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The Journal of Neuroscience, November 15, 1999, 19(22):10036-10043
Embryonic Neurons Adapt to the Inhibitory Proteoglycan Aggrecan
by Increasing Integrin Expression
Maureen L.
Condic1,
Diane M.
Snow2, and
Paul C.
Letourneau3
1 Department of Neurobiology and Anatomy, University of
Utah, School of Medicine, Salt Lake City, Utah 84132-0002, 2 Department of Anatomy and Neurobiology, The University of
Kentucky, Lexington, Kentucky 40536-0298, and 3 Department
of Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
The primary mediators of cell migration during development, wound
healing and metastasis, are receptors of the integrin family. In the
developing and regenerating nervous system, chondroitin sulfate
proteoglycans (CSPGs) inhibit the integrin-dependent migration of
neuronal growth cones. Here we report that embryonic sensory neurons
cultured on the growth-promoting molecule laminin in combination with
the inhibitory CSPG aggrecan rapidly adapt to inhibition. Adaptation is
associated with a two- to threefold increase in the levels of RNA and
surface protein for two laminin receptors, integrin  6 1 and
31, indicating that integrin expression is regulated by aggrecan.
Increased integrin expression is associated both with increases in
neuronal cell adhesion/outgrowth and with decreases in the ability of
aggrecan to inhibit cell adhesion. Directly increasing integrin
expression by adenoviral infection is sufficient to eliminate the
inhibitory effects of aggrecan, indicating that upregulation of
integrin receptors may promote neuronal regeneration in the presence of
inhibitory matrix components.
Key words:
integrin; CSPG; aggrecan; adaptation to inhibition; proteoglycan; regeneration; adenovirus-mediated gene transfer
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INTRODUCTION |
Many matrix components are
considered either growth promoting or inhibiting based on the ability
of purified protein to support or block neurite outgrowth in culture.
It is becoming increasingly evident, however, that the precise nature
of a neuron's response to individual matrix proteins depends on both
the context in which they are encountered and the past history of the
individual neuron (Snow and Letourneau, 1992 ; Condic and Letourneau,
1997). Recent work has shown that a number of molecules can have either
an attractive or a repulsive effect on neuronal outgrowth, depending on
the levels of cyclic nucleotides present in the growth cone (Song et
al., 1998). These results suggest that the history of a growth cone or
the precise balance of molecules that it encounters can greatly
influence the growth response.
Luo and Raper (1994) distinguish between two types of inhibitory
elements for growth cones: molecules that directly impair intrinsic
motility and molecules that interfere with substratum adhesion.
Chondroitin sulfate proteoglycans (CSPGs) are a major class of
inhibitory matrix proteins that have been proposed to act either as
adhesion-inhibiting molecules or through receptors to directly affect
cell motility (Luo and Raper, 1994; Faissner and Steindler, 1995; Hoke
and Silver, 1996). In the adult CNS, the upregulation of CSPGs
after injury is correlated with the failure of axons to extend despite
the continued presence of growth-promoting molecules such as laminin
(for review, see Hoke and Silver, 1996), suggesting that elevated
expression of CSPGs contributes to the inability of neurons to
regenerate after injury.
Aggrecan is one of the major CSPGs expressed in the CNS and PNS of
embryos and adults (Caterson et al., 1983; Oakley and Tosney, 1991;
Schwartz et al., 1996). Aggrecan is inhibitory to the extension of
retinal and sensory neurons in culture (Snow and Letourneau, 1992;
Challacombe et al., 1997). Embryonic neurons can adapt to the
inhibitory effects of aggrecan over time when aggrecan is presented
with growth-promoting molecules such as laminin (Snow et al., 1990a;
Snow and Letourneau, 1992). Adaptation could be accomplished either
through the upregulation of receptors for growth-promoting ligands such
as laminin or through the downregulation of neuronal receptors for
aggrecan. Although the effects of aggrecan are likely to be mediated in
part through a receptor-based mechanism (Snow et al., 1994; Balsamo et
al., 1995; Ernst et al., 1995), aggrecan receptors have not yet been
identified. Neuronal outgrowth is mediated predominantly by receptors
of the integrin family (Letourneau et al., 1994), which bind to a wide
range of ECM and cell-surface ligands. In sensory neurons, neurite
outgrowth on ECM proteins such as laminin is entirely dependent on
integrin function (Tomaselli et al., 1993), suggesting that increased
integrin expression in response to a combination of aggrecan and
laminin may allow neurons to adapt to inhibition and extend on laminin. In this work, we have examined the expression of integrins in response
to aggrecan to determine the role of integrin regulation in neuronal
adaptation to inhibitory proteoglycans.
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MATERIALS AND METHODS |
Cell culture and substratum preparation. dorsal root
ganglia were dissected from embryonic day 12 chicks and dissociated
with brief trypsin treatment (Gomez and Letourneau, 1994). Dissociated cells were preplated on tissue culture plastic in Ham's F12 media containing 10% calf serum for 3 hr at 37°C to remove more adherent non-neuronal cells (Barres et al., 1988). After 3 hr, >95% of cells
remaining in suspension are neuronal. Neurons in suspension were
harvested, rinsed in PBS, and cultured overnight on glass coverslips in
serum-free media as described (Gomez and Letourneau, 1994). NGF was
present in all cultures at 10 ng/ml, concentrations that are saturating
for the TrkA receptor. For overnight cultures, glass coverslips were
coated for 1 hr at room temperature with Engelbreth-Holm-Swarm tumor
laminin (gift of Dr. S. Palm, University of Minnesota) at 20 µg/ml
(LM) or 1 µg/ml (low LM), or Fibronectin (Life Technologies,
Gaithersburg, MD) at 20 µg/ml in PBS. Aggrecan-containing substrata
for culture were prepared by coating coverslips with chick embryonic
limb-bud aggrecan (gift of Dr. David Corrino, Case Western Reserve
University) at 50 µg/ml in PBS for 1 hr, followed by laminin at 20 µg/ml for 1 hr (PG/LM). These treatments resulted in the following
bound concentrations of protein: (LM) 300 ng/cm2 laminin; (low LM) 30 ng/cm2 laminin; (PG/LM) 300 ng/cm2 laminin, 180 ng/cm2 aggrecan. These concentrations are
likely to be above those predicted to form a molecular monolayer.
Binding of laminin and aggrecan to coverslips and 96-well plates
(below) was determined by inclusion of
3H-labeled laminin and
35S-labeled aggrecan at known specific
activities in the coating medium, followed by extraction of the bound
protein with 10% SDS and scintillation counting. Metabolically labeled
chick embryonic aggrecan was obtained from Dr. David Corrino. Laminin
was labeled with 3H as previously
described (Snow and Letourneau, 1992).
RNA and protein analysis. RNA was isolated from
~107 neurons by lysing the cells
in situ in a phenol/SDS buffer (TriReagent, Molecular
Research Center). RNA concentration was determined by UV absorption at
260 nm; 10 µg samples were analyzed on agarose gels and transferred
to nylon membrane by pressure blot (Stratagene, La Jolla, CA) using
standard procedures. Integrin 6, 3, and GAPDH transcripts were
detected with 32P-labeled oligonucleotide
probes. Films and blots were quantitated using a Bio-Rad GS-700 imaging
densitometer, a GS-363 phosphoimager, and NIH image 1.55 analysis software.
Cell surface receptor was labeled with biotin and immunoprecipitated
using published methods and antibodies specific for 6- and
3-integrin (de Curtis et al., 1991). Immunoprecipitated proteins were size-fractionated under reducing conditions on acrylamide gels and
electrophoretically transferred to nitrocellulose membrane using
standard protocols. Biotin-labeled proteins were detected using
streptavidin conjugated to HRP and a chemiluminescent reagent (Pierce,
Rockford, IL) followed by exposure to film. Ratios of protein
determined from quantitation of film were comparable to those obtained
by direct phosphoimaging of chemiluminescently detected proteins (data
not shown).
Cell adhesion assays. Cells were cultured overnight on
either laminin, low laminin, or laminin in combination with low levels of aggrecan (see Cell culture and substratum preparation). Culture on
both PG/LM (see Fig. 3) and low LM (Condic and Letourneau, 1997)
increase integrin expression, apparently by different molecular mechanisms. After culture, neurons were removed from the substratum by
a brief treatment with calcium-free media and gentle scraping in media
containing 10% serum at 4°C to prevent the internalization of
cell-surface integrins. Cells were spun down at 4°C, resuspended, and
maintained at 4°C until plating. To measure cell adhesion, 96-well
plastic plates (Dynatech Immunolon) were coated with laminin (40 µg/ml in PBS for 1 hr), low laminin (2 µg/ml laminin in PBS for 1 hr), or aggrecan and laminin (aggrecan at 50 µg/ml in PBS for 1 hr,
followed by laminin at 40 µg/ml in PBS for 1 hr). On the basis of
binding of isotopically labeled proteins, these concentrations of
applied protein resulted in surface densities of bound protein similar
to those used for culturing (data not shown). The plates were rinsed,
blocked with PBS containing 5 mg/ml BSA and 0.2% sodium azide for 1 hr, and rinsed extensively. Cells were applied at 10,000 cells per well
and allowed to adhere for 3 hr at 37°C. Wells were rinsed once with
100 µl PBS and then incubated for 10 min in PBS containing 2 µg/ml
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (Molecular Probes, Eugene, OR) followed by detection of fluorescein emission using a microplate fluorescence reader (Cytofluor II; Biosearch/Millipore).
To determine the effect on neuronal adhesion of applying proteins to
the substrata in different order (see Fig. 2), aggrecan was bound to
96-well plates before, after, or in combination with laminin. Aggrecan
and laminin binding to the substrata were determined for all applied
concentrations by inclusion of isotopically labeled protein (see Cell
culture and substratum preparation). All three treatments resulted in
identical concentrations of bound laminin (300 ng/cm2) for all concentrations of bound
aggrecan. Plates were prepared as follows. BEFORE: aggrecan was applied
first at concentrations of 1, 2.5, 5, and 50 µg/ml, resulting in
bound concentrations of aggrecan equal to 20, 60, 100, and 180 ng/cm2 followed by rinsing and application
of laminin at 40 µg/ml. AFTER: laminin was applied first at a
concentration of 40 µg/ml, followed by rinsing and application of
aggrecan at a concentration of 10, 25, 100, and 500 µg/ml, resulting
in bound concentrations of aggrecan equal to those achieved by
application of aggrecan before laminin (i.e., 20, 60, 100, and 180 ng/cm2). MIXED: laminin at 40 µg/ml was
mixed with aggrecan at 5, 15, and 25 µg/ml for 30 min before
application to the plates, resulting in bound concentrations of
aggrecan equal to 20, 60, and 100 ng/cm2.
Note that only three concentrations were tested because aggrecan concentrations higher than 25 µg/ml inhibited binding of laminin (data not shown).
Cell outgrowth assays. To determine neurite initiation (see
Fig. 1), cells were cultured on either LM or PG/LM substrata (see Cell
culture and substratum preparation) for 3 or 20 hr, and 10 random
fields were recorded as video images. The total number of cell bodies
and neurites greater than one cell diameter in length were counted for
each field and expressed as the average number of neurites per cell. To
determine the rate of neurite extension, cells were cultured as above
on either LM or PG/LM substrata for 3 or 20 hr and moved to a heated
microscope stage. Dishes were allowed to equilibrate for 30 min, then
images were captured for 1 hr, one image every 5 min. Images were
captured every 5 min to confirm that the path of the neuron was linear over the culture period, so the average rate of growth cone
advance could be determined from the linear distance traversed. The
rate of axon extension was determined from the distance traversed by the growth cone in 1 hr as measured between the positions of the leading edge of the growth cone at the beginning and end of the period
of image acquisition using Image-1 software. To measure outgrowth of
neurons with different levels of integrin expression (see Fig. 4),
neurons were cultured on LM, PG/LM, or low LM substrata overnight, as
described above (Cell adhesion assays), and were subsequently replated
on laminin, low laminin, or PG/LM containing substrata and allowed to
extend axons for 3 hr. Cultures were fixed with 4% paraformaldehyde,
and the percentage of cells with neurites one cell diameter or greater
in length was determined.
Adenoviral infection. Replication-deficient adenoviral
constructs were obtained from the laboratory of Dr. Clayton Buck
(Wistar Institute). The integrin-expressing adenoviral constructs were prepared using standard methods (Graham and Prevec, 1995). Briefly, full-length mouse 1-integrin or -galactosidase (as a control) cDNA were cloned into the pAd.CMV-link plasmid under the control of the
cytomegalovirus immediate early enhancer/promoter element. This
promoter has been shown to yield efficient expression of transgenes in
chick embryonic neurons (Yamagata et al., 1994) and in the nervous
systems of other species. NIH 293 cells were cotransfected with
linearized pAd.CMV-link plasmid (with insert) and the
replication-deficient sub 360 or dl70001 adenoviral backbone. Recombinant virus was collected from plaques, and the inserts were
confirmed by PCR. The virus was subjected to three rounds of plaque
purification to ensure that a single recombinant was selected and
further purified by centrifugation on a cesium gradient. The titer of
the purified recombinant virus was determined with plaque assay.
Embryonic chick neurons were cultured on laminin (300 ng/cm2) substrata and infected overnight
at a viral concentration of 8 × 108 pfu/ml. After 16 hr, the
virus was removed, and neurons were cultured for an additional 48 hr to
insure strong expression of the transgene. Cell surface expression of
the integrin transgene was confirmed by staining cells live with
antibodies that specifically recognize mouse 1-integrin (Ha31/8;
PharMingen, San Diego, CA). All cells in the mouse
1-integrin-infected culture expressed the transgenic protein at the
cell surface. Endogenous 1 is expressed at low levels in both
control and 1-integrin-infected cultures (see Fig. 5B).
-galactosidase expression was confirmed by antibody staining (5 Prime to 3 Prime Biologicals) of fixed and Triton X-100-extracted
cells. Infected neurons were removed from the dishes as described above
(Cell adhesion assays), replated on substrata containing laminin or
PG/LM, and assayed for outgrowth in 3 hr as above (Cell outgrowth
assays). Levels of total 1-integrin expressed at the cell surface
were determined by immunoprecipitation of cell-surface biotinylated
1-protein using an antibody that recognizes both the exogenous
(mouse) and the endogenous (chick) 1 subunits (Chemicon, Temecula,
CA), followed by Western analysis as described (RNA and protein analysis).
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RESULTS |
Neuronal adaptation to inhibition by aggrecan
Embryonic neurons purified from dissected dorsal root ganglia
(Barres et al., 1988) were cultured on substrata containing both the
growth-promoting ECM molecule laminin and low levels of the inhibitory
proteoglycan aggrecan. Neurons were able to adhere to the substratum
and extend axons after 20 hr in culture (Fig.
1A). However, the
outgrowth of neurons cultured in the presence of aggrecan (right
panel) was less profuse than that of cells cultured on
laminin alone (left panel), suggesting that aggrecan affects either the initiation of neurites or the rate of neurite extension once initiated. Observation of cultures at 3 and 20 hr
indicated that the initiation of axons on aggrecan-containing substrata
was delayed relative to controls (p < 0.002, t test) (Fig. 1B). Once initiated, axon
outgrowth was significantly slower on substrata containing aggrecan for
the first several hours of culture (p < 0.001, t test) (Fig. 1C). By 20 hr in culture, however, neurons grown on substrata containing aggrecan had significantly increased their rate of growth such that the rates of axon extension in
the presence or absence of low levels of aggrecan were identical (Fig.
1C). These data indicate that the different substrata are not selecting different neuronal subpopulations with differing abilities to adhere to and extend processes on aggrecan. If neurons that are constitutively able to adhere and extend processes in the
presence of aggrecan were being preferentially selected by aggrecan-containing substrata, then there would be no difference between the behavior of cells measured at 3 versus 20 hr in culture. Rather, the improved performance of neurons on aggrecan-containing substrata over time indicates that neurons are able to adapt to the
inhibitory effects of aggrecan and extend axons under conditions that
initially suppress neurite outgrowth. This adaptation could be mediated
either through the upregulation of receptors for growth-promoting molecules that are also present in the substrata or through the downregulation of neuronal receptors for aggrecan in response to low
levels of the inhibitor.
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Figure 1.
Neurons cultured on substrata containing a low
concentration of the inhibitory proteoglycan aggrecan adapt to
inhibition and extend neurites. A, Neurons were cultured
on substrata containing either laminin alone (left) or
laminin in combination with aggrecan (right). After
16-20 hr in culture, neurons have extended axons on both substrata.
Outgrowth was less profuse and axons were more fasciculated on
substrata containing aggrecan relative to outgrowth on laminin alone.
B, The initiation of neurites is delayed on substrata
containing aggrecan. At 3 hr, significantly fewer cells
(p < 0.002, t test) have
initiated neurites on substrata containing aggrecan relative to cells
on laminin alone. By 6 hr in culture, the number of neurites initiated
on aggrecan-containing substrata has greatly increased, suggesting that
the neurons have adapted to inhibition. Means + SEMs of three
experiments, each measuring at least 174 neurons/condition, are given.
C, The rate of axon extension on laminin at 3 hr in
culture is reduced by the presence of aggrecan
(p < 0.001, t test), but by
20 hr in culture the rate of outgrowth on the two substrata is
identical. Means + SEMs of three experiments, each measuring at least
14 neurons/condition, are given.
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The inhibitory effects of aggrecan on neurite initiation and the rate
of neurite outgrowth were confirmed by the observation that aggrecan
inhibits neuronal cell adhesion to laminin in a dose-dependent manner
(Fig. 2). In the presence of constant
amounts of bound laminin, increasing concentrations of aggrecan
significantly reduced the attachment of neuronal cells. It is not
currently known how aggrecan inhibits neuronal attachment and outgrowth when co-presented with ECM molecules that promote both neuronal adhesion and outgrowth, such as laminin. If aggrecan blocks access of
neurons to laminin, the inhibitory effect of aggrecan on outgrowth should not be strongly influenced by the order of application of the
two proteins to the substratum, as long as comparable amounts of both
molecules are bound in all conditions. However, we observed that the
inhibitory potency of aggrecan was significantly affected by the order
of application of aggrecan and laminin to the substrata (Fig. 2).
Aggrecan adsorbed to the substrata in the presence of soluble laminin
was significantly more inhibitory than comparable amounts of
proteoglycan bound either before or after the application of laminin.
For the highest concentrations of proteoglycan tested, aggrecan bound
after laminin (the condition in which simple masking of laminin should
be, if anything, most pronounced) was less inhibitory than aggrecan
bound before laminin.
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Figure 2.
The adhesion of neurons to substrata containing
constant amounts of bound laminin (300 ng
laminin/cm2) and increasing amounts of bound
aggrecan is affected by the order of application of the two proteins to
the substrata. Aggrecan mixed with laminin in solution (shaded
triangles) is more inhibitory than aggrecan applied before or
after adsorption of laminin to the substrata (Mixed
points are statistically different from either Before or
After at p < 0.05, t
test), suggesting that conformational changes in laminin may underlie
some of the inhibitory effect of aggrecan. At the highest
concentrations tested, aggrecan is more inhibitory when applied before
laminin, supporting the idea that interactions of soluble laminin with
aggrecan result in greater inhibition. Adhesion is given as a
percentage of adhesion to control (laminin only) substrata. Means + SEMs from three experiments are given. When error bars do not appear,
they are smaller than the size of the symbol.
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Changes in integrin expression are associated with adaptation
The molecular basis for neuronal adaptation to aggrecan was
examined by determining the levels of neuronal receptors for laminin in
cells that had been cultured either on laminin alone (300 ng laminin/cm2) or on laminin in combination
with low levels of aggrecan (300 ng
laminin/cm2, 180 ng
aggrecan/cm2). Integrin 61 and
31 are the major laminin receptors in chick ciliary neurons
(Weaver et al., 1995) and contribute significantly to the attachment
(Condic and Letourneau, 1997) and outgrowth (Tomaselli et al., 1993) of
dorsal root ganglion neurons cultured on laminin. Antibodies that block
the function of 1-containing integrins completely block the
attachment and outgrowth of chick sensory neurons on laminin (cf.
Tomaselli et al., 1993) and on laminin in combination with aggrecan
(data not shown), indicating that neuronal outgrowth on laminin, in
either the presence or absence of aggrecan, is integrin dependent.
Cell-surface expression of both integrin 61 and 31 was
significantly increased in neurons encountering laminin in the presence
of aggrecan (Fig. 3B, Table
1). This increase in integrin cell-surface expression was associated with an increase in integrin mRNA (Fig. 3A) and total integrin protein (data not shown),
all of which increase proportionately in neurons cultured on substrata containing laminin and aggrecan. These results suggest that
aggrecan-associated increases in integrin expression are attributable
to either transcriptional upregulation or an increase in the stability
of the integrin message. Both of these mechanisms are distinct from the
posttranslational regulation of integrin expression observed in neurons
experiencing low availability of laminin (Condic and Letourneau, 1997),
where neurons with increased surface expression of integrins show a concomitant decrease in the levels of integrin mRNA and total protein.
The observation that integrin mRNA and protein levels both increase in
the presence of aggrecan indicates that the response of neurons to
aggrecan cannot be explained simply by aggrecan somehow "masking"
laminin to generate a substrate that mimics the effects of low
laminin.
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Figure 3.
Increased expression of integrin 3 and 6 RNA
and cell surface receptor protein in response to aggrecan.
Quantifications of the results are given in Table 1. A,
Northern analysis of integrin total RNA. The expression of RNA for 3
and 6 integrin is increased in neurons cultured on substrata
containing both laminin and aggrecan (LM/PG) relative to
those cultured on laminin alone (LM). The blots
were stripped and reprobed for GAPDH as a loading control.
B, Western analysis of biotinylated cell-surface
integrin protein. Neurons cultured on substrata containing laminin and
aggrecan (LM/PG) show an increase in the expression of
laminin receptors integrin 3 and 6 at the cell surface relative
to those cultured on laminin alone (LM), whereas
the expression of these receptors does not increase in neurons cultured
on substrata containing fibronectin and aggrecan (FN/PG)
when compared with those cultured on fibronectin alone
(FN).
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Increased expression of integrin 3 and 6 in neurons encountering
combined aggrecan and laminin suggests that upregulation of integrin
expression by inhibitory CSPGs may underlie the adaptation of
neurons to aggrecan. Interestingly, for cells cultured on substrata containing fibronectin and aggrecan (Fig. 3B,
FN/PG, Table 1), integrin expression was not altered either
for 61 (a laminin receptor) or for 31 (a receptor for both
laminin and fibronectin), suggesting that the interactions of aggrecan
with laminin and fibronectin are distinct. This conclusion is supported
by the fact that neurons cultured on substrata containing low levels of
aggrecan and laminin adapt to inhibition (Fig. 1), whereas aggrecan
continues to inhibit the outgrowth of neurons on fibronectin after 10 hr or more in culture (Snow et al., 1996).
Neurons with high levels of integrin expression are less inhibited
by aggrecan
If alterations in integrin expression play a role in the
adaptation of neurons to aggrecan, then increased integrin expression must be associated with changes in neuronal behaviors that are important to neurite outgrowth. Both neuronal adhesion and neurite outgrowth on laminin and on laminin/aggrecan substrata were examined in
cells that had been previously cultured on different substrata to
manipulate their levels of integrin expression. To manipulate surface
integrin levels, cells were cultured overnight on laminin, low laminin,
or laminin in combination with low levels of aggrecan (see Cell culture
and substratum preparation). Cultures on both PG/LM (Fig. 3) and low LM
(Condic and Letourneau, 1997) increase integrin expression, apparently
by different molecular mechanisms. Cells expressing high levels of
6- and 3-integrin at the cell surface (i.e., cells cultured
either on low laminin or on laminin in combination with aggrecan) were
significantly more adherent both to laminin and to
laminin/aggrecan-containing substrata (Fig. 4A). Aggrecan was also
less inhibitory to neuronal attachment for cells expressing high levels
of integrin 3 and 6 at the cell surface. For control cells (with
low expression of integrin 3 and 6 at the cell surface (Fig.
4A, black bars), aggrecan in combination
with laminin reduced neuronal attachment to 28% of the levels seen on
laminin alone. In contrast, for neurons with high levels of integrin
expression (white and gray bars), attachment to
aggrecan-containing substrata was considerably closer to that seen on
laminin alone; either 61% (white bars) or 42% (gray bars) of control adhesion. Similarly, neurons
expressing high levels of integrin 3 and 6 at the cell surface
extended neurites more readily on inhibitory (PG/LM-containing)
substrata (Fig. 4B). These data indicate that
increased cell surface integrin expression allows neurons to overcome
inhibition by aggrecan and both attach and extend neurites, despite the
presence of inhibitory matrix components in the extracellular
environment.
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Figure 4.
Increased expression of laminin receptors at the
cell surface is associated with increased neuronal adhesion and
outgrowth. Means + SEMs from three experiments are given.
A, Neurons expressing high levels of integrin 3 and
6 at the cell surface (Low LM Cultured and
PG/LM Cultured) show increased adhesion to laminin and
to PG/LM substrata. *Different from LM cultured at
p < 0.1 (t test). Values for all
other low laminin-cultured and PG/LM-cultured conditions are
significantly different from LM cultured at p < 0.05 or better. B, Neurons expressing high levels of
integrin 3 and 6 at the cell surface extend neurites more readily
on laminin both in the presence and absence of the growth inhibitor
aggrecan. *Different from LM cultured at p < 0.1. **Significantly different from LM cultured at p < 0.005. Where error bars do not appear, they are <1%.
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Overexpression of integrin is sufficient for neuronal adaptation
to aggrecan
To determine the role of increased integrin expression in neuronal
adaptation to aggrecan, we tested the outgrowth of neurons overexpressing a single laminin receptor (integrin 11) in
short-term assays (i.e., time points at which upregulation of
endogenous integrins has not yet occurred). Neurons were cultured on
laminin substrata (300 ng/cm2). On this
substrata, endogenous laminin receptors are expressed at low levels
(Fig. 3, 31, 61; Fig.
5B, 11). Neurons were infected with adenoviral constructs expressing a full-length mouse 1-integrin subunit. In sensory neurons, 11-integrin functions as a laminin and collagen receptor and is thought to be the receptor primarily responsible for adhesion of dorsal root ganglion neurons to
laminin (Tomaselli et al., 1993). Consequently, altering the expression
of this receptor would be predicted to have the greatest effect on
neuronal outgrowth on laminin. In most cells, the 1 subunit is
synthesized in considerable excess (De Strooper et al., 1991; Muller et
al., 1993; Koivisto et al., 1994), and exogenously supplied
-subunits are efficiently expressed as -1 heterodimers at the
cell surface (Hayashi et al., 1991; Felsenfeld et al., 1996; Miyakawa
et al., 1996). Infection with mouse integrin 1-expressing adenovirus
resulted in expression of mouse 1-containing integrins at the
surface of all sensory neurons in culture (Fig. 5A). The levels and time course of infection were manipulated to yield an
approximately twofold increase (2.25 + 0.2) in expression of 1-containing integrins at the cell surface (Fig. 5B).
This increase was similar in magnitude to that which occurs naturally
in neurons that have adapted to aggrecan (Fig. 3B to Fig.
5B). Similar to the "adapted" neurons with high levels
of integrin expression shown in Figure 4, neurons directly manipulated
to express high levels of 1-integrin also showed marked improvement
in outgrowth on substrata containing aggrecan (Fig. 5C). At
3 hr in culture, a time point at which upregulation of endogenous
integrins has not yet occurred and control neurons are strongly
inhibited, neurons expressing high levels of 1-integrin exhibited
outgrowth in the presence of aggrecan that was equivalent to that seen
on laminin alone. These results demonstrate a causal link between
integrin modulation and neuronal adaptation, indicating that increased integrin expression is sufficient to promote the outgrowth of neurons
in the presence of aggrecan.
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Figure 5.
Increased expression of 1-integrin is
sufficient to mediate adaptation of neurons to aggrecan.
A, Neurons infected with adenoviral constructs
expressing a control protein (-Gal,
right) or mouse 1-integrin (left) were stained live
with an antibody that specifically recognizes the mouse 1 subunit.
Arrows indicate the positions of neuronal cells.
B, Overexpression of mouse 1-integrin in chick
neurons results in an approximately twofold increase in total
1-integrin expressed at the cell surface (2.25 + 0.2). Infected
neurons were cultured on LM substrata, where endogenous integrin
expression is low (see Materials and Methods). Cell
surface-biotinylated proteins from mouse 1-integrin-infected
(lane 1) or control, -galactosidase-infected
(lane 2) neurons were immunoprecipitated with an
antisera that recognizes both mouse and chick 1-integrin subunits.
Total 1-integrin expressed at the surface is increased after
infection with 1-expressing adenovirus, reflecting the contribution
of the exogenous mouse protein to total 1 expression. Expression of
endogenous 1 (control cells, lane 2) is quite low
under these conditions. C, Control or
1-integrin-infected neurons were cultured on substrata containing
laminin alone or laminin in combination with aggrecan as in Figure 4.
Control neurons expressing -galactosidase (black
bars) are strongly inhibited in the presence of aggrecan after
3 hr in culture (Figs. 1, 4). At this time in culture, endogenous
increases in integrin expression have not yet occurred. Neurons with
increased laminin-receptor expression (1-expressing; white
bars) are not inhibited by aggrecan, showing outgrowth
equivalent to that seen on laminin alone. Means + SEMs from at least
three experiments are given. *Significantly different from all other
conditions at p < 0.0001 (t
test).
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|
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DISCUSSION |
We have shown that integrin expression at the surface of sensory
neurons increases in response to low levels of the inhibitory proteoglycan aggrecan, an ECM molecule that is not itself a ligand for
integrins. The aggrecan-associated increase in integrin expression occurs through a mechanism that is distinct from that seen in response
to low availability of laminin (Fig. 3) (cf. Condic and Letourneau,
1997), indicating that the effects of aggrecan are unlikely to be
caused by a simple "masking" of laminin by proteoglycan. Increased
integrin expression is correlated with increased neuronal adhesion and
neurite outgrowth in the continued presence of the inhibitor.
Manipulation of integrin expression directly with adenoviral constructs
indicates that high levels of integrin expression are sufficient to
mediate adaptation of neurons to aggrecan. The ability of embryonic
neurons to adapt to aggrecan suggests that modulation of integrin
expression contributes to the regenerative capability of embryonic
neurons in the presence of inhibitory proteoglycans after injury.
Changes in neuronal responsiveness to laminin as a consequence of
exposure to an inhibitory ECM proteoglycan demonstrates that the
"cellular history" of a neuron can be a significant factor in
neuronal interactions with both positive and negative guidance molecules. The regulation of integrins by aggrecan is significant because it demonstrates that proteoglycans can alter expression of
receptors that mediate neuronal interactions with other,
non-proteoglycan components of the ECM and because it suggests a
possible mechanism for promoting the outgrowth of adult neurons in the
presence of inhibitory proteoglycans that are expressed after injury to
the nervous system.
There have been conflicting reports regarding the role that CSPGs play
in regulating neurite outgrowth. CSPG immunostaining in vivo
often localizes to regions of embryos that exclude axons (Snow et al.,
1990b; Oakley and Tosney, 1991; Snow et al., 1991; Brittis et al.,
1992; Landolt et al., 1995), and CSPGs in vitro can inhibit
neurite outgrowth (Snow et al., 1990a; Fichard et al., 1991; Snow and
Letourneau, 1992; Braunewell et al., 1995; Maeda and Noda, 1996;
Challacombe et al., 1997). In contrast, CSPG immunostaining in
vivo is sometimes correlated with tissues that support the growth
of axons (for review, see Pearlman and Sheppard, 1996), and CSPGs in
combination with other matrix proteins can constitute a permissive
substratum for axon outgrowth under some conditions in vitro
(Streit et al., 1993; Faissner et al., 1994) (also see Fig.
1A). One possible explanation for these conflicting results is that different studies have measured the response of neurons
to different species of CSPG. Alternatively, it has been suggested that
the growth-inhibiting or growth-promoting properties of CS-containing
ECM preparations do not reflect CSPG function but are attributable to
the presence of differing CS-binding proteins that in turn mediate the
observed effects on neurite outgrowth (Emerling and Lander, 1996). This
idea is supported by the fact that proteins known to be inhibitory to
neurite outgrowth, including tenascins and thrombospondins, also bind
to CS (Winnemoller et al., 1992; Barnea et al., 1994; Grumet et al.,
1994). A third possibility is that CSPGs do affect neurite adhesion and
outgrowth, but this effect is modulated by the precise combinations or
concentrations of molecules present in the ECM (Snow and Letourneau,
1992). For example, the CSPGs versican and decorin inhibit adhesion of
sensory neurons to fibronectin but not to laminin or collagen
(Braunewell et al., 1995), and both chondroitin sulfate (Dou and
Levine, 1995) and the cell surface proteoglycan NG2 (Dou and Levine,
1994) inhibit sensory neuronal outgrowth on laminin but not on the
growth-promoting cell-surface molecule L1. A final possibility
presented by the current observations is that because inhibitory CSPGs
alter the neuronal response to growth-promoting matrix components over
time, an individual neuron's response to CSPG can change depending on the length of time it has been in contact with CSPG-containing matrix
and the availability of alternative (and perhaps more permissive) substrata for neurite outgrowth.
The adaptive response of neurons to inhibitory matrix components
demonstrated here somewhat complicates the definition of "inhibitory." Clearly, aggrecan can arrest the outgrowth of neurons in acute assays when neurons encounter aggrecan in a discrete location
and have the option of stopping and/or turning to avoid aggrecan-containing regions (Snow et al., 1990a, 1991; Snow and Letourneau, 1992; Challacombe et al., 1997). Our results demonstrate that under conditions in which neurons do not have the option of
extending onto more favorable substrata, they can adjust their surface
expression of integrins to compensate for their environment and extend
neurites, in agreement with previous reports of limited neurite
outgrowth on substrata containing either high amounts of CSPG (Snow et
al., 1990a) or increasing steps of CSPG (Snow and Letourneau, 1992). In
short-term assays, aggrecan inhibits neuronal attachment (Figs. 2,
4A), neurite initiation (Fig. 1B), and rate of neurite outgrowth (Fig. 1C). If only 20 hr
cultures had been examined in isolation, none of these effects would
have been evident because of the adaptive response of the neurons, and
quite different conclusions might have been drawn regarding the
properties of aggrecan in this system. The adaptive ability of neurons
introduces the concept of cellular "history" into our thinking
about how a neuron responds to inhibitory components of the ECM. The
past experience of a growth cone may significantly alter the strength
and even the nature of a neuron's response to what is currently
present in the extracellular environment.
Sensory neurons do not compensate for the inhibitory effects of
aggrecan under all circumstances. When cells are cultured on substrata
containing both fibronectin and aggrecan, the rate of neurite extension
remains quite low relative to cells grown on fibronectin alone after 10 hr or more in culture (Snow et al., 1996). The inability of sensory
neurons to adapt to aggrecan when cultured on fibronectin is consistent
with our observation that expression of the fibronectin receptor
31 is not increased in cells cultured on
aggrecan-fibronectin-containing substrata (Fig. 3, Table 1).
Fibronectin is known to interact with CSPGs including aggrecan. The
CSPG-binding domains of fibronectin are localized to the IIIcs (or
second cell-binding) region of the molecule and to the adjoining type
III repeats (no. 12-14) (Barkalow and Schwarzbauer, 1994; Iida et al.,
1995). The fibronectin IIIcs domain also contains the binding site for
integrin 41 (Iida et al., 1995). Integrins 31 and 51
bind to a different region of the fibronectin molecule, the first
cell-binding (or RGD-containing) domain. It is possible that binding of
31 to fibronectin is not affected by the interactions of
fibronectin with aggrecan at a remote site in the molecule and that the
inhibition of neurite outgrowth observed on substrata containing both
fibronectin and aggrecan is largely caused by alterations in integrin
41 activity that are not compensated for by either integrin
31 or 51.
Inhibitory proteoglycans, including aggrecan, are prominent components
of the neural extracellular matrix during development, and their
expression generally declines postnatally to relatively low levels in
the adult (Meyer-Puttlitz et al., 1995; Lebaron, 1996; Levine and
Nishiyama, 1996; Margolis et al., 1996; Schwartz et al., 1996). After
injury to the CNS, there is a pronounced increase in the expression of
both CSPGs and growth-promoting matrix molecules in regions of damage
(Hoke and Silver, 1996). In "atraumatic" injury models that prevent
the increased expression of proteoglycans normally associated with CNS
damage, adult neurons extend considerable distances through the white
matter of the brain (Davies et al., 1997), suggesting that inhibitory
proteoglycans play a pivotal role in suppressing regeneration. In the
spinal cord, many axons near the site of injury do not retract but
rather exhibit significant sprouting in the initial weeks after injury. These early sprouts, however, do not continue to extend, and their arrested progress is coincident with the time course for increased CSPG
accumulation in and around the site of injury (Li and Raisman, 1994,
1995). In an in vitro model system, McKeon et al. (1995) have shown that CSPG expression associated with cortical injury inhibits axon outgrowth. Treatment with chondroitinase to remove GAG
chains from CSPG molecules results in a significant increase in neurite
extension over scar tissue derived from CNS injury. Interestingly,
co-treatment with both chondroitinase and function-blocking anti-laminin antibodies reverses the growth-promoting effect of chondroitinase alone, suggesting that interactions of neurons with
laminin can support regeneration once the inhibitory activity of CSPGs
has been reduced (McKeon et al., 1995). Consistent with these results
are the findings that inhibitory factors in CNS myelin can be overcome
by the addition of laminin (David et al., 1995).
The results presented here indicate that aggrecan is inhibitory to the
attachment and outgrowth of neurons in culture. Embryonic neurons are
able to compensate for the inhibitory action of aggrecan over time by
increasing their expression of integrins and their responsiveness to
laminin. Increased integrin expression is sufficient to mediate
adaptation of neurons to aggrecan. These results suggest that
manipulation of integrin expression may provide a mechanism for
increasing the regenerative potential of adult neurons after CNS injury.
| |
FOOTNOTES |
Received July 8, 1999; revised Aug. 31, 1999; accepted Sept. 3, 1999.
This work was supported by a grant from the Spinal Cord Research
Foundation and National Institutes of Health (NIH) Grant NS38138-01 to
M.L.C., NIH Grant EY10545 and a grant from the American Paralysis
Association to D.M.S., and NIH Grant HD19950 and a grant from the
Minnesota Medical Foundation to P.C.L. We thank Drs. H. J. Yost,
S. B. Kater, T. Diffenbach, and G. Gallo for critical reading of
this manuscript; A. Cooke, P. Atkinson, K. Roche, and Dr. J. Gurwell
for assistance with experiments; Dr. L. F. Reichardt for integrin
antibodies; and Dr. C. Buck for adenoviral constructs.
Correspondence should be addressed to Dr. M. L. Condic, Department
of Neurobiology and Anatomy, University of Utah, School of Medicine, 50 N. Medical Drive, Salt Lake City, UT 84132-0002. E-mail:
mcondic{at}alta.med.utah.edu.
| |
REFERENCES |
-
Balsamo J,
Ernst H,
Zanin MK,
Hoffman S,
Lilien J
(1995)
The interaction of the retina cell surface N-acetylgalactosaminylphosphotransferase with an endogenous proteoglycan ligand results in inhibition of cadherin-mediated adhesion.
J Cell Biol
129:1391-1401[Abstract/Free Full Text].
-
Barkalow FJ,
Schwarzbauer JE
(1994)
Interactions between fibronectin and chondroitin sulfate are modulated by molecular context.
J Biol Chem
269:3957-3962[Abstract/Free Full Text].
-
Barnea G,
Grumet M,
Milev P,
Silvennoinen O,
Levy JB,
Sap J,
Schlessinger J
(1994)
Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin.
J Biol Chem
269:14349-14352[Abstract/Free Full Text].
-
Barres BA,
Silverstein BE,
Corey DP,
Chun LL
(1988)
Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.
Neuron
1:791-803[ISI][Medline].
-
Braunewell KH,
Pesheva P,
McCarthy JB,
Furcht LT,
Schmitz B,
Schachner M
(1995)
Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth.
Eur J Neurosci
7:805-814[ISI][Medline].
-
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].
-
Caterson B,
Christner JE,
Baker JR
(1983)
Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate.
J Biol Chem
258:8848-8854[Abstract/Free Full Text].
-
Challacombe JF,
Snow DM,
Letourneau PC
(1997)
Dynamic microtubule ends are required for growth cone turning to avoid an inhibitory guidance cue.
J Neurosci
17:3085-3095[Abstract/Free Full Text].
-
Condic ML,
Letourneau PC
(1997)
Ligand induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth.
Nature
389:852-856[Medline].
-
David S,
Braun PE,
Jackson DL,
Kottis V,
McKerracher L
(1995)
Laminin overrides the inhibitory effects of peripheral nervous system and central nervous system myelin-derived inhibitors of neurite growth.
J Neurosci Res
42:594-602[ISI][Medline].
-
Davies SJ,
Fitch MT,
Memberg SP,
Hall AK,
Raisman G,
Silver J
(1997)
Regeneration of adult axons in white matter tracts of the central nervous system.
Nature
390:680-683[Medline].
-
de Curtis I,
Quaranta V,
Tamura RN,
Reichardt LF
(1991)
Laminin receptors in the retina: sequence analysis of the chick integrin alpha 6 subunit. Evidence for transcriptional and posttranslational regulation.
J Cell Biol
113:405-416[Abstract/Free Full Text].
-
De Strooper B,
Van Leuven F,
Carmeliet G,
Van Den Berghe H,
Cassiman JJ
(1991)
Cultured human fibroblasts contain a large pool of precursor beta 1-integrin but lack an intracellular pool of mature subunit.
Eur J Biochem
199:25-33[ISI][Medline].
-
Dou CL,
Levine JM
(1994)
Inhibition of neurite growth by the NG2 chondroitin sulfate proteoglycan.
J Neurosci
14:7616-7628[Abstract].
-
Dou CL,
Levine JM
(1995)
Differential effects of glycosaminoglycans on neurite growth on laminin and L1 substrates.
J Neurosci
15:8053-8066[Abstract].
-
Emerling DE,
Lander AD
(1996)
Inhibitors and promoters of thalamic neuron adhesion and outgrowth in embryonic neocortex: functional association with chondroitin sulfate.
Neuron
17:1089-1100[ISI][Medline].
-
Ernst H,
Zanin MK,
Everman D,
Hoffman S
(1995)
Receptor-mediated adhesive and anti-adhesive functions of chondroitin sulfate proteoglycan preparations from embryonic chicken brain.
J Cell Sci
108:3807-3816[Abstract].
-
Faissner A,
Steindler D
(1995)
Boundaries and inhibitory molecules in developing neural tissues.
Glia
13:233-254[ISI][Medline].
-
Faissner A,
Clement A,
Lochter A,
Streit A,
Mandl C,
Schachner M
(1994)
Isolation of a neural chondroitin sulfate proteoglycan with neurite outgrowth promoting properties.
J Cell Biol
126:783-799[Abstract/Free Full Text].
-
Felsenfeld DP,
Choquet D,
Sheetz MP
(1996)
Ligand binding regulates the directed movement of beta1 integrins on fibroblasts.
Nature
383:438-440[Medline].
-
Fichard A,
Verna J-M,
Olivares J,
Saxod R
(1991)
Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using -D-xyloside.
Dev Biol
148:1-9[ISI][Medline].
-
Gomez TM,
Letourneau PC
(1994)
Filopodia initiate choices made by sensory neuron growth cones at laminin/fibronectin borders in vitro.
J Neurosci
14:5959-5972[Abstract].
-
Graham FL,
Prevec L
(1995)
Methods for construction of adenovirus vectors.
Mol Biotechnol
3:207-220[ISI][Medline].
-
Grumet M,
Milev P,
Sakurai T,
Karthikeyan L,
Bourdon M,
Margolis RK,
Margolis RU
(1994)
Interactions with tenascin and differential effects on cell adhesion of neurocan and phosphacan, two major chondroitin sulfate proteoglycans of nervous tissue.
J Biol Chem
269:12142-12146[Abstract/Free Full Text].
-
Hayashi Y,
Reszka A,
Iguchi T,
Reinish B,
Kawashima T,
Horwitz AF
(1991)
Expression of chicken integrin beta 1 subunit in rat PC12 cells.
Cell Struct Funct
16:135-139[ISI][Medline].
-
Hoke A,
Silver J
(1996)
Proteoglycans and other repulsive molecules in glial boundaries during development and regeneration of the nervous system.
Prog Brain Res
108:149-163[ISI][Medline].
-
Iida J,
Meijne AM,
Spiro RC,
Roos E,
Furcht LT,
McCarthy JB
(1995)
Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and alpha 4 beta 1 integrin.
Cancer Res
55:2177-2185[Abstract/Free Full Text].
-
Koivisto L,
Heino J,
Hakkinen L,
Larjava H
(1994)
The size of the intracellular beta 1-integrin precursor pool regulates maturation of beta 1-integrin subunit and associated alpha-subunits.
Biochem J
300:771-779.
-
Landolt RM,
Vaughan L,
Winterhalter KH,
Zimmermann DR
(1995)
Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth.
Development
121:2303-2312[Abstract].
-
Lebaron RG
(1996)
Versican.
Perspect Dev Neurobiol
3:261-271[ISI][Medline].
-
Letourneau PC,
Condic ML,
Snow DM
(1994)
Interactions of developing neurons with the extracellular matrix.
J Neurosci
14:915-928[ISI][Medline].
-
Levine JM,
Nishiyama A
(1996)
The NG2 chondroitin sulfate proteoglycan: a multifunctional proteoglycan associated with immature cells.
Perspect Dev Neurobiol
3:245-259[ISI][Medline].
-
Li Y,
Raisman G
(1994)
Schwann cells induce sprouting in motor and sensory axons in the adult rat spinal cord.
J Neurosci
14:4050-4063[Abstract].
-
Li Y,
Raisman G
(1995)
Sprouts from cut corticospinal axons persist in the presence of astrocytic scarring in long-term lesions of the adult rat spinal cord.
Exp Neurol
134:102-111[ISI][Medline].
-
Luo Y,
Raper JA
(1994)
Inhibitory factors controlling growth cone motility and guidance.
Curr Opin Neurobiol
4:648-654[Medline].
-
Maeda N,
Noda M
(1996)
6B4 proteoglycan/phosphacan is a repulsive substratum but promotes morphological differentiation of cortical neurons.
Development
122:647-658[Abstract].
-
Margolis RK,
Rauch U,
Maurel P,
Margolis RU
(1996)
Neurocan and phosphacan: two major nervous tissue-specific chondroitin sulfate proteoglycans.
Perspect Dev Neurobiol
3:273-290[ISI][Medline].
-
McKeon RJ,
Hoke A,
Silver J
(1995)
Injury-induced proteoglycans inhibit the potential for laminin-mediated axon growth on astrocytic scars.
Exp Neurol
136:32-43[ISI][Medline].
-
Meyer-Puttlitz B,
Milev P,
Junker E,
Zimmer I,
Margolis RU,
Margolis RK
(1995)
Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of neurocan and phosphacan.
J Neurochem
65:2327-2337[ISI][Medline].
-
Miyakawa Y,
Nishimura T,
Ueyama Y,
Miyake K,
Miyasaka M,
Ikeda Y,
Habu S
(1996)
Cell adhesion via murine alpha4 human beta1 integrin chimera on transfected K562 cells to endothelial cells.
Exp Cell Res
226:75-79[ISI][Medline].
-
Muller AH,
Gawantka V,
Ding X,
Hausen P
(1993)
Maturation induced internalization of beta 1-integrin by Xenopus oocytes and formation of the maternal integrin pool.
Mech Dev
42:77-88[ISI][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[ISI][Medline].
-
Pearlman AL,
Sheppard AM
(1996)
Extracellular matrix in early cortical development.
Prog Brain Res
108:117-134[Medline].
-
Schwartz NB,
Domowicz M,
Krueger RCJ,
Li H,
Mangoura D
(1996)
Brain aggrecan.
Perspect Dev Neurobiol
3:291-306[ISI][Medline].
-
Snow DM,
Letourneau PC
(1992)
Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).
J Neurobiol
23:322-336[ISI][Medline].
-
Snow DM,
Lemmon V,
Carrino DA,
Caplan AI,
Silver J
(1990a)
Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro.
Exp Neurol
109:111-130[ISI][Medline].
-
Snow DM,
Steindler DA,
Silver J
(1990b)
Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: a possible role for a proteoglycan in the development of an axon barrier.
Dev Biol
138:359-376[ISI][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].
-
Snow DM,
Atkinson PB,
Hassinger TD,
Letourneau PC,
Kater SB
(1994)
Chondroitin sulfate proteoglycan elevates cytoplasmic calcium in DRG neurons.
Dev Biol
166:87-100[ISI][Medline].
-
Snow DM,
Brown EM,
Letourneau PC
(1996)
Growth cone behavior in the presence of soluble chondroitin sulfate proteoglycan (CSPG), compared to behavior on CSPG bound to laminin or fibronectin.
Int J Dev Neurosci
14:331-349[ISI][Medline].
-
Song H,
Ming G,
He Z,
Lehmann M,
Tessier-Lavigne M,
Poo M
(1998)
Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides.
Science
281:1515-1518[Abstract/Free Full Text].
-
Streit A,
Nolte C,
Rasony T,
Schachner M
(1993)
Interaction of astrochondrin with extracellular matrix components and its involvement in astrocyte process formation and cerebellar granule cell migration.
J Cell Biol
120:799-814[Abstract/Free Full Text].
-
Tomaselli KJ,
Doherty P,
Emmett CJ,
Damsky CH,
Walsh FS,
Reichardt LF
(1993)
Expression of beta 1 integrins in sensory neurons of the dorsal root ganglion and their functions in neurite outgrowth on two laminin isoforms.
J Neurosci
13:4880-4888[Abstract].
-
Weaver CD,
Yoshida CK,
de Curtis I,
Reichardt LF
(1995)
Expression and in vitro function of beta 1-integrin laminin receptors in the developing avian ciliary ganglion.
J Neurosci
15:5275-5285[Abstract].
-
Winnemoller M,
Schon P,
Vischer P,
Kresse H
(1992)
Interactions between thrombospondin and the small proteoglycan decorin: interference with cell attachment.
Eur J Cell Biol
59:47-55[ISI][Medline].
-
Yamagata M,
Jaye DL,
Sanes JR
(1994)
Gene transfer to avian embryos with a recombinant adenovirus.
Dev Biol
166:355-359[ISI][Medline].
Copyright © 1999 Society for Neuroscience 0270-6474/99/192210036-08$05.00/0
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ABSTRACT
INTRODUCTION
