Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth - PubMed (original) (raw)

Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth

N K Mahanthappa et al. J Neurosci. 1996.

Abstract

Schwann cells proliferate, migrate, and act as sources of neurotrophic support during development and regeneration of peripheral nerves. Recent studies have demonstrated that neuregulins, a family of growth factors secreted by developing motor and peripheral neurons, influence Schwann cell development. In this study, we use three distinct assays to show that glial growth factor 2 (GGF2), a secreted neuregulin, exerts multiple effects on mature Schwann cells in vitro. At doses submaximal for proliferation, GGF2 increases the motility of Schwann cells cultured on peripheral nerve cryosections. Furthermore, in a novel bioassay, focal application of GGF2 causes directed migration in conventional monolayer cultures of directed migration of Schwann cells. At higher doses, GGF2 causes proliferation, as described previously. In a new explant culture system designed to emulate entubulation repair of transected peripheral nerves, GGF2 concentrations greater than necessary to saturate the mitotic response induce the secretion by Schwann cells of activities that promote sympathetic neuron survival and outgrowth. These findings support a model in which neuregulins secreted by peripheral neurons are key components of reciprocal neuron-glia interactions that are important for peripheral nerve development and regeneration.

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Figures

Fig. 1.

Fig. 1.

GGF2-induced enhancement of Schwann cell migration. The maximum extent of Schwann cell migration from neonatal DRG explants onto sciatic nerve sections was measured in the presence of different concentrations of GGF2. A, The extent of migration under control conditions (no additives). At an rhGGF2 concentration submaximal for proliferation, 2.5 ng/ml, the extent of Schwann cell migration is enhanced by ∼63% (B). The maximum distance of Schwann cell migration under different conditions (C) was plotted as fold increase from the basal level (no additives). Bars represent the average ± SEM. BSA (2.5 ng/ml), n = 5; rhGGF2 concentrations of 1 ng/ml,n = 5; 2.5 ng/ml, n = 13; 10 ng/ml,n = 10; and 20 ng/ml, n = 7. Asterisk designates difference from the BSA condition with_p_ < 0.01. Scale bar, 100 μm.

Fig. 2.

Fig. 2.

Beads coated with rhGGF2 promote Schwann cell chemotaxis. Heparin-Sepharose beads were coated with rhGGF2 to varying degrees of saturation (see Table 1) and were incubated with pure Schwann cell cultures for 15 hr. Cultures were stained for BrdU incorporation in parallel with those in Figures 3 and 4, and little staining was observed. Concentrations of rhGGF2 (pg/bead): (A) 0, (B) 0.75, (C) 1.5, (D) 3.0, (E) 6.0, and (F) 12.0. Scale bar, 130 μm.

Fig. 3.

Fig. 3.

Beads coated with rhGGF2 promote Schwann cell DNA synthesis after 40 hr. Cultures were treated as in Figure 2 and were stained after 40 hr. Note the dose-dependent increase in BrdU-stained nuclei and that proliferating cells are localized to the beads.Arrowheads mark some of the few BrdU+cells in B–D. The large arrow in _E_marks a Schwann cell cluster not associated with a bead and lacking BrdU staining. Concentrations of rhGGF2 (pg/bead): (A) 0, (B) 0.75, (C) 1.5, (D) 3.0, (E) 6.0, and (F) 12.0. Scale bar, 130 μm.

Fig. 4.

Fig. 4.

Soluble rhGGF2 promotes Schwann cell DNA synthesis without localization. In parallel with the cultures in Figures 1 and 2, equivalent concentrations of soluble rhGGF2 (see Table 1) were also tested, and the cultures were stained after 40 hr. Concentrations of rhGGF2 (ng/ml): (A) 0, (B) 1.1, (C) 2.2, (D) 4.5, (E) 9.0, and (F) 18.0. Scale bar, 130 μm.

Fig. 5.

Fig. 5.

Schwann cell and neurite outgrowth in a novel explant culture system. Halves of SCGs were cultured in collagen gels within 10 mm lengths of 1.19-mm-diameter polyethylene tubing for 2 d in 1 μg/ml rhGGF2 (A) or 10 ng/ml NGF (B). These cultures were stained for S-100 (A) and tubulin βIII (B) to mark Schwann cells and axons, respectively.Arrowheads in B mark several of the many growth cones visible in such cultures. Scale bar, 100 μm.

Fig. 6.

Fig. 6.

Schwann migration from ganglia increases in the presence of rhGGF2. Tube cultures similar to those in Figure 5 were maintained for 2, 5, and 10 d in the presence of 0 (control), 5, 50, and 500 ng/ml rhGGF2. At the end of the indicated culture period, the tubes were fixed and stained for S-100, and the number of immunoreactive cells was scored at 50 μm intervals (bins) from the explant. Data points represent the average ± SEM;n = 6–7.

Fig. 7.

Fig. 7.

Extraganglionic DNA synthesis commences after 5 d in vitro. Tube cultures similar to those in Figure5 were maintained for 10 d in the presence of 0 (control), 5, 50, and 500 ng/ml rhGGF2. Cultures were fixed and stained for BrdU, and the number of immunoreactive cells was scored at 50 μm intervals (bins) from the explant. Data points represent the average ± SEM;n = 3. Cultures were pulsed with BrdU for 24 hr on either day 9 (top) or day 1 (bottom).

Fig. 8.

Fig. 8.

Ganglionic neurite outgrowth is promoted by rhGGF2. Tube cultures similar to those in Figure 5 were maintained for 10 d in the presence of 0 (control), 5, 50, and 500 ng/ml rhGGF2. Cultures were fixed and stained for tubulin βIII, and the number of neurites was scored at 50 μm intervals (bins) from the explant (noted as “intersections” with the scoring grid). Data points represent the average ± SEM; n = 6–7.

Fig. 9.

Fig. 9.

Neurotrophic factors are produced by Schwann cells in response to rhGGF2. Top, Dissociated SCG neurons were cultured in the indicated concentrations of rhGGF2 and NGF, and the number of tubulin βIII+ neurons was scored after 48 hr. No direct effect of rhGGF2 on SCG neuron survival was observed. Bars represent the average ± SEM;n = 20 fields scored from four independent cultures.Bottom, Similar SCG neuron cultures were established in media conditioned for 5 d by Schwann cells plated at two different densities and treated with the indicated concentrations of rhGGF2 (control = no rhGGF2). Before neurons were plated in conditioned media, all media were diluted by a factor proportional to cell number in the Schwann cell cultures at the time of medium collection (for details, see Results). The number of tubulin βIII+ neurons was scored after 48 hr. Schwann cells produce increasing trophic activity in response to increasing rhGGF2 concentration. Bars represent the average ± SEM; n = 10.

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