngr. Whether this has any physiological relevance has yet to be determined, and the amino-Nogo receptor remains to be identified.…”

Glial inhibition of CNS axon regeneration

“…However, the topology of Nogo-A remains controversial. Although initial evidence places Nogo-66 on the extracellular surface and amino-Nogo facing intracellularly, at least some amino-Nogo can be detected on cell surfaces and both domains are inhibitory to neurite outgrowth 14,15,18,19 (FIG. 2).…”

Stripe assay to examine axonal guidance and cell migration

“…The stripe assay was also applied to investigate neurite outgrowth and guidance behavior related to neuronal regeneration [12][13][14][15] and branch formation 16 . Furthermore, the stripe assay has proven valuable in dissecting signaling cascades initiated downstream of axonal guidance receptors activated by ligands printed in a striped pattern [12][13][14][15] . In addition, the guidance properties of membrane-tethered molecules as well as the short-range action of secreted guidance molecules (such as Slits and Semaphorins) could be mimicked in vitro by this assay 17,18 .…”

“…This protocol was adapted by various research groups for their experimental designs and, for these modifications, we would like to refer throughout the manuscript to the relevant primary research literature 5,6,9,10,14,18,33 . First we describe the guidance behavior of axons from chick retinal ganglion cells (RGCs) growing on carpets consisting of alternating membrane fractions derived from anterior and posterior chick optic tecta (original version, Step 1A of PROCEDURE).…”