 |
 Previous Article | Next Article 
Volume 17, Number 5,
Issue of March 1, 1997
pp. 1642-1659
Copyright ©1997 Society for Neuroscience
Expression 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
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
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; Bandtlow, 1993; Raivich and
Kreutzberg, 1993). Establishment of this supportive microenvironment
requires that nerve undergo the morphological changes of Wallerian
degeneration (Holmes and Young, 1942; Fawcett and Keynes, 1990; Tonge
and Golding, 1993), in which axon segments and myelin distal to a site
of trauma degenerate (Holmes and Young, 1942) and are phagocytosed by
Schwann cells and macrophages (Perry and Brown, 1992). Coincident with axonal degeneration, normally quiescent Schwann cells dedifferentiate and proliferate, an event critically important for the promotion of
axonal regeneration (Hall and Gregson, 1977; Pellegrino et al., 1986;
Fawcett and Keynes, 1990; Nadim et al., 1990). Thus, identification of
the signals regulating Schwann cell dedifferentiation, proliferation,
and migration during Wallerian degeneration is of great
interest.
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; Goodearl et al., 1993). Cloning of GGF cDNAs demonstrated
that these proteins are translated from alternatively spliced mRNAs
transcribed from a single gene (Marchionni et al., 1993) that encodes a
family of growth and differentiation factors, including the heregulins
(Holmes et al., 1992), neu differentiation factor (NDF) (Wen et al.,
1992), acetylcholine receptor-inducing activity (Falls et al., 1993),
and sensory and motor neuron-derived factor (SMDF) (Ho et al., 1995).
Because these factors all induce tyrosine phosphorylation of the
neu (also known as HER2 and c-erbB2) proto-oncogene (Falls et al., 1993; Marchionni et al., 1993), they are
collectively referred to as the neuregulins (NRGs) (Peles and Yarden,
1993). In addition to their in vitro mitogenic effects, the
neuregulins may act as axon-derived signals influencing Schwann cells
during development. Axon-associated neuregulins have been implicated in
promoting neural crest cell differentiation into Schwann cells (Shah et
al., 1994), preventing the apoptotic death of pre- and neonatal Schwann
cells (Dong et al., 1995; Lee et al., 1995; Marchionni, 1995;
Trachtenberg and Thompson, 1996) and as a component of the
"axon-associated mitogen" expressed by neonatal sensory neurons
(Morrissey et al., 1995). In light of these in vitro and
developmental activities, it is highly likely that members of the
neuregulin family promote Schwann cell proliferation during Wallerian
degeneration of adult peripheral nerve, possibly acting as an
axon-derived mitogen.
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). Oligonucleotides for the immunoglobulin-like domain
of neuregulin correspond to nucleotides 169-193 and 481-501 of rat
neu differentiation factor (GenBank accession no. M92430[GenBank]) (Wen et al.,
1992). The common forward oligonucleotide for PCR of the epidermal
growth factor (EGF)-like domains and the adjacent juxtamembrane
segments represents nucleotides 867-895 of rat neuregulin (GenBank
accession no. U02323[GenBank]) (Wen et al., 1994); the common (recognizes
sequences immediately carboxy to juxtamembrane domains 1, 2, and 4)
reverse oligonucleotide for PCR of the same domains corresponds to
nucleotides 1067-1049. The specific "1" juxtamembrane domain
reverse oligonucleotide was designed from the sequence of a rat  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 3
splice variant (GenBank accession no. U02315[GenBank]) (Wen et al., 1994). The
specific "4" juxtamembrane domain reverse oligonucleotide
corresponds to nucleotides 1124-1101 of a 4a splice variant
(GenBank accession no. U02322[GenBank]) (Wen et al., 1994). Oligonucleotides for
PCR of the neuregulin "mesenchymal" N terminus correspond to
nucleotides 358-381 and 535-558 of a rat cDNA (GenBank accession no.
U02318[GenBank]) (Wen et al., 1994). Oligonucleotides used for PCR of rat EGF
receptor correspond to nucleotides 3122-3142 and 3488-3510 of the
mouse sequence (GenBank accession no. U03425[GenBank]) (Luetteke et al., 1994).
Oligonucleotides used for PCR of rat c-neu/erbB2 correspond to
nucleotides 3216-3236 and 3463-3485 of the published rat sequence
(GenBank accession no. X03362[GenBank]) (Bargmann et al., 1986).
Oligonucleotides used for PCR of rat erbB3 correspond to nucleotides
2968-2989 and 3475-3498 of the human HER3/erbB3 sequence (GenBank
accession no. M34309[GenBank]) (Plowman et al., 1990). Oligonucleotides used for
PCR of rat erbB4 correspond to nucleotides 3101-3123 and 3628-3651 of
the human HER4/erbB4 sequence (GenBank accession no. L07868[GenBank]) (Plowman
et al., 1993).
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 subtypes; the diagnostic sizes for specific EGF-like/juxtamembrane domain combinations were determined by direct PCR of bacterial colonies. The identity and orientation of clones were then verified by
cycle sequencing using an automated DNA sequencer (ABI model 373A,
Applied Biosystems, Foster City, CA). If necessary for the purpose of
transcribing riboprobes, fragments from pT7Blue clones were subcloned
into pBluescript KS(+).
The designation of clones isolated in this way and the encoded domains
are as follows: pSLC111, immunoglobulin-like domain of NRG; pSLC118,
2 EGF-like/variable domain; pSLC120, 1 EGF-like/variable domain;
pSLC121, 2 EGF-like/variable domain; pSLC149, 3 EGF-like/variable domain; pSLC153, 4 EGF-like/variable domain; pSLC163, rat EGFR cytoplasmic domain; pSLC162, rat c-neu cytoplasmic cDNA; pSLC125, rat
erbB3 cytoplasmic domain; pSLC112, rat erbB4 cytoplasmic domain. A rat
c-neu probe (neu c(t)pSP6400) was also obtained from the American Type Culture Collection (Rockville, MD). The complete sequence
of the rat EGF, erbB3, and erbB4 receptor cDNAs has been deposited in
GenBank and may be found under accession nos. U52529[GenBank], U52530[GenBank], and
U52531[GenBank], respectively.
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); a NotI oligo-dT primer-adaptor (Promega,
Madison, WI) was used for the first strand synthesis. After treatment
with T4 DNA polymerase and DNA polymerase I (Klenow fragment),
double-stranded cDNA was ligated to EcoRI linkers and then
digested with EcoRI and NotI. cDNAs larger than 500 bp were excised from a 1% agarose gel, purified (Gene-Clean; BIO101, Vista, CA), and ligated to EcoRI/NotI
digested and phosphatased  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). A rat
brainstem/spinal cord library was purchased from Stratagene
(936521).
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).
32P-labeled probe was generated by the random
oligonucleotide priming method (Feinberg and Vogelstein, 1984) with a
commercial kit (Boehringer-Mannheim Biochemicals, Indianapolis, IN) and
hybridized to filters representing a total of 5 × 105
plaque-forming units from each library under high stringency conditions
(50% formamide/5× saline-sodium phosphate-EDTA/5× Denhardt's/0.1% SDS at 42°C). Filters were washed four times in 2× SSC/0.1% SDS at
room temperature and then twice more in 0.2× SSC/0.1% SDS at 68°C.
After isolation of clones by limiting dilution (Maniatis et al., 1990),
plasmid was rescued from phage by coinfecting XL-1 Blue mrf'
bacteria with the phage and ExAssist helper phage (Stratagene) and
subsequent passage of the rescued single-stranded phagemid through SOLR
bacteria (Stratagene). Four neuregulin cDNAs were isolated from the
spinal cord library: pSLC132, a kringle-1a clone; pSLC133, an
SMDF-1a clone; pSLC134, an SMDF-1 cDNA; and pSLC135, an
SMDF-1a cDNA. Fifty-four erbB3 cDNAs were isolated from the
axotomized sciatic nerve library; one of these cDNAs, pSLC138, was
extensively characterized and used for the studies detailed in this
work. A single erbB4 cDNA, pSLC136, was isolated from the spinal cord
library and similarly characterized.
RNA isolation and Northern blot analysis. Total cellular RNA
was isolated from tissues using either the method of Chomczynski and
Sacchi (1987) or a commercial kit (RNeasy Total RNA kit, Qiagen, Chatsworth, CA). RNA concentrations were determined
spectrophotometrically and verified by visualization on agarose gels.
For Northern blot analysis, RNA samples and size standard markers
(Novagen) were separated on 1% agarose/2.2 M formaldehyde
gels, transferred to Sureblot nylon membranes (Oncor, Gaithersburg,
MD), and baked according to the manufacturer's instructions. DNA
probes were gel-purified (Gene-Clean kit, BIO101) and labeled by random
oligonucleotide priming (Feinberg and Vogelstein, 1984) using a
commercial kit (Boehringer-Mannheim). Prehybridization was performed in
buffer containing 45% formamide/5× SSC/10% dextran sulfate/1%
SDS/100 mg/ml salmon sperm DNA/1 mg/ml polyA. Hybridization was
performed in the same buffer with 5× 105 cpm of
probe/ml at 45° for 16-24 hr. Blots were then washed three times in
2× SSC/0.5% SDS at room temperature (20 min/wash) followed by two
washes at 68°C in 0.2× SSC/0.5% SDS (1 hr/wash). Blots were then
either exposed to Kodak XAR-5 film at 70°C with intensifying screens or analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
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-1a cDNA (pSLC135). The GGF kringle
domain probe is a 480 bp NotI fragment from a kringle-1a
cDNA (pSLC132).
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 BrdU antiserum
(1:10,000 dilution in blocking buffer; antibody kindly provided by Dr.
Steven Cohn, Washington University School of Medicine, St. Louis, MO).
The next morning, slides were washed three times in 1× PBS (5 min/wash) and then incubated for 1 hr at room temperature with
gold-conjugated rabbit anti-goat antiserum (Amersham Life Sciences,
Buckinghamshire, England; 1:40 dilution in blocking buffer). Slides
were then washed three times with 1× PBS (5 min/wash) followed by
three 5 min washes in distilled water. Staining was silver-intensified
using a commercial kit (Amersham Life Sciences), and slides were
counterstained with hematoxylin. Slides were dehydrated and mounted
with Permount (Fisher Scientific).
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; Goodearl et al., 1995; Jo et al., 1995; Sandrock et al., 1995).
In keeping with the results of Sandrock et al. (1995), the
pan-neuregulin antiserum also stained the cell bodies of most, if not
all, adult dorsal root ganglion (DRG) neurons. Antibody staining was
not observed in these tissues in the absence of primary antibody and was specifically blocked by preincubation with the immunizing peptide.
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 antiserum (Z0311)
was from DAKO (Carpinteria, CA). SMI 34, a mouse monoclonal
IgG1 antibody recognizing extensively phosphorylated
neurofilament H and M subunits, including those in large and small
peripheral axons, was purchased from Sternberger Monoclonals
(Baltimore, MD). Dichlorotriazinylaminofluorescein (DTAF)- and
Cy3-conjugated secondary antibodies were obtained from Jackson
Immunoresearch Laboratories (West Grove, PA). Tyramide amplification
reagents, including streptavidin-horseradish peroxidase, rhodamine-tyramide, blocking reagent, and amplification diluent were
purchased from DuPont NEN (Renaissance TSA-Direct (Red); Boston,
MA).
Production of recombinant neuregulin. Truncated rat NRG2
representing the EGF-like and juxtamembrane domains
(rNRG2168-240) was produced using the bacterial
expression vector pSLC151, which contains sequence encoding the
indicated amino acids under the control of the T7lac
promoter in pET22b(+) (Novagen). For expression of protein, pSLC151 was
transferred into the BL21(DE3) strain of E. coli. Cultures
were grown to an OD600 of 0.6, and expression was induced
with 1 mM
isopropyl-1-thio--D-galactopyranoside (IPTG). Bacterial
pellets were boiled for 4 min in 0.1 vol of SDS sample buffer (see
below) and then sonicated for 5-10 sec to lyse the cells. Induced
protein of the expected 11 kDa size was readily visualized by Coomassie
blue staining by 1 hr postinduction, with maximal protein expression
achieved by 3 hr postinduction. Experiments described in this work were
performed using total bacterial lysates without further purification of
recombinant neuregulin.
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 20°C until use.
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). In preliminary experiments, it was determined that
peripheral nerve S100 immunoreactivity is readily detectable with
rhodamine-tyramide amplification at a primary antibody dilution of
1:10,000, whereas S100 immunostaining is undetectable in this same
tissue with conventional immunohistochemistry when the primary antibody
is diluted greater than 1:4000. For double-label immunohistochemistry,
anti-S100 antiserum (1:10,000 dilution) diluted in PBS blocking
buffer was first applied to tissue sections overnight at 4°C. After
three PBS washes, biotinylated donkey anti-rabbit antibody diluted in
PBS blocking buffer (1:1000) was applied for 1 hr at room temperature.
Sections were again rinsed three times with PBS and then incubated in
TNB buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M
NaCl, 0.5% DuPont blocking reagent) for 30 min, followed by a 30 min
room temperature incubation with streptavidin-horseradish peroxidase
(1:500 dilution in TNB). After three washes with Tris buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl),
rhodamine-tyramide (diluted 1:1000 in 1× amplification diluent;
DuPont) was applied to tissue sections for 10 min at room temperature.
Sections were then washed three times with Tris buffer, followed by two
washes with PBS. The second primary antibody, diluted in PBS blocking
buffer, was then applied to tissue sections and detected with
DTAF-conjugated donkey anti-rabbit secondary antibody as described
above. Sections were mounted with 1:1 PBS-glycerol before examination.
As a control, the second primary antibody was omitted for some slides,
resulting in an absence of DTAF staining. Subsequent experiments using
conventional double-label immunofluorescence with a mouse anti-S100
antibody (Sigma S2532), in combination with either the erbB or
pan-neuregulin antibodies, produced an immunostaining pattern identical
to that seen with dilutional neglect
immunohistochemistry.
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;
Orr-Urtreger et al., 1993; Meyer and Birchmeier, 1994; Ho et al., 1995)
and motor neurons of the spinal cord ventral horn (Falls et al., 1993; Marchionni et al., 1993; Orr-Urtreger et al., 1993; Chen et al., 1994;
Corfas et al., 1995; Ho et al., 1995) express NRGs, no information is
available regarding NRG mRNA expression in the nerve itself. Therefore,
we used the reverse transcription (RT)-PCR to compare the expression of
NRG splice variant mRNAs in axotomized sciatic nerve (reflecting both
cell types endogenous to the nerve and responding macrophages) with
that of tissues containing the sensory (L4, L5, and L6 DRG) and motor
(lumbar enlargement of the spinal cord) neuron populations projecting
axons into this nerve. Because functional neuregulin requires an intact
EGF-like domain to bind to its receptor(s) and mediate the biological
activity of the factor (Wen et al., 1994), we designed our initial PCR
primers to span this domain and the adjacent juxtamembrane domain,
which modulates the secretion and/or release of the factor (Wen et al., 1994). Rat cDNA clones of the EGF-like and juxtamembrane domains were
generated from axotomized nerve (nerve distal to a site of surgical
transection 16 hr, 3 d, and 7 d postaxotomy) and lumbar spinal cord and DRG (7 and 10 d postaxotomy) templates. After initial screening and grouping (for details, see Materials and Methods), sequence analysis confirmed that the isolated clones contain
intact, and therefore presumably functional, EGF-like domains.
Neuregulin splice variants were readily detectable in axotomized nerve
with this analysis. Although it should be noted that the ratios of
clonal isolation obtained in these experiments may vary somewhat from
the absolute abundance of each splice variant within a tissue, it is
nonetheless apparent that neuregulins within axotomized nerve are
represented by a population of splice variants (predominantly 2
isoforms) quite distinct from that detected in lumbar DRG and spinal
cord (exclusively 1, 2, 3, and 4 splice variants; Fig.
1A). These findings, considered
together with the typical exclusion of mRNA from the axons of
vertebrate neurons, argue that the neuregulin mRNAs detected in
axotomized nerve cannot have been entirely derived from the remnants of
degenerating axons in the sciatic nerve and thus are likely to
originate from cells endogenous to nerve.
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).
Distinctly different subsets of neuregulin splice variants were
identified in axotomized sciatic nerve as opposed to the regions of the
nervous system projecting axons into this same structure. Regions
encompassed by these cDNAs include the EGF-like common domain
(EGF), EGF-like variable domains ( or ),
the juxtamembrane domains (numbers 1-4), and the
transmembrane domain (TM). Note that splice
variants with a "3" juxtamembrane domain terminate within the
juxtamembrane domain (indicated by a dark bar at the C
terminus). Ratios beneath each juxtamembrane
domain indicate the number of times a clone with each
/-juxtamembrane combination was isolated relative to the number
of clones generated with primers "A" and
"B." Ratios in parentheses
indicate the same values for clones generated using the
"A" primer in combination with "C" (4 juxtamembrane-specific) or "D" (3 juxtamembrane-specific) reverse primers. B, Schematic
representation of currently known members of the GGF and SMDF
neuregulin subfamilies. Although the encoded proteins contain domains
in common [e.g., the EGF-like common domain (EGF)],
each NRG subfamily is distinguished from one another by unique N
termini [represented by SP (signal peptide) and
Kringle for GGF and the SMDF N terminus (Apolar)
above]. Only truncated and presumably secreted (3) variants have
been described previously for GGF (Marchionni et al., 1993) and SMDF
(Ho et al., 1995). In contrast, the clones described in this work
represent transmembrane precursors (1a variants) of SMDF and GGF;
the presence of all domains illustrated has been confirmed by sequence
analysis. These transmembrane precursors may either be cleaved to
release soluble factor (Burgess et al., 1995) or potentially remain
embedded in the membrane to mediate juxtacrine interactions (Bosenberg
and Massague, 1993). Domains not defined above are as follows:
Ig-like, Immunoglobulin-like; Glyco,
glycosylation region; , EGF-like variable; 1 and
3, juxtamembrane domains; Cytoplasmic,
cytoplasmic domain common to all neuregulin transmembrane precursors;
a, one of three potential neuregulin C termini.
[View Larger Version of this Image (14K GIF file)]
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; Ben-Baruch and Yarden,
1994; Ho et al., 1995)]. This results in the production of NRG splice variants with unique N termini and allows the neuregulins to be divided
into three corresponding subfamilies, each defined by their unique N
terminus. To obtain subfamily-specific probes, the neuregulin
EGF-like/juxtamembrane domain clones and a clone of the neuregulin
immunoglobulin-like domain (see Materials and Methods) were used to
screen normal and axotomized sciatic nerve and spinal cord cDNA
libraries for full-length clones of neuregulin. Although no cDNAs were
obtained from the sciatic nerve libraries, four neuregulin clones were
isolated from the spinal cord library and mapped, and selected regions
were sequenced to establish their identity. All of these cDNAs were
found to encode previously undescribed transmembrane precursors within
the GGF and SMDF neuregulin subfamilies (for the structures of these
clones and a delineation of the GGF and SMDF subfamilies, see Fig.
1B).
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-1a variant) cDNA (Fig.
2A). This probe, which contains the
EGF-like domain common to all NRG splice variants, detected up to three mRNA species with sizes of 2.0, 3.5, and 7.5 kb, with the 2.0 kb mRNA
predominating in all tested tissues. Neuregulin mRNA was readily
detectable in tissues containing neurons projecting into the sciatic
nerve (lumbar DRG and spinal cord), as well as in brain and skeletal
muscle. Expression of neuregulin transcripts in noninjured nerve was
compared with that in nerve distal to a site of surgical transection
7 d postaxotomy. It is interesting that although neuregulin mRNA
was undetectable in noninjured nerve, neuregulin mRNA is induced by
7 d postaxotomy to levels comparable to that seen in spinal cord,
a tissue previously reported to demonstrate some of the highest levels
of neuregulin mRNA expression in the body (Wen et al., 1992). As a
control, this blot also contained RNA from the rat JS1 schwannoma cell
line, a line derived from a tumor induced by in utero
chemical mutagenesis (Schubert et al., 1974; Kimura et al., 1990). The
absolute highest abundance of neuregulin mRNA that we observed was in
the JS1 cell line (Fig. 2A).
Fig. 2.
Neuregulin mRNA expression in sciatic nerve is
induced by axotomy. A, A cDNA fragment
(Probe) spanning the neuregulin transmembrane (TM), EGF-like
(EGF1), immunoglobulin-like
(Ig-like), and Kringle domains of a GGF
(kringle-1a) neuregulin splice variant was used to probe a Northern
blot (15 µg of total cellular RNA/lane). Up to three mRNA species
(2.0, 3.5, and 7.5 kb) are seen in this exposure. This blot has been
purposefully overexposed to demonstrate the presence of the 7.5 kb
transcript. B, A total of 10 µg per lane of cellular
RNA from noninjured (Control) sciatic nerve and the nerve segment distal to a site of surgical transection taken 1, 3, 5, 7, 18, or 30 d postaxotomy was probed with the same probe used
in A. Repeat experiments performed with a mixture of
GGF, SMDF, and NDF N-terminal probes confirmed these observations and also demonstrated levels of neuregulin mRNA expression at 10 d postaxotomy similar to those at 7 and 18 d. A
photograph of the ethidium bromide-stained gel before
transfer is presented beneath the Northern blot to
demonstrate the uniform loading of these samples; equivalent transfer
was confirmed by examining the membrane under ultraviolet illumination.
C, D, Northern blots of total cellular RNA (10 µg/lane) isolated from the lumbar enlargement of the spinal cord
(SpCord), the nerve segment distal to a site of surgical
transection 7 d postaxotomy (7d Nerve), JS1 rat
schwannoma cells (JS1), gastrocnemius/soleus muscle
(Muscle), Lung, small intestine
(Sm Int), and large intestine (Lg Int)
were probed with the indicated probes, which are specific for the N
termini of GGF (C) or SMDF (D) neuregulin
splice variants. Except for the 7 d postaxotomy nerve, all tissues
were collected from animals that had not undergone any surgical
manipulation. Expected positions of the 2.0, 3.5, and 7.5 kb mRNAs are
indicated. Note that the GGF probe hybridizes to the 2.0 kb transcript,
whereas the SMDF probe recognizes both the 3.5 and 7.5 kb mRNAs.
Although SMDF expression was most prominent in JS1 cells, longer
exposures of this blot also demonstrated mRNAs of the same size in
spinal cord (data not shown). Again, photographs of the
ethidium bromide-stained gels before transfer are presented
below each autoradiograph.
[View Larger Version of this Image (57K GIF file)]
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; Wen et al., 1992). The specificity of
affinity-purified antibodies isolated from these antisera was tested by
Western blot analysis of lysates from bacteria expressing the rat
NRG2 EGF-like and juxtamembrane domains
(rNRG2168-240) (Ben-Baruch and Yarden, 1994). The
pan-neuregulin antibodies recognized a band of ~11 kDa, the expected
size of the bacterially produced NRG fusion protein, in lysates of
IPTG-induced cells carrying the neuregulin expression plasmid, but not
in similarly induced cells of the same bacterial strain expressing an
unrelated antigen, -galactosidase (Fig.
3A); the pan-neuregulin antiserum also
recognizes two less intense larger bands, which presumably represent
aggregation products of the recombinant protein. Western blot analysis
of lysates of spinal cord and DRG from noninjured animals and 5 d postaxotomy sciatic nerve showed labeling of several protein bands ranging in size from 45 to 100 kDa (Fig. 3B). Preincubation
of the pan-neuregulin antiserum with the immunizing peptide blocked detection of the indicated proteins (Fig. 3B, compare
1 to 2). The sizes of the specifically blocked
protein species in spinal cord are 100, 75, and 52 kDa, which is
similar to the immunoblotting results reported previously with other
antisera in lysates of this tissue (Sandrock et al., 1995). In
preliminary experiments (see also below), antisera raised against other
epitopes present in specific neuregulin subsets (transmembrane
precursors with "a" C termini, secreted forms with "3"
juxtamembrane domains) specifically recognized proteins of the same
size as detected with our pan-neuregulin antibody.
Fig. 3.
Western blot analysis of neuregulins in axotomized
sciatic nerve. A, Lysates of bacteria expressing either
truncated recombinant rat NRG2 (rNRG2168-240) or an
unrelated antigen (-galactosidase) were separated on a 15% gel (25 µg/lane) and probed with the pan-neuregulin antiserum (1 µg/ml).
The times after IPTG induction and the identity of the induced proteins
are indicated above the lanes. The positions of the
detected rNRG2168-240 (arrow, left) and
size markers (arrows, right) are indicated.
B, The specificity of the pan-neuregulin antiserum was
verified by preincubating antiserum (1 µg/ml) for 24 hr at 4°C with
10 µg/ml of the immunizing peptide. The pattern of bands detected in
immunoblots was then compared for blots probed with the pan-neuregulin
antiserum preincubated with no added peptide (left) or
the immunizing peptide (right). The sources of the
lysates tested (spinal cord and DRG from noninjured animals, 5 d
postaxotomy nerve) are indicated above each lane (45 µg protein loaded/lane). Size markers are indicated on the
left side of the left panel, and specific
bands are indicated on the right side of the panel. The
bands indicated are the major forms seen in this experiment. With
longer exposure, no additional specific bands were seen in the DRG or
spinal cord lysates, but in the 5 d postaxotomy nerve lysate, two
other bands (100 and 75 kDa) became detectable in this and other
experiments. C-E, Expression of neuregulin protein was
examined in lysates of noninjured (Normal) nerve
and the nerve segment distal to a site of surgical transection 1, 3, 5, 7, 10, 18, or 30 d postsurgery (45 µg protein loaded/lane) using
the pan-neuregulin antiserum (C), an antibody directed
against the C terminus of "a" transmembrane precursors
(D), and an antibody directed against a peptide from the
"3" juxtamembrane domain of GGF (E). Size markers
are indicated on the left side of the top panel of C, with specific bands indicated by
arrows on the right side of each panel in
C, D, and E. The
bottom panel in C is a longer exposure of
a second immunoblot performed to highlight the 45 and 55 kDa bands. In
D, note also that the "a" neuregulin antibody
recognizes a 55 kDa band beginning 1-3 d postaxotomy; although this
band is specifically blocked by preincubation of the antiserum with
immunizing peptide (data not shown), it may be too small to represent a
complete NRG transmembrane precursor, and its precise identity is
currently unclear. In E, a slightly larger band is seen
in the 7 and 10 d postaxotomy nerve lysates. Although this band is
specifically blocked by preincubation with the immunizing peptide, it
was not observed in immunoblots performed with the pan-neuregulin
antiserum.
[View Larger Version of this Image (41K GIF file)]
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).
[View Larger Version of this Image (42K GIF file)]
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) and cat (Pellegrino et al., 1986; Oaklander
et al., 1987), a similar detailed analysis of postaxotomy Schwann cell
mitotic indices in the rat has not been performed (Friede and
Johnstone, 1967). Because it is well recognized that there are
species-specific temporal variations in the onset and peak of Schwann
cell mitogenesis (Lubinska, 1964), it was necessary that we establish
these parameters for the rat. Crush or transection injury of the
sciatic nerve was performed on three to five adult male Harlan Sprague
Dawley rats per time point and, at the same times used for our RNA and
protein isolations, animals were pulsed with BrdU to label cells in the
DNA synthesis phase of the cell cycle. Labeled nuclei within the
sciatic nerve distal to the side of axotomy were identified by
immunohistochemistry, and the number of labeled cells seen in animals
with crush injuries, which allows axonal regeneration, was compared
with that seen in rats with transection injuries, in which axonal
regrowth was prevented. Preliminary counts of labeled Schwann cell
nuclei showed similar time courses of induction for DNA synthesis in
crush and transection injuries (data not shown). Although virtually no
BrdU incorporation was identified in noninjured nerve and 1 d
postinjury, a marked increase in the number of labeled Schwann cell
nuclei was evident at 3 d and reached a peak 5-7 d after injury
(Fig. 5A-E). By 10 d postinjury, a
decline in the number of Schwann cells synthesizing DNA was evident,
and 18 and 30 d into the course of Wallerian degeneration, only
scattered labeled nuclei were seen (Fig. 5F).
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.
[View Larger Version of this Image (39K GIF file)]
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) (S.L.C., personal observations). Therefore, we
determined the Schwann cell labeling index (number of labeled Schwann
cell nuclei/total number of Schwann cell nuclei; for criteria used to
identify Schwann cells, see Materials and Methods) in nerve 1, 3, and
5 d postaxotomy to establish the peak in mitotic activity. Because
we had determined that neuregulin mRNA was persistently elevated
through even the later phases of Wallerian degeneration, we also
determined the Schwann cell labeling index 18 d postaxotomy. Studies were performed on serial sections from animals that had undergone surgical transection of the nerve with prevention of regeneration to avoid any complications that might be introduced by
regenerating axon sprouts reentering more distal segments of the nerve.
To detect any confounding variability that might be introduced by
anterograde spread of mitotic activity, such as has been described in
the cat (Oaklander et al., 1987), cell counts were performed at 3 mm
intervals along the surgically transected nerve. Schwann cell labeling
indices in noninjured nerve (Fig. 5G, Table
1), as well as the distal segment of nerve, 1 d
postaxotomy (data not shown) were <0.01%. By 3 d postaxotomy,
however, labeling indices throughout the nerve distal to the site of
transection (excluding the distal stump) were 14-15% (Fig.
5G, Table 1). Schwann cell labeling indices obtained in
distal nerve segments 5 d postaxotomy were lower (10-11%; Fig.
5G, Table 1) than those determined 3 d postaxotomy.
Mitotic activity is markedly lower by 18 d postaxotomy with a
labeling index of ~2% seen throughout the distal segment of the
nerve (Fig. 5G, Table 1). We conclude that the peak of
Schwann cell mitotic activity occurs 3 d postaxotomy, a result
similar to that obtained previously with explants of degenerating rat
nerve maintained in vitro (Clemence et al., 1989). The onset
of Schwann cell proliferation therefore coincides with the initial
induction of neuregulin mRNA synthesis. In the later phases of
Wallerian degeneration (18 and 30 d postaxotomy), although elevated levels of neuregulin mRNA are still present, Schwann cell
mitotic activity is markedly decreased.
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; Ben-Baruch and Yarden, 1994). Although the
human (Xu et al., 1984; Haley et al., 1987), mouse (Avivi et al., 1991;
Luetteke et al., 1994), and chicken (Flickinger et al., 1992) EGF
receptor genes have been cloned, and erbB2 has been cloned
from several species, including the rat (Bargmann et al., 1986), only
human erbB3 and erbB4 receptor cDNAs had been
isolated when we began this work (Plowman et al., 1990, 1993).
Therefore, we used RT-PCR with axotomized sciatic nerve, DRG and spinal
cord templates to isolate partial cDNAs of the rat EGF,
erbB3, and erbB4 receptors, and sequenced these
clones to establish their relationship to the human genes. Each of the
rat clones was found to be closely related to their human and (when
sequence was available) mouse orthologs (Luetteke et al., 1994; Moscoso
et al., 1995). The rat erbB3 and erbB4 clones
were used to screen the nerve and spinal cord libraries. More extensive
cDNAs were isolated for each receptor and characterized for use in our
studies (see Materials and Methods).
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). It
has been demonstrated previously that the erbB2 receptor is expressed
by Schwann cells (Cohen et al., 1992; Jin et al., 1993). To determine
whether erbB3 or erbB4 receptors are expressed in sciatic nerve, RNA
from noninjured and postaxotomy nerve, the JS1 schwannoma cell line,
and other nervous system structures were probed for expression of the
corresponding mRNAs. Although erbB4 was undetectable in both noninjured
and surgically transected nerve, erbB3 mRNA was readily detectable in
noninjured sciatic nerve and was induced six- to sevenfold by 7 d
postaxotomy (data not shown). Furthermore, very high levels of erbB3
mRNA were present in the JS1 schwannoma line, suggesting that Schwann cells might be the source of erbB3 mRNA in peripheral nerve.
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). Intriguingly, we also found that the erbB3
receptor is induced with a time course parallel to that of erbB2. The
expression of both erbB2 and erbB3 persists at an elevated level until
18 d postaxotomy, when a slight decline is evident for both
receptors (more so for erbB3 than erbB2). EGF receptor is also induced
by axotomy, peaking at 10 d after transection. However, it is
interesting that although both erbB2 and erbB3 are present at high
levels in the JS1 schwannoma cell line, the EGF receptor is
undetectable in this cell line. This result, coupled with preliminary
immunohistochemical studies showing that the most intense EGF receptor
immunoreactivity in axotomized nerve is associated with epineurial
cells (S.S.K. and S.L.C., unpublished observations), suggests that a
cell type(s) other than Schwann cells may be the source of much of the
EGF receptor protein in nerve.
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).
[View Larger Version of this Image (59K GIF file)]
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 and neurofilaments (SMI 34, a mouse monoclonal recognizing heavily phosphorylated H and M
chains found in peripheral axons), respectively. Three different
antibodies to different neuregulin epitopes were used in these studies
and found to give identical immunohistochemical results (see
below).
|
