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Development 114, 17-29 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
17
In vitro control of neuronal polarity by glycosaminoglycans
F. LAFONT1, M. ROUGET2, A. TRILLER3, A. PROCFUANTZ1 and A. ROUSSELET1
URA 1414, Ecole Normale Supirieure, Dtveloppement et Evolution du Systeme Nerveux, 46 rue d'Ulm, 75230 Paris Cedex 05,
France
2
Laboratoire de Biologic Cellulaire et Tissulaire, Faculty de Mtdecine, 27 bvd Jean Moulin, Marseille 05, France
3
INSERM U261, Institut Pasteur, 26 rue du Dr. Roux, 75724 Paris Cedex 15, France
Summary
We have studied the effects of proteoglycans (PGs) and
glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or
Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C
chondroitin sulfate (CSc) and bovine mucosa dermatan
sulfate (DS) added to embryonic rat neurons strongly
enhanced total neurite growth after 48 h in vitro. No
trophic effects were seen when PGs treated with a
mixture of glycanases were used. PGs, CSc and HS not
only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon
whereas, in contrast, DS increased dendrite growth.
GAGs bound to the cell surface and were rapidly
internalized, a feature that correlated well with the
absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the
separate regulation of neuronal polarity by HS and DS
were not elucidated, we found that, as opposed to HS,
DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of
microtubule-associated protein 2 (MAP2), a specific
dendritic marker. Thisfindingconfirms and extends our
previous observations on the role of adhesion in the
regulation of dendrite growth.
Introduction
ment is still unclear. However, several reports indicate
that they can regulate neuronal maturation, either
directly or in association with other neurotrophic
molecules. For example, Muir et al. (1989) reported
that a specific proteoglycan could inhibit the neurite
growth activity of laminin. Conversely, proteoglycans
acting as growth factor receptors (e.g. TGF/3 or basic
FGF) might have a strong growth-promoting activity
(Neufeld et al., 1987; Burgess and Maciag, 1989;
Cheifetz and Massague\ 1989; Gordon et al., 1989;
Ruoslahti, 1989; Kiefer et al., 1990).
This capacity of proteoglycans and glycosaminoglycans (GAGs) to bind, concentrate and eventually
internalize several basic growth factors is linked both to
the conformation of the carbohydrate chains and to
their high content of negative charges. Thus, it is not
surprising that GAGs, in the absence of their protein
cores, can themselves be biologically active entities
(Verna et al., 1989). However, the exact role and mode
of action of proteoglycans and, in particular, the actual
function of the complex sugars which, in many cases,
constitute the most abundant part of the molecule have
not been elucidated.
In previous studies we have used an experimental in
vitro model that allows us to study in a quantitative
Extracellular matrix (ECM) molecules play a major
role in the development of the nervous system. Two
well-characterized ECM molecules are laminin and
fibronectin, which participate in the attachment, migration and differentiation of peripheral and central
neurons, both in vitro and in vivo (for a review see
Sanes, 1989). More recently, several studies have
indicated that proteoglycans (PGs) secreted in the
extracellular space or linked to the neuronal membrane
by a true transmembrane segment or a glycolipid
anchor participate in neuronal differentiation (Matthew
et al., 1985; Cole et al., 1986; Fransson, 1987; HantazAmbroise et al., 1987; Dow et al., 1988; Ruoslahti,
1988; Werz and Schachner, 1988).
Proteoglycans are composed of a protein core on
which long chains of various sulfated sugars are
attached, sulfated dermatan, heparan and chondroitin
being the most common found in brain PGs (Margolis
and Margolis, 1989; Herndon and Lander, 1990). In
spite of their developmentally regulated expression
within the mammalian brain (Oohira et al., 1986;
Herndon and Lander, 1990), the exact role of these
proteoglycans and glycosaminoglycans during develop-
Key words: neurons, cell culture, polarity,
glycosaminoglycans, adhesion.
18
Control of neuronal polarity by glycosaminoglycans
manner the effect of various compounds on the growth
of neurites and on the development of neuronal
polarity. We have established that the growth of axons
and dendrites could be regulated separately (DenisDonini et al., 1984; Chamak et al., 1987) and that ECM
molecules present in astrocyte-conditioned media are
instrumental in this regulation (Rousselet et al., 1988,
1990). Moreover, we have demonstrated that the
influence of different matrix molecules on neuronal
shape and polarity (i.e., the growth of axons or
dendrites) varied depending on how they were presented to the cells. In particular, it is clear that
substratum-bound and soluble ECM factors induce
different patterns of growth and polarity (Chamak and
Prochiantz, 1989; Lochter et al., 1991).
Since, in addition to laminin and fibronectin, proteoglycans are also present in astrocyte-conditioned medium, the present studies analysed the potential
influence of these molecules on neuronal growth and
polarity. We report here that astrocyte-derived proteoglycans and proteoglycans from the EHS tumor stimulate axonal growth in vitro. Moreover, we demonstrate
that this influence of PGs on neuronal polarity can be
reproduced by the addition in the culture medium of
small concentrations of heparan or chondroi'tin sulfates
and thus does not seem to depend on the presence of
the PG core protein. Very interestingly, we find that
dermatan sulfate, as opposed to heparan sulfate, has a
strong tendency to stimulate dendrite elongation and
that the action of these two glycosaminoglycans is
associated with their binding to the nerve cells and their
subsequent internalization.
Materials and methods
minutes after seeding. Mesencephalic astrocytes were prepared by culturing the cells, seeded at the density of 3.5 x 105
cells cm"2, for 3 weeks in the presence of 8% FCS. It is worth
noting that, although most of these experiments presented
here were done with mesencephalic neurons, similar results
were obtained with neurons from cortex, spinal cord or
striatum.
Morphological analysis
Cells were cultured for 2 days, fixed with 2.5% glutaraldehyde
in PBS pH 7.4 for 30 minutes at room temperature, washed
twice with PBS and stained with toluidine blue (0.2% in 1%
Na2CC>3). Stained cells were air dried and examined with an
optical microscope (Leitz). For each experiment 50 to 100
neurons were digitalized and analysed with a morphological
analysis software (IMSTAR, France). All statistics (Student's
Mest) were done with the help of the Statview II program
(Abacus Concepts, Inc.).
Quantification of cell survival
Cells were dissociated and plated as described above. The
number of live cells per well at time zero was estimated by
trypan blue exclusion under the microscope just before
plating and taken as 100%. The percentages of survival after
different times in culture were also calculated by the trypan
blue exclusion procedure.
Quantification of cell adhesion
Cellular adhesion was quantified according to the method of
Dow et al., (1988). Briefly, cells were seeded at 20,000 cells
per well and then the multiwell plates were turned up side
down. The inverted plates were incubated for 1 hour at 4°C
and subsequently 2 hours at 37°C. The cells were then fixed
(2.5% glutaraldehyde in PBS), rinsed with water, air dried,
stained with 0.1% cristal violet in borate buffer pH 9 (Kueng
et al., 1989), washed several times and air dried. Cristal violet
bound to the cell nuclei was solubilized in 10% acetic acid and
the optical density of the solution in each well determined at
570 nm with an automatic multiwell-plate spectrophotometer.
Culture media, DMEM and F12 were from Gibco. Penicillin,
streptomycin, insulin, transferin, progesterone, putrescine,
selenium sodium salt and the different GAGs (bovin kidney
heparan sulfate; bovin mucosa dermatan sulfate and shark
cartilage type C chondroitin sulfate) were purchased from
Sigma. Protease-free heparinase from Flavobacterium heparinum, heparitinase and chondroitinase ABC from Proteus
vulgaris were from Seikagaku Kogyo Co, Ltd (Miles).
Antibodies against microtubule-associated protein 2 (MAP2)
and Neurofilament-H (NF-H) were kind gifts of Drs A.
Fellous and P. Levitt, respectively. The polyclonal antibodies
against laminin and high molecular weight core protein of the
HSPG from the EHS tumor, as well as the tumor itself, were
provided by Dr. M. Vigny. TRTTC-linked second antibodies
were from Biosis. Biotinylated antibodies and labelled
streptavidin were from Amersham.
Immunocytochemistry
Cells seeded on 16 mm diameter glass coverslips coated with
1.5 fig ml" 1 polyornithine were cultured for 3 days, fixed for 1
hour at 4°C with paraformaldehyde (4% in PBS), rinsed in
PBS-glycine 5 mM pH 7.4, incubated (1 hour 37°C) with antiMAP2 antibodies (1/400) in PBS plus 10% FCS and 0.01%
saponin (buffer A), washed 3 times and further incubated for
1 hour at 37°C with anti-NF-H antibodies (1/50). After 3
washes cells were incubated (1 hour, 37°C) with Texas redconjugated anti-rabbit Igs (1/200), washed 3 times, incubated
with biotinylated anti-mouse Igs (1/200), washed 3 times,
incubated (1 hour, 37°C) with fluorescein-conjugated streptavidin (1/200), washed 3 times in PBS and twice in water,
mounted in mowiol and observed with a Leitz epifluorescence
microscope. All washes and dilutions were done in buffer A.
Cell culture
All cell culture protocols were as described previously
(Rousselet et al., 1988, 1990). In brief, cells were mechanically dissociated from rat embryonic mesencephalon (ED 14),
seeded at a density of 25 x 103 cells cm~2 (morphological and
imunocytochemical analysis) or 105 cells cm (ELISA and
adhesion assays) on plastic tissue culture wells (Nunc)
precoated with 1.5 ng ml" 1 D.L-poIyornithine (A/r 40,000,
Sigma) and cultured in chemically denned medium (CDM).
Astrocyte or EHS proteoglycans (1 and 4 /jg ml" 1 respectively) and the various GAGs^(10 fig ml"1) were added 30
ELISA on whole cells
The ELISA was performed according to the method of Munn
and Cheung, 1989. Briefly, cells were seeded in 96 microwell
plates precoated with polyornithine (1.5 fig ml" 1 ), cultured
for 2 days, fixed (1 hour, room temperature) with 4%
paraformaldehyde (in PBS plus 1 mM Ca , Mg2 ),rinsedin
PBS-glycine 5 mM pH 7.4, and incubated for 1 hour at 37°C in
PBS plus FCS 10%. Anti-MAP2 (1/4000 in PBS plus 10%
FCS) antibodies or non-immune rabbit IgGs were added for 1
hour at 37°C. After several washes in PBS plus 0.1% FCS,
cells were further incubated with a peroxidase-labelled second
Lafont and others
12
antibody (1 hour, 37°C in PBS plus 10% FCS) and washed
several times with PBS. Peroxidase activity was revealed with
o-phenylenediamine-dihydrochloride and quantified spectrophotometrically at 470 nm. The number of living cells present
in sister wells was estimated by direct counting or by the
cristal violet method (Kueng et al., 1989).
Proteoglycan purification
Mesencephalic astrocytes cultured for 3 weeks in the presence
of 10% FCS were washed several times with DMEM-F12 and
cultured for another 2 days in the absence of serum.
Conditioned medium adjusted to 6 M urea was adsorbed on a
DEAE cellulose column (DE52) and equilibrated in the
following buffer: 0.2 M NaCl, 6 M urea, 0.05 M Tris-HCl pH
7.4. The column was washed in the same buffer until no
proteins were detected in the effluent and then glial PGs were
eluted with 0.7 M NaCl, 6 M urea and 0.05 M Tris-HCl pH
7.4. In some cases conditioning was achieved in the presence
of [35S]sulfate salt (50 ,uCi ml"1) in sulfate-free DMEM-F12.
The purity of the preparation was verified by SDS-PAGE
analysis (4% acrylamide). Glial PGs were dialysed against
DMEM-F12 for 24 hours at 4°C before use. All purification
steps were performed in aseptic conditions and in the
presence of PMSF (1 mM).
PGs from the EHS tumor were purified according to the
method of Hassel et al. (1985). Briefly, tumors propagated in
Balb/c mice for 3 weeks were harvested, rinsed in saline
extracting medium plus 6 M urea and loaded on a DE 52 anion
exchange column equilibrated with 6 M urea, 0.15 M NaCl,
0.04 M EDTA, 8 mM NEM, 2 mM PMSF and 50 mM TrisHCl pH 6.8. Bound proteins were eluted with different steps
of 0.15, 0.30, 0.70, 1 and 1.5 M NaCl in the equilibration
buffer. The fractions dialysed against PBS were analyzed by
SDS-PAGE, and those containing pure PGs, as checked by
electrophoretic profiles were kept frozen at — 80°C until use.
After slow defrosting, PGs were either filtered with a 0.22 [im
Millipore filter or sterilized with a drop of chloroform.
Homogeneity of the PGs was checked by electrophoresis on
SDS-polyacrylamide gels followed either by silver staining
(BioRad-kit) or immunoblotting. As indicated in Fig. 1, all
stained material remained in the form of large molecular
weight entities unable to penetrate a 6% gel (lane 1), unless
part of the sugars were enzymatically degraded by heparitinase (lane 2). Immunoblotting with anti-laminin antibodies
(lane 3) failed to reveal any contamination by these matrix
molecules (the most abundant in EHS tumor).
Enzymatic treatments
Proteoglycans (400 fig) were incubated for 2 hours at 37°C
with heparinase or heparitinase (0.1 U ml"1) or chondroi'tinases (0.05 U ml" 1 ), in 1 ml of DMEM-F12 culture medium
supplemented with 25 mM sodium acetate. Proteolytic
treatments were achieved overnight at 37°C with trypsine (1
mg ml"1) or proteinase K (1 mg ml"1) and stopped with 10%
FCS. Digested PGs were added to the cultures to give the
final concentration of 4 fig ml" 1 . In control experiments, the
enzymes were directly added in the culture wells at the
appropriate concentrations. The degradation of PGs was
monitored by SDS-PAGE.
Electron microscopy
Cells were fixed in glutaraldehyde (3% in PBS) for 1 hour at
room temperature, washed several times with PBS and postfixed in osmium tetroxyde (2% in PBS) for 30 minutes at room
temperature. After dehydration they were embedded in
Epon, cut, collected on grids, contrasted with uranyl acetate
and lead citrate, and viewed with a Jeol 2000 electron
19
3 4
I
%
200>
11 61>
Si ~u
Fig. 1. SDS-PAGE and
immunoblottings of PGs.
Lanes 1 and 2: purified
EHS-PGs treated (lane 2)
or not (lane 1) with
heparitinase, silver
staining. Lanes 3 and 4:
anti-laminin
immunoblotting of PGs
(lane 3) and laminin (lane
4). Positions of the
relative molecular mass
markers are indicated on
the left side of the figure
(xlO" 3 ).
microscope (Vuillet et al., 1984; Autillo-Touati et al., 1988).
For immuno-electron microscopy, cells fixed with 4% paraformaldehyde were incubated with the anti-core protein of
the HSPG from the EHS tumor (1/400 in PBS) for 1 hour at
37°C, washed, incubated with peroxidase-linked anti-rabbit
IgGs (1/400 in PBS) for another hour and washed several
times before addition of diaminobenzydine (0.2 mg mP 1 ) and
H2O2 (0.003%) in Tris 50 mM, pH 7.8 for 5 minutes at room
temperature. The cells were then washed thoroughly, postfixed in OsO4, dehydrated, embedded in Epon, cut, collected
and viewed.
GAGs biotinylation
GAGs biotinylated with biotin hydrazid (Pierce) according to
the manufacturer's procedure were exhaustively dialysed
against 1 M NaCl in PBS and against PBS. Labelling and
absence of free biotin hydrazid were checked by PAGEelectrophoresis under the conditions used for DNA analysis
(Maniatis), followed by blotting, incubation of the blots with
peroxidase-streptavidin and development of the reaction with
DAB (0.2 mg ml"1* and H2O2 (0.003%).
Confocal microscopy
Data were obtained with a confocal scanning laser microscope
Phoibos 1000 (Sarastro, Stockholm). Excitation was obtained
with an Argon ion laser set at 514 nm for TRITC excitation
and the emitted light was filtered with an appropriate long
pass filter (530 nm). The background noise was reduced and
the contrast enhanced by applying a median gaussian filter to
the original data. Pseudocolor images coding for fluorescence
emission were obtained from a linear look-up table. It
decreased from red to yellow, green and blue.
Results
Effects of PGs: oh rteuronal morphology
Mixed proteoglycans (PGs) isolated from astrocyte
20
Control of neuronal polarity by glycosaminoglycans
Table 1. Percentages of surviving neurons after 3, 18,
30 or 48 hours in culture in different conditions
Time in culture (hours)
Conditions
Control (CDM)
PGs
HS
DS
CSc
3
18
84±5
81±6
86±4
81 ±4
84±3
81±6
80±3
84±4
m±7
30
75 ±12
81±7
82±7
83±7
81 ±7
V
'
t*
48
1
75±9
70±3
78±6
74±3
78±3
Mesencephalic neurons were plated onto polyornithine-coated
substratum. The trypan blue method was used at different times to
visualize living cells. Cells from three independent fields (9xlO~3
cm) were counted directly under the microscope. Values are the
results of 2 independent experiments. Neuronal survival was not
modified significantly in any of the tested conditions.
conditioned medium or from the EHS tumor were
added to the cells 30 minutes after seeding and their
morphological effects were analysed after 2 days. In the
conditions of chemically denned medium (CDM) plus
or minus PGs (or GAGs) all cells were neuronal in
nature, as demonstrated by their labeling with antibodies directed against the neurofilament triplet or
neural specific enolase (not shown, see Rousselet et al.,
1988). As shown in Table 1 for EHS PGs (and several
GAGs), the addition of these molecules did not affect
neuronal survival, which remained constant for 48
hours. In fact at the time of neuronal analysis, more
that 90% of the cells attached 3 hours after plating were
still alive.
Fig. 2 illustrates the morphological influence of the
addition of 4 fig ml" 1 EHS proteoglycans, a concentration that did not affect cell viability (Table 1). In
control conditions (Fig. 2A), cell bodies are spread with
short neurites and numerous cytoplasmic veils. The
presence of the proteoglycans induced vigourous
neurite growth, most neurons being asymmetric with a
single long neurite. In addition, these long neurites
tended to fasciculate with one another (Fig. 2B). The
percentage of neurons with longest neurite length
exceeding 50 ^m increased from 10% in the control
wells to more than 80% (Fig. 2C) in the presence of
PGs. In view of the high neuronal survival (Table 1), it
can be assumed that the morphological changes illustrated in Fig. 2 do not reflect the selective survival of
specific neuronal subpopulations with distinct morphological traits.
We observed similar effects of astrocyte proteoglycans (G-PGs) on neurite growth as indicated in Table 2
(Exp. 1). Indeed, astrocyte and EHS tumor proteoglycans (EHS-PGs) increased total neurite length, but the
most striking result was the fivefold increase in the
length of the longest neurite. Since PGs extracted from
the tumor and from the astrocytes gave identical
results, all following experiments were performed with
the tumor proteins, which were easier to purify in high
quantities.
•A
s
C •
i
120
100 -0%
100
D
L+.CDM
•
L+, CDM+EHS-PGs
200
Length
Fig. 2. Effects of EHS tumor PGs on neuronal
morphology. (A) Mesencephalic neurons cultured for 2
days in CDM. Note the spreading of the cell bodies and
the large numbers of lamellipodia and veils. (B)
Mesencephalic neurons cultured for 2 days in CDM plus
PGs (4 ng ml" 1 ). Note the round cell bodies and the
tendency of the long axon-like neurites to fasciculate. (C)
Cumulative quantitative analysis of the length of the
longest neurite (L+) in chemically defined medium (CDM)
or in CDM plus PGs. This figure pools the results of 3
independent experiments in which 50 neurons per
experiment were analysed in each condition.
300
F, Lafont and others
21
Fig. 3. Double-immunostaining (same field) of mesencephalic neurons cultured with or without PGs. Mesencephalic
neurons cultured in CDM (A,B) or in CDM plus PGs (C,D) were reacted after 3 days in culture with anti-neurofilament
(axon-specific) (B,D) or anti-MAP2 (dendrite-specific) antibodies (A,C).
PGs favor axonal elongation
We were interested in determining whether the long
neurites growing in the presence of PGs were in fact
axons as suggested by their morphology. In order to
examine this directly, cells were fixed and labeled with
antibodies specific for either somatodendritic structures
(i.e., anti microtubule-associated protein 2, MAP2) or
young axons (i.e., the highly phosphorylated isoforms
of neurofilament proteins, NF-H) (Pennypacker et al.,
1991). These double-staining experiments illustrated in
Fig. 3 were achieved after 3 days in vitro. Clearly, the
long neurites induced by PGs can be stained only with
the axon-specific antibody (Fig. 3D) and not with the
anti-MAP2 antibody (Fig. 3C). The absence of MAP2
staining in the long neurites present after 3 days in
culture with EHS-PGs confirms the axon-like nature of
these neurites and indicates that the axons are sufficiently differentiated not to contain significant
amounts of the MAP2 antigen (Higgins et al., 1988).
To examine the localization of added EHS-PGs on
neurons in culture, the cells were incubated with an
antibody directed against the core protein of the high
molecular weight form of the heparan sulfate proteoglycan and the product of the immunoreaction was
analysed by electron microscopy. As illustrated in Fig.
4, EHS-PGs bound to the cell membrane and the core
protein epitopes recognized by the antibody were not
internalised (Fig. 4A). When neurons were in close
contact with another cell (Fig. 4B), no staining was
observed at the interface of the cells, indicating that the
antibody actually recognized added EHS-PGs and did
not stain endogenous molecules. In fact, no labeling
was seen when EHS-PGs were not previously added to
the culture (not. shown). Note that the staining was
present at the surfaces of both cell bodies and axons as
shown in Fig. 4C.
Effects of purified GAGs on neuronal morphology
PGs are composed of a core protein on which different
types of glycosaminoglycans (GAGs) are attached. To
test directly the role of GAGs on neuronal morphogenesis, bovine kidney heparan sulfate (HS), shark cartilage chondroitin sulfate c (CSc) and a bovine mucosa
chondroitin sulfate b (dermatan sulfate, DS) were
added to the cultures at a concentration of 10 /zg ml" 1
which, as demonstrated by preliminary experiments,
gave the best effects but did not impair neuronal
22
Control of neuronal polarity by glycosaminoglycans
Table 2. Morphometric analysis of neurons cultured
for 2 days in different conditions
Neuritic length (pm)
L+
Lt
PN
Exp. 1
(PGs)
CDM
G-PGs
EHS-PGs
16±6
76±3**
83±7"
37±16
97±7*»
110±22**
2.2±0.3
1.5±0.2
1.8±0.2
Exp. 2
(soluble GAGs)
CDM
HS
DS
CSc
23±5
78±1*
52±5*
84±13*
56±16
102±l*
142±19*
123±20*
2.4±0.6
1.6±0.1
3.0±0.2
2.1±0.2
Exp. 3
(bound GAGs)
CDM
HS
DS
CSc
30 ±3
33±3
25±3
32±3
42±4
53±6
42±6
45±5
2.1±0.1
1.4±0.1
1.0±0.1
1.0±0.1
Mesencephalic neurons were cultured for 2 days, fixed and
stained. For each condition, 50 isolated neurons were drawn. Total
neurite length (Lt), length of the longest neurite (L+) and the
number of primary neurites (PN) were determined for each
neuron. The results are pooling from 4 independent experiments.
The significance of the difference (s.e.m.) were estimated by the
Student's / test: */><0.05; "P<0.01.
Fig. 4. Binding of PGs to mesencephalic neurons as
revealed by electron microscopy. Neurons were cultured
for 2 days in the presence of EHS-PGs, fixed and labelled
with anti-high molecular weight PG core protein antibodies
labelled with peroxidase. Cells were processed for electron
microscopy as described in Methods. The black precipitate
surrounding the cells (arrowheads) is indicative of the
presence of the neuron-bound PGs. Note that the PGs are
present on the cell body (A) and on the neurites (C). On
the contrary, none of them are detectable at the cell
contacts (B, little arrows). Bar, 1 pan.
survival (Table 1). After 2 days, the cells were analysed
for their morphologies.
As illustrated in Fig. 5, the three sugars did not exert
identical effects on neurite growth. The neuronal
morphologies induced by HS (Fig. 5B) and the CSc
(Fig. 5D) were almost identical to that observed in the
presence of the PGs. Cell bodies were small and
rounded, total neurite growth was increased twofold
and this increase corresponded to the preferential
development of a single axon-like neurite which
accounted for more than 75% of total neurite growth
(Table 2, Exp. 2). Compared to HS and CSc, DS had an
even stronger effect on total neurite growth (threefold
increase), but this effect consisted of a strong enhancement of dendrite-like growth, since the axon-like
compartment contributed to one third only of the total
neuritic arbor (Table 2, Fig. 5C). None of the sugars
had any significant effect (compared to CDM) when
bound on the polyornithine coating before cell plating,
thus indicating that they were active in their soluble
form only (Table 2, Exp. 3).
To confirm the importance of the sugar moieties on
PG-induced neuronal morphogenesis, EHS PGs were
treated with heparinase and chondroitinase ABC or
heparitinase. Control experiments in which the enzymes were added to the cultures showed that heparinase and chondroitinase had no effect on cell morphology. The partial cleavage of heparan sulfate
polymers and the removal of chondroitin chains
checked by polyacrylamide gel electrophoresis completely abolished the effect of PGs on total neurite
growth and on the growth of the axon-like longest
neurite as shown in Fig. 6 in the case of heparinase plus
chondroitinase ABC. In contrast, treating the PG
preparation with either trypsin or proteinase K did not
abolish PG-induced neurite growth (not shown) confirming that GAGs by themselves have an interesting
effect on neuronal growth and polarity.
HS and DS regulate neuronal polarity
In the following sections, we shall only compare the
activities of dermatan and heparan sulfate that gave the
more distinct morphological differences. However, we
observed very few differences between HS and CSc in
their abilities to induce axonal growth preferentially.
Fig. 7 shows the double immunostaining- of neurons
cultured for 3 days in the presence of HS (A,B) or DS
(C,D). The long neurites present in HS were almost
Fig. 8. Confocal microscopy analysis of GAGs distributions. Biotinylated HS (A,C) or DS (B,D) were added to the cell
culture for 1 hour (A,B) or 18 hours (C,D). Cells were fixed with paraformaldehyde (4%) and reacted with streptavidintexas-red. Confocal sections of the neurons shown in this figure correspond to a cut through the mid-height of the cells.
Bar, 1 fsm.
F. Lafont and others
23
v
•
•
•
%
.
t
. * V.
Fig. 5. Influence of purified GAGs on neuronal morphology. Mesencephalic neurons were cultured for 2 days in CDM
(A), HS (B), DS (C) or CSc (D). Note that the three GAGs promote neurite growth, but that neuronal morphology
strongly depends on the nature of the GAGs added to the culture.
always (98% of the cases) immunodecorated by the
axon-specific antibody but showed no or little staining
with the anti-MAP2 antibody. In DS, the cells were
multipolar with most neurites labelled with the antiMAP2 antibody (Fig. 7C). Very surprisingly, although
the longest neurites were in majority axons, an
important proportion of them (39%) had marked
dendrite biochemical characteristics such as the absence
of axonal NF-H and rather large quantities of MAP2
antigen (Fig. 7).
These different effects of the two sugars on the
polarity of developing embryonic neurons, combined
with the fact that the sugars had to be given in a soluble
form (Table 2), led us to compare their cellular
distributions. To do so the sugars were biotinylated and
added to the cells 30 minutes after plating. The cultures
were fixed 1 or 18 hours later and the distribution of HS
(Fig. 8A,C) or DS (Fig. 8B,D) revealed with fluorescent streptavidin was observed with a confocal
microscope.
One hour after plating, the distributions of the 2
sugars were quite different. DS were rapidly internalized thus making it difficult to observe any labeling of
the cell membrane, (Fig. 8B). In contrast, although also
rapidly internalized, HS molecules could be observed
both inside the cell and at its surface (Fig. 8A). It is
interesting to note that the HS distribution at the cell
surface is not uniform and indicates some kind of
asymmetric organization of the binding sites. Another
difference beween the two GAGs is the accumulation of
HS in the nucleus, a phenomenon that was not observed
consistently in the case of DS (Fig. 8C).
Polarity, adhesion and MAP2 synthesis
In previous reports, we proposed that while axons were
able to grow in low adhesion conditions, dendrite
gTowth was only possible in high adhesion conditions
(Chamak and Prochiantz, 1989; Rousselet et al., 1990;
Prochiantz, 1990). This prompted us to semi-quantify
neuronal adhesion 2 hours following plating in CDM or
in the presence of PGs, HS or DS. These data were
compared with neuronal spreading and with the
capacity to synthesize MAP2.
As demonstrated in Fig. 9A, neuronal spreading
(apparent surface of the soma) measured 2 days
following plating was highest in CDM and reduced in
the presence of both GAGs. Although not as efficient
24
Control of neuronal polarity by glycosaminoglycans
J
<
V
as intact PGs (not shown), HS was much more efficient
than DS in reducing neuronal spreading. In addition,
we found a good correlation between the amounts of
MAP2 present in the cells after 2 days in culture and
adhesion 2 hours after plating (Fig. 9B,C). Finally,
neuronal morphologies of neurons grown for 2 days in
the presence of HS or DS were examined in electron
microscopy. Fig. 10 illustrates that neurons grown in the
presence of HS are rounded and seem to be loosely
attached to the substratum (Fig. 10A) whereas in the
presence of DS the soma are flattened and present a
long and continuous attachment to the culture dish
(Fig. 10B). It is noteworthy that the general shape of
the nuclei (rounded or flattened) reflected that of the
cell bodies.
Discussion
V•
B
120
Total Neuntic Length
El Longest Neuritic Length
PGs
PG-mixt
Fig. 6. Effects of sugar removal on PGs activity. PGs were
added intact (A) or after degradation with chondroi'tinase
ABC plus heparinase (B). Quantitative estimations of total
neurite length and of the length of the longest neurite are
shown in panel C where the results of 3 independent
experiments have been pooled and 150 neurons analysed in
control conditions (PGs) or after treatment with the
mixture of glycanases (PG-mixt). Symbols relate to the
significance of the differences with the results obtained
with intact PGs. (s.e.m., Student's Mest). * P<0.02.
In this report, we demonstrate that PGs purified either
from astrocyte-conditioned medium or from the EHS
tumor have a strong influence on neurite growth and,
more specifically, on axonal growth. This PG-induced
axonal growth is associated with a decrease both in
adhesion and in the synthesis of MAP2. The trophic and
morphogenetic influence of PGs is abolished when the
glycoproteins are enzymatically deglycosylated whereas
it is not affected by the hydrolysis of the core protein
with trypsin or proteinase K. This importance of the
sugar moieties (GAGs) is further confirmed by experiments in which specific sugars, chondroitin-, dermatanor heparan-sulfate, were directly added to the cultures.
Interestingly enough, it was found that although all
GAGs tested strongly promote neurite growth, the type
of neurite produced in majority (axon versus dendrites)
is highly dependent on the chemical structure of the
sulfated carbohydrate chains. This finding illustrated in
this report for E14 rat post-mitotic mesencephalic
neurons, remains valid for neurons prepared from other
brain regions (spinal cord, cortex and striatum, in
particular) between E13 and E18.
The culture conditions used in these experiments
allow neuronal survival, and typically result in cultures
with a cell population more that 99% neuronal. Thus, it
is unlikely that the effects of the different PGs and.
GAGs are mediated through the few non-neuronal cells
present in the culture. Furthermore, the low cellular
concentration and the very rapid effects of PGs and
GAGs (e.g. adhesion was measured 2 hours after
seeding) strongly suggest that PGs and GAGs act
directly at the level of their target cells. In particular,
although we cannot preclude it entirely, it is unlikely
that the effects of PGs and GAGs require a long-range
diffusion of molecules synthesized by the neurons.
Rather, we favor a hypothesis by which these molecules
would trigger a chain of intracellular events by acting on
receptors or by increasing the efficiency of some
autocrine phenomenons.
A crucial point in the interpretation of our results is
the possible selective survival, in the different conditions, of specific subpopulations presenting defined
F. Lafont and others
25
Fig. 7. Influence of purified GAGs on neuronal polarity. Mesencephalic neurons were cultured for 3 days in the presence
of HS (A,B) or DS (C,D). Axons and dendrites were identified by double-immunostaining (samefield)with the antineurofilament (B,D) and anti-MAP2 (A,C) antibodies.
morphological traits, e.g. presence of a long axon-like
neurite. We consider this possibility very unlikely on
the basis of the following considerations. Firstly,
cellular survival after 2 days was over 90% whereas
more than 80% of the cells presented the same
morphology (e.g. length of the longest neurite greater
than 50 /zm). Secondly, morphological examinations
were done after 2 and 6 days yielding identical
qualitative and quantitative results although cell survival was lower in the older cultures (not shown).
Thirdly, variations in cell adhesion (a critical factor in
cell polarity) were measured 2 hours after seeding when
all cells were still alive. Finally, in another model of
polarity induction through adhesion, we have shown
that the effects were fully reversible (Chamak and
Prochiantz, 1989). From this, we infer that the effects of
PGs and GAGs are instructive and do not reflect a
selective mechanism.
Antibodies against MAP2 or against highly phosphorylated isoforms of high molecular weight NF
proteins (NF-H) were used to characterize dendrites
and axons respectively. MAP2 has been shown to be a
good dendritic marker, (Matus et al., 1986; Higgins et
al., 1988) and the amounts of MAP2 quantified by an
ELISA assay on fixed cells correlated well with the
immunological staining. The use of the neurofilament
proteins as axonal markers can be more problematic
because of the late synthesis of some axon-specific
isoforms (Foster et al., 1987). However, in good
accordance with the results of Pennypacker et al. (1991)
the anti NF-H antibody used in this study allowed us to
discriminate between axons and dendrites in 3-day-old
cultures, as demonstrated by double immunostaining
experiments.
Another point of concern is the purity of the
proteoglycan preparations. In particular, these molecules are known to interact strongly with several
factors endowed with potent morphogenetic properties
such as laminin, fibronectin, basic FGF and TGF/3
(Ruoslahti, 1988). The EHS proteoglycans used in this
study were purified following well established procedures (Hassel et al., 1985) and their analysis on SDSpolyacrylamide gels showed no obvious contaminants.
Since we were not interested in purifying distinct
subsets of PGs, no CsCl fractionation was achieved,
thus the PG mixture we work with is comparable to the
one described by others (Kato et al., 1978; Fujiwara et
al., 1984). However, the fact that the anion exchange
26
Control of neuronal polarity by glycosaminoglycans
200
DS
Fig. 9. Adhesion and MAP2 expression in the presence of
PGs or GAGs. (A) Soma surface (fan2) of neurons
cultured in presence or abscence of GAGs. For each
condition, 100 isolated neurons were analysed with the
IMSTAR morphometrical analysis software. (B) Adhesion
was measured after 2 hours of cell culture on inverted
plates, by the cristal violet staining procedure as described
in Methods. Values presented were calculated from three
independent experiments. (C) MAP2 expression estimated
by a whole-cell ELISA test. Values were calculated from
three independent experiments. Symbols relate to the
significance of the difference with the values obtained in
CDM. * /><0.01
column was equilibrated in 6 M urea diminishes the
probability of the presence of contaminating molecules
in our preparations. This is in fact well demonstrated by
the absence of contaminating laminin, the most abun-
dant matrix molecules in the EHS tumor, clearly
illustrated in the Western blot of Fig. 1. Finally, even
though the presence of small amounts of highly active
contaminating factors can never be entirely precluded,
the fact that the morphogenetic effects observed with
such proteoglycans were lost after hydrolysis with
protease-free sugar-degrading enzymes and could be
replicated with purified GAGs eliminates simple explanations based on a contamination by any of the factors
mentioned above.
The physiological significance of the morphogenetic
effects of PGs and GAGs is underlined by the fact that
these molecules are synthesized in the nervous system,
in particular the brain (Margolis and Margolis, 1989;
Herndon and Lander, 1990). In good agreement with
published results on the structure of brain-derived PGs
(Fransson, 1987; Hoffman and Edelman, 1987; Ratner
et al., 1988; Margolis and Margolis, 1989), we verified
that astrocytes in culture release PGs in which chondroitin- and heparan-sulfate are present. The distribution of PGs during development, as analysed by
several investigators, has shown that these molecules
are not only developmentally regulated, but that their
distribution coincides with specific pathways either
permissive or repulsive for the migration of cells and the
elongation of growth cones (Perris et al., 1991).
An important result of our studies is the capability of
pure sugars to modify neuronal growth and morphology. Such a morphogenetic influence of the GAGs
has been observed in another model (Verna et al.,
1989). However, to our knowledge, the analysis of how
different GAGs can act in a distinct manner on the
development of neuronal polarity had not been studied
before. Of particular interest are the converse activities
of dermatan- and heparan-sulfate, which promote
dendrite and axon growth, respectively. The induction
of axon growth associated with a decrease in adhesion
and in MAP2 synthesis confirms previous results
demonstrating that, in contrast to dendrites, axons,
because of their high axoplasmic viscosity, are able to
grow in low adhesion conditions (Chamak and Prochiantz, 1989; Rousselet et al., 1990; Prochiantz, 1990).
Although our study is limited to the nervous system, it
can be underlined that, in view of the similarities
between the mechanisms of polarity establishment and
maintainance in several cell types, the results reported
here may be of larger physiological significance (Dotti
and Simon, 1990).
Our observations on the possible physiological
importance of GAGs synthesis and distribution, combined with the fact that PGs treated with heparinase,
heparitinase and chondroitinase ABC lose all their
growth-inducing properties raise the question of the
respective roles of the core proteins and of the sugar
moities in PGs physiology. Although confirming the
importance of PGs and GAGs in neuronal differentiation, the results reported here certainly highlight the
importance of the GAGs moiety at the expenses of core
proteins. This statement can anyhow be corrected by
the fact that our culture conditions (low cell density,
CDM), in which control neurons have little growth
F. Lafont and others
27
Fig. 10. Electron micrographs of mesencephalic
neurons grown in the presence of GAGs. Cells
cultured for 2 days on Petriperm dishes in the
presence of HS (A) and DS (B) were fixed
with glutaraldehyde and osmium tetroxide and
processed for electron microscopy as described
in Methods. The embedded cells were cut in a
plane perpendicular to the culture substratum.
Note the ball-shape of the loosely attached cell
body of the neurons grown in the presence of
HS (contact with the substratum is indicated by
arrowheads), as compared to the flat cell body
of neurons cultured in DS. Bar, 1 //m.
activity, would not have allowed us to discover an
inhibitory action of the core proteins on actively
elongating neurites as was reported for NGF-treated
PC12 cells (Snow et al., 1990; Oohira et al., 1991).
It is also possible that it is in culture only that the
presence of the core protein is of little importance. This
might be due to the fact that pure sugar chains are able
to bind to the neurons and to mimic the action of intact
PGs. It is indeed rather unlikely that, in vivo, proteinfree sugar chains can be either secreted into the medium
or present at the neuronal surface. Thus, it can be
proposed that core proteins act as a means of exposing
the sugars at the neuronal surface or of facilitating their
secretion into the intercellular space. Moreover it can
be speculated that depending on the type of core
proteins, PGs could be targeted to specific neuronal
compartments, e.g. dendrites or axons. Indeed,
Rapraeger and his collaborators have demonstrated a
specific targeting of PGs to the apical or the basolateral
compartments of the epithelial cell (Rapraeger et al.,
1986). This possibility is strengthened by the fact that
several PGs are anchored to the cell surface by a
glycolipid-link (Herndon and Lander, 1990) known to
act as an apical target signal in polarized cells, neurons
included (Lisanti et al., 1989; Dotti et al., 1991).
Interestingly enough in this context, Dotti and Simon
(1990) have demonstrated the equivalence between
axons and the apical compartment of epithelial cells.
The mechanism by which GAGs exert their trophic
and polarizing actions must be considered. This question was partially addressed in the present study, in
particular in the experiments showing that they can
modulate neuronal adhesion and MAP2 expression.
More experimental work, however, will be needed to
understand the actual physiological role of these
molecules. Although we cannot eliminate the possi-
bility that PGs and GAGs are true growth factors acting
autonomously after binding specific receptors, it is
more likely that these molecules modulate the activity
of cell and substratum adhesion molecules as demonstrated in the case of NCAM or laminin (Cole et al.,
1986; Muir et al., 1989).
More generally, in view of the high affinity for
heparin of several growth factors, such as bFGF or
TGF0, it is possible that the addition of GAGs or PGs
increases the trophic influences of such molecules either
by augmenting their ability to diffuse (Flaumenhaft et
al., 1990) or their binding capacities at the cell surface.
Heparin-activated capture of growth factor has been
proposed, at least in the case of bFGF, to be required
for the further activation of the high affinity tyrosinekinase-linked receptor (Yayon et al., 1991; Rapraeger
et al., 1991). In addition, PGs and GAGs might be
involved in the specific internalization and intracellular
targeting of several factors (Ruoslahti, 1989; Baldin et
al., 1990). This latter point is substantiated by our
observation that biotinylated GAGs are rapidly internalized in culture. This rapid internalization of GAGs
contrasts with the apparent absence of internalization
of EHS-PGs. This latter observation has to be taken
with caution since the antibody used was directed
exclusively against the core protein, leaving open a
possible internalization of the GAGs.
Finally, the fact that HS stimulates axonal growth
whereas DS allows the growth of all neurites with a
strong positive effect on dendrite development sustains
the idea that specific domains or even receptors might
exist at the cell surface that recognize distinct GAGs
sequences. This last point is substantiated by our
confocal microscopy experiments which suggest that DS
and HS do not behave in the same way when added to
neurons in culture. In particular, it can be noted that
28
Control of neuronal polarity by
glycosaminoglycans
HS, although internalized and targeted to the nuclei,
are found associated with the cell surface in a clearly
non-random disposition and seemingly underline an
asymmetric organization of the nerve cell even before
neurite growth. The possible significance of this
distribution and the possible link between the existence
of separate domains at the surface of the cell and the
development of neuronal polarity is being presently
investigated.
We thank Dr. K. Moya for helpful suggestions and for his
careful reading of the manuscript. The help of Dr. M. Vigny
in the design of some experiments is also acknowledged. Mrs
H. Debroas and C. Valenza are acknowledged for their
skillful technical assistance. This work was supported by
CNRS, FIDIA-France and grants from AFM, DRET (89-200)
and MRT (89 C 0701).
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{Accepted 11 October 1991)