The Journal of Neuroscience, August 13, 2003, 23(19):7269-7280
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Hypertrophic Neuropathies and Malignant Peripheral Nerve Sheath Tumors in Transgenic Mice Overexpressing Glial Growth Factor 
3 in Myelinating Schwann Cells
Richard P. H. Huijbregts,1
Kevin A. Roth,1
Robert E. Schmidt,2 and
Steven L. Carroll1
1Division of Neuropathology, Department of
Pathology, The University of Alabama at Birmingham, Birmingham, Alabama
35294-0017, and 2Division of Neuropathology,
Department of Pathology, Washington University School of Medicine, St. Louis,
Missouri 63110
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Abstract
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The neuregulin-1 (NRG-1) family of growth and differentiation factors
exerts a variety of effects on Schwann cells and their precursors during
nervous system development; however, NRG-1 effects on adult Schwann cells are
poorly defined. Several lines of evidence suggest that NRG-1 actions on adult
Schwann cells are distinct from those observed during development. To test
this hypothesis, we generated transgenic mice overexpressing the NRG-1 isoform
glial growth factor 
3 (GGF3) in myelinating Schwann cells [protein
zero (P0)GGF3 mice]. P0-GGF3 mice develop
resting tremors, gait abnormalities, decreased hindlimb strength, and
paralysis by 
7 months of age. Sciatic nerves from these animals show a
hypertrophic neuropathy characterized by demyelination, remyelination, and
"onion bulb" formation. Development of this hypertrophic
neuropathy is preceded by Schwann cell hyperplasia that is prominent in
1-month-old mice and present but decreased in 2- and 4-month-old animals.
P0-GGF3 mice also develop peripheral ganglion-associated
malignant peripheral nerve sheath tumors. Motor, sensory, and sympathetic
ganglia from 1-, 2-, and 4-month-old P0-GGF3 mice uniformly
contain intraganglionic, likely preneoplastic, Schwann cell proliferations.
Examination of bromodeoxyuridine incorporation and caspase-3 activation in
sciatic nerves and trigeminal ganglia indicates that Schwann cell hyperplasia
in P0-GGF3 mice reflects increased proliferation rather than
decreased apoptosis. These observations are consistent with the hypothesis
that GGF3 induces proliferation of adult Schwann cells and demyelination
of peripheral nerve axons. Furthermore, overexpression of this NRG-1 isoform
frequently induces neoplastic Schwann cell proliferation within PNS ganglia,
suggesting that NRG-1 may contribute to human Schwann cell neoplasia.
Key words: Schwann cell; erbB receptor; schwannoma; neuropathy; neuregulin; glial growth factor
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Introduction
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The phenotype of Schwann cells and their precursors is modulated during
development by members of the neuregulin-1 (NRG-1) family of growth and
differentiation factors. NRG-1 proteins, which include glial growth factor
(GGF), neu differentiation factor, heregulin, and acetylcholine
receptor-inducing activity, are alternatively spliced membrane-bound or
soluble molecules derived from a single gene
(Fischbach and Rosen, 1997
).
The soluble NRG-1 isoform GGF3 promotes glial differentiation of neural
crest cells (Shah et al.,
1994), the pluripotent precursors from which Schwann cells arise,
and prevents these cells from differentiating into neurons. NRG-1 isoforms
also regulate the survival, proliferation, and differentiation of Schwann cell
precursors, the glial elements derived from neural crest cells
(Dong et al., 1995), and prime
these cells to express factors necessary for myelination. From late
embryogenesis through the neonatal period, axonally derived NRG-1 proteins
"match" the number of Schwann cells and axons by promoting the
proliferation, migration, and survival of axon-associated committed immature
Schwann cells (Topilko et al.,
1996; Felts, 1999;
Adlkofer and Lai, 2000;
Garratt et al., 2000a). It is
thus apparent that NRG-1 is critically important throughout Schwann cell
development, with the precise responses elicited by these factors depending on
the developmental stage of the glia.
Although NRG-1 is also expressed by neurons projecting into the adult PNS
(Chen et al., 1994;
Bermingham-McDonogh et al.,
1997), little is known regarding NRG-1 actions on adult Schwann
cells. Several lines of evidence suggest that NRG-1 has in vivo
effects on adult Schwann cells differing from those observed during
development. Adult Schwann cells, unlike embryonic Schwann cells, depend on
autocrine signals for survival (Cheng et
al., 1998), having lost a requirement for axonally derived NRG-1
(Meyer et al., 1997;
Morris et al., 1999;
Wolpowitz et al., 2000); these
observations are consistent with an absence of Schwann cell apoptosis in
nerves from older rodents, both in the uninjured state and after nerve
transection (Grinspan et al.,
1996). Furthermore, GGF3 induces demyelination in
established Schwann cell-neuron cocultures
(Zanazzi et al., 2001); this
contrasts with the observation that Schwann cell-targeted ablation of the
NRG-1 receptor subunit erbB2 inhibits the initial myelination of axons during
embryogenesis and early postnatal life
(Garratt et al., 2000b).
Additional clues to potential NRG-1 actions on adult Schwann cells are
provided by studies in injured peripheral nerves, in which the expression of
multiple NRG-1 isoforms has been found to be induced coincident with the onset
of Schwann cell proliferation (Carroll et
al., 1997). Tyrosine phosphorylation of Schwann cell erbB
receptors is increased with a similar time course
(Kwon et al., 1997),
suggesting that NRG-1 promotes Schwann cell mitogenesis in the regenerating
adult nerve. These observations argue that NRG-1 continues to regulate the
phenotype of adult Schwann cells. NRG-1 actions on adult Schwann cells,
however, likely differ from those encountered earlier in life. To test this
hypothesis, we have generated transgenic mice that overexpress the NRG-1
isoform GGF3 in mature myelinating Schwann cells.
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Materials and Methods
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Animal care and surgical procedures. Mice were cared for in
accordance with the guidelines of the NIH Guide for the Care and Use of
Laboratory Animals. All animal experiments were approved by the
Institutional Animal Care and Use Committee of the University of Alabama at
Birmingham.
Mice were housed in standard cages with filter lids. Water and food were
available ad libitum. Animals were killed by either decapitation or
cardiac perfusion after being anesthetized with Metofane (Schering-Plough
Research Institute, Union, NJ) or Nembutal (pentobarbital; Abbott Labs, North
Chicago, IL).
Construction of a protein zero-GGF3 transgene. A
cDNA containing the entire coding sequence for GGF3 was constructed from
three distinct clones, an approach that allowed a codon encoding a proline in
our partial GGF3 cDNA (GenBank accession number AF194996
[GenBank]
; S. L. Carroll
et al., unpublished data) to be replaced with sequences encoding the leucine
more commonly present at this position. As the first step in this process, an
SphI-EcoRV fragment from the 3' end of a GGF1a
cDNA (pSLC132; GenBank accession number AF194993
[GenBank]
) was replaced with an
SphI-EcoRI-Klenow fragment from the 3' end of the
GGF3 clone pSLC343. The N-terminal sequence of the GGF3 cDNA was
then reassembled by replacing a BamHI-NotI fragment in this
intermediate construct with a BamHI-NotI fragment from a
genomic clone (pSLC384) containing sequences encoding the entire kringle
domain. The complete GGF3 cDNA sequence was cloned into vector pGEM-11f
(+) (Promega, Madison, WI) to produce pSLC405. The complete cDNA was then
released from pSLC405 by digestion with BamHI and HindIII.
The protruding ends of the GGF3 cDNA were blunted using DNA polymerase I
(Klenow fragment), and the cDNA was ligated to AscI linkers (New
England Biolabs, Beverly, MA). Subsequently, the GGF3 cDNA was subcloned
into pBluescript II KS (+) cut with EcoRV to produce pSLC409. The
GGF3 cDNA was released from pSLC409 by partial digestion with
AscI and then cloned into AscI-digested mouse protein
zero-TOTA (mP0TOTA; Feltri et
al., 1999) to produce the transgene construct pSLC410. The
linearized fragment used to generate transgenic animals was released by
digesting pSLC410 with PacI and SpeI, which liberates the
P0 sequence containing the inserted GGF3 cDNA from the vector
backbone.
Generation of transgenic mice. To generate transgenic animals, the
linearized transgene construct was injected into fertilized C57BL/6 x
SJL hybrid F1 oocytes, which were reimplanted into pseudopregnant
Swiss Webster recipients. The resulting offspring were screened using PCR with
genomic DNA isolated from tail preps. The primers used for these screens were
a forward primer (5'-ATCCACATCAACATCCACG-3') corresponding to
nucleotides 1115-1133 (relative to the initiation ATG) of the GGF3
sequence and a reverse primer(5'-AAGTTGCTGAGAGACCAC-3')
corresponding to nucleotides 123-106 relative to the initiation ATG in exon 1
of the murine major myelin P0 sequence. Transgenic founders and
their offspring were backcrossed to C57BL/6J x SJL/J F1
hybrids (The Jackson Laboratory, Bar Harbor, ME).
Semiquantitative reverse transcription-PCR. Total RNA was isolated
from tissue or cultured cells with Trizol (Invitrogen, San Diego, CA). After
DNase treatment (Promega), RNA was reverse transcribed with Superscript II
reverse transcriptase (Invitrogen). A portion of the DNase-treated sample was
not reverse-transcribed and was used to verify an absence of genomic DNA
contamination. These cDNAs served as templates for semiquantitative reverse
transcription (RT)-PCR. Primers for detection of transgene transcripts were
designed using Primer Express II software (Applied Biosystems, Foster City,
CA). The GGF3 forward primer (5'-CCTCTGCCAACATCACCATTG-3')
corresponds to nucleotides 1164 -1174 of the GGF3 rat cDNA sequence
(GenBank accession number AF194996
[GenBank]
). The GGF3 reverse primer
(5'-TGACGGGTTTGACAGGTCCT-3') corresponds to nucleotides 1414 -1393
of the GGF3 rat cDNA sequence. To facilitate normalization of GGF3
mRNA levels, cyclophilin mRNA levels were also assayed. The cyclophilin
forward primer (5'-CAAGACTAGGTGGCTGGATGG-3') corresponds to
nucleotides 393-413 in the rat cyclophilin cDNA sequence
(Danielson et al., 1988). The
cyclophilin reverse primer (5'-TAAAATGCCCGCAAGTCAAAGAAA-3')
corresponds to nucleotides 561-538 in the rat cyclophilin cDNA sequence. PCR
was performed using a PerkinElmer Life Sciences (Emeryville, CA) GeneAmp 2400
thermocycler. PCR conditions and empirical determination of the linear range
of the PCR reaction for each primer pair were established as described
(Carroll and Frohnert, 1998)
using cDNA isolated from JS1 cells transiently transfected with plasmid
pSLC458, which contains the GGF3 construct under the control of the
cytomegalovirus immediate early promoter. Cycle parameters, after an initial 3
min melt at 94°C, were 30 sec at 94°C followed by 30 sec at 60°C
and 1 min at 72°C. The midpoints of the logarithmic range of amplification
were determined to be 27 cycles for GGF3 and 28 cycles for cyclophilin;
consequently, amplification for assays of GGF3 and cyclophilin mRNA
levels in sciatic nerves was performed using 27 and 28 cycles,
respectively.
Western blot analyses. Protein from the sciatic nerve was isolated
using Trizol (Invitrogen) according to the manufacturer's protocol. The
denatured protein pellet was resuspended in 100 mM Tris-HCl, pH
6.8, and 1% SDS, heated at 55°C and sonicated twice (30 sec/sonication
with a 1 min cooling interval between sonications) in a bath sonicator.
Samples were centrifuged at 20,000 x g for 5 min. The
supernatant was removed and assayed for protein content. Twenty micrograms of
protein were resolved by SDS-PAGE, immunoblotted onto a polyvinylidene
difluoride membrane, and probed with a rabbit polyclonal pan-neuregulin
antibody (Carroll et al.,
1997) followed by a horseradish peroxidaseconjugated donkey
anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA).
Immunoreactive species were detected by chemiluminescence (SuperSignal Pico
chemiluminescence kit; Pierce, Rockford, IL).
Preparation of plastic sections for light and electron microscopic
analyses. Tissues were immersion-fixed in 3% glutaraldehyde in Sorensen's
buffer for 1-2 hr at room temperature and then postfixed in this same fixative
overnight at 4°C. Sciatic nerve segments and ganglia were then postfixed
in OsO4, dehydrated through graded alcohols, and embedded in
Epon-Araldite or Spurr's medium. Semithin sections of nerves cut perpendicular
to the orientation of the axonal fibers were stained with toluidine blue for
light microscopic examination. For electron microscopy, thin-layer sections
were stained with uranyl acetate and lead citrate. Electron microscopy was
performed using a Philips 200 electron microscope.
Bromodeoxyuridine incorporation studies. Mice were injected
intraperitoneally with bromodeoxyuridine (BrdU) and 5-fluorouracil (5-FU; 60
mg/kg BrdU and 6 mg/kg 5-FU) for 90 min before killing. BrdU-pulsed animals
were perfused transcardially with 4% paraformaldehyde in PBS and then
postfixed in 4% paraformaldehyde at 4°C. The sciatic nerve, trigeminal
nerve, selected PNS ganglia, jejunum (a positive control for BrdU
incorporation), and, when applicable, tumor tissue were isolated.
Tissues were embedded in paraffin, and 5-6 µm sections were prepared and
mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA).
Sections were deparaffinized in Citrisolve (Fisher Scientific) and rehydrated
through isopropanol to water and then PBS. Citrate antigen retrieval was
performed by steaming slides for 20 min in a rice steamer and then allowing
slides to cool to room temperature for 20 min. Endogenous peroxidase activity
was blocked by treating slides with 3% H2O2 for 5 min,
followed by washes performed first with water and then with PBS. Sections were
incubated for 15-30 min in tyramide signal amplification blocking buffer
(PerkinElmer Life Sciences). After blocking, slides were incubated with
goat-anti-BrdU antiserum (1:200,000 dilution in blocking buffer; antiserum
kindly provided by Dr. Steven Cohn, Washington University School of Medicine)
overnight at 4°C. After three rinses in PBS, sections were incubated at
room temperature for 1 hr in blocking buffer containing donkey anti-goat-HRP
(1:1000 dilution; Jackson ImmunoResearch). After three washes in PBS, sections
were incubated with cyanine 3-tyramide in Plus Amp buffer (PerkinElmer Life
Sciences) for 30 min. Sections were then washed two times with PBS (5
min/wash), followed by a wash with PBS containing 0.04 µg/ml bisbenzamide
to label nuclei within the sections. Sections were then washed twice more with
PBS and mounted in PBS/glycerol, (1:1 v/v).
Digital images from immunostained preparations were acquired using a Zeiss
(Thornwood, NY) Axioskop fluorescence microscope and analyzed using Image-Pro
Plus acquisition and analysis software. Nuclei within the endoneurium
(excluding those within the endoneurial vasculature) were identified as
Schwann cells on the basis of the morphology of their nuclei (oval,
blunt-ended nuclei oriented longitudinally relative to the long axis of the
nerve); we and others have previously found these criteria to be quite
reliable for the identification of Schwann cells in histologic sections
(Asbury, 1967;
Carroll et al., 1997). Schwann
cells actively synthesizing DNA were identified by the presence of BrdU
immunoreactivity colocalizing with bisbenzamide staining.
Immunohistochemistry for activated caspase-3, S100, and
collagen type IV. These immunostains were performed on paraffin sections
of nerves. Activated caspase-3 was detected using a rabbit polyclonal antibody
specific for this antigen (Cell Signaling, Inc.) with tyramide signal
amplification immunohistochemistry performed as described above. S100
immunoreactivity was detected using a rabbit polyclonal anti-S100
primary antibody (Dako, Carpinteria, CA; 1:200 dilution) and a Cy3-conjugated
anti-rabbit secondary antibody (Jackson ImmunoResearch). Immunostaining for
collagen type IV was performed using a mouse monoclonal primary antibody
specific for this antigen (clone PHM-12, 1.5 µg/ml; Ventana Medical
Systems, Tucson, AZ) followed by a horseradish peroxidase-conjugated secondary
antibody, with immunoreactivity detected by diaminobenzidine deposition.
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Results
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Generation of transgenic mice overexpressing the NRG-1 isoform GGF
3 in myelinating Schwann cells
To test the hypothesis that NRG-1 is capable of inducing Schwann cell
dedifferentiation, proliferation, or both in the noninjured peripheral nerve,
we produced transgenic mice constitutively overexpressing NRG-1 in this
tissue. We chose to overexpress GGF3, an NRG-1 splice variant that is
directly secreted without the need for release by a transmembrane
domain-specific protease, in the peripheral nerve. The transgene for these
experiments (referred to subsequently as the P0-GGF3
transgene) was constructed by inserting sequences encoding GGF3 into a
genomic clone of the major peripheral myelin P0 gene, which has
been modified so that the cDNA can be inserted at the P0 initiation
codon (Fig. 1A). This
vector has been found to reliably produce high-level expression in myelinating
Schwann cells, with low-level expression first detectable at postnatal day 5
(P5) and high-level expression evident by P15 and persisting into adulthood
(Feltri et al., 1999).
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Figure 1. Mice carrying the P0-GGF3 transgene overexpress GGF3
in sciatic nerve. A, Schematic representation of the
P0-GGF3 transgene in which the expression of a rat GGF3
cDNA is directed by 6.0 kb of 5' flanking sequence and intragenic
regulatory elements from the mouse myelin P0 locus contained in
mP0TOTA (Feltri et al.,
1999). Black boxes indicate exons of the P0 gene, with
the structural domains of the GGF3 cDNA indicated as white boxes. The
GGF domains are as follows: SP, signal peptide; Kringle, kringle domain; EGF,
EGF-like domain; , EGF -variant domain; 3, 3 variant juxtamembrane
domain; and 3'-UTR, the GGF3 3' untranslated region. The
P0 start codon in mP0TOTA has been replaced with an
AscI site, and the GGF3 cDNA has been inserted into this site.
B, Semiquantitative RT-PCR analyses of RNA isolated from the sciatic
nerves of P0-GGF3 mice (line 33) demonstrate that GGF3
mRNA is overexpressed in this tissue. Semiquantitative RT-PCR was performed
using primers specific for GGF3 and the housekeeping gene cyclophilin
with RNA isolated from the sciatic nerves of 4-month-old transgenic mice
(Transgenic) or age-matched, nontransgenic controls (Control). Signals for
GGF3 were normalized to cyclophilin levels in the same specimen.
GGF3/cyclophilin ratios are presented as bars, with SEM indicated for
each bar. The GGF3/cyclophilin ratio from a nontransgenic mouse (139) is
arbitrarily defined as 1, with the level of expression in individual mice
normalized to this reference. Numbers under each bar are the identifiers of
specific animals. C, Immunoblot analyses of protein from the sciatic
nerves of P0-GGF3 mice demonstrate that this tissue contains
elevated levels of neuregulin-1 protein. Twenty micrograms of sciatic nerve
protein from 2-month-old transgenic mice and nontransgenic littermates (line
33) were resolved, blotted, and probed with an antibody specific for the NRG-1
EGF-like common domain [a pan-neuregulin antibody
(Carroll et al., 1997)]. The
arrow to the left indicates the position of a 60 kDa antigen that is expressed
at increased levels in two different transgenic mice (Transgenic) relative to
a nontransgenic control (Control). Arrows to the right indicate the positions
of size markers.
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Four P0-GGF3 founders (founders 33-35 and 44) were
identified and showed little evidence of neurologic or other abnormalities
until they were 7 months of age (see below). However, two of these
founders produced no transgenic progeny, whereas a third produced only a
single transgenic pup, which could not be further propagated
(Table 1). Transgenic offspring
were readily obtained from the fourth founder (founder 33). To assess
transgene expression in the progeny of P0-GGF3 founder 33,
semiquantitative RT-PCR analyses were performed on RNA isolated from the
sciatic nerves of 4-month-old transgenic animals and their nontransgenic
littermates. GGF mRNA levels in the sciatic nerves of nontransgenic mice were
barely detectable, with levels at the extreme lower end of our assay range
(Fig. 1B). In
contrast, GGF mRNA levels in line 33 P0-GGF3 mice were much
higher than in their nontransgenic littermates. Because of the very low levels
of GGF mRNA in the nontransgenic sciatic nerve, we can only estimate that GGF
mRNA levels in the sciatic nerves of 4-month-old P0-GGF3
animals are at least 4- to 10-fold higher than in age-matched nontransgenic
controls. When probed with an antibody specific for the epidermal growth
factor (EGF)-like common domain present in all NRG-1 isoforms
(Carroll et al., 1997),
sciatic nerve lysates from P0-GGF3 mice of line 33 were found
to contain increased levels of a 60 kDa NRG-1-like antigen
(Fig. 1C). The size of
this NRG-1-like antigen is very similar to that previously reported for GGF-II
(59 kDa; Goodearl et al.,
1993; Marchionni et al.,
1993), indicating that full-length GGF3 protein is
overexpressed in sciatic nerves from these transgenic animals. Levels of
expression of GGF3 mRNA and protein showed some variation between
individuals of line 33; because GGF3 expression in these animals is
directed by the regulatory elements of the P0 gene, and GGF3
has been found to repress P0 expression
(Cheng and Mudge, 1996),
variable GGF expression in P0-GGF3 mice may reflect cyclic
induction and repression of transgene expression.
Mice overexpressing GGF3 in myelinating Schwann cells develop
hindlimb paralysis associated with hypertrophic neuropathy
At 7 months of age, founders 33-35 developed gait abnormalities and
started dragging their hind legs. When picked up by their tails, these
founders clasped their feet tightly to their bellies rather than demonstrating
a normal outstretching of the hind legs and splaying of the toes. Hindlimb
muscular strength was diminished, as demonstrated by a decreased ability to
grasp with their hind feet. As their condition progressed, founders 33-35
developed ptosis, a hunched posture, and a prominent resting tremor. In
addition, founder 34 demonstrated severe priapism toward the end of his
course. In founder 44, hind leg weakness was the most prominent symptom, which
eventually developed into complete hindlimb paralysis
(Fig. 2A). No
abnormalities of urination or defecation were present in any founder,
indicating that spinal cord function was grossly intact. We subsequently
observed similar neurologic abnormalities in the progeny of founder 33.
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Figure 2. Gait abnormalities and hindlimb paralysis are caused by a hypertrophic
neuropathy in P0-GGF3 founders 44 and 35. A,
Hindlimb paralysis, evident as leg dragging while ambulating, in
P0-GGF3 founder 44. B-D, Plastic semithin cross
sections of sciatic nerves from transgenic founders 35 (B) and 44
(C) compared to an age-matched nontransgenic control (D).
These 1 µm sections have been stained with toluidine blue. Numerous onion
bulbs are evident in nerves from both founders (e.g., B, arrowhead).
Furthermore, numerous large axons, which are thinly myelinated (B,
arrows), are present. Occasional actively degenerating axons are also seen
(C, arrowheads). Magnification (B-D), 1000x; scale
bars (B-D), 25 µm. E, Electron micrograph showing a
lipid-laden macrophage in the sciatic nerve from founder 44. Magnification, 6
· 103x. F, Electron micrograph demonstrating
a demyelinated large axon in sciatic nerve from founder 44. Magnification, 15
· 103x.
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To investigate the anatomic basis of the neurologic abnormalities in these
animals, necropsy examinations were performed on founders 44 and 35 and some
of the progeny of founder 33. No gross abnormalities were identified in these
studies with the exception of atrophy of the hindlimb musculature. One
micrometer plastic semithin cross sections of the sciatic nerves were
prepared, stained with toluidine blue, and examined by light microscopy.
Sciatic nerves from founders 35 (Fig.
2B) and 44 (Fig.
2C) as well as mice from line 33 (see below) showed
multiple abnormalities characteristic of a severe hypertrophic neuropathy
including numerous well developed "onion bulbs" (e.g., see
Fig. 2B, arrowhead).
Most of the remaining large axons were thinly myelinated
(Fig. 2B, arrows), and
occasional structures morphologically consistent with actively degenerating
axons were present (Fig.
2C, arrowheads). None of these changes were ever observed
in sciatic nerves from age-matched nontransgenic controls
(Fig. 2D).
Electron microscopic examination of nerves from founders 35 and 44
confirmed the presence of well developed onion bulbs. Furthermore, these
studies demonstrated the presence of endoneurial lipid-laden macrophages
(Fig. 2E) and
occasional (<0.1%) large unmyelinated ("naked") axons
(Fig. 2F). Sciatic
nerves from P0-GGF3 transgenic mice therefore showed evidence
of both demyelination (lipid-laden macrophages and naked axons) and
remyelination (thinly myelinated large axons). As the same hypertrophic
neuropathy occurs in founders 44 and 35 and animals from line 33, we conclude
that this neuropathy results from GGF3 overexpression rather than
line-specific integration site effects.
The hypertrophic neuropathy in P0-GGF3 transgenic
mice develops in adulthood
The development of neurologic abnormalities at 7 months of age
suggests that the progression of clinical findings in P0-GGF3
transgenic mice is paralleled by the development of anatomic changes. To test
this hypothesis, sciatic nerves were collected from P0-GGF3
transgenic (line 33) and wild-type control mice at 1 (seven transgenic and two
wild-type), 2 (eight transgenic and two wild-type), 4 (four transgenic and one
wild-type), and 7 (four transgenics and one wild-type) months of age. One
micrometer plastic semithin cross sections were prepared from these tissues,
stained with toluidine blue, and examined by light microscopy. At 1 month of
age, there was no evidence of hypertrophic neuropathy in the sciatic nerves of
P0-GGF3 mice (Fig.
3A), with myelination in this tissue similar to that seen
in age-matched controls (data not shown). By 2 months of age, however,
rudimentary onion bulbs were detected in the sciatic nerves of some transgenic
animals (Fig. 3B,
arrows). By 4 months of age, both onion bulbs and many large, thinly
myelinated axons were present, abnormalities that were even more evident in
7-month-old animals (Fig.
3C). Onion bulb formation in particular was very
prominent in many 7-month-old transgenic mice of line 33, much as was seen in
founders 35 and 44 (see above). None of these pathologic findings were evident
in the sciatic nerves of wild-type control animals.
Although the findings described above were commonly evident in the sciatic
nerves of P0-GGF3 mice of line 33, the time course with which
these pathologic changes developed was variable in some animals, with
dysfunction developing more slowly in some individuals and more rapidly in
others. One exceptional mouse had already developed severe hindlimb paralysis
by the age of 2.5 months.
Schwann cell hyperplasia is evident in the sciatic nerve of
P0-GGF3 transgenic mice by 1 month of age
Neuregulin-1 promotes the survival
(Grinspan et al., 1996) and
proliferation (Topilko et al.,
1996; Felts, 1999;
Adlkofer and Lai, 2000;
Garratt et al., 2000a) of
neonatal Schwann cells. If NRG-1 has either of these effects on adult Schwann
cells, we would expect that Schwann cell hyperplasia would be evident in the
nerves of P0-GGF3 mice. To test this hypothesis, longitudinal
paraffin sections of sciatic nerves collected from the same
P0-GGF3 transgenic and age-matched control animals described
above were stained with hematoxylin and eosin and examined by light
microscopy. At all ages examined (1, 2, 4, and 7 months), the sciatic nerves
of P0-GGF3 mice contained more Schwann cell nuclei per
high-power field (0.3 mm2) than nerve from nontransgenic
littermates (Fig. 4A).
Interestingly, however, the density of Schwann cell nuclei also appeared to
decrease with increasing age in both transgenic and control animals. To
confirm these impressions, counts of the number of Schwann cell nuclei per 0.3
mm2 were performed on sections of sciatic nerves from 1-, 2-, 4-,
and 7-month-old transgenic and nontransgenic mice. Nerves from 1-, 2-, and
4-month-old P0-GGF3-overexpressing mice contained
significantly (p 
0.01) higher Schwann cells densities than
age-matched nontransgenic littermates (Fig.
4B). Although highly variable, the number of Schwann cell
nuclei per 0.3 mm2 was also increased in 7-month-old
P0-GGF3 mice.
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Figure 4. A, Representative hematoxylin- and eosin-stained longitudinal
sections of sciatic nerve from P0-GGF3 transgenic mice
(Transgenic) and age-matched nontransgenic controls (Control) at 1, 2, 4, and
7 months of age; ages, indicated to the left of each row, apply both to the
images in that row and the adjacent bar graph. Note that sciatic nerves from
transgenic animals have higher Schwann cell densities at all ages than seen in
age-matched controls. In both transgenic and control mice, however, the number
of Schwann cell nuclei per 0.3 mm2 decreases with age.
Magnification, 40x; scale bars, 50 µm. B, Bars indicate the
average number of Schwann cell nuclei per high-power field at (top to bottom)
1, 2, 4, and 7 months of age in sciatic nerves from P0-GGF3
transgenic mice (Transgenic) and age-matched nontransgenic controls (Control).
SD is indicated for each bar. Numbers below each bar are animal identifiers.
C, Alterations in the average number of Schwann cell nuclei per
high-power field (0.3 mm2) with age in P0-GGF3
transgenic mice (TG) and nontransgenic littermates (CON). SEM is indicated for
each bar. The age of the animals is indicated below each pair of bars.
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These counts also confirmed that the average density of Schwann cell nuclei
decreased with age in P0-GGF3 animals. Sections of sciatic
nerves from transgenic mice contained 400-550 nuclei/0.3 mm2
at 1 month of age, 200 -275 nuclei/0.3 mm2 at 2 months, 150 -225
nuclei/0.3 mm2 at 4 months, and 75-225 nuclei/0.3 mm2 at
7 months (Fig. 4C). An
age-associated decrease in the average density of Schwann cell nuclei was also
observed in sciatic nerves from control mice; this decrease was much more
modest, however, than that observed in P0-GGF3 mice.
Measurements of the cross-sectional area of sciatic nerves from 1-month-old
(11 transgenic and 3 control), 2-month-old (12 transgenic and 4 control), and
4-month-old (5 transgenic and 9 control) P0-GGF3 mice and
nontransgenic littermates showed that the average nerve area increased with
age, with the nerve area in 4-month-old animals being 150% of that
observed in 1-month-old mice. At all three ages, however, there was no
significant difference in the average cross-sectional area of sciatic nerves
from transgenic and nontransgenic mice [e.g., nerve area in 4-month-old
transgenic mice, 216,814 ± 35531 µm2 (mean ± SD of
the mean); in 4-month-old nontransgenic mice, 197,226 ± 32658
µm2]. These observations suggest that at least one factor
contributing to the age-related decrease in Schwann cell nuclear density in
P0-GGF3 mice is the endoneurial accumulation of elements
(e.g., the Schwann cell processes forming onion bulbs and endoneurial
collagen) in the increasingly pathologic nerve, which produces a progressively
wider separation of Schwann cell nuclei. A similar age-related decrease in
Schwann cell nuclear density occurs in sciatic nerves from nontransgenic
animals and probably reflects the endoneurial accumulation of myelin and other
molecules associated with maturation of the nerve. Because the average
cross-sectional area of sciatic nerves from 4-month-old transgenic and
nontransgenic mice is not significantly different, it seems likely that the
hypertrophy observed for individual Schwann cell-axon units in
P0-GGF mice is offset by the loss of myelin and axons that
occurs as the pathology progresses, resulting in a minimal overall change in
nerve diameter.
To verify that the cells producing hyperplasia in nerves from
P0-GGF3 mice are indeed Schwann cells, sciatic nerve sections
from 1-month-old transgenic animals and nontransgenic littermates were stained
for the Schwann cell marker S100 and collagen type IV, a protein
component of the basal laminae; collagen type IV deposition is found around
Schwann cells (Baron-Van Evercooren et al.,
1986; Lorimier et al.,
1992) and endothelial cells
(Lorimier et al., 1992) but
not around other cell types within the endoneurium. S100
immunoreactivity was associated with both the normal Schwann cells in
nontransgenic nerves (data not shown) and the hyperplastic elements within
nerves from P0-GGF3 mice
(Fig. 5A). Collagen
type IV immunoreactivity was evident as faint brown staining outlining Schwann
cell-axon units and as stronger endothelial-associated staining in nerves from
nontransgenic mice (Fig.
5B). In contrast, collagen type IV immunoreactivity was
prominent in nerves from P0-GGF3 mice
(Fig. 5C) and was
frequently observed to invest the hyperplastic cells within this tissue
(Fig. 5C, arrows).
Because endothelial cells are not S100-positive, the S100 and
collagen type IV immunoreactivity associated with hyperplastic elements in
P0-GGF3 nerve identifies these cells as Schwann cells.
Mice overexpressing GGF3 in myelinating Schwann cells develop
malignant peripheral nerve sheath tumors
In addition to the neurologic abnormalities described above, founders 33
and 34 demonstrated decreasing responsiveness associated with bulging eyes
that first became evident at 6 -7 months of age. When autopsied, these mice
were found to have large intracranial masses at the base of the skull. The
masses were centered on the trigeminal ganglion and displaced the brain
upward; both gross and microscopic examinations showed no evidence that these
masses invaded the brain. Microscopic examination of the mass from founder 34
(Fig. 6A) showed this
tumor to be a hypercellular neoplasm composed of Schwann cell-like spindled
cells containing elongated nuclei. Mitoses and areas of tumor necrosis were
common in this neoplasm. Although containing small areas histologically
similar to the tumor from founder 34, the majority of the intracranial
neoplasm from founder 33 was much more hypercellular and contained even more
numerous mitotic figures (Fig.
6B). Ganglionic neurons were found entrapped within both
neoplasms, confirming their association with the trigeminal ganglion.
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Figure 6. P0-GGF3 transgenic mice develop tumors resembling human
malignant peripheral nerve sheath tumors. A, B, Representative
sections of trigeminal ganglion-associated tumors from
P0-GGF3 founders 34 (A) and 33 (B). The
asterisk in A indicates a focus of tumor necrosis. Magnification,
40x; scale bar, 50 µm. C, Section of the neoplasm shown in
B immunostained for the Schwann cell marker S100 (red
staining). This preparation has been counterstained with the nuclear marker
bisbenzamide. Magnification, 63x; scalebar 20 µm. D, Section
of the neoplasm shown in B immunostained for the basement membrane
protein collagen type IV. Collagen type IV immunoreactivity is seen as brown
staining that invests individual tumor cells. This preparation has been
counterstained with hematoxylin to demonstrate individual tumor cells.
Magnification, 63x; scale bar, 20 µm. E, F, Transmission
electron micrographs of a neoplasm developing in an offspring of founder 33.
Note that individual tumor cells are surrounded by a basal lamina (shown at
higher magnification in F), with loops of basal lamina material
frequently seen extending away from the tumor cells (F, arrowhead).
Magnification: E, 10,000x; F, 40,000x.
G, Expression of the P0-GGF3 transgene is maintained
in the MPNST-like tumors developing in P0-GGF3 mice. The
transgene mRNA is detected as a 410 bp PCR product in cDNA from the tumor
shown in E and F (Tumor) but not in cDNAs from age-matched
nontransgenic trigeminal nerves (Trigeminal). The transgene product is not
detected in the absence of reverse transcription (- lanes), indicating that
the PCR product obtained in these experiments is not derived from
contaminating genomic DNA. H, Tumors from P0-GGF3
mice express high levels of the NRG-1 receptor subunits erbB2 and erbB3. ErbB2
(left) and erbB3 (right) are detected as 185 kDa immunoreactive species in
lysates of the tumor shown in E and F (Tumor) and in
trigeminal nerve (Trigeminal) collected from 5- and 7-month-old
P0-GGF3 mice.
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Similar neoplasms were found in three of the older (6- to 10-month-old)
progeny of P0-GGF3 founder 33. These neoplasms, like the
tumor found in founder 33, were markedly hypercellular lesions containing
numerous mitotic figures. Two of these tumors were associated with the
trigeminal ganglion. The third neoplasm was within the sciatic nerve,
producing diffuse enlargement of the nerve that began at the exit of the nerve
roots from the vertebral canal and extended along the upper half of the nerve.
No such neoplasms were ever observed in the nontransgenic littermates of these
mice. Again, because neoplasms were found in founder 34 as well as several
mice from line 33, we conclude that the development of these tumors results
from transgene-directed GGF3 overexpression rather than integration site
effects.
The morphologic features of the tumors developing in
P0-GGF3 mice are highly similar to those of human malignant
peripheral nerve sheath tumors (MPNSTs), a type of neoplasm that may occur
either sporadically or as part of the hereditary cancer predisposition
syndrome neurofibromatosis type 1 (Gutmann
et al., 1997; Parada,
2000; Gutmann,
2001). To confirm this impression, immunohistochemical studies and
ultrastructural examination of the MPNST-like neoplasms occurring in
P0-GGF3 mice were performed in accordance with recent
consensus recommendations for the evaluation of neoplasms occurring in
transgenic mice (Weiss et al.,
2002). Extensive areas within the tumors were immunoreactive for
the Schwann cell marker S100 (Fig.
6C), indicating that these neoplasms, like human MPNSTs,
were Schwann cell tumors. Furthermore, individual tumor cells were surrounded
by immunoreactivity for collagen type IV, a basal lamina protein that
demonstrates a similar distribution in human MPNSTs
(Leong et al., 1997;
Vang et al., 2000).
Ultrastructural examination demonstrated that the tumor cells had broad,
interdigitating processes (Fig.
6E) densely laden with ribosomes and endoplasmic
reticulum. Tumor cells were frequently invested by a basal lamina that at
times extended away from the cell surface as loops
(Fig. 6F, arrowhead).
Long-spacing collagen (Luse body) was also identified within the tumor. These
ultrastructural features are highly similar to those of human MPNSTs
(Taxy et al., 1981;
Erlandson and Woodruff, 1982;
Arpornchayanon et al., 1984;
Herrera and Pinto de Moraes,
1984; Erlandson,
1985; Dickersin,
1987; Takeuchi and Ushigome,
2001). Considered together, the histologic, immunohistochemical,
and ultrastructural features of the neoplasms developing in
P0-GGF3 mice meet the World Health Organization criteria
(Woodruff et al., 2000) for
classification as MPNSTs. Per consensus recommendations
(Weiss et al., 2002), these
neoplasms are referred to below as genetically engineered murine (GEM)
MPNSTs.
If NRG-1 overexpression promotes the continued growth of the GEM MPNSTs
developing in P0-GGF3 mice, then expression of the
P0-GGF3 transgene should be maintained in these neoplasms,
and the tumor cells should express the receptors necessary for NRG-1
responsiveness. Examining the expression of the P0-GGF3
transgene using transgene-specific primers, we found that GGF3 mRNA was
readily detectable in trigeminal-associated tumor from transgenic mice but not
in trigeminal nerve from nontransgenic littermates
(Fig. 6G).
Furthermore, the NRG-1 receptor subunits erbB2 and erbB3 were expressed in GEM
MPNST (Fig. 6H). A
comparison of the levels of erbB2 and erbB3 protein in GEM MPNSTs and
trigeminal nerves from 5- and 7-month-old P0-GGF3 mice
indicated that both erbB kinases were markedly overexpressed in the tumor
relative to the levels evident in nerves not containing grossly evident
neoplasm; because it was not possible to detect erbB protein in the trigeminal
nerve while simultaneously avoiding saturation of the signals from the tumor,
we could not accurately quantitate the relative levels of expression in these
tissues. Nonetheless, it is apparent that both GGF3 and its erbB
receptors are expressed in GEM MPNSTs, suggesting that these molecules promote
tumor cell proliferation via autocrine or paracrine effects.
Localized Schwann cell hyperplasia is present in multiple peripheral
nervous system ganglia in P0-GGF3 transgenic mice
The majority of the MPNST-like neoplasms in P0-GGF3 mice
are associated with the trigeminal ganglion. This observation suggested that
preneoplastic lesions might be specifically associated with this structure in
P0-GGF3 mice. To test this hypothesis, we examined trigeminal
nerves and associated ganglia from P0-GGF3 transgenic mice
and age-matched wild-type controls at 1, 2, 4, and 7 months of age. As in the
sciatic nerve, increased numbers of Schwann cell nuclei were uniformly evident
within the trigeminal ganglia of P0-GGF3 animals at all ages
examined (Fig. 7). However,
unlike the proliferations in sciatic nerve, Schwann cell hyperplasia was
predominantly associated with the ganglia, with much less hypercellularity
present in adjacent regions of the trigeminal nerve. Furthermore,
hypercellular collections of Schwann cells were frequently seen extending from
the areas of hyperplasia within the ganglion along the surface of the
trigeminal nerve. As in the sciatic nerve, Schwann cell density appeared to
decrease with increasing age within the trigeminal ganglion. Because of the
nonuniform distribution of Schwann cells within the trigeminal nerve, however,
it was not possible to quantitate with accuracy the average Schwann cell
density in this structure.
On the basis of these findings, we examined sympathetic (superior cervical,
superior mesenteric, and celiac) and sensory (dorsal root) ganglia from
P0-GGF3 mice for evidence of similar hyperplastic lesions. We
found that Schwann cell hyperplasia was evident in sympathetic ganglia, much
as seen in the trigeminal ganglia. Such changes were never seen in sympathetic
ganglia from age-matched nontransgenic controls. Schwann cell hyperplasia was
also present in dorsal root ganglia (DRG) from transgenic animals;
furthermore, in some animals, although there was no evidence of massive tumors
arising in the DRG, these structures were grossly enlarged and showed
histologic findings similar to those of the MPNST-like neoplasms described
above (data not shown). In both the sympathetic and sensory ganglia, Schwann
cell hyperplasia was most prominent within the ganglion, with much lower
cellularity evident in the adjacent nerve; again, hypercellular collections of
Schwann cells were observed extending from the ganglion along the surface of
the adjacent nerve. Occasionally, microscopic lesions histologically similar
to the tumors identified in this line of P0-GGF3 mice were
seen within peripheral ganglia. We conclude that persistent Schwann cell
hyperplasia, possibly representing preneoplastic lesions, is uniformly present
in motor, sympathetic, and sensory ganglia within the PNS of
P0-GGF3 transgenic mice.
Increased Schwann cell DNA synthesis is evident in sciatic nerves and
trigeminal ganglia from P0-GGF3 transgenic mice
The increased number of Schwann cell nuclei evident in the sciatic nerves
and trigeminal ganglia of P0-GGF3 mice could reflect
increased proliferation, enhanced survival, or a combination of these two
factors. To investigate the possibility that the increase in Schwann cell
numbers is attributable to increased mitogenesis, 1-, 2-, and 4-month-old
transgenic mice and age-matched nontransgenic controls were injected
intraperitoneally with 60 mg/kg BrdU and 6 mg/kg 5-FU (to enhance BrdU
incorporation) and killed 90 min later. Sciatic and trigeminal nerves from
these animals were fixed, paraffin-embedded, and immunolabeled for
incorporated BrdU. Consistent with earlier observations in the sciatic nerves
of 2-month-old mice (Asbury,
1967), BrdU-immunoreactive Schwann cell nuclei were rare in
sciatic nerves from 1-, 2-, and 4-month-old wild-type mice
(Fig. 8A-C). In
contrast, multiple BrdU-positive nuclei were seen in sciatic nerve sections
from 1-, 2-, and 4-month-old P0-GGF3 mice
(Fig. 8D-F, arrows).
Similar increases in the number of BrdU-immunoreactive Schwann cell nuclei
were evident within the trigeminal ganglia of P0-GGF3
transgenic mice.
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Figure 8. DNA synthesis in sciatic nerves from P0-GGF3 transgenic
and wild-type mice. A-C, Representative section from a 1-month-old
wild-type control (1 mo Con) mouse. D-F, Representative section from
a 1-month-old P0-GGF3 transgenic (1 mo TG) mouse. Left
column, Staining for the nuclear dye bisbenzamide. Middle column,
Immunostaining for incorporated BrdU. Right column, Merging of images in the
left and middle columns. Arrows in E and F indicate
BrdU-positive Schwann cell nuclei. Scale bars, 50 µm. G, BrdU
labeling indices determined in the sciatic nerves of P0-GGF3
transgenic mice and age-matched wild-type controls. The percentage of Schwann
cell nuclei labeled with BrdU is indicated on the left, with bars indicating
the average percentage labeled and the SEM. The age of the animals is
indicated below the bars. Sections of jejunum from each animal were also
immunostained (data not shown); BrdU incorporation in the proliferative zones
of the crypts of Lieberkuhn served as a positive control for BrdU delivery.
BrdU labeling indices for sciatic nerves from P0-GGF3
transgenic mice and age-matched wild-type controls were compared using one-way
ANOVA, followed by Tukey'sposthoctest, withp < 0.05
considered statistically significant. *p << 0.001 relative to
nontransgenic controls.
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To quantitate these differences, BrdU labeling indices (the number of
BrdU-positive Schwann cell nuclei divided by the total number of Schwann cell
nuclei) were determined for sciatic nerves and trigeminal ganglia from 1-, 2-,
and 4-month-old P0-GGF3 transgenic mice and age-matched
non-transgenic controls. In the sciatic nerve, BrdU labeling indices were
significantly higher in P0-GGF3 mice than in wild-type
controls at 1 and 2 months of age (Fig.
8G). However, BrdU labeling indices in sciatic nerve from
P0-GGF3 mice demonstrated an age-related decrease, with an
average labeling index of 1.2% in 1-month-old animals and a 0.2% labeling
index in 4-month-old mice; this decrease occurred despite continued high-level
expression of GGF3 in the sciatic nerve out to 4 months of age (see
Fig. 1B). Likewise,
BrdU labeling indices in the trigeminal ganglia of both 1- and 2-month-old
P0-GGF3 mice were much higher than that observed in
age-matched wild-type controls (data not shown). We conclude that Schwann cell
DNA synthesis is increased in both the sciatic nerve and trigeminal ganglion
of mice overexpressing GGF3 in myelinating Schwann cells. It is
currently unclear why Schwann cell DNA synthesis decreases with increasing age
in P0-GGF3 mice despite continued GGF3 expression.
To assess the possibility that overexpression of GGF3 increases
Schwann cell numbers by repressing apoptosis, we stained sciatic and
trigeminal nerve sections from 1-, 2-, and 4-month-old
P0-GGF3 transgenic and wild-type mice with an antibody
specific for activated caspase-3, the predominant effector caspase in the
nervous system. We found only very rare Schwann cells that were immunoreactive
for activated caspase-3, showed apoptotic nuclear features, or both in
wild-type mice; these findings are consistent with previous studies in which
it has been found that developmental Schwann cell apoptosis is essentially
complete in rats before postnatal day 21 and that apoptosis is virtually
absent in normal sciatic nerve from adult rats
(Grinspan et al., 1996). We
also found only rare activated caspase-3-immunoreactive Schwann cells in nerve
sections from P0-GGF3 transgenic mice. We conclude that the
levels of Schwann cell apoptosis in the sciatic and trigeminal nerves of 1-,
2-, and 4-month-old mice are exceedingly low; hence, inhibition of baseline
Schwann cell apoptosis likely makes a minimal contribution to the increased
cellularity observed in mice overexpressing GGF3 in peripheral
nerve.
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Discussion
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To determine what effects NRG-1 elicits in mature Schwann cells, we
generated transgenic mice constitutively overexpressing the NRG-1 isoform
GGF3 in noninjured peripheral nerves. After an initial period of
myelination that occurs coincident with prominent Schwann cell hyperplasia,
these mice develop a hypertrophic neuropathy, a condition produced by
repetitive rounds of demyelination and remyelination. Many
P0-GGF3 mice also develop ganglion-associated neoplasms
similar to human MPNSTs, a tumor type that occurs sporadically or in
association with neurofibromatosis type 1. These MPNST-like neoplasms likely
arise from preneoplastic lesions uniformly present in motor, sympathetic, and
sensory ganglia from P0-GGF3 animals. Considered together,
these findings have important implications regarding NRG-1 actions on mature
Schwann cells in normal and injured peripheral nerve and the possible
involvement of NRG-1 in the pathogenesis of peripheral nerve sheath
tumors.
All four P0-GGF3 founders and the progeny of founder 33
developed a hypertrophic neuropathy characterized by onion bulb formation with
evidence of active demyelination and remyelination. Myelination is relatively
normal in 1-month-old P0-GGF3 mice, with pathology first
becoming evident at 2 months of age and worsening thereafter. The ability
of GGF3 to induce demyelination in the noninjured sciatic nerve of
P0-GGF3 transgenic mice is consistent with a previous report
that this NRG-1 isoform promotes demyelination in myelinated cocultures of
mature Schwann cells and DRG neurons
(Zanazzi et al., 2001).
GGF3-mediated repression of myelin protein expression is also consistent
with the development of onion bulbs in the sciatic nerves of
P0-GGF3 mice; alternative cycles of induction and repression
of GGF3 expression, which is controlled in these animals by the
regulatory elements of the P0 gene, would be expected to produce
the repetitive cycles of demyelination and remyelination that produce these
pathologic structures. Thus, both in vivo and in vitro
studies demonstrate that GGF3 induces demyelination in differentiated
adult Schwann cells. The NRG-1 effects on myelination in this setting are
likely distinct from those reported in the neonatal period, in which ablation
of the erbB2 locus inhibits the initial establishment of myelination
(Garratt et al., 2000b). The
occurrence of demyelination in P0-GGF3 mice is thus
consistent with the hypothesis that NRG-1 stimulates demyelination when its
expression is induced after injury of the adult nerve.
Sciatic nerve pathology in P0-GGF3 mice is similar to that
of human hypertrophic neuropathies such as hereditary sensory and motor
neuropathy I (HMSN I; Charcot-Marie-Tooth disease). Nonetheless, it is
unlikely that the NRG-1 locus is mutated in HMSN I. Most cases of HMSN I
develop as a consequence of mutations in the peripheral myelin protein 22,
connexin 32, P0, and EGR2 genes
(Young and Suter, 2001).
Furthermore, although rare forms of HMSN I exist for which the gene locus has
not yet been identified, none of the mapping studies performed to date have
implicated the NRG-1 locus in the pathogenesis of these neuropathies. These
observations do not exclude a role for NRG-1 in the development of HMSN I.
Indeed, given the effects this growth factor has on myelination, it is quite
possible that NRG-1 plays a role in the pathogenesis of human hypertrophic
neuropathies.
In addition to demyelination, we found that GGF3 stimulates in
vivo Schwann cell mitogenesis. Sciatic nerves from
P0-GGF3 mice as young as 1 month of age show a marked
increase in Schwann cell nuclear density relative to nontransgenic controls.
Although average Schwann cell densities decreased with age in both transgenic
and nontransgenic mice, Schwann cell numbers in P0-GGF3 mice
were significantly higher than in control mice out to 7 months of age. This
GGF3-mediated increase in Schwann cell numbers was accompanied by
increased bromodeoxyuridine incorporation, indicating that stimulation of
Schwann cell proliferation was a major mechanism increasing Schwann cell
numbers in P0-GGF3 mice. In contrast, there was little
evidence of Schwann cells immunoreactive for activated caspase-3, a major
mediator of nervous system programmed cell death, in sciatic nerves from 1-,
2-, and 4-month-old wild-type or transgenic mice. We cannot yet rule out the
possibility that transgenic overexpression of GGF3 diminishes the extent
of programmed cell death normally occurring during the first postnatal week
(Grinspan et al., 1996),
thereby contributing to an initial increase in Schwann cell numbers.
Nonetheless, our findings in older mice are consistent with the hypothesis
that NRG-1 stimulates Schwann cell mitogenesis in injured adult nerve. It is
not yet clear whether the promitogenic action of NRG-1 in this setting is
distinct from its demyelinative effects.
Constitutive overexpression of GGF3 was also associated with the
development of MPNST-like tumors in two P0-GGF3 lines. This
finding is consistent with several previous observations linking the
NRG-1-erbB signaling pathway to Schwann cell neoplasia. First, Brockes et al.
(1986) detected high to
intermediate levels of a GGF-like activity in a subset of human neurofibromas
(the precursor lesion from which many MPNSTs arise) and low levels of this
activity in the single MPNST they examined. Furthermore, malignant schwannomas
(a MPNST variant) induced by in utero treatment of rats
(Perantoni et al., 1987;
Nikitin et al., 1991), mice
(Buzard et al., 1999b), or
hamsters (Buzard et al., 1999a)
with the mutagen N-ethyl-N-nitrosourea (EtNU), frequently
carry a form of the NRG-1 receptor subunit erbB2 with an activating point
mutation. Last, we have found that JS1 cells
(Schubert et al., 1974;
Kimura et al., 1990), a
Schwann cell line derived from an EtNU-induced MPNST, express high levels of
several NRG-1 isoforms (including GGF variants), erbB3, and a nonmutated form
of erbB2 (Frohnert et al.,
2003). JS1 cells demonstrate constitutive tyrosine phosphorylation
of these erbB kinases, which can be blocked pharmacologically, resulting in a
marked decrease in DNA synthesis. These observations, considered together with
our finding that overexpression of GGF3 induces MPNST formation, are
consistent with the hypothesis that constitutive activation of the NRG-1-erbB
signaling pathway induces Schwann cell neoplasia in rodents. The role of these
signaling events in the pathogenesis of human Schwann cell neoplasms remains
to be determined.
Although GGF3 overexpression contributes to the development of
MPNST-like tumors in P0-GGF3 mice, it is unlikely that this
is the only molecular event involved in this process. Several factors support
this conclusion, including the nonuniform development of these neoplasms in
the PNS (see below), the occurrence of MPNSTs in only some animals, and the
tendency of these tumors to be found in older animals. At present, it is
unclear whether these additional genetic events include those previously
identified in human MPNSTs such as inactivation of the NF1
(Perry et al., 2001),
Rb (Birindelli et al.,
2001), p53 (Menon et
al., 1990; Birindelli et al.,
2001), and CDKN2A/p16
(Kourea et al., 1999;
Nielsen et al., 1999) genes.
These additional mutations likely occur in cells within the hyperplastic
lesions uniformly present in the motor, sensory, and sympathetic peripheral
ganglia of P0-GGF3 mice. Several features suggest that the
hyperplastic lesions in ganglia are preneoplastic, including their relatively
high degree of cellularity, their association with the ganglion (see below),
and the tendency of cells in these regions to extend outward along the
epineurium.
In humans, MPNSTs are most frequently found in large nerves such as the
sciatic nerve. However, human MPNSTs also develop in cranial nerves, most
commonly in the trigeminal ganglion or its peripheral branches
(Urich and Tien, 1998). Like
this minor subset of human MPNSTs, the majority of the MPNSTs developing in
P0-GGF3 mice arose within trigeminal ganglia. A similar
distribution is seen for MPNSTs induced in rats and mice by in utero
carcinogenesis with EtNU (Druckrey et al.,
1966; Wechsler et al.,
1979; Anderson et al.,
1989; Buzard et al.,
1999b). The preferential association of EtNU-induced tumors with
the trigeminal ganglion has been suggested to reflect the developmental state
of this structure at embryonic day 15, the time of EtNU exposure
(Nikitin et al., 1991).
However, because NRG-1 overexpression in P0-GGF3 mice likely
occurs with a different time course from EtNU exposure, other possibilities
must be considered. First, PNS ganglia may contain a distinct Schwann cell
subtype particularly sensitive to NRG-1 stimulation. Second, MPNSTs and their
precursor lesions may arise from some other NRG-responsive glial lineage
specific to ganglia such as satellite cells; alternatively, MPNSTs may develop
from immature Schwann cell-like elements or stem cells analogous to those
recently identified in the adult gut
(Kruger et al., 2002) that are
persistently present in perinatal or adult ganglia. Last, other factors unique
to the ganglionic microenvironment (e.g., costimulation by growth factors
released from ganglionic neurons) may alter Schwann cell responses to NRG-1
stimulation.
In conclusion, overexpression of GGF3 in noninjured peripheral nerves
induces Schwann cell proliferation and demyelination, ultimately resulting in
the development of both a hypertrophic neuropathy similar to
Charcot-Marie-Tooth disease and neoplasms resembling the human malignant
peripheral nerve sheath tumors that occur both sporadically and in association
with neurofibromatosis type 1. The effects produced by GGF3
overexpression are therefore consistent with the hypothesis that NRG-1
isoforms mediate both demyelination and Schwann cell proliferation after
injury of an adult peripheral nerve. Given its phenotype, the
P0-GGF3 transgenic mouse will be highly useful in future
studies further examining NRG-1 actions on nontransformed mature Schwann cells
in vivo and the role NRG-1 plays in Schwann cell neoplasia.
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Footnotes
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Received Dec. 17, 2002;
revised Jun. 3, 2003;
accepted Jun. 6, 2003.
This work was supported by National Institutes of Health Grants R37 DK19645
(R.E.S.), R01 AG10299 (R.E.S.), and R01 NS37514(S.L.C.). We thank Stephanie
Byer, Lee Millican, Lucie Beaudet, and Cecelia Latham for expert technical
assistance.
Correspondence should be addressed to Dr. Steven L. Carroll, Division of
Neuropathology, Department of Pathology, The University of Alabama at
Birmingham, 1720 Seventh Avenue South, SC843, Birmingham, AL 35294-0017.
E-mail:
carroll{at}path.uab.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/237269-12$15.00/0
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Abstract
Introduction
