A Synthetic Peptide Ligand of Neural Cell Adhesion
Molecule (NCAM), C3d, Promotes Neuritogenesis and Synaptogenesis
and Modulates Presynaptic Function in Primary Cultures of Rat
Hippocampal Neurons*
Darya
Kiryushko
§,
Thomas
Kofoed¶,
Galina
Skladchikova
,
Arne
Holm¶,
Vladimir
Berezin
, and
Elisabeth
Bock
From the
Protein Laboratory, Institute of Molecular
Pathology, Panum Institute Bldg. 6.2, Blegdamsvej 3C, DK-2200,
Copenhagen N, Denmark, the § Laboratory of Biophysics and
Bioelectronics, Dniepropetrovsk State University, Naukovii pr. 13, 49050, Dniepropetrovsk, Ukraine, and the ¶ Chemistry Department,
Royal Agricultural and Veterinary University, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark
Received for publication, November 14, 2002, and in revised form, December 23, 2002
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ABSTRACT |
The neural cell adhesion molecule (NCAM) plays a
key role in morphogenesis of the nervous system and in remodeling of
neuronal connections accompanying regenerative and cognitive processes. Recently, a new synthetic ligand of NCAM, the C3-peptide, which binds
to the NCAM IgI module, has been identified by means of combinatorial chemistry (Rønn, L. C. B, Olsen, M.,
Ostergaard, S., Kiselyov, V., Berezin, V., Mortensen, M. T.,
Lerche, M. H., Jensen, P. H., Soroka, V., Saffell, J. L., Doherty, P., Poulsen, F. M., Bock, E., Holm, A., and
Saffells, J. L. (1999) Nat. Biotechnol. 17, 1000-1005). In vitro, the dendrimeric form of C3, termed
C3d, disrupts NCAM-mediated cell adhesion, induces neurite outgrowth, and triggers intracellular signaling cascades similar to those activated by homophilic NCAM binding. The peptide may therefore be
expected to regulate regeneration and synaptic plasticity. Here we
demonstrate that in primary cultures of hippocampal neurons: 1) C3d
induces a sustained neuritogenic response, the neuritogenic activity of
the compound being dependent on the dose, starting time, and duration
of peptide application; 2) the peptide triggers the neuritogenic
response by forming an adhesive substratum necessary for NCAM-mediated
neurite formation and elongation; 3) C3d promotes synapse formation;
and 4) C3d modulates the presynaptic function, causing a transient
increase of the function at low (2 and 5 µM) doses and a
reduction when applied at a higher concentration (10 µM).
The effect of the peptide is dependent on the activation of the
fibroblast growth factor receptor. We suggest that C3d may
constitute a useful lead for the development of
compounds for treatment of various neurodegenerative disorders.
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INTRODUCTION |
The neural cell adhesion molecule
(NCAM)1 plays a key role
in morphogenesis of the nervous system (2) and in remodeling of
neuronal connections associated with regenerative and cognitive processes (for review, see Ref. 3). The extracellular part of NCAM
binds to a variety of ligands, the most important being the NCAM
molecule itself. Homophilic NCAM binding is thought to involve the
first five immunoglobulin (IgI-IgV) modules of NCAM with a double
reciprocal interaction between the IgI and IgII modules of two
interacting NCAM molecules (4, 5). Upon homophilic NCAM binding,
intracellular signaling cascades, including the Ras-mitogen-activated protein kinase (Ras-MAPK) and phospholipase C
-associated pathways, are activated (6), resulting in neurite outgrowth. Antibody interventive studies have shown NCAM necessary for
the induction and maintenance of long term potentiation and for stable
memory retention in vivo (7, 8), suggesting that NCAM is
involved in synaptic plasticity.
The modulation of processes of neuronal differentiation and plasticity
through NCAM has been impeded by the absence of small synthetic
agonists mimicking homophilic or heterophilic interactions of NCAM.
However, recently, a new synthetic ligand of NCAM, the C3-peptide, has
been identified by means of combinatorial chemistry. The dendrimeric
tetramer of this peptide (C3d) binds to the IgI module of NCAM with a
dissociation constant similar to that of the natural homophilic ligand,
the IgII module (1, 4). In vitro, C3d disrupts NCAM-mediated
cell adhesion, induces neurite outgrowth, and triggers intracellular
signaling cascades similar to those activated by physiological
homophilic NCAM binding (1, 6). In vivo, C3d has been
demonstrated to induce amnesia in a passive avoidance learning paradigm
in the adult rat and to prevent NCAM internalization in a 3-4-h period
following task acquisition (9), indicating that the peptide is capable
of modulating synaptic function.
In this study, we investigate the kinetics of the neuritogenic effect
of C3d and show that by providing an adhesive substratum for neurite
promotion, the peptide induces both a persistent neuritogenic response
from primary hippocampal neurons and the formation of functional
synapses, with synaptogenesis accelerated in cultures grown on C3d
versus control (poly-L-lysine) substratum. We
also show that C3d affects the presynaptic function in primary
hippocampal neurons in a concentration- and time-dependent
manner, and this effect is dependent on the activation of FGF receptor.
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EXPERIMENTAL PROCEDURES |
Preparation of Peptides--
The C3 undecapeptide (ASKKPKRNIKA)
and the control peptide C3ala, in which Lys-6 and Arg-7 were
substituted with Ala, were synthesized as dendrimers composed of four
monomers coupled to a lysine backbone by Prof. Arne Holm (Royal
Agricultural and Veterinary University, Copenhagen, Denmark).
Primary Cultures of Rat Hippocampal Neurons--
Hippocampal
neurons were prepared from embryonic day 19 rat embryos as described in
(10). Neurons were seeded at a density of 10,000 cells/cm2 in eight-well LabTek tissue culture
chambers with a growth surface of Permanox plastic (Nunc, Roskilde,
Denmark) or fibronectin (Sigma). For coating, slides were incubated
with fibronectin diluted in H2O and dried overnight at room
temperature in a flow bench to reach a final surface concentration of
0.01-20 µg/cm2. For C3d immobilization, culture chambers
were preincubated with various concentrations of the peptide for 2 h at 37 °C. Before cell seeding, culture chambers were washed in
phosphate-buffered saline and blocked with 1% bovine serum albumin
(BSA). After plating, cultures were maintained at 37 °C, 5%
CO2 in Neurobasal medium containing 20 mM
HEPES, 100 units/ml penicillin, 100 µg/ml streptomycin, and
0.4% w/v BSA (Sigma), supplemented with B27 (all from Invitrogen).
Analysis of Neurite Outgrowth--
For analysis of neurite
outgrowth, cultures were fixed with 4% v/v formaldehyde, stained for
20 min with 0.04% w/v Coomassie Blue R 250 in 45% v/v ethanol and
45% v/v acetic acid, and subsequently analyzed employing
computer-assisted microscopy as described previously (11). In each
experiment, the length of neurites from 150-200 neurons was determined.
Analysis of NCAM Expression--
The expression of NCAM was
analyzed by immunoblotting. To estimate the amount of NCAM in
hippocampal cells 0-30 min after trypsinization, 106 cells
in suspension were collected for lysis after cell dissociation in DNase
I. The cells were pelleted at 1000 × g for 2 min and resuspended in lysis buffer (10 mM Tris-HCl, 2% v/v Triton
X-100, pH 8, containing a mixture of protease inhibitors: 0.8 µM aprotinin, 50 µM bestatin, 15 µM E-64, 20 µM leupeptin, and 10 µM pepstatin A; set III, Calbiochem). For analysis of
NCAM expression at 1-24 h after dissociation, hippocampal
neurons were plated in 60-mm tissue culture dishes (Nunc) at a density
of 40,000 cells/cm2 and maintained in culture for 1, 2, 3, 6, or 24 h. Cells were scraped off in 0.5 ml of Neurobasal growth
medium, pelleted at 1000 × g for 2 min, and
solubilized in lysis buffer. Cell extracts were briefly sonicated
(10-15 s, 40 watts), clarified by centrifugation at 20,000 × g for 5 min at 4 °C, and treated with Endo-N (1 µg/ml, recombinant MBP-fusion protein, Protein Laboratory, Institute of
Molecular Pathology, Panum Institute, Copenhagen, Denmark) to remove
the NCAM polysialylation. SDS-PAGE and immunoblotting were performed as
described in Ref. 12. Protein bands were visualized using enhanced
chemiluminescence substrate Western Dura (Pierce) and processed with
the GenTools software package (Syngene, Cambridge, UK).
Immunofluorescence--
Visualization of filamentous actin
(F-actin) was performed as described in Ref. 13. For double
immunostaining for synaptophysin and GAP43, cell cultures were fixed in
4% w/v paraformaldehyde in sodium phosphate buffer (0.1 M
NaH2PO4, 50 mM sucrose, 0.4 mM CaCl2, pH 7.1) for 30 min at room
temperature, blocked with 10% v/v normal goat serum, 1% w/v BSA in
phosphate-buffered saline for 1 h, and washed with 1% BSA
containing 0.2% w/v saponin. For double immunostaining for the
postsynaptic density protein PSD-95 and NCAM, cells were fixed in
methanol/acetone (1:1 v/v) for 5 min at
20 °C and washed with 1%
BSA. Neurons were subsequently incubated with the following primary
antibodies diluted in buffer: antisynaptophysin mouse monoclonal
antibody (IgG; diluted 1:500, Sigma) and rabbit anti-rat-GAP43
polyclonal antibodies (1:1000, prepared as described previously (14)),
or anti-PSD-95 monoclonal antibody (IgG; 1:500, clone 6G6, Affinity
Bioreagents, AH Diagnostics) and rabbit anti-rat-NCAM polyclonal
antibody (1:1000, Protein Laboratory). Incubation with primary
antibodies was performed at 4 °C overnight. Bound antibodies were
detected with Texas Red-conjugated goat anti-mouse IgG (1:100,
Molecular Probes, Eugene, OR), or fluorescein-conjugated goat
anti-rabbit IgG (1:200, Molecular Probes). Slides were mounted using
Prolong Antifade mounting medium (Molecular Probes) and scanned with a
MultiProbe 2001 Laser Scanning Confocal Microscope (Amersham
Biosciences) equipped with an oil immersion ×60 1.4 NA or ×100 1.4 NA
objective (Nikon, Tokyo, Japan).
Mass Spectrometry and Determination of Amount of C3d in Culture
Medium--
To determine the amount of C3d in culture medium at
different times after addition of the peptide, 200-µl samples
containing 1 or 10 µM of the peptide dissolved in
Neurobasal medium, were incubated in eight-well LabTek tissue culture
chamber slides at 37 °C, 5% CO2 in the absence or
presence of plated hippocampal neurons. At various times (0, 1, 6, or
24 h), aliquots were collected. Mass spectroscopic determination
of the amount of C3d in the samples was performed using an Esquire
liquid chromatography/mass spectrometer. The mass spectrometer was an
Esquire ion trap MS analyzer (Bruker Daltonik GmbH, Bremen, Germany)
connected to an HP1100 system (Agilent Technologies, Palo Alto, CA)
with a Vadyac low trifluoroacetic acid reverse phase column (21 × 150 mm). The solvent system comprised two solvents: (a)
water with 0.02% v/v trifluoroacetic acid and (b)
acetonitrile/water (90:10) with 0.02% trifluoroacetic acid. A gradient
of B was steadily increasing from 0 to 50% over 30 min of acquisition
with a flow rate of 0.250 ml/min. Angiotensin II (Bachem,
Bubendorf, Switzerland, 10 µM) was used as internal standard to control the volume of the injected peptide and was added to
each sample to a final concentration of 1 µM. The total injection volume was 50 µl. Quantification of the C3d amount was performed by isolation of the MS signal at 1073,8 Da [M + 5H]5+ and angiotensin II at 1046.0 Da [M + H]+. The total counts for each sample were found by
integration of the isolated MS signal.
To quantify the rates of C3d clearance from the culture medium based on
the obtained experimental data (concentrations of C3d after various
times of incubation), a mathematical model was developed (see
"Appendix"). The model adequately described the experimentally obtained time course of C3d clearance from the culture medium and was
used for estimation of rate constants of C3d degradation and
sedimentation (see "Results").
Labeling and Quantitative Analysis of Functionally Active
Synapses--
FM1-43 labeling of functional synapses was performed as
described previously by Ryan et al. (15). Briefly, 10 days
in vitro (DIV) hippocampal neurons were incubated in the
presence of 2 µM of the fluorescent styryl membrane probe
FM1-43 (Molecular Probes) and 90 mM KCl for 60 s
followed by washing 3-4 times in normal saline for 5 min each to
remove surface-bound FM1-43. Normal saline contained 137 mM NaCl, 5.4 mM KCl, 0.8 mM
MgSO4, 1.3 mM CaCl2·2H2O, 0.4 mM
KH2PO4, 4 mM NaHCO3,
0.3 mM Na2HPO4, 5.6 mM D-glucose, 20 mM HEPES, pH adjusted to 7.2 with
NaOH. After loading, a field containing labeled punctae-like areas
(0.3-3 µm2) was chosen. Pixel intensities from each
object were averaged to obtain a measure of the local fluorescence
intensity in the individual presynaptic terminal. To evaluate the rate
of turnover of synaptic vesicles, the synapses loaded with FM1-43 were
destained by 150-s stimulation with 90 mM KCl in
FM1-43-free solution. Fluorescence images of FM1-43-loaded synapses
were obtained with a MultiProbe 2001 laser scanning confocal
microscope, ×60 1.4 NA objective, at an excitation wavelength of 488 nm. Images were quantified using the ImageSpace software package
(Amersham Biosciences). To evaluate the rate of FM1-43 unloading, the
experimentally obtained time courses of synaptic destaining were
approximated with a single exponential function, exp(
kt)
where k is the rate of destaining and t is the
time elapsed after the start of KCl application.
Statistics and Graphical Presentations--
Statistics and
graphical presentations were carried out using the Origin version 5.0 software package (OriginLab, Northampton, MA). Statistical evaluations
were performed using a two-sided Student's t test. The
results are given as mean ± S.E. Unless otherwise stated
asterisks indicate the statistical significance: *, p < 0.05, **, p < 0.01, ***, p < 0.001 as compared with control.
 |
RESULTS |
Time Course of NCAM Expression after Trypsinization--
The
induction of neurite outgrowth from hippocampal neurons by C3d takes
place upon its binding to NCAM-IgI (1). Thus, the amount of NCAM
expressed on the cell surface at the time when C3d is added to the
culture medium might be of importance for its neuritogenic activity. We
therefore first analyzed the expression of the two NCAM
isoforms, 180 kDa NCAM and 140 kDa NCAM, at different times after
trypsinization of hippocampal cells (see "Experimental Procedures"
for details). In Fig. 1, it can be seen
that the amount of 180 kDa NCAM and 140 kDa NCAM was significantly less
at 0-3 h as compared with 24 h in vitro. During this
period, the level of expression of 180 kDa NCAM and 140 kDa NCAM
amounted to ~20% and ~50%, respectively, as compared with
the level of expression at 24 h in vitro. Thus, the
receptor for C3d binding, although reduced, was present even shortly
after trypsinization.

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Fig. 1.
Expression of 180 kDa NCAM and 140 kDa NCAM
isoforms in primary hippocampal neurons at different times after cell
dissociation. Unless stated otherwise, the results are
given as mean ± S.E.; four independent experiments were
performed. As shown in A, hippocampal neurons were collected
for lysis 0-24 h after cell dissociation. Cell extracts prepared in
SDS-containing lysis buffer were subjected in duplicate to SDS-PAGE and
immunoblotted using polyclonal rabbit anti-rat NCAM antibodies.
B, quantification of expression of 180 kDa NCAM ( ) and
140 kDa NCAM ( ) isoforms of experiments performed as shown in
A. The amount of each isoform is expressed relative to the
level of expression at 24 h after cell dissociation, which was set
to 100%. Four independent experiments were performed.
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C3d Induces Persistent Neurite Outgrowth from Hippocampal
Neurons--
Treatment of hippocampal neurons with C3d for 24 h
has been shown to induce a neuritogenic response with a bell-shaped
dose-response relationship, the maximal activity being observed at a
peptide concentration of 0.54 µM (1). Accordingly, in our
experiments, 1 µM C3d induced profound neurite outgrowth
(Fig. 2). The time responses of neurite
outgrowth induced by various doses of the peptide (0.3, 1, 3 µM, Fig. 3A)
were very similar, with the strongest induction of neurite outgrowth
being observed at 1 µM C3d.

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Fig. 2.
Confocal images of rat hippocampal neurons
grown in low density culture for 1, 2, or 3 days in the absence
(A, 3 DIV) or presence (B-D, 1, 2, or 3 DIV, respectively) of 1 µM
C3d. Immunostaining for filamentous (F) actin is
shown.
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Fig. 3.
The neuritogenic activity of C3d, time- and
dose-response studies. A, effect of 0.3 µM ( ), 1 µM ( ), and 3 µM ( ) C3d on the formation of neurites from primary
hippocampal neurons depending on time in vitro. ,
control. In each experiment, the length of neurites from 150-200
neurons was estimated at each time point. B, dependence of
neurite outgrowth stimulated by 0.3 µM ( ), 1 µM ( ), and 3 µM ( ) C3d on the time of
C3d addition after seeding of cells. For each point, the average
neurite length was measured 24 h after the beginning of C3d
treatment. C, effect of the C3d-peptide on neurite
outgrowth: dependence on time of exposure. Cultures were treated with
0.3 µM ( ), 1 µM ( ), 3 µM ( ), 10 µM ( ), and 30 µM ( ) C3d for 1, 2, 4, 8, or 24 h from the time
of seeding. The average neurite length was measured 24 h after
seeding. Interpolating curves were derived based on five independent
experiments. D, dependence of time of exposure to C3d
resulting in maximal neuritogenic response on the applied concentration
of C3d.
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The neuritogenic effect of C3d also depended on the time of application
after cell seeding and the total time of exposure to the peptide. In a
first set of experiments, C3d was added at different times after cell
seeding (0-24 h, Fig. 3B) and, for each time point, the
average length of neurites was estimated after 24 h of exposure to
the peptide. For all peptide concentrations, the amplitude of the
neuritogenic response was highest if the peptide was applied 0-2 h
after plating. When treatment with C3d started 24 h after seeding,
no neuritogenic effect was observed (Fig. 3B), indicating
that the later treatment with C3d starts after plating, the smaller was
the magnitude of the neuritogenic response induced by the NCAM ligand.
In a second set of experiments, hippocampal neurons were treated with
C3d in various concentrations for 1, 2, 4, 8, or 24 h starting
from the time of seeding (time 0), and subsequently the average neurite
length was estimated at 24 h in vitro (Fig. 3C). It can be seen that the maximal neuritogenic activity
of C3d varied significantly depending both on exposure time and on the
peptide concentration. To determine the maxima of the obtained experimental curves, sets of experimental data were interpolated with
the Bezier polynomes, and the time of exposure to C3d resulting in a
maximal neuritogenic response at any given concentration was plotted
against the employed dose (Fig. 3D). From the graph, it can
be seen that the higher the concentration of C3d, the shorter was the
time of exposure resulting in a maximal neuritogenic stimulation.
Taken together, these results highlight two aspects of the neuritogenic
effect of C3d: 1) C3d triggering of intracellular signaling pathways
may only be possible shortly after cell seeding, with a "time
window" in the range of 2 h, and 2) C3d may promote neurite
extension by forming an adhesive peptide substratum. In the latter
case, the same "optimal" surface concentration of C3d could be
achieved either by a short exposure to high concentrations of the
peptide or by longer exposures to lower doses. This would explain the
observation that the optimal time of exposure to C3d decreases with the
rise of peptide concentration (Fig. 3D).
C3d Exerts Its Effect by Forming an Adhesive Substratum for Neurite
Promotion--
To test the above hypothesis, hippocampal neurons were
grown in culture chambers coated with C3d. The immobilized peptide strongly stimulated neurite outgrowth in a dose-dependent
manner (Fig. 4A). In contrast
to soluble C3d, the dose-response curve for immobilized peptide did not
demonstrate a decrease at high peptide concentrations, reaching instead
a plateau at [C3d] = 3 µM, probably due to a saturation
of the binding sites on the plastic surface at this concentration (Fig.
4A). Moreover, addition of soluble C3d to cultures of
hippocampal cells inhibited neurite outgrowth induced by
immobilized C3d (not shown). These results indicated that at
high C3d concentrations, the soluble peptide presumably interfered with
cell-substrate adhesion, thus preventing neurite promotion.

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Fig. 4.
The neuritogenic activity of C3d, effects of
coating and repetitive treatment. A, the effect of coating
culture dishes with C3d on neurite outgrowth ( ) as compared with the
effect of C3d addition to the medium ( ). B, the effect of
repetitive treatment with C3d on neurite development after 48 or
72 h in vitro. Empty columns, C3d added at
0 h; shadowed columns, C3d added at 0 and 24 h;
filled columns, C3d added at 0, 24, and 48 h.
C, the effect of repetitive treatment with 1 µM C3d on C3d- and fibronectin-induced neurite outgrowth
after 48 h in vitro. , control; , C3d added at
0 h; , C3d added at 0 and 24 h. Three independent
experiments were performed.
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If this was the case, one might expect that readdition of the peptide
to the culture medium would competitively inhibit C3d-induced neurite
outgrowth. Indeed, treatment of hippocampal neurons twice with C3d at
times 0 and 24 h led to a marked decrease of the neuritogenic response when estimated at 48 h. The effect was even more
pronounced when C3d was applied at times 0, 24, and 48 h, and the
response was measured at 72 h as compared with control cultures
treated with C3d only once, at the time of seeding (Fig.
4B).
To test whether the observed effect was C3d-specific, we plated
hippocampal neurons onto a fibronectin substratum (0.02-20 µg/cm2) and treated them with 1 µM C3d at
time 0 or at times 0 and 24 h. The average length of neurites was
measured after 48 h. Cultures grown on fibronectin, but not
treated with C3d, were used as controls. From Fig. 4C, it
can be seen that fibronectin stimulates neurite outgrowth in a
dose-dependent manner with a threshold of 0.05 µg/cm2. C3d added at time 0 induced an additional
neuritogenic effect over that exerted by fibronectin. A second
application of the C3d (24 h) abolished the neuritogenesis induced by
the peptide without affecting the rate of fibronectin-induced neurite
outgrowth (Fig. 4C). Thus, repetitive treatment with C3d
specifically modulated C3d-, but not fibronectin-induced, neurite outgrowth.
These results indicate that C3d induces the neuritogenic response not
only by triggering intracellular signaling pathways but also by forming
an adhesive substratum and subsequent interaction with NCAM expressed
on the plasma membrane. Soluble C3d might competitively disrupt this
interaction, resulting in a significant decrease of the
neuritogenic response.
Pharmacodynamic Properties of C3d in Hippocampal Cell
Culture--
To directly confirm the above hypothesis, we
investigated the time course of the C3d concentration in culture medium
with and without hippocampal cells using mass spectroscopy. The time courses of the peptide concentration in the medium,
[C3d]f, normalized to the corresponding initial values (1 and
10 µM), represented biphasic curves with a fast initial
[C3d]f fall at 0-6 h of the incubation followed by a slower
decrease at 6-24 h (Fig. 5). The rate of
C3d clearance from the culture medium in the absence of cells was
higher for 1 µM than for 10 µM C3d. This
indicated that at least two mechanisms with different time scales
contributed to the [C3d]f decrease in the culture medium over
time. We presumed that the process of fast initial (0-2 h) C3d
clearance from the culture medium reflected the adsorption of the
peptide to the plastic surface since the slower disappearance of
10 µM rather than 1 µM C3d at short times suggested the presence of a saturating clearance process, which would
not be the case if the peptide simply was degraded in the medium. At
longer (6-24 h) times, a slower clearance was observed, which probably
reflected C3d degradation. To estimate the rates of these processes, we
constructed a mathematical model comprising both mechanisms (see
"Appendix"). The model adequately described the experimental data
(Fig. 5, solid curves), allowing us to estimate time
constants of C3d degradation (
1) and sedimentation
(
2), which were found to be
1 = 50 h
for both peptide doses and
2 = 5.2 h for 1 µM C3d or
2 = 0.4 h for 10 µM C3d (see "Appendix" for details).

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Fig. 5.
The time course of C3d concentration in the
culture medium ([C3d]f). For each time point,
[C3d]f was normalized to that at 0 h
([C3d] ). Filled circles,
[C3d] = 10 µM;
filled squares, [C3d] = 1 µM. Smooth solid curves represent the
numerical solution of Eq. 1 (see "Appendix") with the following
parameters: k1 = 0.02 h 1, k2 = 0.05 1/µM·h. HN, linear approximation
of the time course of clearance of 1 µM C3d in the
presence of hippocampal neurons. Five independent experiments were
performed.
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In the presence of hippocampal neurons, 1 µM C3d was
cleared from the culture medium significantly faster (Fig. 5). However, the time course of the C3d clearance could not be approximated by the
biexponential curve since the peptide was degraded faster during the
first hours in culture than at 6-24 h. This indicated that C3d was
also cleaved by the cells, the rate of cell-induced C3d degradation
decreasing by time in culture. This might be due to the binding of C3d
to the cell receptors (NCAM) followed by cleavage of the peptide by
cell proteases or shedding and/or endocytosis of NCAM together with the
bound peptide at short times after cell dissociation.
C3d Promotes Synapse Formation in Primary Hippocampal
Neurons--
Since C3d peptide induced persistent neurite outgrowth
terminating in the formation of a neuronal network approximately at day
5 in vitro, it seemed important to elucidate how rapidly the induced neuritogenic response resulted in formation of functional synapses. This process has been shown to correlate strongly with the
acquisition of focal accumulations of synaptic vesicle proteins such as
synaptophysin (17). Thus, synaptophysin-positive spots provide an
accurate quantifiable estimate of presynaptic terminals (18). We
investigated the time course of expression of synaptophysin in primary
hippocampal neurons plated onto substrata consisting of C3d, the Ig2
module of NCAM, or poly-L-lysin (PLL). Spontaneous neuritogenesis on PLL was used as a control. Cells were immunostained for synaptophysin after 3, 4, 5, 6, and 8 days in culture. Fig. 6 shows double immunostaining for the
neuronal membrane protein GAP43 and the presynaptic marker,
synaptophysin, of neurons grown either on C3d (A, C) or PLL (B, D)
substratum at 5 (A, B) and 8 (C, D) days in vitro. At 5 DIV,
cells grown on the two substrata had similar morphology (Fig. 6,
A and B). However, punctate patches of
synaptophysin immunoreactivity on cell bodies and dendrites were more
numerous in cultures grown on C3d than on control substratum (compare
Fig. 6, A and B). At 8 DIV, no difference was
seen (compare Fig. 6, C and D). To obtain a
quantitative estimate of the rate of synapse formation, we evaluated
the number of synaptophysin-positive spots/unit of neurite length in
the cultures, grown on the two substrates. From Fig.
7A, it can be seen that
hippocampal neurons cultivated on either C3d or the NCAM-Ig2 module
developed synapses significantly faster than cells cultivated on PLL.
The difference in the number of formed synapses was considerable at
3-6 DIV but disappeared by 8 DIV (Fig. 7A). The time course
of expression of the postsynaptic density marker PSD-95 in cultures
grown on C3d was similar to that of synaptophysin with a punctate
PSD-95 immunoreactivity appearing at approximately 5 DIV (not shown). The observed effect could not be attributed to the enhanced encounter rate between neurites in C3d- versus PLL-stimulated cells
since at any time in vitro, the average neurite length in
cultures grown on PLL somewhat exceeded that in cultures grown on C3d
or Ig2 (Fig. 7B). Thus, both the natural NCAM ligand,
NCAM-Ig2, and the peptide mimetic C3d promoted synapse formation in
primary hippocampal neurons.

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Fig. 6.
Time course of
synaptophysin expression in hippocampal neurons cultured
onto C3d (A and C) or
poly-L-lysin (B and D)
substrata. Double immunostaining of hippocampal neurons at 5 DIV
(A and B) and 8 DIV (C and
D) with anti-synaptophysin (red) and rabbit
anti-rat GAP43 antibodies (green).
Insets, synaptophysin immunoreactivity, derived
from corresponding micrographs. Bars, 10 µm.
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Fig. 7.
Quantification of the time course of
synaptophysin expression (A) and neurite outgrowth
(B) in primary hippocampal neurons grown on
poly-L-lysin ( ), Ig2-NCAM ( ), or C3d ( )
substrata. The data were normalized to the expression of
synaptophysin on poly-L-lysin at 8 DIV (A) or to
the average neurite length on poly-L-lysin at 8 DIV
(B).
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|
C3d Affects the Presynaptic Function in Primary
Hippocampal Neurons--
NCAM is supposed to participate in synaptic
connectivity by mediating adhesion between pre- and postsynaptic sites
and modulating a series of intracellular signaling cascades (see Ref. 3
for review). Accordingly, recent studies suggest that NCAM is involved in synaptic plasticity (19), which in part depends on the presynaptic events mediating the release of neurotransmitter, termed the
presynaptic function (20). The presynaptic function reflects the
probability of transmitter release in response to presynaptic
depolarization, and it can be directly imaged using the fluorescent
styryl dye FM1-43 (21). The dye is taken up into synaptic vesicles in
presynaptic terminals in an activity-dependent manner
(loading) as a result of endocytosis following presynaptic stimulation
and transmitter release. Subsequent rounds of exocytosis caused by
presynaptic stimulation lead to the release of dye from the presynaptic
boutons (unloading). Thus, the rate of FM1-43 unloading provides a
direct index of presynaptic function (15, 20).
To study the effect of C3d on the presynaptic function in primary
hippocampal neurons, cells were grown on a PLL substratum for 10 days
and then treated with different concentrations of C3d (2, 5, and 10 µM, dissolved in culture medium) for 3 or 48 h.
Control cultures were treated with culture medium only. Both in control
and treated cultures, FM1-43 fluorescence appeared as puncta-like
synaptic spots along dendrites (Fig. 8).
C3d significantly changed the kinetics of destaining of synapses with
KCl. Fig. 8 shows confocal images of FM1-43-stained hippocampal
cultures, treated with different doses of C3d before (A-C)
and 100 s after (D-F) the start of stimulation with
high KCl. It can be seen that both in control cultures (A
and D) and in cultures treated with 2 µM C3d
(B and D), the fluorescence of synaptic puncta
was significantly lowered after 100 s of stimulation with KCl.
Conversely, for 10 µM C3d, the decrease in fluorescence
intensity after 100 s of KCl treatment was very small (compare
C with F). From the obtained time courses, we
calculated the rate of FM1-43 unloading (Fig. 9A) and the size of the
recycling vesicle pool (Fig. 9B) at various C3d
concentrations applied for 3 or48 h. The second parameter represented a
difference in fluorescence,
F, between fully stained and
destained (300 s after the start of KCl stimulation) synaptic puncta
(15, 22). From Fig. 9, it can be seen that after 3 h of
incubation, both parameters exhibited a slight increase with 2 and 5 µM C3d, whereas a higher peptide concentration (10 µM) caused a more than 2-fold decrease in the rate of
FM1-43 unloading and the size of the recycling vesicle pool. At 48 h of incubation with C3d, a slight increase was observed for the rate
of synapse destaining at 2 µM C3d and a pronounced
inhibition at 10 µM C3d. As regards
F, only
10 µM C3d had an inhibitory effect after 48 h of
incubation. Neither the rate of synapse destaining nor
F was affected by the treatment of cultures with 10 µM
C3ala, the control peptide with two alanine substitutions and no
neuritogenic activity (1) (Fig. 9, A and B,
insets). These results indicate that the presynaptic
function was transiently increased by low to moderate concentrations of
C3d and suppressed by high peptide doses.

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Fig. 8.
Confocal images of FM1-43-stained hippocampal
cultures, treated with 2 or 10 µM
C3d for 3 h, before (A-C) and 100 s after
(D-F) start of stimulation with high KCl.
A and D, control cultures;
B and E, 2 µM C3d; C and
F, 10 µM C3d. Bars, 5 µm.
|
|

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Fig. 9.
C3d changes the total number of synaptic
vesicles released. A, dependence of the rate of puncta
destaining on the applied C3d concentration after 3 ( ) or 48 h
( ) of incubation with the peptide. Inset, the effect of
the blocking of FGFR on the rate of puncta destaining. C3d in a
concentration of 10 µM was applied for 3 h either in
the absence (C3) or in the presence (C3 + SU) of FGF receptor inhibitor SU5402, 50 µM.
The application of the control peptide with two alanine substitutions
(C3ala, 10 µM) or SU5402 alone (SU,
50 µM) had no effect. B, dependence of the
difference in fluorescence intensity ( F) between fully stained and
fully destained puncta on the applied C3d concentration after 3 ( )
or 48 h ( ) of incubation with the peptide. Inset,
the effect of the blocking of FGFR on the F. C3d in a
concentration of 10 µM was applied for 3 h either in
the absence (C3) or in the presence (C3d + SU) of FGF receptor inhibitor SU5402, 50 µM.
The application of the control peptide with two alanine substitutions
(C3ala, 10 µM) or SU5402 alone (SU,
50 µM) had no effect.
|
|
Since C3d, like NCAM itself, has been shown to activate intracellular
signaling acting via FGFR, we checked whether blocking of this receptor
would prevent the decrease in synaptic function caused by treatment
with high peptide concentrations. Indeed, the inhibitory effect induced
by 3 h of application of 10 µM C3d on both
F and the rate of synapse destaining was rescued by about 80% in the presence of the inhibitor of FGFR 1, SU5402, the
application of SU5402 alone being without effect (Fig. 9, A
and B, insets). Thus, C3d-induced modulation of
presynaptic function was dependent on FGFR.
 |
DISCUSSION |
The dynamic regulation of adhesion is critical for proper axon
growth and has been shown to take place through either
internalization/recycling (23) or shedding (24) of CAMs expressed on
the cell surface, whereas the ligand is constituted by substrate
molecules. We suggest that C3d exerts its neuritogenic effect by
forming an adhesive substratum promoting the neurite extension. First,
the neuritogenic effect of C3d depended on the time of application with
little or no neuritogenic response if the peptide was added more than 4 h after plating. The possible explanation of the observed effect of C3d is probably that neurite induction by NCAM ligands can only be
achieved when these are presented on a growth surface or associated to
the extracellular cell matrix. If C3d is added 2-4 h after seeding,
when the cells are attached to the plastic substratum and, thus, no
peptide substratum can be formed underneath, poor neuritogenesis is observed.
Second, the time course of C3d clearance from the culture medium
indicates the presence of surface adsorption with different time scales
for 1 and 10 µM C3d (Fig. 5). Low doses of the peptide may fail to form a C3d substratum sufficient for neurite promotion, whereas high concentrations of C3d will result in a saturation of the
peptide substratum and thus in a residual amount of the peptide
remaining in solution. This residual soluble peptide may competitively
disrupt the interaction between cellular NCAM and substratum-bound C3d,
thereby preventing effective neurite extension. The hypothesis is
consistent with the bell-shaped dose-response relationship for C3d
added at time 0 and is supported by the observation that the magnitude
of the neuritogenic response to C3d depends on the time of exposure to
the ligand. Indeed, at a low concentration (0.3 µM), C3d
forms a slowly saturating substratum without notable interference with
the cell-substratum adhesion. This makes an optimal exposure time as
long as 24 h. When the concentration of the peptide is higher
(3-30 µM) and decreases more slowly, a competitive
inhibition of C3d-induced neurite outgrowth is seen due to deadhesion
caused by residual soluble peptide in the medium. Therefore, the
strongest neuritogenic response for high (3-30 µM) C3d
doses is achieved when using short exposure times (1-4 h, Fig. 3),
enough for the formation of a substratum for neurite outgrowth while
avoiding interference of the soluble C3d with cellular adhesion.
According to the proposed hypothesis, repetitive application of C3d
would competitively abolish C3d-induced neurite outgrowth but not
outgrowth triggered by ligands of other adhesion molecules. Indeed, in
our experiments, C3d-triggered neuritogenesis was inhibited by
repetitive treatment with the peptide, whereas the neuritogenic response to fibronectin, a ligand of integrins, was unaffected (Fig.
4C). Thus, C3d, like NCAM itself, exerted its neuritogenic effect not only by triggering intracellular signal transduction pathways as shown before (6) but also by providing a substratum for
cellular adhesion, the latter being crucial for the induction of
neurite outgrowth.
Neurite outgrowth, initiated by C3d, resulted in the formation of
neuronal network and functional synapses (Fig. 6, A and C). Moreover, hippocampal neurons cultivated on C3d or Ig2
substrata showed accelerated synapse formation as compared with that in cultures grown onto PLL (Fig. 7). The formed synapses were functional by 6-8 DIV, as judged from the fact that they accumulated the fluorescent lipophilic dye FM1-43 during KCl-induced depolarization and
released it upon subsequent KCl-induced synaptic firing. Thus, C3d
affects the rates of both neurite outgrowth and synapse formation.
C3d has been shown to inhibit the processes of memory acquisition (9),
probably by affecting synaptic plasticity. The latter is known to be
partially dependent on the rate of presynaptic endocytosis (25). We
found that when applied in low to moderate concentrations (2-5
µM), C3d had a modest transient stimulatory effect on
presynaptic function after 3 h of incubation, whereas a high
concentration of the peptide steadily suppressed it. To our knowledge,
this is the first demonstration of an NCAM-mediated modulation of the
presynaptic function.
Synaptic plasticity in primary hippocampal neurons is associated with
cAMP/protein kinase A-dependent activation of the
transcription factor CREB, which in turn regulates gene expression
during long term potentiation (26), in particular enhancing the number
of active presynaptic terminals (27). Recent data suggest that the
cAMP/protein kinase A pathway is activated upon homophilic NCAM
stimulation (28) and upon depolarization-induced Ca2+
influx through L-type Ca2+ channels (29), resulting in CREB
phosphorylation. Treatment with C3d itself induced phosphorylation of
CREB in PC12 cells and primary hippocampal neurons (29) and increased
the intracellular Ca2+ concentration by ~20
nM at a dose of 5.4 µM (30). We suggest that
Ca2+ influx and activation of protein kinase A induced by
C3d may activate CREB, enhancing the expression of synaptic proteins, which results in synaptic strengthening.
At high concentrations, C3d has been shown to steadily increase the
intracellular Ca2+ concentration in primary hippocampal
neurons up to 170-180 nM (30), probably significantly
changing the submembrane Ca2+ concentration in presynaptic
terminals. Such a rise of [Ca2+]i may affect
interactions between CAMs and the cytoskeleton and/or activate the
intracellular protease calpain, causing rapid uncoupling of synaptic
contacts (16). The inhibitory effect of C3d on presynaptic function was
due to its signaling properties, not to a deadhesion effect per
se, since this inhibition was rescued by blocking FGF receptor
activation (Fig. 9), an essential component of NCAM-mediated signal transduction.
In conclusion, the recently identified synthetic peptide-ligand of
NCAM, C3d, induces persistent neurite outgrowth by forming an adhesive
substratum for neurite extension and promotes synapse formation in
primary hippocampal neurons. Furthermore, NCAM stimulation by C3d
modulates the presynaptic function in long term neuronal cultures,
predominantly through an interaction of NCAM with the FGF receptor. The
detailed mechanism of NCAM-mediated modulation of the presynaptic
function will need further clarification.
 |
FOOTNOTES |
*
The work was supported by grants from the Danish Medical
Research Council, The Lundbeck foundation, Vera og Carl Johan
Michaelsens Legat, The Danish Cancer Society, and the EU program (Grant
QLK6-CT-1999-02187).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
45-35-32-73-35; Fax: 45-35-36-01-16; E-mail: bock@plab.ku.dk.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M211628200
 |
ABBREVIATIONS |
The abbreviations used are:
NCAM, neural
CAM;
CAM, cell adhesion molecule;
FGF, fibroblast growth factor;
FGFR, FGF receptor;
Ras-MAPK, Ras-mitogen-activated protein kinase;
CREB, cAMP- response element-binding protein;
PLL, poly-L-lysin;
BSA, bovine serum albumin;
DIV, days in
vitro.
 |
APPENDIX |
In this section, we describe the determination of the rates
of C3d sedimentation and degradation in culture medium. Assuming that
the decrease in C3d concentration in the culture medium is caused by
two basic mechanisms (see "Results"), 1) C3d sedimentation due to
surface adsorption and 2) C3d degradation in the volume, we propose a
model sufficient for studies of local dynamics of [C3d] in the
culture medium. The local dynamics of C3d concentration in the medium
(Cf) and the concentration of the bound C3d in the
thin layer near the substratum (Cb) are described by
the following simultaneous ordinary differential equations,
|
(Eq. 1)
|
where C
is the
equilibrium concentration of the bound C3d in the thin layer near the
substratum, µM; k1 is the rate of
C3d degradation, 1/h; k2 is the rate
of C3d sedimentation, 1/µM·h; the initial
conditions are Cf(0) = C
,
Cb(0) = 0. Based on the biphasic shape of the
obtained experimental curves, we assumed that at short times (0-2 h),
the sedimentation process dominates over degradation. Therefore,
considering k1
0, we can rewrite Eq. 1 as
shown in Eq. 2.
|
(Eq. 2)
|
The solutions of this set are shown in Eqs. 3 and 4:
|
(Eq. 3)
|
and
|
(Eq. 4)
|
From the experimental data, at t = t1 = 1 h, for 1 µM C3d,
Cf(t = 0) = C
= 1 µM, Cf(t = t1) = C
= 0.84 µM; and
for 10 µM C3d, Cf(t = 0) = C
= 1 µM, Cf(t = t1) = C
= 9.2 µM.
Therefore, from Eqs. 3 and 4, we can write the equations shown in Eqs.
5 and 6.
|
(Eq. 5)
|
|
(Eq. 6)
|
With given values of the parameters
C
,
C
,
C
, C
, t1, a
solution of Eq. 5 for k2 does not exist. From
Eq. 6 comes Eq. 7.
|
(Eq. 7)
|
Solving Eq. 7 numerically, we obtain the following approximations:
C
= 0.654 µM, k2 = 0.26 1/µM·h. Defining the time constant of
sedimentation
2 so that
Cb(t =
2) = C
(e
1)/e, from Eq. 7, we derive Eq. 8.
|
(Eq. 8)
|
From Eq. 8,
2 = 5.2 h for 1 µM
C3d, and
2 = 0.4 h for 10 µM C3d.
Thus, at longer times (6-24 h), it is mainly the degradation process
that contributes to the time course of 10 µM C3d
clearance. Based on this, we can determine the unknown rate of
degradation k1. Considering that
Cb
C
at
t > 6 h, from Eq. 1, we obtain
Cm(0) = C
, Cb(0) = C
, which gives the
solution Cm = C
·exp(
k1t), Cb = C
. From this
equation we can derive the rate of C3d degradation in the culture
medium k1 using the experimental data. Defining
t6 = 6 h, t24 = 24 h, Cm(t6) = C
= 8.0 µM,
Cm(t24) = C
= 5.6 µM, obtain
k1 = ln(C
/C
)/ (t24
t6) = 0.02 h
1. Thus, the
time constant of C3d degradation in the culture medium in the absence
of cells is
1 = 1/k1 = 50 h
for both peptide concentrations, and the time constants of
sedimentation
2 = 5.2 h for 1 µM C3d and
2 = 0.4 h for 10 µM C3d.
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