ErbB2 signaling in Schwann cells is mostly dispensable for maintenance of myelinated peripheral nerves and proliferation of adult Schwann cells after injury

Abstract

Neuregulin/erbB signaling is critically required for survival and proliferation of Schwann cells as well as for establishing correct myelin thickness of peripheral nerves during development. In this study, we investigated whether erbB2 signaling in Schwann cells is also essential for the maintenance of myelinated peripheral nerves and for Schwann cell proliferation and survival after nerve injury. To this end, we used inducible Cre-loxP technology using a PLP-CreERT2 allele to ablate erbB2 in adult Schwann cells. ErbB2 expression was markedly reduced after induction of erbB2 gene disruption with no apparent effect on the maintenance of already established myelinated peripheral nerves. In contrast to development, Schwann cell proliferation and survival were not impaired in mutant animals after nerve injury, despite reduced levels of MAPK-P (phosphorylated mitogen-activated protein kinase) and cyclin D1. ErbB1 and erbB4 do not compensate for the loss of erbB2. We conclude that adult Schwann cells do not require major neuregulin signaling through erbB2 for proliferation and survival after nerve injury, in contrast to development and in cell culture.

The floxed erbB2 locus is efficiently recombined by the Cre recombinase in Schwann cells of adult peripheral nerves. A, Schematic representation of the erbB2^flox allele with two loxP sites (triangle) flanking three exons (top). The erbB2Δ allele (bottom) is obtained after Cre-induced recombination of erbB2^flox. The arrows indicate primers used for PCR analysis. Exons are indicated by boxes and lettered (Garratt et al., 2000). B, PCR detecting the recombined erbB2Δ allele in genomic DNA from adult sciatic nerves of three untreated (lanes 1–3) and three TM-treated (lanes 4–6) mutant animals (PLP-CreERT2 erbB2^lacZ/flox). C, Semiquantitative PCR detecting the non-recombined erbB2^flox allele in genomic DNA from adult sciatic nerves of three untreated (lanes 1–3) and three TM-treated (lanes 4–6) mutant animals (PLP-CreERT2 erbB2^lacZ/flox). GAPDH serves as internal control. D, Western blot analysis of pooled lysates prepared from sciatic nerves of control (con) and mutant (mut) mice. Control nerves were from three PLP-CreERT2 erbB2^wt/flox; mutant nerves were from three PLP-CreERT2 erbB2^lacZ/flox mice. Mice had been treated with TM for 5 consecutive days at the age of 10 weeks and analyzed 2 months later. The membrane was probed with antibodies specific for erbB2 and β-actin. Note that erbB2 is undetectable in mutant nerves.

Myelinated peripheral nerves are maintained in the absence of erbB2. These are images of transverse semithin sections of adult mouse nerves stained with toluidine blue. The tibial, peroneal, and sural branches of the sciatic nerve are shown for one control and one mutant mouse, 10 weeks after TM induction. Scale bar, 10 μm.

ErbB2-mediated signaling is not required for injury-induced proliferation of Schwann cells. A, Images of longitudinal sections of uninjured (a, b) and the distal stumps of injured (4dpT; c, d) sciatic nerves, double-labeled with an anti-BrdU monoclonal antibody (green) and a rabbit antiserum against the Schwann cell marker S100β (red). Nerves were taken from control (a, c) and mutant (b, d) animals; control nerves were from PLP-CreERT2 erbB2^wt/flox; mutant nerves were from PLP-CreERT2 erbB2^lacZ/flox mice. All mice had been treated with TM for 5 consecutive days at 10 weeks of age. Surgery was performed 10 d after the last TM injection. Note that the number of BrdU-positive nuclei is similar in mutants and control animals at 4dpT. Contralateral, unlesioned nerves contain no labeled cells. Scale bar, 40 μm. B, Quantitative analysis of cell proliferation in control (con) (gray bar) and mutant (mut) (black bar) sciatic nerves 4dpT and 12dpT. Data are mean values obtained from three animals per time point and genotype. There is no significant difference between the control and mutant nerves by Student’s t test. Error bars indicate SD.

A, Western blot analysis of lysates prepared from uninjured nerves (lanes 1 and 2) and from adult sciatic nerves 4dpT (lanes 3 and 4). Control (con) nerves were from PLP-CreERT2 erbB2^wt/flox, and mutant (mut) nerves were from PLP-CreERT2 erbB2^lacZ/flox mice. All mice had been treated with TM for 5 consecutive days at 10 weeks of age, and surgery was performed 10 d after the last TM injection. Membranes were probed with antibodies specific for erbB2, erbB3, and β-actin. B, Quantitative analysis relative to β-actin signals. The corrected densitometry data from each group were compared, arbitrarily setting the erbB2 signal for unlesioned control adult nerves to 1.0. Nerves of four mice per genotype were pooled and two pools were analyzed (Exp. 1 and 2). C, ErbB4 and erbB1 do not compensate for the loss of erbB2. Western blot analysis of uninjured (lanes 1 and 2) and injured sciatic nerves (4dpT; lanes 3 and 4), from adult control and mutant mice as described in Figure 3A. Membrane was probed with antibodies specific for erbB4, erbB1, and β-actin. Protein extract of cultured oligodendrocytes (OLs) expressing both erbB4 and erbB1 was used as a positive control (lane 5).

Lowered levels of activated MAPK and cyclin D1 in erbB2-conditional null nerves. A, Western blot analysis of pooled uninjured (lanes 1 and 2) and injured (4dpT; lanes 3 and 4) sciatic nerves from adult control and mutant mice as described in Figure 4A. Membrane was probed with antibodies specific for MAPK-P (top), total MAPK (middle), and β-actin (bottom). B, The amount of MAPK-P was corrected for unequal total MAPK signals. The corrected densitometry data from each group were compared, arbitrarily setting the MAPK-P signal for unlesioned control adult nerves to 1.0. Nerves of four mice per genotype were pooled and two pools were analyzed (Exp. 1 and 2). C, Western blot analysis of pooled injured sciatic nerves (4dpT) from adult control (lane 1) and mutant (lane 2) mice. The membrane was probed with antibodies specific for cyclin D1 (CycD1) and β-actin. D, Quantification of the signals shows a significant decrease in cyclin D1 levels in injured mutant nerves. Nerves of four mice per genotype were pooled, and two pools were analyzed (Exp. 1 and 2). con, Control; mut, mutant.