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Previous Article | Next Article
Volume 17, Number 5, Issue of March 1, 1997 pp. 1642-1659
Copyright ©1997 Society for NeuroscienceExpression of Neuregulins and their Putative Receptors, ErbB2 and ErbB3, Is Induced during Wallerian Degeneration
Steven L. Carroll1, Michele L. Miller1, Paul W. Frohnert1, Susanne S. Kim1, and John A. Corbett2 1 Division of Neuropathology, Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, and 2 Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104
ABSTRACT
Schwann cell dedifferentiation and proliferation is a prerequisite to axonal regeneration in the injured peripheral nervous system. The neuregulin (NRG) family of growth and differentiation factors may play a particularly important role in this process, because these axon-associated molecules are potent Schwann cell mitogens and differentiation factors in vitro. We have examined Schwann cell DNA synthesis and the expression of NRGs and their receptors, the erbB membrane tyrosine kinases, in rat sciatic nerve, sensory ganglia, and spinal cord 0-30 d postaxotomy. Analysis of NRG cDNAs from these tissues revealed several novel splice variants and showed that cells endogenous to injured nerve express NRG mRNAs. A selective induction of mRNAs encoding the glial growth factor (GGF) subfamily of NRGs occurs in nerve beginning 3 d postaxotomy and thus coincides with the onset of Schwann cell DNA synthesis. In later stages of Wallerian degeneration, however, Schwann cell mitogenesis markedly decreases, whereas elevated GGF expression persists. Of the four known erbB kinases, Schwann cells express both erbB2 and erbB3 receptors over the entire interval studied. Expression of erbB2 and erbB3 is coordinately induced in response to axotomy, indicating that Schwann cell responses to NRGs may be modulated by changes in receptor density. Neuregulin (including transmembrane precursors) and erbB protein are associated with Schwann cells postaxotomy. Thus, in contrast to the concept of NRGs as axon-associated mitogens, our findings suggest that NRGs produced by Schwann cells themselves may be partially responsible for Schwann cell proliferation during Wallerian degeneration, probably acting via autocrine or paracrine mechanisms.
Key words: neuregulins; glial growth factor; Schwann cell; erbB receptor; Wallerian degeneration; autocrine; paracrine
INTRODUCTION
Axotomy of peripheral nerve induces the development of a microenvironment that supports axonal regeneration (Aguayo et al., 1978
; Bray et al., 1987
In vitro proliferation studies using neonatal rat sciatic nerve Schwann cells have led to the identification of a group of potent Schwann cell mitogens known as the glial growth factors (GGFs) (Brockes et al., 1980
In this report, we determined (1) whether NRG mRNA and protein are present in sciatic nerve before and during the period of maximal Schwann cell DNA synthesis; (2) which NRG isoforms are present and what their likely sources are; (3) whether Schwann cells bear the receptors necessary for responsiveness to NRGs; and (4) whether the types of erbB receptors expressed change as Schwann cells cease proliferation and move into a new, quiescent differentiated state. Although we have found that these molecules are indeed expressed in a pattern consistent with a role as mediators of Schwann cell proliferation, several of our results were unexpected and indicate that the role of neuregulins in Wallerian degeneration is likely to be complex.
MATERIALS AND METHODS
Animals and surgical procedures. Adult male Harlan Sprague Dawley rats (200-300 gm) were obtained from Sasco (Omaha, NE). All animals were handled in accordance with the guidelines of National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Rats were anesthetized by inhalation of methoxyfluorane (Metofane). Unilateral axotomies were produced by exposing the sciatic nerve in the midgluteal region, transecting it at the sciatic notch, and reflecting the distal segment caudally to prevent regeneration. Crush injuries were similarly produced by crushing the nerve at the sciatic notch with jeweler's forceps. Wounds were carefully sutured shut and animals were allowed to recover. Water and food were provided ad libitum until the rats were killed.
PCR and subcloning. Unless otherwise dictated by the desired target sequence, oligonucleotides were designed with the aid of PrimerSelect software (Windows version 3.01a, DNAStar, Madison, WI). Our designation of neuregulin domains follows the suggestion of Peles and Yarden (1993) 1 splice variant clone isolated with the common EGF-like/juxtamembrane oligonucleotides (clone pSLC120) and has the sequence AGTTCCTCCGCTTCCATAAAT. The specific "3" juxtamembrane domain reverse oligonucleotide corresponds to nucleotides 731-710 of a
Single-stranded cDNA for use as PCR templates in the initial cloning of the neuregulin EGF-like/juxtamembrane domain cDNAs and erbB probes was synthesized from poly(A+) RNA isolated with oligo-dT cellulose chromatography; the reactions were performed in a 20 µl reaction using random hexamers as primers and Moloney murine leukemia virus reverse transcriptase (Superscript Plus, Life Technologies, Gaithersburg, MD). After completion of the reactions, specimens were diluted to 100 µl with double-distilled water, boiled for 5 min, and stored at 20°C until use. Two microliters of each cDNA was used as a PCR template. PCR was performed for 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1-2 min (depending on the length of the expected product). Products were then either gel-purified, treated with Klenow fragment of DNA polymerase I for 30 min at 37°C and cloned into the EcoRV site of pBluescript KS(+) (Stratagene, La Jolla, CA), or chloroform-extracted and cloned without further purification into the EcoRV site of the T-vector pT7Blue (Novagen, Madison, WI). Neuregulin clones were screened with DdeI restriction digestion to distinguish
and
The designation of clones isolated in this way and the encoded domains are as follows: pSLC111, immunoglobulin-like domain of NRG; pSLC118,
Construction and screening of cDNA libraries. Equal amounts of poly(A+) RNA isolated from 16 hr, 3 d, and 7 d postaxotomy nerve segments (both proximal and distal to the axotomy site) were used to synthesize cDNA by the technique of Gubler and Hoffman (1983) ZAPII (Stratagene) arms. Ligated phage was packaged (Gigapack Gold, Stratagene) and plated on Escherichia coli (XL-1 Blue strain, Stratagene). A total of 1.2 × 106 primary recombinants was produced in three separate syntheses, pooled, and amplified (Maniatis et al., 1990
The axotomized nerve and spinal cord libraries were plated at high density (50,000 plaques/150 mm plate), and duplicate filter lifts were prepared from each plate (Maniatis et al., 1990
RNA isolation and Northern blot analysis. Total cellular RNA was isolated from tissues using either the method of Chomczynski and Sacchi (1987)
The mesenchymal N terminus probe used for our Northern blot analyses is a 201 bp PCR fragment generated from a small intestine template and verified by sequence analysis (pSLC178). The SMDF N terminus probe is a 1050 bp fragment from a SMDF-
Bromodeoxyuridine incorporation studies. The sciatic nerve of 200-300 gm adult male Harlan Sprague Dawley rats was exposed and transected or crushed at the sciatic notch as described above (see Animals and surgical procedures). At specified times after injury, animals were injected intraperitoneally with a mixture of 15 mg/ml bromodeoxyuridine (BrdU; Sigma B-5002, St. Louis, MO) and 1.5 mg/ml 5-fluorouracil (Sigma F-0503) in 7 mM NaOH/0.85% NaCl (4 ml/kg). Ninety minutes after injection of the BrdU solution, animals were killed, and the tissues of interest were dissected and placed in Bouin's fixative overnight at 4°C. Tissues were then embedded in paraffin, and serial, 5 µm sections were cut and mounted on SuperFrost Plus slides (Fisher Scientific, Pittsburgh, PA).
Every fifth section from each set of slides was deparaffinized, rehydrated through graded alcohols to 1× PBS, and then microwaved for 3 min in the same buffer. After a 15 min incubation in blocking buffer (1% bovine serum albumin/0.2% powdered milk/0.3% Triton X-100), sections were incubated overnight at 4°C with goat
For analysis, slides were visualized with epi-illumination and bright-field optics. Coded slides were photographed at 400× magnification, and the number of labeled and total nuclei was determined by an independent observer; labeled cells outside the endoneurium and those within the vasculature were excluded from these counts. Schwann cells were readily verified in these analyses by their oval, blunt-ended nuclei oriented longitudinally relative to the long axis of the nerve; these criteria have been found to be reliable in earlier analyses of peripheral nerve DNA synthesis (Bradley and Asbury, 1970
Production of a pan-neuregulin polyclonal antibody. A 13 amino acid peptide (sequence FTVKDLSNPSRYL) corresponding to a portion of the rat neuregulin EGF-like common domain was synthesized on a multiple-antigen peptide (MAP) carrier core (Research Genetics, Huntsville, AL). Primary immunization of New Zealand white rabbits was performed by subcutaneous injection of MAP peptide emulsified with an equal volume of complete Freund's adjuvant, with booster injections performed 2, 6, and 10 weeks after the primary injection (0.5 mg peptide/injection emulsified with incomplete Freund's adjuvant). Antipeptide antibody titers were monitored by ELISA using MAP peptide-coated plates (1 µg/well). Three rabbits mounted high antipeptide antibody titers; 10 week postimmunization sera from these animals were pooled, and antipeptide antibodies were purified from this serum pool by passage over a peptide/Sepharose affinity column. High-titer fractions, as identified by ELISA, were pooled, aliquoted, and stored at 4°C. Only affinity-purified pan-neuregulin antiserum was used in the experiments described in this work.
In addition to the characterization of the pan-neuregulin antibodies by Western blotting and immunohistochemistry described in Results (see below), the staining pattern of this antiserum was examined in adult skeletal muscle and dorsal root ganglia and compared with the patterns reported previously with other anti-neuregulin antibodies. In skeletal muscle, small intramuscular nerves and muscle surface "plaques" morphologically consistent with neuromuscular junctions were intensely stained in a pattern very similar to earlier reports (Freeman et al., 1994
Antisera and immunohistochemical reagents. Rabbit polyclonal antibodies and the corresponding peptides to the EGF receptor (sc-03), erbB2/c-neu (sc-284), erbB3 (sc-285), erbB4 (sc-283), neuregulin transmembrane precursors with an "a" C terminus (sc-348), and neuregulin splice variants with a "3" juxtamembrane domain ("secreted" forms; sc-347) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-S100
Production of recombinant neuregulin. Truncated rat NRG
Immunoblotting. Tissue and cell line homogenates for immunoblotting were prepared by homogenizing tissue in 19 vol of ice-cold HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 250 mM sucrose) containing 2 µg/ml aprotinin and 2 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined with a modified Lowry method (DC Protein Assay, Bio-Rad, Hercules, CA). Samples were separated on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (0.45 µm; Hoefer, San Francisco, CA). Transfer was accomplished by electroblotting overnight at 40 mA in 25 mM Tris, pH 8.3, 0.192 M glycine, and 20% methanol. After transfer, equivalent transfer was verified by Coomassie blue staining of residual protein in the gel. Membranes were blocked at 4°C overnight or for 2 hr at room temperature with 5% nonfat dry milk in 1× TBST (0.15 M NaCl, 10 mM Tris, pH 8.0, 0.05% Tween 20, 0.002% sodium azide). Primary antibodies were diluted with 1% nonfat dry milk in 1× TBST. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories) was used at a 1:7000 dilution in 1× TBST. Immunoreactive species were identified by enhanced chemiluminescence (Amersham).
Immunohistochemistry. At specified times after axotomy, rats were anesthetized by Metofane inhalation and perfused transcardially with room temperature 0.85% saline followed by room temperature 4% paraformaldehyde in 1× PBS. Tissues of interest were dissected and post-fixed overnight at 4°C in 4% paraformaldehyde in 1× PBS. After a rinse in ice-cold 1× PBS, tissues were infiltrated at 4°C for 48 hr with 10% sucrose in 1× PBS and then embedded in TBS tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC). Eight micrometer cryostat sections were thaw-mounted on SuperFrost Plus slides (Fisher Scientific) and stored at
For conventional single- and double-label immunohistochemistry, nonspecific binding was blocked by incubation for 20 min at room temperature with PBS blocking buffer (0.1 M PBS, pH 7.4, 1% bovine serum albumin, 0.2% powdered milk, 0.3% Triton X-100). Primary antibodies diluted in PBS blocking buffer were applied overnight at 4°C. After three PBS rinses, sections were incubated for 1 hr at room temperature with DTAF- (1:100 dilution) and/or Cy3- (1:200 dilution) conjugated secondary antibodies diluted in PBS blocking buffer. Sections were then washed three times with PBS and mounted in 1:1 PBS/glycerol. Control sections were incubated without primary antibodies to identify potential nonspecific binding of the secondary antibodies. In addition, the specificity of the observed immunostaining was confirmed by preincubating antisera with either immunizing peptide or a nonrelated peptide in concentrations spanning four orders of magnitude (10 µg/ml to 10 ng/ml of peptide); in all instances, preabsorption at the higher concentrations of immunizing peptide abolished the staining pattern of the primary antibody, with lower peptide concentrations demonstrating progressively stronger (albeit still diminished) immunostaining. Preincubation with nonrelated peptide had no effect on the staining pattern of the primary antibodies.
For double-label immunohistochemical experiments in which both primary antibodies were raised in rabbit, the recently developed technique of "dilutional neglect" immunohistochemistry was used (Shindler and Roth, 1996
Conventional immunofluorescence images were visualized using a Zeiss-Axioskop microscope equipped for epifluorescence. Confocal immunofluorescent images were obtained using a Molecular Dynamics Sarastro 2000 laser scanning unit coupled to a Zeiss Axioskop microscope equipped with a Zeiss Plan ApoChromat 63×, 1.4 numerical aperture objective. For both rhodamine/DTAF and Cy3/DTAF double-labeled preparations, images were acquired simultaneously using a 488/568 nm primary beam splitter and a 565 nm secondary beam splitter with the longer wavelength light being passed through a 600DF40 filter and the shorter through a 530DF30 filter on a second photomultiplier tube. It was confirmed that this optical setup resulted in no fluorescent bleedthrough between channels. Images were processed on a Silicon Graphics workstation using the Imagespace software package (Molecular Dynamics). Processing consisted of independent linear scaling of the red and green images, with either no further processing or a single passage through a Gaussian filter.
RESULTS
Isolation and characterization of neuregulin cDNAs expressed in axotomized sciatic nerve and neuronal populations projecting axons into this nerve
If the neuregulins promote in vivo Schwann cell mitogenesis during Wallerian degeneration, their availability to Schwann cells should coincide with the onset of DNA synthesis and persist throughout the subsequent period of maximal proliferation. Although it is known that DRG sensory neurons (Marchionni et al., 1993
Fig. 1. Identification of neuregulin splice variants expressed in axotomized sciatic nerve and the corresponding neuronal populations. A, Poly(A+) RNA was isolated from a pool of surgically transected sciatic nerve distal to the site of transection collected 16 hr, 3 d, and 7 d postinjury and a pool of L4, L5, and L6 dorsal root ganglia and lumbar spinal cord collected 7 and 10 d postaxotomy. Each RNA pool was reverse-transcribed to cDNA with random hexamer primers and used as templates for PCR with primers, the positions of which are indicated by arrows (modified from Ben-Baruch and Yarden, 1994
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In addition to alternative splicing, neuregulin variability is generated further through the probable utilization of at least three alternative promoters [the "mesenchymal," "GGF," and "SMDF" promoters (Peles and Yarden, 1993 GGF expression is induced in axotomized sciatic nerve
Although our RT-PCR analyses indicated that NRG transcripts are generated by cells within injured nerve, this technique is exquisitely sensitive, and the resulting clones could reflect the contribution of a very minor cell population within this tissue. To make an initial assessment of whether appreciable levels of neuregulin mRNAs are expressed in normal and axotomized sciatic nerve and the neuronal populations projecting axons into this nerve, RNA samples were probed by Northern blotting using a cDNA fragment encoding the extracellular domain of a GGF (kringle-
Fig. 2. Neuregulin mRNA expression in sciatic nerve is induced by axotomy. A, A cDNA fragment (Probe) spanning the neuregulin transmembrane (TM), EGF-like (EGF
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To more fully characterize the time course of neuregulin mRNA induction in axotomized sciatic nerve, surgical transection of the sciatic nerve was performed under conditions that do not allow axonal regeneration (see Materials and Methods). At specified times (1-30 d postaxotomy), tissues were dissected and used to prepare total cellular RNA; this time course spans the period in which the quiescent Schwann cells of the intact adult nerve dedifferentiate, traverse their period of maximal proliferation, and again withdraw from the cell cycle, entering a new quiescent, dedifferentiated state (see below). A blot of noninjured and postaxotomy nerve RNA over this time course was hybridized to the GGF extracellular domain probe described above (Fig. 2B). Although NRG mRNA was undetectable in noninjured nerve and only faintly seen by 1 d post-transection, NRG mRNA with the same size distribution seen in Figure 2A was readily detectable by 3 d postaxotomy and continued to increase in abundance to 30 d postaxotomy.
Although our results thus far indicated that NRG synthesized within axotomized nerve represented a major potential source of this Schwann cell mitogen, these experiments did not allow us to determine which of the major neuregulin subfamilies these transcripts represented. Replicate RNA blots containing RNA from 7 d postaxotomy nerve, JS1 cells, and a panel of tissues expected to express one or more NRG splice variants with each N terminus were therefore probed with cDNA fragments encoding each of the distinct N termini. A GGF-specific probe recognized primarily the 2.0 kb mRNA with prominent GGF expression identified in axotomized sciatic nerve, spinal cord, skeletal muscle, and JS1 schwannoma cells (Fig. 2C). Lower levels of GGF transcripts are also present in small and large intestine. In contrast, the SMDF N terminus probe recognized the 3.5 and 7.5 kb NRG transcripts in JS1 cells (Fig. 2D) and, at much lower levels, in spinal cord (data not shown; see Fig. 2 legend). The mesenchymal N terminus was not detected in axotomized nerve with Northern blots (data not shown), although a weak signal for these isoforms was detected in subsequent RT-PCR analyses (data not shown). We conclude that GGF forms of NRG represent the major set of the splice variants induced in peripheral nerve by axotomy. Characterization of a pan-neuregulin antiserum and demonstration that neuregulin protein is induced in sciatic nerve postaxotomy
Although we found that neuregulin mRNA is induced in sciatic nerve by axotomy, this did not establish when neuregulin protein is potentially available to Schwann cells or with what cell types this protein is associated. The array of neuregulin splice variants raises the questions of whether specific isoforms might be induced postaxotomy and whether different neuregulin splice variants might have distinct cellular distributions. To facilitate study of all neuregulin splice variants, we raised a "pan-neuregulin" antiserum by immunizing rabbits with a peptide sequence found in the EGF-like common domain of all neuregulin isoforms, but not in other molecules with an EGF domain (Holmes et al., 1992
Fig. 3. Western blot analysis of neuregulins in axotomized sciatic nerve. A, Lysates of bacteria expressing either truncated recombinant rat NRG
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Protein was prepared from noninjured sciatic nerve and surgically transected nerve at the same time points used for our RNA analyses, and immunoblots of these lysates were probed with the pan-neuregulin antibodies, as well as with antibodies directed against the subset of neuregulin transmembrane precursors with an "a" C terminus and "secreted" neuregulins (isoforms with a "3" juxtamembrane domain). Analysis of the normal and injured nerve lysates with the pan-neuregulin antiserum demonstrated a complex pattern of immunoreactive species that changed with increasing time after axotomy. Three NRG species (100, 70, and 55 kDa) are detectable in noninjured nerve (Fig. 3C, top). Because NRG mRNA is detectable only with difficulty in noninjured nerve [undetectable by Northern analysis (see above), but detectable by RT-PCR] (S.L.C., unpublished observations), these proteins may be derived from axonally associated NRGs or a relatively stable population of molecules produced by cells endogenous to the nerve. After axotomy, the relative abundance of the 55 and 70 kDa NRG-like forms diminishes rapidly. By 3 d postaxotomy, three other NRG-like immunoreactive proteins (45, 75, and 90 kDa) are increasing in abundance. The 75 and 90 kDa proteins continue to increase in abundance through 30 d. The 45 kDa species, however, peaks in abundance 3 d postaxotomy and gradually declines thereafter, last detectable at 18 d postaxotomy (for a longer exposure demonstrating these changes, see Fig. 3C, bottom). A 55 kDa immunoreactive protein is again detected 7-10 d postaxotomy and persists to 30 d after injury (Fig. 3C, bottom). The identity of at least a portion of the protein in some of these induced bands was clarified further by immunoblotting with two additional antisera. An antibody directed against neuregulin precursors with an "a" C terminus specifically labeled proteins at the same positions as the 90 and 100 kDa species recognized by the pan-neuregulin antiserum (Fig. 3D), as well as a 55 kDa protein (see figure legend). Similarly, antiserum recognizing the "3" juxtamembrane domain specifically detected a protein migrating at the same position as the 75 kDa polypeptide labeled by the pan-neuregulin antiserum (Fig. 3E). These results demonstrate that NRG-like immunoreactive proteins are induced by 3 d postaxotomy, thereby coinciding with the onset of Schwann cell DNA synthesis. At later times, however, further alterations in the sizes of the detected NRG-like species occur, suggesting that Schwann cells may be exposed to differentially spliced and/or processed NRG isoforms over the period studied in this work. Neuregulin induction is not detected in spinal cord and DRG postaxotomy
The expression of NRG transcripts by cells within the nerve does not obviate the possibility that axon-associated NRG might contribute further to Schwann cell proliferation during regeneration. If NRGs are presented to Schwann cells on regenerating axonal growth cones, it is possible that an increase in NRG protein levels might be seen in the affected neuronal populations. Therefore, we examined the expression of NRG protein in lumbar (L4, L5, and L6) DRG and the lumbar enlargement of the spinal cord 0-30 d after surgical transection of the sciatic nerve using our pan-neuregulin antiserum. In contrast to axotomized nerve, analysis of protein expression in the lumbar DRG and spinal cord showed minimal evidence of neuregulin induction by sciatic nerve transection (Fig. 4A,B). Western blot studies of normal and postaxotomy DRG and spinal cord with the "secreted" ("3" juxtamembrane) and "a" neuregulin transmembrane precursors, as well as RNA (Northern blot, semiquantitative RT-PCR) analyses, also showed, at best, minimal evidence of alterations in neuregulin expression (data not shown). These results argue that if presentation of axon-associated NRG does play a role in regulating Schwann cell mitogenesis, it is not accompanied by a marked increase in the accumulation of neuronal NRG.
Fig. 4. Western blot analysis of neuregulins in postaxotomy spinal cord and DRG. The pan-neuregulin antiserum was used to probe immunoblots of lysates of lumbar dorsal root ganglia (L4 and L5 DRG; A) and spinal cord (B) collected from noninjured (Normal) and axotomized animals at the indicated times postaxotomy (45 µg/ml protein loaded/lane). No induced neuregulin proteins were identified in either tissue. The position of specific neuregulin proteins is indicated (arrows).
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Induction of Schwann cell DNA synthesis in axotomized sciatic nerve correlates with the onset of GGF induction
Although the induction of Schwann cell DNA synthesis in surgically transected sciatic nerve has been carefully analyzed in the adult mouse (Bradley and Asbury, 1970
Fig. 5. DNA synthesis by Schwann cells in axotomized sciatic nerve. A-F, Photomicrographs of BrdU-labeled Schwann cell nuclei in sciatic nerve crushed at the sciatic notch. Nuclei actively synthesizing DNA during the BrdU pulse were detected by silver-enhanced immunogold staining and visualized with epi-illumination. Shown are representative fields from noninjured nerve (A) and nerve 15 mm distal to a site of crush injury 1 d (B), 3 d (C), 5 d (D), 7 d (E), and 18 d (F) after injury. All photomicrographs are taken at 400× magnification. Scale bar, 50 µm. G, Schwann cell labeling indices 3, 5, and 18 d after surgical transection. Mean labeling indices ± SEs are indicated by the horizontal bars. Positions relative to the site of injury are indicated below each bar, as well as by the schematic diagram at the bottom of the figure. N.D., Not done.
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Although the largest number of labeled nuclei was found 5 d postaxotomy, it was not clear that this time represented the peak of mitotic activity, because the total number of Schwann cells also increases dramatically between 3 and 5 d after injury (Abercrombie and Johnson, 1946
Table 1. Schwann cell DNA synthesis in axotomized nerve
Time postaxotomy Position No. of sections examined No. of Schwann cell nuclei labeled/examined Average labeling index SE 3 d Noninjured 28 2 /706 0.16% 0.15% 6 mm proximal 30 9 /468 2.3% 1.1% 3 mm proximal 30 54 /920 10.0% 0.9% Neuroma 18 431 /1980 22.2% 1.3% Stump 32 329 /2134 16.6% 1.7% 3 mm distal 26 89 /718 13.1% 1.5% 6 mm distal 30 107 /796 14.6% 1.7% 9 mm distal 20 55 /477 11.9% 1.3% 12 mm distal 16 59 /417 14.4% 1.2% 15 mm distal 12 50 /353 14.5% 1.1% 5 d Noninjured 24 0 /670 0 0 6 mm proximal 4 0 /117 0 0 3 mm proximal 22 6 /595 0.9% 0.3% Neuroma 34 257 /2479 10.5% 0.7% Stump 28 224 /2146 10.5% 0.5% 3 mm distal 18 76 /699 11.1% 1.3% 6 mm distal 22 69 /794 10.4% 2.0% 9 mm distal 30 98 /966 10.7% 1.4% 12 mm distal 20 82 /795 10.2% 1.5% 15 mm distal 6 20 /243 8.1% 1.4% 18 d Noninjured 12 1 /910 0.1% 0.1% 6 mm proximal ND 3 mm proximal ND Neuroma 34 117 /6652 1.9% 0.2% Stump 36 82 /4080 1.9% 0.2% 3 mm distal 28 61 /3006 2.3% 0.5% 6 mm distal 28 43 /2779 1.6% 0.2% 9 mm distal 36 52 /3193 1.8% 0.3% 12 mm distal 34 50 /3651 1.4% 0.3% 15 mm distal ND ND, Not determined. The erbB2 and erbB3 neuregulin receptors are coordinately induced by axotomy of peripheral nerve
Although neuregulin expression is clearly induced in sciatic nerve by axotomy and coincides with the onset of Schwann cell DNA synthesis, it remained to be determined whether Schwann cells bear the erbB receptors necessary for responsiveness to neuregulins in vivo. The erbB family of receptors consists of four genes in man known as erbB1 (the EGF receptor), erbB2 (HER2/c-neu), erbB3 (HER3), and erbB4 (HER4/tyro2) (for review, see Peles and Yarden, 1993
Although all NRG splice variants share the common characteristic of stimulating erbB2 tyrosine phosphorylation, it has become evident that the products of the closely related proto-oncogenes erbB3 and erbB4 are the specific receptors for the NRGs and that erbB2 transphosphorylation occurs when ligand binding induces erbB3 or erbB4 to form a heterodimer with erbB2 (Carraway and Cantley, 1994
The effects of sciatic axotomy on the expression of all four erbB receptors were examined by probing immunoblots of lysates from noninjured nerve and the segment of nerve distal to a site of surgical transection over the same period examined for neuregulin expression. Although erbB4 was undetectable at all time points examined, erbB1, erbB2, and erbB3 proteins were present in sciatic nerve at all time points studied (Fig. 6). The erbB2 receptor protein is upregulated in response to sciatic axotomy beginning 5 d postaxotomy, in keeping with previous observations at the mRNA level (Cohen et al., 1992 
Fig. 6. erbB2 and erbB3 are coordinately induced in sciatic nerve by surgical transection. Western blots of tissue lysates prepared from noninjured (Control) and the segment of sciatic nerve distal to a site of surgical transection (1-30 d postaxotomy), as well as whole-cell lysates of JS1 cells, were probed with antibodies specific for each of the erbB membrane tyrosine kinases (indicated below each panel). Although erbB1 (EGF receptor), erbB2, and erbB3 are readily detectable in sciatic nerve, erbB4 was never seen in axotomized nerve over this time course (note the easily visualized band in the brain lysate). Western blot analyses were also used to confirm that erbB4 was absent in noninjured nerve and JS1 cells (data not shown).
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Immunoreactive neuregulin and erbB protein in normal and postaxotomy nerve localizes to Schwann cells
Although our analyses of neuregulin and erbB receptor mRNA and protein in sciatic nerve clearly demonstrated that increased expression of these molecules is induced by axotomy, their cellular source remained unclear. Therefore, we localized neuregulin-, erbB2-, and erbB3-like immunoreactivity in noninjured sciatic nerve and nerve segments distal to a site of surgical transection 3, 5, and 7 d postaxotomy. To assess possible cellular associations of these molecules, Schwann cells and axons were identified in these preparations by staining with antibodies for S100
An initial characterization verified that the pan-neuregulin antiserum reproduced previously described patterns of staining in skeletal muscle and dorsal root ganglia (see Materials and Methods). These antibodies were found to label both noninjured and axotomized nerve. In noninjured nerve, immunoreactivity was visualized as fluorescent streaks. Double-label indirect immunofluorescence demonstrated that the pan-neuregulin immunoreactivity was associated with Schwann cells (Fig. 7A,B) and did not overlap with that for neurofilaments (Fig. 7C). The absence of axonal neuregulin immunoreactivity in the more proximal segments of noninjured nerve is consistent with previous reports (Sandrock et al., 1995 
Fig. 7. Neuregulin-like immunoreactivity in normal and surgically transected sciatic nerve visualized by indirect immunofluorescence. A-C, Confocal images of longitudinal sections of noninjured sciatic nerve double-labeled with the pan-neuregulin antiserum [DTAF (green) label in A and Cy3 label (orange-red) in C] and antibodies to either the Schwann cell marker S100
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If the erbB2 and erbB3 receptors mediate the Schwann cell response to axon-associated neuregulin, it is expected that these molecules would be expressed on the Schwann cell membrane facing the axon to facilitate reception of the neuronal signal. We therefore immunolocalized erbB protein on cross-sections of noninjured nerve and examined its relationship to the axon, as highlighted by staining for neurofilaments. Both erbB2 and erbB3 were readily detectable in this tissue with a virtually identical distribution. Immunoreactivity for erbB receptors was detected as intense rings of immunofluorescence with fainter central staining (Fig. 8A,B). No staining was observed in the absence of primary antibodies, and preincubation of the primary antisera with the immunizing peptides completely blocked immunostaining, whereas preincubation with a nonrelated peptide had no effect (data not shown). Colocalization with immunoreactivity for neurofilaments demonstrated that the outer rim of erbB2 and erbB3 staining was separated from the axon by an unstained region, presumably representing the myelin sheath of the Schwann cell (Fig. 8C,D); this rim of erbB receptor staining completely overlapped that of S100 
Fig. 8. Indirect immunofluorescent localization of erbB2 and erbB3 in normal and surgically transected sciatic nerve. A, B, Confocal images of cross sections of noninjured nerve decorated with antibody to erbB2 (A) or erbB3 (B). Staining is visualized as rings of orange-red staining surrounding a region of central staining. Scale bars: 20 µm in A and 10 µm in B. C, D, Confocal images of cross sections of noninjured nerve double-labeled with antibody to neurofilament [DTAF (green) label] and either erbB2 (C) or erbB3 [D; erbB antibodies visualized with Cy3-labeled secondary antibodies (orange-red)]. Note the unstained region separating the outer rings of erbB receptor immunostaining from the central axonal (neurofilament) staining. As a control, double-label immunohistochemistry for S100
[View Larger Version of this Image (115K GIF file)]
To establish that the increase in erbB2 and erbB3 protein observed beginning 5 d postaxotomy resulted from increased Schwann cell expression of these molecules, we also examined erbB receptor immunoreactivity in axotomized sciatic nerve (3, 5, and 7 d postaxotomy). The majority of cells in the endoneurium stained with an essentially identical pattern for both erbB2 and erbB3, with the most intense staining seen 5 and 7 d postaxotomy (Fig. 8E). In keeping with earlier descriptions of erbB2 immunostaining in axotomized nerve (Cohen et al., 1992
DISCUSSION
The neuregulins have been demonstrated to be potent Schwann cell mitogens in vitro (Brockes et al., 1980
The onset of Schwann cell DNA synthesis in surgically transected sciatic nerve 3 d postaxotomy coincides with the initial accumulation of NRG mRNAs and at least some forms of NRG protein. NRG mRNA is undetectable in noninjured nerve by RNA blot analysis, with significant accumulation beginning 3 d after the axon is separated from the cell body and persisting to 30 d postaxotomy, a time well after the axon has degenerated and been phagocytosed. Furthermore, our RT-PCR analyses indicate that the neuregulin EGF-like/juxtamembrane domains expressed in sciatic nerve differ significantly from those seen in the lumbar DRG and spinal cord. We conclude that NRG mRNAs induced in response to peripheral nerve injury are derived from cells within the nerve itself. Potential cellular sources of NRG mRNAs in axotomized nerve include fibroblasts, perineurial cells, Schwann cells, mast cells, and macrophages. In keeping with an earlier report that schwannomas express extremely high levels of a GGF-like activity (Brockes et al., 1986
There is strong evidence that axon-associated neuregulins promote Schwann cell differentiation and proliferation in the developing peripheral nervous system (Shah et al., 1994
One of our more surprising results was the finding that elevated neuregulin mRNA and protein levels persist for as long as 30 d postaxotomy, a time well after the Schwann cell labeling index has decreased to a small fraction of its peak value. Precisely how a decrease in Schwann cell mitogenesis is accomplished in the face of continued elevated levels of NRGs is currently unclear. Possibilities include a compartmentalization of NRGs that precludes interaction with the Schwann cell erbB receptors, an acquired resistance (desensitization) to NRG stimulation after the initial period of mitogenesis, and a predominance of other NRG actions (e.g., maintenance of a new state of differentiation, prevention of apoptotic death after separation from axons) in the later phases of Wallerian degeneration. We would also caution that it is possible that the neuregulins produced by Schwann cells may have actions on other cell types within injured nerve. At least some of these effects could potentially be modulated by varying the types of neuregulin splice variants expressed over the first 30 d of Wallerian degeneration. It will therefore be of great interest to analyze in detail the structure of the neuregulin isoforms expressed in sciatic nerve over the course of the events we have defined in this work.
In contrast to the mesenchymal neuregulin isoforms (Wen et al., 1994
A transition between different neuregulin receptor subtypes is yet another potential mechanism for modulating NRG actions during Wallerian degeneration of peripheral nerve. However, our demonstration that only erbB2 and erbB3 receptors are expressed in noninjured nerve and throughout the entire period of Wallerian degeneration that we studied argues that an erbB2/erbB3 heterodimer most likely represents the functional neuregulin receptor in Schwann cells and is consistent with an earlier report that these receptors are expressed by cultured human Schwann cells (Levi et al., 1995
Analysis of neuregulin and erbB receptor expression in peripheral nerve undergoing Wallerian degeneration has provided several new insights that have modified our view of this complex and intriguing process. In addition to identifying previously unexpected routes of interaction for the neuregulins, our analyses have also allowed us to narrow the field of candidate molecules that must be manipulated in the next stage of this work, i.e., interrupting signaling through the Schwann cell neuregulin/erbB pathway to establish whether activation of this pathway is required for Schwann cell mitogenesis after axotomy. Although mice with genetic ablation of the neuregulin (Meyer and Birchmeier, 1995
FOOTNOTES
Received Sept. 27, 1996; revised Dec. 2, 1996; accepted Dec. 10, 1996.
This work was supported in part by Grant 36-37#4 from the American Cancer Society (I.R.G.) and a Howard Hughes Medical Institute Pilot Research Grant (S.L.C.). We thank Drs. Kevin Roth, Robert Schmidt, Eugene Johnson, William Snider, and Josh Sanes for helpful comments on this manuscript. We especially thank Dr. Roth for communicating the details of his "dilutional neglect" immunohistochemical technique before publication and Bill Coleman for assistance with confocal microscopy. We also gratefully acknowledge Angela Schroeder for assistance with figure preparation.
Correspondence should be addressed to Dr. Steven L. Carroll, Department of Pathology, Division of Neuropathology, Box 8118, 660 South Euclid Avenue, Washington University School of Medicine, St. Louis, MO 63110.
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