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Glial growth factor/neuregulin inhibits Schwann cell myelination and induces demyelination.

Zanazzi G, Einheber S, Westreich R, Hannocks MJ, Bedell-Hogan D, Marchionni MA, Salzer JL - J. Cell Biol. (2001)

Bottom Line: Glial growth factor (GGF), a neuregulin-1 isoform, significantly inhibited myelination by preventing axonal segregation and ensheathment.Two other Schwann cell mitogens, fibroblast growth factor-2 and transforming growth factor-beta, inhibited myelination but did not cause demyelination, suggesting this effect is specific to the neuregulins.GGF treatment of myelinating cultures also induced phosphorylation of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and a 120-kD protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, New York University Medical Center, New York, New York 10016, USA.

ABSTRACT
During development, neuregulin-1 promotes Schwann cell proliferation and survival; its role in later events of Schwann cell differentiation, including myelination, is poorly understood. Accordingly, we have examined the effects of neuregulin-1 on myelination in neuron-Schwann cell cocultures. Glial growth factor (GGF), a neuregulin-1 isoform, significantly inhibited myelination by preventing axonal segregation and ensheathment. Basal lamina formation was not affected. Treatment of established myelinated cultures with GGF resulted in striking demyelination that frequently began at the paranodes and progressed to the internode. Demyelination was dose dependent and accompanied by dedifferentiation of Schwann cells to a promyelinating stage, as evidenced by reexpression of the transcription factor suppressed cAMP-inducible POU; a significant proportion of cells with extensive demyelination also proliferated. Two other Schwann cell mitogens, fibroblast growth factor-2 and transforming growth factor-beta, inhibited myelination but did not cause demyelination, suggesting this effect is specific to the neuregulins. The neuregulin receptor proteins, erbB2 and erbB3, are expressed on ensheathing and myelinating Schwann cells and rapidly phosphorylated with GGF treatment. GGF treatment of myelinating cultures also induced phosphorylation of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and a 120-kD protein. These results suggest that neuronal mitogens, including the neuregulins, may inhibit myelination during development and that activation of mitogen signaling pathways may contribute to the initial demyelination and subsequent Schwann cell proliferation observed in various pathologic conditions.

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Expression and tyrosine phosphorylation of GGF receptors. (A) Expression and phosphorylation of GGF receptors. DRG neurons, Schwann cells, premyelinated (premy), and myelinated (my) cocultures were lysed directly (−) or after 2.5 min of 200 ng/ml GGF treatment (+). 15 μg of each lysate was fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti–erbB2, anti–erbB3, or antiphosphotyrosine antibodies. ErbB2 and erbB3 were expressed by Schwann cells (c and d) and in the cocultures (e–h). Note that while GGF activated p185 in all cultures containing Schwann cells, p120 was only activated in the myelinated cocultures (h); a constitutively phosphorylated band of ∼140 kD was present in all the lanes. (B) Time course of phosphorylation. Myelinated cocultures were lysed either directly or at varying times after treatment with 200 ng/ml GGF. Lysates were fractionated and blotted as described in A, and probed with antiphosphotyrosine antibodies. Both p185 and p120 were rapidly tyrosine phosphorylated and continued to be phosphorylated after 30 min of GGF treatment. (C) Expression and phosphorylation of GGF receptors in demyelinating cocultures. Myelinated cocultures were maintained for 3 d in media without or with 20 or 200 ng/ml GGF. Protein lysates were prepared, fractionated, blotted, and probed as described in A. While there was a dose-dependent decrease in erbB2 (top), erbB3 levels were unchanged (middle). (Bottom) There was a dose-dependent increase in p185 tyrosine phosphorylation in the cocultures treated with GGF for 3 d.
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Figure 7: Expression and tyrosine phosphorylation of GGF receptors. (A) Expression and phosphorylation of GGF receptors. DRG neurons, Schwann cells, premyelinated (premy), and myelinated (my) cocultures were lysed directly (−) or after 2.5 min of 200 ng/ml GGF treatment (+). 15 μg of each lysate was fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti–erbB2, anti–erbB3, or antiphosphotyrosine antibodies. ErbB2 and erbB3 were expressed by Schwann cells (c and d) and in the cocultures (e–h). Note that while GGF activated p185 in all cultures containing Schwann cells, p120 was only activated in the myelinated cocultures (h); a constitutively phosphorylated band of ∼140 kD was present in all the lanes. (B) Time course of phosphorylation. Myelinated cocultures were lysed either directly or at varying times after treatment with 200 ng/ml GGF. Lysates were fractionated and blotted as described in A, and probed with antiphosphotyrosine antibodies. Both p185 and p120 were rapidly tyrosine phosphorylated and continued to be phosphorylated after 30 min of GGF treatment. (C) Expression and phosphorylation of GGF receptors in demyelinating cocultures. Myelinated cocultures were maintained for 3 d in media without or with 20 or 200 ng/ml GGF. Protein lysates were prepared, fractionated, blotted, and probed as described in A. While there was a dose-dependent decrease in erbB2 (top), erbB3 levels were unchanged (middle). (Bottom) There was a dose-dependent increase in p185 tyrosine phosphorylation in the cocultures treated with GGF for 3 d.

Mentions: Finally, we characterized the effects of GGF on neuregulin receptors and downstream signaling pathways. Detergent lysates were prepared directly from Schwann cell, DRG neuron, premyelinated, and myelinated cocultures or after treating these cultures with 200 ng/ml of GGF for 2.5 min. Lysates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti–erbB2, anti–erbB3, or antiphosphotyrosine antibodies. As previously reported (Levi et al. 1995; Canoll et al. 1996), erbB2 and erbB3 were robustly expressed in Schwann cells (Fig. 7 A). GGF increased the tyrosine phosphorylation of p185, which corresponds to the expected mol wt of erbB2 and erbB3 (Fig. 7 A). In contrast, minimal erbB2 and erbB3 were detected by Western blotting of neurons, although low-level erbB2 expression was observed by immunofluorescence (data not shown). Consistent with this limited erbB expression, no phosphorylation of p185 was observed after GGF treatment of the DRG neuron cultures.


Glial growth factor/neuregulin inhibits Schwann cell myelination and induces demyelination.

Zanazzi G, Einheber S, Westreich R, Hannocks MJ, Bedell-Hogan D, Marchionni MA, Salzer JL - J. Cell Biol. (2001)

Expression and tyrosine phosphorylation of GGF receptors. (A) Expression and phosphorylation of GGF receptors. DRG neurons, Schwann cells, premyelinated (premy), and myelinated (my) cocultures were lysed directly (−) or after 2.5 min of 200 ng/ml GGF treatment (+). 15 μg of each lysate was fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti–erbB2, anti–erbB3, or antiphosphotyrosine antibodies. ErbB2 and erbB3 were expressed by Schwann cells (c and d) and in the cocultures (e–h). Note that while GGF activated p185 in all cultures containing Schwann cells, p120 was only activated in the myelinated cocultures (h); a constitutively phosphorylated band of ∼140 kD was present in all the lanes. (B) Time course of phosphorylation. Myelinated cocultures were lysed either directly or at varying times after treatment with 200 ng/ml GGF. Lysates were fractionated and blotted as described in A, and probed with antiphosphotyrosine antibodies. Both p185 and p120 were rapidly tyrosine phosphorylated and continued to be phosphorylated after 30 min of GGF treatment. (C) Expression and phosphorylation of GGF receptors in demyelinating cocultures. Myelinated cocultures were maintained for 3 d in media without or with 20 or 200 ng/ml GGF. Protein lysates were prepared, fractionated, blotted, and probed as described in A. While there was a dose-dependent decrease in erbB2 (top), erbB3 levels were unchanged (middle). (Bottom) There was a dose-dependent increase in p185 tyrosine phosphorylation in the cocultures treated with GGF for 3 d.
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Figure 7: Expression and tyrosine phosphorylation of GGF receptors. (A) Expression and phosphorylation of GGF receptors. DRG neurons, Schwann cells, premyelinated (premy), and myelinated (my) cocultures were lysed directly (−) or after 2.5 min of 200 ng/ml GGF treatment (+). 15 μg of each lysate was fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti–erbB2, anti–erbB3, or antiphosphotyrosine antibodies. ErbB2 and erbB3 were expressed by Schwann cells (c and d) and in the cocultures (e–h). Note that while GGF activated p185 in all cultures containing Schwann cells, p120 was only activated in the myelinated cocultures (h); a constitutively phosphorylated band of ∼140 kD was present in all the lanes. (B) Time course of phosphorylation. Myelinated cocultures were lysed either directly or at varying times after treatment with 200 ng/ml GGF. Lysates were fractionated and blotted as described in A, and probed with antiphosphotyrosine antibodies. Both p185 and p120 were rapidly tyrosine phosphorylated and continued to be phosphorylated after 30 min of GGF treatment. (C) Expression and phosphorylation of GGF receptors in demyelinating cocultures. Myelinated cocultures were maintained for 3 d in media without or with 20 or 200 ng/ml GGF. Protein lysates were prepared, fractionated, blotted, and probed as described in A. While there was a dose-dependent decrease in erbB2 (top), erbB3 levels were unchanged (middle). (Bottom) There was a dose-dependent increase in p185 tyrosine phosphorylation in the cocultures treated with GGF for 3 d.
Mentions: Finally, we characterized the effects of GGF on neuregulin receptors and downstream signaling pathways. Detergent lysates were prepared directly from Schwann cell, DRG neuron, premyelinated, and myelinated cocultures or after treating these cultures with 200 ng/ml of GGF for 2.5 min. Lysates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti–erbB2, anti–erbB3, or antiphosphotyrosine antibodies. As previously reported (Levi et al. 1995; Canoll et al. 1996), erbB2 and erbB3 were robustly expressed in Schwann cells (Fig. 7 A). GGF increased the tyrosine phosphorylation of p185, which corresponds to the expected mol wt of erbB2 and erbB3 (Fig. 7 A). In contrast, minimal erbB2 and erbB3 were detected by Western blotting of neurons, although low-level erbB2 expression was observed by immunofluorescence (data not shown). Consistent with this limited erbB expression, no phosphorylation of p185 was observed after GGF treatment of the DRG neuron cultures.

Bottom Line: Glial growth factor (GGF), a neuregulin-1 isoform, significantly inhibited myelination by preventing axonal segregation and ensheathment.Two other Schwann cell mitogens, fibroblast growth factor-2 and transforming growth factor-beta, inhibited myelination but did not cause demyelination, suggesting this effect is specific to the neuregulins.GGF treatment of myelinating cultures also induced phosphorylation of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and a 120-kD protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, New York University Medical Center, New York, New York 10016, USA.

ABSTRACT
During development, neuregulin-1 promotes Schwann cell proliferation and survival; its role in later events of Schwann cell differentiation, including myelination, is poorly understood. Accordingly, we have examined the effects of neuregulin-1 on myelination in neuron-Schwann cell cocultures. Glial growth factor (GGF), a neuregulin-1 isoform, significantly inhibited myelination by preventing axonal segregation and ensheathment. Basal lamina formation was not affected. Treatment of established myelinated cultures with GGF resulted in striking demyelination that frequently began at the paranodes and progressed to the internode. Demyelination was dose dependent and accompanied by dedifferentiation of Schwann cells to a promyelinating stage, as evidenced by reexpression of the transcription factor suppressed cAMP-inducible POU; a significant proportion of cells with extensive demyelination also proliferated. Two other Schwann cell mitogens, fibroblast growth factor-2 and transforming growth factor-beta, inhibited myelination but did not cause demyelination, suggesting this effect is specific to the neuregulins. The neuregulin receptor proteins, erbB2 and erbB3, are expressed on ensheathing and myelinating Schwann cells and rapidly phosphorylated with GGF treatment. GGF treatment of myelinating cultures also induced phosphorylation of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and a 120-kD protein. These results suggest that neuronal mitogens, including the neuregulins, may inhibit myelination during development and that activation of mitogen signaling pathways may contribute to the initial demyelination and subsequent Schwann cell proliferation observed in various pathologic conditions.

Show MeSH
Related in: MedlinePlus