Neuregulins constitute a family of related growth factors that play important roles in Schwann cell development and maturation. We investigated the involvement of beta-neuregulin in Schwann cell migration, using a simple in vitro bioassay. Pure Schwann cells were prepared from the sciatic nerves of 5-day-old rats and were grown in defined medium, with or without serum, until a monolayer of confluent cells was formed. A cell-free area was then generated by inflicting a scratch resulting in a 1-mm-wide gap. Schwann cell migration within the gap was monitored microscopically at given time intervals and was quantified using an image analysis system. The extent of cell proliferation was estimated by BrdU incorporation, and cell migration was quantified both in the absence and presence of cytosine arabinoside. We found that, in the absence of serum, beta-neuregulin at a dose submaximal for proliferation increased the rate of Schwann cell migration by 84%. A more moderate effect was observed when beta-neuregulin was applied in the presence of serum which, however, is by itself responsible for increased Schwann cell motility. To assess the signal transduction pathways involved in this procedure we used one inhibitor of MAPK, PD098059, two inhibitors of PI-3-kinase, wortmannin, and LY0294002, and three different PKC inhibitors. Of these PD098059 inhibited the neuregulin-induced enhancement in Schwann cell migration by 40%, the two PI-3-kinase inhibitors yielded an approximately 20% inhibition while the PKC inhibitors were ineffective. Our data indicate that the action of beta-neuregulin on Schwann cell motility is primarily mediated via the MAPK pathway.

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identi®ed at the surface of cultured SCs by chimera coated¯uorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the ®rst, migration is monitored at speci®c time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras signi®cantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycantype protein tyrosine phosphatase z/b (RPTPz/b) antibodies identi®ed the presence of RPTPz/b on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPz/b from SC lysates. Altogether these data point to RPTPz/b is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.

Neuregulins constitute a family of related growth factors that play important roles in Schwann cell development and maturation. We investigated the involvement of beta-neuregulin in Schwann cell migration, using a simple in vitro bioassay. Pure Schwann cells were prepared from the sciatic nerves of 5-day-old rats and were grown in defined medium, with or without serum, until a monolayer of confluent cells was formed. A cell-free area was then generated by inflicting a scratch resulting in a 1-mm-wide gap. Schwann cell migration within the gap was monitored microscopically at given time intervals and was quantified using an image analysis system. The extent of cell proliferation was estimated by BrdU incorporation, and cell migration was quantified both in the absence and presence of cytosine arabinoside. We found that, in the absence of serum, beta-neuregulin at a dose submaximal for proliferation increased the rate of Schwann cell migration by 84%. A more moderate effect was observed when beta-neuregulin was applied in the presence of serum which, however, is by itself responsible for increased Schwann cell motility. To assess the signal transduction pathways involved in this procedure we used one inhibitor of MAPK, PD098059, two inhibitors of PI-3-kinase, wortmannin, and LY0294002, and three different PKC inhibitors. Of these PD098059 inhibited the neuregulin-induced enhancement in Schwann cell migration by 40%, the two PI-3-kinase inhibitors yielded an approximately 20% inhibition while the PKC inhibitors were ineffective. Our data indicate that the action of beta-neuregulin on Schwann cell motility is primarily mediated via the MAPK pathway.

To probe the effects of possible inhibitors or enhancers of in vivo myelination, we have modified a technique widely used in studies of the developing neuromuscular system that involves incorporation of test compounds into a silicon rubber solution, which solidifies on contact with air. U-shaped rubber implants are inserted around the sciatic nerve of 1-day-old rats and left in place for 24-48 h. Sections from the region of the nerve lying within the implant, with or without the test compound, are then immunolabeled, examined with in situ hybridization or electron microscopy. Application of EDTA (440 microg/implant) in this way strongly suppressed the levels of the myelin-associated molecules protein P0, myelin basic protein (MBP), and galactocerebroside (Galc). mRNA levels for P0 and the myelin-related transcription factor Krox-20 were also reduced, further supporting association of the EDTA-induced effect with the myelinating Schwann cells. In contrast, no obvious differences were observed in either neurofilament (NF) protein or glial fibrillary acidic protein (GFAP) expression, suggesting absence of influence on axons or nonmyelinating Schwann cells. Despite the severely altered molecular composition of myelin in the presence of EDTA, examination in the electron microscope did not reveal any apparent ultrastructural changes in the myelin sheaths or nerve development. This work introduces a novel method for studying nerve development and shows that EDTA, which chelates divalent cations such as Ca(2+) and Mg(2+), strongly and selectively reduces levels of molecules, which, on postnatal days 1-4, are expressed in myelinating cells at much higher levels than in cells not engaged in myelination.

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