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The Journal of Neuroscience, July 15, 1998, 18(14):5203-5211
Neuronal Matrix Metalloproteinase-2 Degrades and Inactivates a
Neurite-Inhibiting Chondroitin Sulfate Proteoglycan
Jian
Zuo1,
Toby A.
Ferguson1,
Yosbani J.
Hernandez1,
William G.
Stetler-Stevenson2, and
David
Muir1
1 Departments of Pediatrics (Neurology Division) and
Neuroscience, University of Florida Brain Institute and College of
Medicine, Gainesville, Florida 32610-0296, and
2 Extracellular Matrix Pathology Section, Laboratory of
Pathology, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 20892
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ABSTRACT |
Chondroitin sulfate proteoglycans (CSPGs) are implicated in the
regulation of axonal growth. We previously reported that the neurite-promoting activity of laminin is inhibited by association with
a Schwann cell-derived CSPG and that endoneurial laminin may be
inhibited by this CSPG as well [Zuo J, Hernandez YJ, Muir D (1998)
Chondroitin sulfate proteoglycan with neurite-inhibiting activity is
upregulated after peripheral nerve injury. J Neurobiol 34:41-54]. Mechanisms regulating axonal growth were studied by using
an in vitro bioassay in which regenerating embryonic
dorsal root ganglionic neurons (DRGn) were grown on sections of normal adult nerve. DRGn achieved slow neuritic growth on sections of normal
nerve, which was reduced significantly by treatment with metalloproteinase inhibitors. Similar results were obtained on a
synthetic substratum composed of laminin and inhibitory CSPG. DRGn
expressed the matrix metalloproteinase, MMP-2, which was transported to
the growth cone. Recombinant MMP-2 inactivated the neurite-inhibiting
CSPG without hindering the neurite-promoting potential of laminin.
Similarly, neuritic growth by DRGn cultured on normal nerve sections
was increased markedly by first treating the nerve sections with MMP-2.
The proteolytic deinhibition by MMP-2 was equivalent to and nonadditive
with that achieved by chondroitinase, suggesting that both enzymes
inactivated inhibitory CSPG. Additionally, the increases in neuritic
growth resulting from treating nerve sections with MMP-2 or
chondroitinase were blocked by anti-laminin antibodies. From these
results we conclude that MMP-2 provides a mechanism for the
deinhibition of laminin in the endoneurial basal lamina and may play an
important role in the regeneration of peripheral nerve.
Key words:
chondroitin sulfate proteoglycan; matrix
metalloproteinase; neuronal regeneration; neurite inhibitor; basal
lamina; peripheral nerve; laminin; cryoculture
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INTRODUCTION |
Injury to mammalian peripheral nerve
is followed by Wallerian degeneration in the distal nerve segment,
which involves the clearing of severed axons and myelin. Despite this
extensive remodeling, the primary structure of endoneurial basal
laminae persists, providing an essential scaffolding for nerve
regeneration (Satinsky et al., 1964 ; Ide et al., 1983). However,
modifications to the molecular composition of the basal lamina occur
that, as part of the degenerative process, appear to be a prerequisite
for axonal regeneration (Salonen et al., 1987; Giannini and Dyck, 1990;
Fu and Gordon, 1997). Using a series of nerve anastomoses, Langley and
Anderson (1904) reported that transected axons do not regenerate along
a normal nerve. More recently, the nonpermissive status of normal nerve
was confirmed by Bedi and coworkers (1992), who found that adult
sensory axons fail to grow neurites on sections of normal adult nerve.
These findings suggest that, although rich in molecules that promote axonal regeneration in vitro, Schwann cell surfaces and
their associated basal lamina in normal nerve fail to support axonal growth.
It is now generally accepted that extracellular matrices (ECM) of the
mammalian nervous system can present to neurons both stimulatory and
inhibitory cues for axonal growth. Recent studies of nonpermissive
nervous tissues show that several families of cell surface and ECM
macromolecules have potential growth inhibitory properties (for review,
see Faissner and Steindler, 1995; Kolodkin, 1996). In particular,
chondroitin sulfate proteoglycans (CSPGs) are abundant in the adult
nervous system and evoke a strong avoidance reaction by a variety of
neuronal subtypes in vitro (Muir et al., 1989a; Snow et al.,
1991). CSPGs permeate boundary structures confronted by developing and
regenerating axons (Oakley and Tosney, 1991; Brittis et al., 1992;
Pindzola et al., 1993). Normal adult peripheral nerve contains
neurite-inhibiting activity associated with CSPG, which is increased
further in the distal nerve after injury (Zuo et al., 1998). CSPG
colocalizes with laminin within the Schwann cell basal lamina and
appears to have a wider distribution within the endoneurial compartment
in degenerating nerve (Kuecherer-Ehret et al., 1990; Tona et al.,
1993). Because of its considerable regenerative capacity, it may seem
counter-intuitive that peripheral nerve contains neurite inhibitors.
One possibility is that growth inhibitors help to stabilize axons in
normal nerve. By blocking the neurite-promoting potential of laminin in
the endoneurial basal lamina, inhibitory CSPG may prevent axons from
sprouting collaterals, especially at nodes of Ranvier. This implies
that nerve sheaths actually might suppress axonal growth under normal conditions. However, this suppression somehow must be reversed in nerve
regeneration.
The growth cone, the leading structure of growing axons, senses
guidance cues from the surrounding environment and implements directed
outgrowth. Axonal regeneration requires extensive growth cone motility
and infiltration within damaged and degenerating nervous tissue.
Substantial evidence now indicates that neurons secrete
matrix-degrading enzymes and actively remodel surrounding ECM substrata
(Monard, 1988; Pittman and Buettner, 1989; Fambrough et al., 1996).
Proteinases, including plasminogen activators and matrix
metalloproteinases (MMPs), are expressed by peripheral neurons and
released by growth cones, implicating the growing tip of axons in the
proteolysis of matrix components (Pittman and Williams, 1988; McGuire
and Seeds, 1990; Muir, 1994; Nordstrom et al., 1995; Hayden and Seeds,
1996). MMPs are believed to be the physiologically relevant mediators
of ECM degradation and matrix remodeling (Matrisian, 1992). MMPs also
participate in the proteolytic processing of both ECM and cell surface
components, which can result in alterations of cell properties such as
attachment and migration (Chantry et al., 1992; DiStefano et al., 1993;
Ray and Stetler-Stevenson, 1995; Giannelli et al., 1997). Thus, the function of MMPs, at first thought to be limited to the catabolism and
cellular infiltration of ECM, also includes an important regulatory mechanism for the proteolytic activation of cryptic molecular domains.
In the present report, axonal growth by regenerating embryonic dorsal
root ganglionic neurons (DRGn) cultured on sections of normal adult
nerve and on a synthetic substratum composed of laminin and inhibitory
CSPG was found to be dependent on metalloproteinase activity. DRGn
expressed MMP-2, which inactivated inhibitory CSPG and unmasked the
neurite-promoting activity of associated laminin. From these studies we
conclude that MMP-2 provides a mechanism for the deinhibition of
laminin in the endoneurial basal lamina.
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MATERIALS AND METHODS |
Assay of neuritic growth. Dorsal root ganglia from
day 8 chick embryos were dissociated enzymatically, and the DRGn were
purified by differential cell attachment as described (Manthorpe et
al., 1983; Muir et al., 1989b). Assays of neurite outgrowth and
inhibition were performed as reported previously (Muir et al., 1989a;
Zuo et al., 1998). Briefly, substrata were established by treating polyornithine-coated 96-well plates with of a maximally stimulating concentration of EHS tumor laminin-1 (1-2 µg/ml, 50 µl/well)
premixed with the neurite-inhibiting CSPG, NIF, at the concentrations
indicated. DRGn were seeded (103 neurons per well)
in a defined medium containing N2 supplements, 1% heat-treated bovine
serum albumin, and 10 ng/ml nerve growth factor
(DMEM/N2+NGF). DMEM/N2 is a modification of the
defined medium N2 of Bottenstein and Sato (1979) in which the base
medium is replaced with DMEM only. It was shown previously that
cysteine (a common component of classical media) is a potent inhibitor
of metalloproteinases (Muir, 1994). Recent formulations of DMEM (Life
Technologies, Grand Island, NY) have replaced cysteine with cystine
(dicysteine), which does not inhibit metalloproteinases. Neurite
outgrowth was scored by phase-contrast microscopy after 4 hr by
counting the percentage of neurons bearing processes >4 cell body
diameters. Under these conditions the maximal neuritic response to
laminin was  75% of neurons bearing neurites. For the assay of
inhibitory activity, we defined one neurite inhibitory unit (NIU) as
the sample concentration required to reduce growth to 50% of the
maximal response to laminin. Approximately 75% inhibition of laminin
activity was achieved by 2 NIU/ml of inhibitory CSPG (see below) and
maximal inhibition (96%) by >4 NIU/ml in these assays. The high
buoyant density inhibitory CSPG, designated neurite-inhibiting factor (NIF), used in this study was isolated from RN22 rat schwannoma conditioned medium as described previously (Muir et al., 1989a). Inhibitory CSPG with similar properties was obtained from Schwann cell
medium and from rat nerve (Zuo et al., 1998). RN22 are a more abundant
source than Schwann cells, and the nerve preparation may contain CSPG
contaminants contributed by cells other than Schwann cells. Also,
metabolic radiolabeling was most effective in cell culture. Therefore,
RN22-derived NIF was chosen for use in this study.
Neurite assays also were used to test for proteolytic inactivation of
NIF, either when in solution or when substratum-bound with laminin. NIF
was incubated in appropriate buffers with active forms (see below) of
recombinant MMP-2 (2 µg/ml), recombinant MMP-3 (2 µg/ml), or
chondroitinase ABC (0.1 U/ml) (Sigma, St. Louis, MO). Recombinant human
proMMP-2 was prepared as described elsewhere (Fridman et al., 1992).
Recombinant rat proMMP-3 (Machida et al., 1989) was a gift from Dr. G. Ciment (Oregon Health Science University). For all applications,
recombinant proMMP-2 and proMMP-3 were activated by treatment with 1 mM p-aminophenylmercuric acetate at 37°C for 2 hr and then dialyzed against 50 mM Tris-HCl, pH 7.8. The
protease inhibitor phenanthroline initially was dissolved at 0.5 M in 50% ethanol and used at a final concentration of 1 mM.
Cryoculture assay. Cryoculture is a neurite outgrowth assay
in which neurons are cultured directly on unfixed nerve sections (Carbonetto et al., 1987). Rat sciatic nerves were removed under general anesthesia and rapidly frozen in dry ice. Nerve segments (1 cm)
were cryosectioned (14 µm) and then mounted on sterile polyornithine-coated glass coverslips and stored at  20°C. Before cryoculture, mounted sections were treated for 4 hr at 37°C with (1)
activated MMP-2 (10 µg/ml) in 25 mM Tris-HCl, pH 7.8, containing 2.5 mM CaCl2; (2)
chondroitinase ABC (0.1 U/ml) (Sigma) in 50 mM Tris-HCl, pH
8.0, containing 50 mM NaCl; or (3) buffer control. DRGn
(4000/section) were seeded directly onto the nerve sections in
DMEM/N2+NGF. As indicated, the metalloproteinase
inhibitors cysteine (250 µM) and
3-(N-hydroxycarbamoyl)-2(R)-isobutylpropionyl-L-tryptophan methylamide (GM6001; 50 µM) (prepared as described by
Grobelny et al., 1992) were added to the culture medium. Assays also
were performed by using function-blocking antibodies to laminin.
Antibodies were added to the culture medium 1 hr after the DRGn were
first seeded to allow for attachment to the nerve sections. Polyclonal antibody raised against human placental laminins (Telios
Pharmaceuticals, La Jolla, CA) was used at a 1:50 dilution. This
polyclonal antibody cross-reacts with and blocks the neurite-promoting
activity of rat laminin-1 and laminin-2 (Engvall et al., 1986). Normal
rabbit serum was used as a control for this polyclonal laminin
antibody.
At 48 hr after seeding DRGn, assays were terminated by fixing the
cryoculture sections with 4% paraformaldehyde in 0.1 M
phosphate buffer. Chick neurons were immunolabeled selectively with
monoclonal anti-chick neural cell adhesion molecule (N-CAM) (Watanabe
et al., 1986), using biotinylated anti-mouse IgG and extravidin-FITC conjugate (Sigma). Immunolabeled neurites were viewed by
epifluorescence microscopy, and lengths were scored in acquired digital
images. The hybridoma cell line 5e, producing monoclonal N-CAM
antibody, was obtained from the Developmental Studies Hybridoma Bank
(University of Iowa; National Institute of Child Health and Human
Development contract NO1-HD-2-3144). The 5e cell line was grown in
medium supplemented with IgG-depleted fetal bovine serum (2%) and
OptiMAB (Life Technologies). Anti-chick N-CAM antibody was isolated
from culture supernatant by protein-G affinity chromatography and used at 10 µg/ml.
Degradation of radiosulfate-labeled inhibitory CSPG.
[35S]NIF was prepared by metabolic labeling
of RN22 cultures and isolated from conditioned medium as described
previously (Muir et al., 1989a). [35S]NIF was
incubated with MMP-2 and chondroitinase ABC as described above for
bioassays. For SDS-PAGE, samples were prepared under highly reducing
and denaturing conditions (Lowe-Krentz and Keller, 1984) and
electrophoresed on 5% polyacrylamide minigels. Autoradiographic gels
were dried, coated with scintillant (EnHance, New England Nuclear,
Boston, MA), and exposed to x-ray film for 7 d before automated
development.
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RESULTS |
Metalloproteinase-dependent neurite outgrowth on reconstituted
NIF-laminin substrata
NIF is a CSPG first isolated from RN22 schwannoma culture medium
that inhibits the neurite-promoting activity of laminin in a
concentration-dependent manner (Muir et al., 1989a). We first reported
that the inhibition of neuritic growth on a laminin substratum treated
with a maximally inhibiting concentration of NIF (8 NIU/ml) was
persistent and nearly complete. In later work we found that free
cysteine (a common component of culture media) is a potent inhibitor of
metalloproteinase activity and that levels of cysteine in many
classical culture media significantly inhibited metalloproteinase activity in vitro (Muir, 1994). In the present study a
defined culture medium (DMEM/N2+NGF) was formulated
without cysteine to minimize metalloproteinase inhibition (see
Materials and Methods). All DRGn bioassays included NGF in the culture
medium. In a standard 4 hr bioassay, neuritic growth on a NIF-laminin
substratum was virtually the same with or without cysteine in the
medium. However, in the absence of cysteine, gradual neuritic growth
was observed on NIF-laminin that was not seen previously by neurons
grown in the presence of cysteine. To quantitate this effect, we
established a NIF-laminin substratum, using a submaximal concentration
of NIF (2 NIU/ml) and a maximal concentration of laminin (1 µg/ml).
On this substratum 19% of the DRGn had extended neurites ( 4 cell
body diameters) after 4 hr in medium with and without cysteine. This
response corresponded to ~75% inhibition of the neurite outgrowth on
laminin only. Results are shown in Figure
1. Over 48 hr, neuritic growth on this
NIF-laminin substratum steadily increased to 67% in the absence of
cysteine but remained mainly inhibited in the presence of cysteine.
Cysteine did not alter the neuritic growth on laminin alone (Fig. 1).
These results suggest that the time-dependent neuritic growth observed
on NIF-laminin was dependent on a metalloproteinase activity. On the
basis of this initial observation, we hypothesized that DRGn express a
metalloproteinase that inactivates the inhibitory proteoglycan NIF and,
as a result, that unmasks the neurite-promoting potential of
NIF-inhibited laminin. We refer to this effect as deinhibition.
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Figure 1.
MMP-dependent neurite outgrowth on NIF-laminin.
DRGn were seeded on laminin and NIF-laminin substrata in
DMEM/N2+NGF medium with and without the
metalloproteinase inhibitor cysteine (250 µM). The
NIF-laminin substratum consisted of laminin (1 µg/ml) and NIF (2 NIU/ml) (a submaximal concentration of NIF). The percentage of neurons
bearing a neurite >4 cell body diameters was scored at 4, 24, and 48 hr after seeding. Determinations were made by scoring 50 neurons per
culture well; data represent the means of duplicate determinations made
in each of two independent assays (n = 4). SD  5.4.
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Metalloproteinase-dependent neurite outgrowth on
nerve sections
In the culture assays described above, inhibited substrata were
established by the recombination of laminin-1 (isolated from mouse EHS
tumor) and RN22-derived NIF. An apparently identical inhibitory CSPG
was isolated from cultured rat Schwann cells and normal adult rat nerve
(Zuo et al., 1998). In addition, we reported findings indicating that
the neurite-promoting activity of laminin in the endoneurial basal
lamina of adult nerve is inhibited by association with this CSPG. In
the nerve, laminin-rich basal laminae form continuous sleeves around
axon-Schwann cell units that appear in longitudinal section as an
array of parallel tracks. Embryonic neurons grown on fresh-frozen nerve
sections extend neurites along these basal lamina tracks. This growth
pattern clearly demonstrates that neurites probe for and follow
neurite-promoting or, at least, permissive pathways within the tissue
section substratum. Adult DRGn fail to extend neurites on sections
of normal adult nerve (Bedi et al., 1992); therefore, in the present
study embryonic DRGn were used to examine the inhibitory properties of
this tissue.
Neuritic growth by embryonic DRGn on normal nerve sections was gradual
(120 µm/48 hr) (see Table 1; Fig.
4A) and was more reminiscent of the long-term growth
observed on a NIF-laminin substratum (as described in Fig. 1) than
growth on laminin alone (which exceeded 1000 µm/48 hr). Furthermore,
neurite outgrowth on nerve sections was decreased by cysteine, also
like that on a reconstituted NIF-laminin substratum. To avoid
potential toxicity at higher cysteine levels and to improve the
specificity of metalloproteinase inhibition, we used the dipeptide
metalloproteinase inhibitor GM6001 in subsequent cryoculture assays.
The addition of GM6001 to the culture medium decreased neuritic growth
in a concentration-dependent manner over the range of 1-100
µM. Compared with controls, the average neurite length in
a 48 hr assay was decreased to 32.0 ± 37.5 by a near-maximal
concentration of GM6001 (50 µM) (ANOVA; p < 0.0001). GM6001 has very low toxicity, and neuritic growth on
laminin-coated plastic was unaffected by concentrations several-fold higher. From these results we conclude that neuritic growth by embryonic DRGn on normal adult nerve sections involves a
metalloproteinase activity.
Regenerating DRGn express and transport MMP-2
We previously reported zymographic (gelatin overlay
electrophoresis) and Western immunoblot analyses demonstrating that
MMP-2 is the predominant MMP secreted by DRGn cultures grown in the presence of NGF (Muir, 1994). To support the contention that MMP-2 played a role in deinhibiting substrata in the assays above, we examined whether MMP-2 was present at the growing tip of neurites. Immunocytochemical labeling of permeabilized DRGn cultures showed that
MMP-2 was present within neuronal cell bodies, neurites, and growth
cones, including filopodia (Fig.
2A,B). Staining of unpermeabilized neurons indicated that MMP-2 also might be present on
the cell surface (data not shown). Low contrast and the uncertainty that fine growth cone membranes remained impervious to antibodies made
it difficult to determine the distribution of MMP-2 on the surface of
the growing tip. DRGn cultures contain a variety of sensory neuron
subtypes; however, the vast majority of DRGn grown in the presence of
NGF was intensely MMP-2-positive. MMP-2 expression by DRGn cultured on
different substrata (including NIF-laminin) was not noticeably
different. These results show that MMP-2 is transported to the growth
cone of DRGn during in vitro regeneration.
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Figure 2.
Immunofluorescent microscopy of cultured DRGn
(A) and a selected growth cone labeled with MMP-2
antibody (B). DRGn were grown for 24 hr on
laminin-coated chamber slides and then immunolabeled with anti-MMP2/475
antibody (2 µg/ml) and FITC-conjugated anti-rabbit secondary
antibody.
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Recombinant MMP-2 inactivates NIF and deinhibits
NIF-laminin substrata
Incubating NIF with concentrated medium collected from DRGn
cultures resulted in a nearly complete loss of the inhibitory activity
of NIF, that is, its ability to inhibit the neurite-promoting activity
of laminin (data not shown). To confirm the effect of MMP-2 on NIF
activity, we incubated NIF with various concentrations of recombinant
MMP-2 and then assayed for neurite-inhibiting activity. Because MMP-3
is expressed by PC12 cells treated with NGF and by certain embryonic
neurons in vivo (Machida et al., 1989; Nordstrom et al.,
1995), the effects of MMP-3 on NIF activity were examined also.
Treatment with MMP-2 decreased NIF activity in a
concentration-dependent manner (Fig.
3A). By comparison, MMP-3 was
much less potent, and only a partial loss of NIF activity was observed
even at high concentrations of activated enzyme. This finding was
somewhat surprising because MMP-3 is known to degrade a variety of
proteoglycans and to have a broader substrate specificity than MMP-2
(Wilhelm et al., 1987). However, these results indicate that NIF
activity is particularly vulnerable to proteolytic inactivation by
MMP-2.
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Figure 3.
Treatment of NIF with MMP-2 caused a
concentration-dependent loss of neurite-inhibiting activity.
A, NIF was incubated first with serial concentrations
(0.01-10 µg/ml) of recombinant human MMP-2 or MMP-3. The equivalent
of 8 NIU/ml of NIF (a near-maximal concentration before MMP treatment)
was mixed with laminin (2 µg/ml), and then the mixture was applied
(50 µl/well) to culture 96-well plates coated with polyornithine. The
control condition consisted of 8 NIU/ml of untreated (fully active) NIF
mixed with laminin (2 µg/ml). B, NIF (8 NIU/ml) was
mixed with laminin (2 µg/ml) and applied to culture wells, forming a
highly inhibitory substratum (Control). Then this
substratum was treated with MMP-2 (2 µg/ml), with the inclusion of
the MMP inhibitor phenanthroline as indicated. For both
A and B, neurons were seeded on the
resulting substrata, and 4 hr later the percentage of neurons bearing a
neurite >4 cell body diameters was scored. Determinations were made by
scoring 50 neurons per culture well, and data represent the means of
duplicate determinations made in each of two independent assays
(n = 4). A, SD 4.4;
B, SD 5.0.
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In the experiments above, NIF was inactivated by MMP-2 before
recombination with laminin and application to the culture substratum. We also examined the ability of MMP-2 to alter the neurite-promoting properties of a NIF-laminin substratum already established on tissue
culture plastic. Results are given in Figure 3B. Treatment of a maximally inhibited NIF-laminin substratum with nanomolar concentrations of MMP-2 inactivated NIF and restored the
neurite-promoting activity of the laminin component. This deinhibition
by MMP-2 did not occur in the presence of the metalloproteinase
inhibitor phenanthroline. Thus, MMP-2 proteolytic activity inactivated
NIF without hindering the neurite-promoting activity of laminin.
Treatment of laminin-1 and laminin-2 (when in solution or when
substratum-bound) with MMP-2 or MMP-3 neither increased nor decreased
their neurite-promoting activity.
MMP-2 deinhibits nerve sections in cryoculture assay
Embryonic DRGn cultured on nerve sections extended neurites
along exposed basal laminae with an average length of 120 µm in a
2 d period (Table 1; Fig.
4A). To examine if
modification of the nerve substratum by MMP-2 influenced neuritic
growth, we pretreated sections with recombinant MMP-2 before
cryoculture. Pretreatment with MMP-2 resulted in a 50% increase in
neurite outgrowth by DRGn (Table 1; Fig. 4B). Neurite
growth continued to be closely associated with the basal lamina in
sections treated with MMP-2, indicating that MMP-2 deinhibited the
basal lamina of normal nerve. In addition, neuritic growth was
increased to a similar extent on nerve sections treated with
chondroitinase ABC (Table 1). The sequential treatment of nerve
sections with MMP-2 and chondroitinase did not have a significant
additive effect over treatment with either enzyme alone. These results,
combined with the substrate specificity of chondroitinase, strengthen
the likelihood that both chondroitinase and MMP-2 degraded the same
inhibitory component, a CSPG. It is important to note that NIF activity
is expressed only by the intact CSPG and that NIF can be inactivated by
degradation of either its chondroitin sulfate chains or its core
protein (Muir et al., 1989a).
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Figure 4.
Neuronal cryoculture on peripheral nerve treated
with MMP-2. Shown is a 2 d neuritic growth by embryonic chick DRGn
cultured on fresh-frozen sections of adult rat sciatic nerve. Neurites
follow the longitudinal paths of exposed basal laminae. In the control
condition (A), the DRGn were cultured on
(untreated) fresh-frozen sections of normal adult rat sciatic nerve in
DMEM/N2+NGF medium. In B, nerve
sections were pretreated with MMP-2 (10 µg/ml) before the seeding of
the neurons. Chick neurons were labeled immunofluorescently, using a
species-specific monoclonal antibody to N-CAM, and images were captured
by digital microphotography. Scale bar, 100 µm.
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The neurite-promoting activity of laminin is deinhibited by
treatment of nerve sections with MMP-2 or chondroitinase
Several groups have reported that neuritic growth on normal nerve
sections is not inhibited by antibodies that block laminin function
(Sandrock and Matthew, 1987; Anton et al., 1994; Agius and Cochard,
1998). This finding indicates that the baseline neuritic growth on
nerve sections is not mediated by laminin and that laminin present in
normal nerve is not functionally active. Our aim was to determine
whether the increases in neuritic growth seen on normal nerve treated
with MMP-2 and chondroitinase occurred in response to laminin.
Cryoculture experiments on enzyme-treated sections were performed in
the presence of antibodies that block neuritic growth in response to
laminin. Neuritic growth was increased slightly on nerve sections in
the presence of normal rabbit serum, which served as a baseline control
for the antibody conditions (Fig. 5).
Neuritic growth on sections was increased by ~50% on sections
deinhibited with either MMP-2 or chondroitinase, and this effect was
reduced to baseline in the presence of the laminin antibodies. From
these results we conclude that MMP-2 and chondroitinase deinhibit the
neurite-promoting activity of endoneurial laminin.
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Figure 5.
Embryonic chick DRGn were cultured on fresh-frozen
sections of normal adult rat sciatic nerve pretreated with buffer only
(Untreated, control), MMP-2, or chondroitinase ABC
(Ch'ase), as described in Materials and Methods. DRGn
were seeded on the nerve sections and allowed to attach for 1 hr before
treatment with normal rabbit serum (NRS, control) or
anti-laminin antibodies ( -Ln). Assays were terminated
after 48 hr of growth by aldehyde fixation. The chick DRGn were
immunolabeled with an anti-chick N-CAM monoclonal antibody, and the
length of the single longest neurite per neuron was measured. In random
fields >50 neurons were scored on each of the multiple sections in
three separate experiments. Data represent the means and SEM expressed
as a percentage of the untreated control value. *p 0.005; **p < 0.0001. Statistical significance
was determined by Student's t test.
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Proteolytic degradation of NIF core protein
To confirm the proteolytic degradation of NIF by MMP-2,
we treated [35S]NIF with recombinant MMP-2 and
then examined it by gel electrophoresis (Fig.
6). [35S]NIF was
prepared by metabolic labeling of its chondroitin sulfate chains by the
inclusion of inorganic radiosulfate in the RN22 culture medium (see
Materials and Methods). Intact [35S]NIF appeared
on autoradiographic gels as a broad band with a molecular mass of
400 kDa (lane 1). After incubation with MMP-2, the 400 kDa autoradiographic band was shifted and included a trail of
35S-labeled polypeptides (lane 2). NIF
degradation by MMP-2 was prevented in the presence of phenanthroline,
confirming that the degradation resulted from metalloproteinase
activity (lane 3). MMP-2 degradation of NIF was attributed
exclusively to core protein proteolysis because
[35S]glycosaminoglycans remained associated with
polypeptide fragments. In contrast, the
[35S]glycosaminoglycan (and thus autoradiographic
tag) of NIF was stripped from the core protein by chondroitinase ABC
digestion (lane 4). Western immunoblotting of NIF
treated with chondroitinase revealed a core protein of ~150-200 kDa
(see Zuo et al., 1998).
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Figure 6.
Autoradiographic analysis of proteolytic
degradation of [35S]NIF by MMP-2. Metabolically
radiosulfate-labeled NIF was incubated with MMP-2 before SDS-PAGE under
reducing conditions. Intact [35S]NIF (3 µg)
appeared on autoradiograms as a broad band with a molecular mass of
400 kDa (lane 1). The electrophoretic mobility of
[35S]NIF (3 µg) was shifted after proteolysis by
MMP-2 (20 ng in 10 µl; lane 2). Inclusion of
phenanthroline (1 mM) prevented the degradation of
[35S]NIF (lane 3).
[35S]chondroitin sulfate chains of
[35S]NIF were cleaved by chondroitinase ABC (0.02 U in 10 µl), resulting in the complete loss of autoradiographic
profile (lane 4).
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DISCUSSION |
Numerous studies indicate that certain extracellular proteoglycans
are inhibitory molecules that, at high concentration relative to
growth-promoting signals, may regulate or repress axonal growth. We
previously characterized a neurite-inhibiting CSPG, named NIF, produced
by cultured rat RN22 schwannoma and Schwann cells (Muir et al., 1989a).
NIF reversibly binds to and inhibits the neurite-promoting activity of
laminin. NIF-laminin complexes isolated from culture medium fail to
promote neuritic growth when they are substratum-bound. However, both
NIF and laminin are recovered in active forms by dissociative
fractionation of NIF-laminin complexes. Additionally, the
neurite-promoting activity of laminin is restored by degrading substratum-bound NIF-laminin complexes with chondroitinase (Zuo et
al., 1998). NIF activity is expressed only by the intact proteoglycan, and its inhibitory activity is abolished by cleavage of its chondroitin sulfate chains by chondroitinase or by limited proteolysis of its core
protein. On the basis of the premise that a NIF-laminin substratum can
be deinhibited enzymatically, we set out to determine whether neurons
secrete enzymes that inactivate NIF in vitro and in
peripheral nerve tissue.
We first discovered that embryonic DRGn grown on a NIF-laminin
substratum for extended periods failed to grow neurites in media
containing the metalloproteinase inhibitor cysteine. In medium without
cysteine, neurites initially were delayed but gradually elongated on
the NIF-laminin. Because it was known to us that DRGn (treated with
NGF) express the metalloproteinase MMP-2 (Muir, 1994), experiments were
performed to determine whether MMP-2 can degrade NIF and deinhibit
NIF-laminin substrata. Principal findings from our study include the
following: (1) Neuritic growth by embryonic DRGn on a synthetic
NIF-laminin substratum and on normal nerve tissue sections is
dependent mainly on a metalloproteinase. (2) MMP-2 is secreted by DRGn
and is transported to the growing tips of regenerating neurites. (3)
MMP-2 degrades and inactivates NIF but does not affect the
neurite-promoting activity of laminin. (4) Neuritic growth by DRGn
cultured on nerve tissue sections is increased markedly by first
treating the nerve sections with MMP-2, and the deinhibiting effect of
MMP-2 is equal to and nonadditive with that of chondroitinase. (5)
Laminin activity is masked in normal adult nerve, and treatment of
nerve tissue sections with MMP-2 or chondroitinase unmasks the
neurite-promoting activity of endoneurial laminin. These findings
support the conclusion that MMP-2 expression provides a mechanism for
growth cones to deinhibit the growth-promoting properties of laminin
residing in the endoneurial basal lamina.
The culture of neurons on tissue sections (cryoculture) has been used
to study mechanisms of neuritic growth and to better understand the
neurite-promoting status of peripheral and central nervous tissues.
Neurite outgrowth on tissue sections is clearly directed by the
organization of tissues in the underlying substrata. Growth cones have
an exquisite ability to probe their substratum and decipher cues that
influence their direction of growth. Experimentally, the pattern of
neuritic growth on tissue sections reveals the functional state of
molecular signals within the extracellular environment. Furthermore,
cryoculture offers the unique opportunity to examine the
neurite-promoting properties of normal tissues devoid of degenerative
and inflammatory elements. By using sections of normal nerve, this
bioassay also can help to assess determinants of neuronal
differentiation and stabilization. Moreover, it is then possible to
study alterations that occur during the transition from a steady-state
to a regenerative one. In nerve injury this transition may have
important consequences for the success of axonal regeneration and
functional recovery. Langley and Anderson (1904) first reported that
transected axons do not regenerate along a normal nerve. More recently,
the repressive status of normal nerve has been confirmed in the
cryoculture model wherein adult DRG axons fail to grow neurites on
sections of normal adult nerve (Bedi et al., 1992). These findings
suggest that, although rich in laminin, Schwann cell surfaces and their
associated basal lamina in normal nerve fail to support and actively
may inhibit axonal growth. This repressive disposition may be
responsible for the initial delay in axonal regeneration that occurs
after nerve injury (see Tapia et al., 1995). Although axons sprout
numerous collaterals within hours of injury, significant elongation is delayed initially and then accelerates to reach a constant rate within
3 d (Wyrwicka, 1950). Regenerating axon sprouts arise rapidly from
the first node of Ranvier proximal to the site of injury and
immediately interface with established tissue components that have not
yet undergone significant Wallerian degeneration (for review, see Fu
and Gordon, 1997). The transition of the endoneurial basal lamina from
an inhibited state to a promoting one is likely an important event
governing the onset of axonal elongation. Extracellular matrices are
relatively stable structures, and modifications of basal lamina
composition are not likely to occur rapidly. On the other hand, whereas
axon sprouting and immediate elongation occur without requiring the
cell body to participate, the ability to overcome inhibitory signals
may be transcription/translation-dependent. The requirement for axonal
transport of regulatory proteins to the site of nerve injury would
incur a considerable delay. In this regard, we previously have reported
that the expression of MMP-2 by cultured DRGn is induced by NGF (Muir,
1994). NGF levels are very low in normal sciatic nerve. However, local
NGF levels rapidly increase near the site of nerve injury (Heumann et
al., 1987). Although it presently is unknown whether MMP-2 is
upregulated in response to NGF in vivo, it is interesting to
speculate that proteolytic mechanisms are an important part of the
regenerative responses to NGF.
MMP-2 is present at the growing tip of neurites, and it is now
known that MMP-2 is activated and can reside on the cell surface (Sato
et al., 1996). Therefore, axonal growth may involve spatial and
temporal regulation of ECM degradation at the cell surface. This
suggests that the growing axon may invoke a process of
focalized proteolysis similar to that proposed for the
pericellular degradation brought to bear at the leading edge of
migrating and invading cells during wound healing and tumor metastasis
(Basbaum and Werb, 1996). On the basis of past and present findings, we
propose that MMP-2 at the growing tip of axons may have two
interrelated functions: one involving degradation of collagenous matrix
barriers (see Muir, 1994) and another more selective process of
degrading inhibitory CSPG. MMP-2 action at the growth cone may
represent a discrete and focal mechanism by which neurons alter their
environment in the distal nerve.
Differences in the growth responses of embryonic and adult DRGn on
nerve tissue sections have been reported (Bedi et al., 1992; Agius and
Cochard, 1998). It has been postulated that the age-dependent decline
in axonal growth may be attributed to a relative insensitivity of
embryonic neurons to growth inhibitors. This explanation clearly does
not apply to the response of embryonic DRGn to CSPG in
vitro. Furthermore, our findings indicate that growth by embryonic
DRGn, like that reported for adult neurons, also is inhibited
significantly by CSPG within the endoneurium of normal adult nerve
sections. Although other intrinsic mechanisms may influence the growth
of embryonic and adult neurons on nerve sections, our studies raise the
possibility that the expression of enzymes such as MMP-2 enables
embryonic DRGn to overcome the inhibition by CSPG, which otherwise
represents an insurmountable barrier to axonal growth. MMP-2 expression
and activity are highly regulated, and further experiments are required
to determine whether adult DRGn fail to inactivate inhibitory CSPG
under the conditions of these assays or if the mechanism of
deinhibition by MMP-2 can be elicited by growth factors other than NGF.
It is interesting that various neurotrophins influence the growth of
DRGn on different tissue substrata and that, in contrast to previous
findings with NGF alone, mature DRGn treated with a combination of NGF,
NT-3, and BDNF extended neurites on sections of normal adult sciatic nerve (Tuttle and Matthew, 1995; Golding et al., 1996).
Using confocal microscopy to discriminate the association of
neurites with discrete aspects of the basal lamina, Agius and Cochard
(1998) observed that embryonic DRGn neurites did not obtain access to
the laminin-rich inner structures of endoneurial tubes in normal nerve
sections. Accordingly, the moderate neuritic growth observed in this
condition was attributed to components other than laminin. Our findings
using function-blocking laminin antibodies confirm previous reports
that growth on normal nerve sections is not mediated by laminin. The
degenerative process that occurs in nerve distal to the site of injury
has a significant impact on the ability of nerve to support axonal
regeneration and, furthermore, may involve an ability of growth cones
to access the laminin-rich aspects of the endoneurial basal laminin
(Bedi et al., 1992; Danielsen et al., 1995; Agius and Cochard, 1998).
Similarly, we found that the treatment of normal nerve sections with
MMP-2 markedly increases the ability of nerve tissue to support
neuritic growth and that this increased occurred in response to laminin
because it was blocked by laminin antibodies.
Several in vitro and in vivo models demonstrate
that the entire distal nerve has an increased ability to support axonal
growth after Wallerian degeneration (Bedi et al., 1992; Danielsen et al., 1995). After injury, Schwann cells become activated and
participate in the remodeling of the endoneurial basal lamina.
Degradation and remodeling of extracellular matrices are certainly
important aspects of the regenerative process, and MMP-2 is expressed
not only by regenerating neurons but also by Schwann cells and invading macrophages (Muir, 1995; Yamada et al., 1995; La Fleur et al., 1996).
Accordingly, an additional supportive role for these cells in the
regenerative process may include the general conversion of the
endoneurial basal lamina from a repressive to a facilitative substratum
for axonal growth. We propose that MMP-2 plays a key role in this
fundamental process. It very well may be that the proteolytic
competence of regenerating nervous tissues and resident neurons
determines the extent and course of axonal regeneration. Studying the
expression of matrix-degrading enzymes in response to injury and the
proteolytic vulnerability of inhibitory components also might help to
explain the contrasting regenerative responses of injured peripheral
and central nervous tissues at various developmental stages.
| |
FOOTNOTES |
Received April 8, 1998; accepted April 30, 1998.
Support was provided by grants awarded to D.M. from the National
Institutes of Health (NS31255) and the Florida State Brain and Spinal
Cord Injury Rehabilitation Trust Fund. We thank Dr. R. E. Galardy
for the gift of GM6001.
Correspondence should be addressed to Dr. David Muir, Pediatric
Neurology, Box 100296, University of Florida College of Medicine, Gainesville, FL 32610.
| |
REFERENCES |
-
Agius E,
Cochard P
(1998)
Comparison of neurite outgrowth induced by intact and injured sciatic nerves: a confocal and functional analysis.
J Neurosci
18:328-338[Abstract/Free Full Text].
-
Anton ES,
Sandrock AJ,
Matthew WD
(1994)
Merosin promotes neurite growth and Schwann cell migration in vitro and nerve regeneration in vivo: evidence using an antibody to merosin, ARM-1.
Dev Biol
164:133-146[Web of Science][Medline].
-
Basbaum CB,
Werb Z
(1996)
Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface.
Curr Opin Cell Biol
8:731-738[Web of Science][Medline].
-
Bedi KS,
Winter J,
Berry M,
Cohen J
(1992)
Adult rat dorsal root ganglion neurons extend neurites on predegenerated but not on normal peripheral nerves in vitro.
Eur J Neurosci
4:193-200[Web of Science][Medline].
-
Bottenstein JE,
Sato GH
(1979)
Growth of a rat neuroblastoma cell line in serum-free supplemented medium.
Proc Natl Acad Sci USA
76:514-517[Abstract/Free Full Text].
-
Brittis PA,
Canning DR,
Silver J
(1992)
Chondroitin sulfate as a regulator of neuronal patterning in the retina.
Science
255:733-736[Abstract/Free Full Text].
-
Carbonetto S,
Evans D,
Cochard P
(1987)
Nerve fiber growth in culture on tissue substrata from central and peripheral nervous systems.
J Neurosci
7:610-620[Abstract].
-
Chantry A,
Gregson N,
Glynn P
(1992)
Degradation of myelin basic protein by a membrane-associated metalloprotease: neural distribution of the enzyme.
Neurochem Res
17:861-867[Web of Science][Medline].
-
Danielsen N,
Kerns JM,
Holmquist B,
Zhao Q,
Lundborg G,
Kanje M
(1995)
Predegeneration enhances regeneration into acellular nerve grafts.
Brain Res
681:105-108[Web of Science][Medline].
-
DiStefano PS,
Chelsea DM,
Schick CM,
McKelvy JF
(1993)
Involvement of a metalloprotease in low-affinity nerve growth factor receptor truncation: inhibition of truncation in vitro and in vivo.
J Neurosci
13:2405-2414[Abstract].
-
Engvall E,
Davis GE,
Dickerson K,
Ruoslahti E,
Varon S,
Manthorpe M
(1986)
Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.
J Cell Biol
103:2457-2465[Abstract/Free Full Text].
-
Faissner A,
Steindler D
(1995)
Boundaries and inhibitory molecules in developing neural tissues.
Glia
13:233-254[Web of Science][Medline].
-
Fambrough D,
Pan D,
Rubin GM,
Goodman CS
(1996)
The cell surface metalloprotease/disintegrin Kuzbanian is required for axonal extension in Drosophila.
Proc Natl Acad Sci USA
93:13233-13238[Abstract/Free Full Text].
-
Fridman R,
Fuerst TR,
Bird RE,
Hoyhtya M,
Oelkuct M,
Kraus S,
Komarek D,
Liotta LA,
Berman ML,
Stetler-Stevenson WG
(1992)
Domain structure of human 72 kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.
J Biol Chem
267:15398-15405[Abstract/Free Full Text].
-
Fu SY,
Gordon T
(1997)
The cellular and molecular basis of peripheral nerve regeneration.
Mol Neurobiol
14:67-116[Web of Science][Medline].
-
Giannelli G,
Falk-Marzillier J,
Schraldi O,
Stetler-Stevenson WG,
Quaranta V
(1997)
Induction of cell migration by matrix metalloproteinase-2 cleavage of laminin-5.
Science
277:225-228[Abstract/Free Full Text].
-
Giannini C,
Dyck PJ
(1990)
The fate of Schwann cell basement membranes in permanently transected nerves.
J Neuropathol Exp Neurol
49:550-563[Web of Science][Medline].
-
Golding JP,
Shewan D,
Berry M,
Cohen J
(1996)
An in vitro model of the rat dorsal root entry zone reveals developmental changes in the extent of sensory axon growth into the spinal cord.
Mol Cell Neurosci
7:191-203[Web of Science][Medline].
-
Grobelny D,
Poncz L,
Galardy RE
(1992)
Inhibition of human skin fibroblast collagenase, thermolsin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids.
Biochemistry
31:7152-7154[Medline].
-
Hayden SM,
Seeds NW
(1996)
Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia.
J Neurosci
16:2307-2317[Abstract/Free Full Text].
-
Heumann R,
Korsching S,
Bandtlow C,
Thoenen H
(1987)
Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection.
J Cell Biol
104:1623-1631[Abstract/Free Full Text].
-
Ide C,
Tohyama K,
Yokota R,
Nitatori T,
Onodera S
(1983)
Schwann cell basal lamina and nerve regeneration.
Brain Res
288:61-75[Web of Science][Medline].
-
Kolodkin AL
(1996)
Growth cones and the cues that repel them.
Trends Neurosci
19:507-513[Web of Science][Medline].
-
Kuecherer-Ehret A,
Graeber MB,
Edgar D,
Thoenen H,
Kreutzberg GW
(1990)
Immunoelectron microscopic localization of laminin in normal and regenerating mouse sciatic nerve.
J Neurocytol
19:101-109[Web of Science][Medline].
-
La Fleur M,
Underwood JL,
Rappolee DA,
Werb Z
(1996)
Basement membrane and repair of injury to peripheral nerve: defining a potential role for macrophages, matrix metalloproteinases, and tissue inhibitor of metalloproteinases-1.
J Exp Med
184:2311-2326[Abstract/Free Full Text].
-
Langley JN,
Anderson HK
(1904)
The union of different kinds of nerve fibres.
J Physiol (Lond)
31:365-391.
-
Lowe-Krentz LJ,
Keller JM
(1984)
Disulfide-bonded aggregates of heparan sulfate proteoglycans.
Biochemistry
23:2621-2627[Medline].
-
Machida CM,
Rodland KD,
Matrisian L,
Magun BE,
Ciment G
(1989)
NGF induction of the gene encoding the protease transin accompanies neuronal differentiation in PC12 cells.
Neuron
2:1587-1596[Web of Science][Medline].
-
Manthorpe M,
Engvall E,
Ruoslahti E,
Longo FM,
Davis GE,
Varon S
(1983)
Laminin promotes neuritic regeneration from cultured peripheral and central neurons.
J Cell Biol
97:1882-1890[Abstract/Free Full Text].
-
Matrisian LM
(1992)
The matrix-degrading metalloproteinases.
BioEssays
14:455-463[Web of Science][Medline].
-
McGuire PG,
Seeds NW
(1990)
Degradation of underlying extracellular matrix by sensory neurons during neurite outgrowth.
Neuron
4:633-642[Web of Science][Medline].
-
Monard D
(1988)
Cell-derived proteases and protease inhibitors as regulators of neurite outgrowth.
Trends Neurosci
11:541-544[Web of Science][Medline].
-
Muir D
(1994)
Metalloproteinase-dependent neurite outgrowth within a synthetic extracellular matrix is induced by nerve growth factor.
Exp Cell Res
210:243-252[Web of Science][Medline].
-
Muir D
(1995)
Differences in proliferation and invasion by normal, transformed, and NF1 Schwann cell cultures are influenced by matrix metalloproteinase expression.
Clin Exp Metastasis
13:303-314[Web of Science][Medline].
-
Muir D,
Engvall E,
Varon S,
Manthorpe M
(1989a)
Schwannoma cell-derived inhibitor of the neurite-promoting activity of laminin.
J Cell Biol
109:2353-2362[Abstract/Free Full Text].
-
Muir D,
Gennrich C,
Varon S,
Manthorpe M
(1989b)
Rat sciatic nerve Schwann cell microcultures: responses to mitogens and production of trophic and neurite-promoting factors.
Neurochem Res
14:1003-1012[Web of Science][Medline].
-
Nordstrom LA,
Lochner J,
Yeung W,
Ciment G
(1995)
The metalloproteinase stromelysin-1 (transin) mediates PC12 cell growth cone invasiveness through basal laminae.
Mol Cell Neurosci
6:56-68[Web of Science][Medline].
-
Oakley RA,
Tosney KW
(1991)
Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo.
Dev Biol
147:187-206[Web of Science][Medline].
-
Pindzola RR,
Doller C,
Silver J
(1993)
Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during development and after root and sciatic nerve lesions.
Dev Biol
156:34-48[Web of Science][Medline].
-
Pittman RN,
Buettner HM
(1989)
Degradation of extracellular matrix by neuronal proteases.
Dev Neurosci
11:361-375[Web of Science][Medline].
-
Pittman RN,
Williams AG
(1988)
Neurite penetration into collagen gels requires Ca2+-dependent metalloproteinase activity.
Dev Neurosci
11:41-51.
-
Ray JM,
Stetler-Stevenson WG
(1995)
Gelatinase A activity directly modulates melanoma cell adhesion and spreading.
EMBO J
14:908-917[Web of Science][Medline].
-
Salonen V,
Peltonen J,
Roytta M,
Virtanen I
(1987)
Laminin in traumatized peripheral nerve: basement membrane changes during degeneration and regeneration.
J Neurocytol
16:713-720[Web of Science][Medline].
-
Sandrock AW,
Matthew WD
(1987)
Identification of a peripheral nerve neurite growth-promoting activity by development and use of an in vitro bioassay.
Proc Natl Acad Sci USA
84:6934-6938[Abstract/Free Full Text].
-
Satinsky D,
Pepe FA,
Liu CN
(1964)
The neurilemma cell in peripheral nerve degeneration and regeneration.
Exp Neurol
9:441-451.
-
Sato H,
Takino T,
Kinoshita T,
Imai K,
Okada Y,
Stetler-Stevenson WG,
Seiki M
(1996)
Cell surface binding and activation of gelatinase A induced by expression of membrane-type-1-matrix metalloproteinase (MT1-MMP).
FEBS Lett
385:238-240[Web of Science][Medline].
-
Snow DM,
Watanabe M,
Letourneau PC,
Silver J
(1991)
A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth.
Development
113:1473-1485[Abstract].
-
Tapia M,
Inestrosa NC,
Alvarez J
(1995)
Early axonal regeneration: repression by Schwann cells and a protease?
Exp Neurol
131:124-132[Web of Science][Medline].
-
Tona A,
Perides G,
Rahemtulla F,
Dahl D
(1993)
Extracellular matrixin regenerating rat sciatic nerve: a comparative study on the localization of laminin, hyaluronic acid, and chondroitin sulfate proteoglycans, including versican.
J Histochem Cytochem
41:593-599[Abstract].
-
Tuttle R,
Matthew WD
(1995)
Neurotrophins affect the pattern of DRG neurite growth in a bioassay that presents a choice of CNS and PNS substrates.
Development
121:1301-1309[Abstract].
-
Watanabe M,
Frelinger ALD,
Rutishauser U
(1986)
Topography of N-CAM structural and functional determinants. I. Classification of monoclonal antibody epitopes.
J Cell Biol
103:1721-1727[Abstract/Free Full Text].
-
Wilhelm SM,
Collier IE,
Kronberger A,
Eisen AZ,
Marmer BL,
Grant GA,
Bauer EA,
Goldberg GI
(1987)
Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells.
Proc Natl Acad Sci USA
84:6725-6729[Abstract/Free Full Text].
-
Wyrwicka W
(1950)
On the rate of regeneration of the rat sciatic nerve in the white mouse.
Acta Biol Exp (Warsz)
15:147-153.
-
Yamada T,
Miyazaki K,
Koshikawa N,
Takahashi M,
Akatsu H,
Yamamoto T
(1995)
Selective localization of gelatinase A, an enzyme degrading beta-amyloid protein, in white matter microglia and in Schwann cells.
Acta Neuropathol (Berl)
89:199-203[Medline].
-
Zuo J,
Hernandez YJ,
Muir D
(1998)
Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury.
J Neurobiol
34:41-54[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18145203-09$05.00/0
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|
P. H. Larsen, J. E. Wells, W. B. Stallcup, G. Opdenakker, and V. W. Yong
Matrix Metalloproteinase-9 Facilitates Remyelination in Part by Processing the Inhibitory NG2 Proteoglycan
J. Neurosci.,
December 3, 2003;
23(35):
11127 - 11135.
[Abstract]
[Full Text]
[PDF]
|
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C. A. Krekoski, D. Neubauer, J. B. Graham, and D. Muir
Metalloproteinase-Dependent Predegeneration In Vitro Enhances Axonal Regeneration within Acellular Peripheral Nerve Grafts
J. Neurosci.,
December 1, 2002;
22(23):
10408 - 10415.
[Abstract]
[Full Text]
[PDF]
|
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|
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C. A. Webber, J. C. Hocking, V. W. Yong, C. L. Stange, and S. McFarlane
Metalloproteases and Guidance of Retinal Axons in the Developing Visual System
J. Neurosci.,
September 15, 2002;
22(18):
8091 - 8100.
[Abstract]
[Full Text]
[PDF]
|
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E. Llano, G. Adam, A. M. Pendas, V. Quesada, L. M. Sanchez, I. Santamaria, S. Noselli, and C. Lopez-Otin
Structural and Enzymatic Characterization of Drosophila Dm2-MMP, a Membrane-bound Matrix Metalloproteinase with Tissue-specific Expression
J. Biol. Chem.,
June 21, 2002;
277(26):
23321 - 23329.
[Abstract]
[Full Text]
[PDF]
|
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A. Szklarczyk, J. Lapinska, M. Rylski, R. D. G. McKay, and L. Kaczmarek
Matrix Metalloproteinase-9 Undergoes Expression and Activation during Dendritic Remodeling in Adult Hippocampus
J. Neurosci.,
February 1, 2002;
22(3):
920 - 930.
[Abstract]
[Full Text]
[PDF]
|
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H. Hayashita-Kinoh, H. Kinoh, A. Okada, K. Komori, Y. Itoh, T. Chiba, M. Kajita, I. Yana, and M. Seiki
Membrane-Type 5 Matrix Metalloproteinase Is Expressed in Differentiated Neurons and Regulates Axonal Growth
Cell Growth Differ.,
November 1, 2001;
12(11):
573 - 580.
[Abstract]
[Full Text]
[PDF]
|
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C. A. Krekoski, D. Neubauer, J. Zuo, and D. Muir
Axonal Regeneration into Acellular Nerve Grafts Is Enhanced by Degradation of Chondroitin Sulfate Proteoglycan
J. Neurosci.,
August 15, 2001;
21(16):
6206 - 6213.
[Abstract]
[Full Text]
[PDF]
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M. L. Condic
Adult Neuronal Regeneration Induced by Transgenic Integrin Expression
J. Neurosci.,
July 1, 2001;
21(13):
4782 - 4788.
[Abstract]
[Full Text]
[PDF]
|
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L. B. Siconolfi and N. W. Seeds
Induction of the Plasminogen Activator System Accompanies Peripheral Nerve Regeneration after Sciatic Nerve Crush
J. Neurosci.,
June 15, 2001;
21(12):
4336 - 4347.
[Abstract]
[Full Text]
[PDF]
|
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|
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|
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M. S. Ramer, I. Duraisingam, J. V. Priestley, and S. B. McMahon
Two-Tiered Inhibition of Axon Regeneration at the Dorsal Root Entry Zone
J. Neurosci.,
April 15, 2001;
21(8):
2651 - 2660.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
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X. Wang, J. Jung, M. Asahi, W. Chwang, L. Russo, M. A. Moskowitz, C. E. Dixon, M. E. Fini, and E. H. Lo
Effects of Matrix Metalloproteinase-9 Gene Knock-Out on Morphological and Motor Outcomes after Traumatic Brain Injury
J. Neurosci.,
September 15, 2000;
20(18):
7037 - 7042.
[Abstract]
[Full Text]
[PDF]
|
|
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|
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J. Jaworski, I. W. Biedermann, J. Lapinska, A. Szklarczyk, I. Figiel, D. Konopka, D. Nowicka, R. K. Filipkowski, M. Hetman, A. Kowalczyk, et al.
Neuronal Excitation-driven and AP-1-dependent Activation of Tissue Inhibitor of Metalloproteinases-1 Gene Expression in Rodent Hippocampus
J. Biol. Chem.,
October 1, 1999;
274(40):
28106 - 28112.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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L. Y. S. Oh, P. H. Larsen, C. A. Krekoski, D. R. Edwards, F. Donovan, Z. Werb, and V. W. Yong
Matrix Metalloproteinase-9/Gelatinase B Is Required for Process Outgrowth by Oligodendrocytes
J. Neurosci.,
October 1, 1999;
19(19):
8464 - 8475.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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H. Nagase and J. F. Woessner Jr.
Matrix Metalloproteinases
J. Biol. Chem.,
July 30, 1999;
274(31):
21491 - 21494.
[Full Text]
[PDF]
|
|
|
|
|
|
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S. J. Price, D. R. Greaves, and H. Watkins
Identification of Novel, Functional Genetic Variants in the Human Matrix Metalloproteinase-2 Gene. ROLE OF Sp1 IN ALLELE-SPECIFIC TRANSCRIPTIONAL REGULATION
J. Biol. Chem.,
March 2, 2001;
276(10):
7549 - 7558.
[Abstract]
[Full Text]
[PDF]
|
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S. Kiryu-Seo, M. Sasaki, H. Yokohama, S. Nakagomi, T. Hirayama, S. Aoki, K. Wada, and H. Kiyama
Damage-induced neuronal endopeptidase (DINE) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers
PNAS,
April 11, 2000;
97(8):
4345 - 4350.
[Abstract]
[Full Text]
[PDF]
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Top
Abstract
Introduction
