1 Department of Cellular and Structural Biology, University of Colorado School
of Medicine, and University of Colorado Cancer Center, Denver, CO 80262,
USA
2 Departmento de Química Biológica, Facultad de Ciencias
Químicas, Universidad Nacional de Córdoba, and CIQUIBIC,
CONICET, Córdoba, Argentina
3 Instituto Investigación Médica Mercedes y Martín Ferreyra
(INIMEC-CONICET), Córdoba, Argentina
* Author for correspondence (e-mail: squiroga{at}dqbfcq.uncor.edu)
Accepted 20 November 2002
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Summary |
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Key words: Axonal growth, Growth cone, Membrane expansion, Regulated exocytosis, IGF-1, IGF-1 receptor, BDNF, KIF2
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Introduction |
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Our laboratories have developed two assays to study plasmalemmal expansion,
an important parameter of neurite assembly. One of these, a cell-free assay
using isolated growth cones, had demonstrated earlier that membrane insertion
at the growth cone can be triggered by depolarization and Ca2+
influx (Lockerbie et al.,
1991; Wood et al.,
1992
). The second assay involves the incorporation of fluorescent
lipid into PPVs and the study of their dynamics in growth cones of live
neurons in culture.
The present report examines the ability of the receptors for IGF-1 and BDNF to regulate plasmalemmal expansion at the growth cone and addresses the question of whether the two growth factors promote neurite outgrowth by similar or different mechanisms.
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Materials and Methods |
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Cell culture
Cultures of dissociated hippocampal pyramidal cells from embryonic rat
brain were prepared as described previously
(Mascotti et al., 1997;
Cáceres et al., 1986
).
Cells were plated onto polylysine-coated glass coverslips and maintained in
DMEM plus 10% horse serum for 1 hour. The coverslips with the attached cells
were then transferred to 60-mm Petri dishes containing serum-free medium plus
the N2 mixture (Bottenstein and Sato,
1979
). To allow neuronal survival and growth, this medium contains
a high level of insulin sufficient to stimulate the insulin as well as the
IGF-1 receptors. Cultures were maintained in a humidified 37°C incubator
with 5% CO2.
Immunofluorescence
Cells were fixed for 1 hour at room temperature with 4% (w/v)
paraformaldehyde in phosphate-buffered saline (PBS) containing 4% (w/v)
sucrose. Cultures were washed with PBS, permeabilized with 0.1% (v/v) Triton
X-100 in PBS for 30 minutes and again washed in PBS. After labeling with a
first primary antibody (1-3 hours at room temperature) and washing with PBS,
cultures were stained with labeled secondary antibody (fluorescein- or
rhodamine-conjugated; 1 hour at 37°C) and washed with PBS. The same
procedure was repeated for the second primary and secondary antibodies.
F-actin was labeled with Texas-Red-conjugated phalloidin. In most cases, the
cells were observed with a Zeiss Axiovert microscope equipped with
epifluorescence optics. Fluorescence images were digitized directly into a
Metamorph/Metafluor Image Processor (Universal Imaging Corporation, West
Chester, PA) and printed using Adobe PhotoShop. For confocal imaging, we used
a Zeiss Axiovert 200M epifluorescence microscope equipped for digital
deconvolution (Marianas System, Intelligent Imaging Innovations).
Preparation of GCPs
Growth cones particles (GCPs) were prepared as described
(Pfenninger et al., 1983;
Lohse et al., 1996
). In brief,
brains of 18-day-gestation fetal rats were homogenized. A low-speed
supernatant (LSS) was prepared, loaded onto a discontinuous sucrose density
gradient with steps of 0.83 M and 2.66 M sucrose, and spun to equilibrium at
242,000 gmax. The fraction at the load-0.83 M
interface (designated `A') contained the isolated growth cones or GCPs.
Remaining particulate elements of the LSS were collected at the 0.83-2.66 M
interface (fraction BC).
Membrane expansion assays
GCP binding of 125I-labeled wheat germ agglutinin (WGA) was used
as a measure of membrane area. WGA was radioiodinated using chloramine T and
carrier-free Na125I (Hunter and
Greenwood, 1962) in the presence of 0.2 M hapten sugar,
N-acetylglucosamine (GlcNAc). Labeled lectin was purified by
size-exclusion chromatography on Sephadex G-25 and affinity chromatography on
a GlcNAc column (Pierce Chemical, Rockford, IL). The resulting
125I-WGA had a specific activity of
0.8 µCi
µg-1 protein. This was diluted with unlabeled WGA so that each
assay tube contained
300,000 cpm at a final concentration of 5 µM WGA.
Under these conditions, WGA binding was linear; non-specific binding,
determined in the presence of 0.2 M GlcNAc, was
5%. For expansion assays,
1.3 ml of ice-cold GCP-containing band A was mixed first with 0.5 ml cold
2x dilution buffer (100 mM sucrose, 20 mM glucose, 200 mM NaCl, 10 mM
KCl, 2.4 mM NaH2PO4, 44 mM HEPES, 2.4 mM
MgCl2, pH 7.3) and, after 20 minutes, with a further 0.8 ml of the
same buffer. After an additional 20 minutes on ice, 100 µl aliquots of the
suspension were added to assay tubes containing 100 µl L-15 culture medium
plus factor, saponin or vehicle. Samples were equilibrated on ice for 30
minutes and then warmed up in a water bath to 36°C (controls were kept on
ice), typically for 6 minutes, and subsequently chilled in ice slurry for 5
minutes. Ice-cold 125I-WGA, diluted in L-15 as described above, was
added to each tube and incubation continued for 15 minutes on ice. Samples
were diluted with 200 µl cold L-15 and 300 µl aliquots loaded onto 0.5
ml cushions of 0.4 M sucrose in PBS, in siliconized conical tubes. These were
spun at 37,500 gmax for 1 hour and then frozen. The
tips containing the pellets were cut off and counted in a
-radiation
counter. Every experimental set included in triplicate (i) 0°C controls
and (ii) samples containing 0.01% (w/v) saponin that were warmed up to
36°C for 6 minutes. This allowed us to determine external labeling in
control conditions and total labeling after permeabilization, respectively.
The difference between the two measurements was the size of the internal pool,
which was used (separately for each set) as the bias for expressing membrane
externalization.
Neurons were cultured as described above, incubated for 12 hours in N2
medium without insulin but containing 50 ng ml-1 BDNF, and labeled
for 30 minutes with BODIPY-ceramide at room temperature
(Pagano et al., 1991;
Paglini et al., 2001
). After a
2.5 hour `chase' at 37°C (in the absence of label), the dissipation of the
fluorescent spots (red channel) at the growth cones was examined with the
microscope (equipped with a heated stage) in control conditions or after the
addition of 20 nM IGF-1. Other cultures were kept in the absence of exogenous
growth factors and then challenged with 50 ng ml-1 BDNF.
Gel electrophoresis and western blot
Proteins were separated by SDS/polyacrylamide-gel electrophoresis
(SDS-PAGE) (Laemmli, 1970).
The concentration of acrylamide of the resolving gel varied from 7.5% to 11%.
The resolved proteins were transferred to polyvinylidene difluoride (PVDF)
membranes (Millipore) in Tris-glycine buffer containing 20% methanol. The
filters were dried, washed with Tris-buffered saline (TBS; 10 mM Tris pH 7.5,
150 mM NaCl) and blocked for 1 hour in TBS containing 5% bovine serum albumin
(BSA). For probing blots with a single antibody, the filters were incubated
with the primary antibody in PBS containing 0.05% Tween 20 for 2 hours at room
temperature. After washing with TBS containing 0.05% Tween 20, the filters
were incubated with horseradish-peroxidase-conjugated secondary antibody
(Promega, Madison, WI) for 1 hour at room temperature. After washing, the
blots were developed using a chemiluminescence detection kit (ECL, Amersham
Life Sciences, Arlington Heights, IL).
TrkB activation in GCPs
The GCP suspension retrieved from the gradient was mixed gently with an
equal volume of `intracellular buffer' (20 mM Hepes pH 7.3, 50 mM KCl, 5 mM
NaCl, 6 mM MgCl2) and permeabilized with 0.02% ß-escin. These
GCPs were incubated for 5 minutes in the presence or absence of BDNF (0.2 nM)
on ice and then, upon the addition of 1 mM ATP, warmed up to 37°C for 1
minute or 5 minutes. The reaction was terminated by chilling samples and
adding 1% Triton X-100 plus 3 mM vanadate and a protease-inhibitor cocktail.
After 10 minutes on ice, samples were centrifuged at 30,000 g
for 1 hour to pellet Triton-insoluble cytoskeletal elements. For
immunoprecipitation of the receptor, the supernatant was incubated with
anti-TrkB antibody (5 µg ml-1) for 2 hours at 4°C before
adding protein-A/G-coated beads. The precipitates were resolved by SDS-PAGE
and blotted onto PVDF membrane. After quenching, blots were double-probed with
mouse anti-P-tyr mAb and rabbit anti-TrkB primary antibodies. Blots were
washed and then incubated with both Cy5-conjugated anti-mouse and Cy3-labeled
anti-rabbit secondary antibodies. Finally, blots were imaged with a Typhoon
9200 scanner and data collected in the red and green channels to allow for
simultaneous quantification of TrkB and Ptyr.
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Results |
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Live neurons in culture
These experiments depend on the expression of the appropriate receptors on
the growth cones under investigation. The presence of
ßgc-containing IGF-1 receptor in hippocampal neurons in
culture has been reported (Mascotti et
al., 1997). The distribution of TrkB and F-actin in growth cones
of such neurons is illustrated in Fig.
1A. This confocal image shows almost complete overlap of the two
labels, indicating the presence of TrkB in the growth cone periphery,
including the finest filopodia.
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To study membrane addition, hippocampal neurons cultured in the presence of
50 ng ml-1 BDNF or in the absence of exogenous growth factors (see
Materials and Methods) were pulse-labeled at room temperature with
BODIPY-ceramide, a fluorescent sphingomyelin and glucosylceramide precursor,
and then chased for 2.5-3 hours at 37°C
(Pagano et al., 1991). This
resulted in intense fluorescence of the Golgi complex and in the presence of
variously sized fluorescent compartments in the perikaryon, along the neurites
and, eventually, in the axonal growth cone
(Fig. 2A,B).
Fig. 3 shows the labeled
compartments in the growth cone in a confocal image at higher resolution.
BODIPY-ceramide has a concentration-dependent emission spectrum, with a
maximum at 515 nm (green) at low concentration. In the growth cone in
Fig. 3A, green label is evident
in the plasma membrane, extending into the filopodia and lamellipodia. At high
concentration, BODIPY-ceramide forms excimers with an emission maximum at
620 nm (red). This enables selective visualization of Golgi-derived
vesicles. Hence, all membrane addition experiments were recorded in the red
channel (Figs 2,
4). We attempted to correlate
the Golgi-derived compartments in growth cones with structures detectable in
phase-contrast images (Fig.
3B). However, there was no obvious correlation between the
elements labeled red and specific structures discernible under phase
contrast.
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In pulse-chase experiments, in control medium or upon challenge with 50 ng
ml-1 BDNF, the fluorescent spots in the distal axon persisted for
extended periods of time (>14 minutes). Upon challenge with IGF-1 (20 nM),
however, fluorescent puncta in growth cones dissipated relatively rapidly
(Fig. 4), whereas those in the
perikaryon and axon shaft seemed unaffected by the challenge. This observation
was quantified by counting fluorescent spots in each growth cone as a function
of time after onset of the challenge and by expressing the numbers normalized
to 1 at the onset of challenge (Fig.
5). Fluorescent puncta under control and BDNF conditions exhibited
a half life (t1/2) of >14 minutes, whereas the
t1/2 for IGF-1 was only 6 minutes following
challenge. In other words, IGF-1, but not BDNF, accelerated the dissipation of
ceramide label from plasmalemmal precursor compartments at the growth
cone.
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Isolated growth cones
To determine directly and quantitatively whether growth factors regulated
membrane addition in growth cones, we used fetal forebrain GCPs in our
cell-free membrane expansion assay
(Lockerbie et al., 1991). This
membrane expansion assay measures membrane externalization from the internal
vesicular pool as an increase in exposed glycoconjugates. First, we determined
the internal pool of WGA binding sites in GCPs by comparing intact,
unstimulated GCPs to permeabilized fractions. We observed that the internal
pool varied from
20% to
50% of total binding sites from experiment
to experiment (mean±s.d.=34.7 ±13.5%; n=7). Therefore,
this pool was measured for each experiment, and the changes in externally
exposed WGA binding sites were expressed as a percentage of that pool.
Fig. 6 shows dose-response
curves of these changes for IGF-1 and BDNF. It is evident that BDNF did not
affect WGA labeling over a wide concentration range. By contrast, IGF-1 caused
a dose-dependent increase in WGA labeling that reached
40% of the
internal pool at
1 nM. The effect was blocked on ice and was time
dependent, with an optimal response between 5 minutes and 10 minutes at
36°C (data not shown). It follows that IGF-1, but not BDNF, stimulates
externalization of WGA binding sites in forebrain GCPs.
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The apparent lack of a growth cone response to BDNF raises the question of
whether its receptor, TrkB, is present and/or functionally intact. In order to
address this question, we prepared subcellular fractions from 18-day fetal rat
brains and probed western blots with antibodies to TrkA, TrkB and TrkC
(generous gift of D. R. Kaplan). All Trk proteins were detectable (data not
shown) (Knüsel et al.,
1994) but TrkB immunoreactivity was the most abundant.
Fig. 4 shows TrkB
immunoreactivity at
140 kDa, the size of the full-length receptor
(Chao, 1992
). TrkB was
enriched increasingly as one moved from homogenate to LSS and then to its
derivative fraction, the GCPs. Densitometric analysis of a representative
experiment indicated an increase of >80% in GCPs compared with the
homogenate. The low-speed pellet (LSP), containing large cell fragments,
exhibited a similar amount of immunoreactivity to the homogenate. By contrast,
the LSS subfraction BC was depleted to about one-half of the immunoreactivity
seen in LSS. Because of the primarily axonal origin of GCPs
(Saito et al., 1992
;
Lohse et al., 1996
), these
blots indicate enrichment of intact TrkB in axonal growth cones isolated from
rat forebrain. To ascertain that TrkB was indeed activatable in GCPs, we
stimulated the receptor with BDNF, immunoprecipitated it using an anti-TrkB
antibody and probed blots with both anti-TrkB and anti-P-tyr antibodies, using
two different chromophores as reporter groups
(Fig. 7B). TrkB labeling (Cy3
chromophore; top) reveals that equal amounts of the receptor were loaded in
each lane of this gel. Tyrosine phosphorylation of the receptor (Cy5
chromophore; bottom), however, is quite different in the three lanes:
Comparison of controls at 1 minute and 5 minutes indicates some kinase
activity without the addition of exogenous BDNF. In the presence of BDNF (5
minutes), however, there is a 2.5-times increase in P-tyr relative to the
corresponding control. Thus, TrkB in GCPs can indeed be activated by BDNF.
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Effects of BDNF on neurite growth and distribution of
ßgc
The lack of BDNF stimulation of plasmalemmal expansion in our assays
prompted us to examine the effects of BDNF on neurite growth in our cultured
hippocampal neurons. To assess the degree of differentiation, a neuron was
considered to be in stage 3 when the length of one of the processes (the axon)
exceeded the average length of the other processes (the minor processes) by at
least 20 µm (Cáceres et al.,
1986; Dotti et al.,
1988
); without a discernible axon, neurons were in stage 2. In the
cultures grown without BDNF,
65% of the neurons were in stage 2 after 36
hours of differentiation in vitro (DIV). In the presence of 50 ng
ml-1 of BDNF, however, almost 80% of the cells were in stage 3 (36
hours of DIV), indicating that BDNF significantly accelerated the rate of axon
growth and neuronal polarization in our experimental system. This was borne
out in measurements of axon length (Table
1). BDNF nearly doubled axon length over a 36-hour growth period.
These differences are evident in Fig.
8A,C vs Fig.
8E,G.
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We have reported previously (Quiroga et
al., 1995) that ßgc is highly enriched in GCPs
isolated from fetal brain. In hippocampal neurons in culture, however, growth
cone enrichment of ßgc was transient and decreased
significantly before the cells began dendritic differentiation. This suggested
that the lack of a trophic factor in the cultures might have been responsible
for the decline in ßgc. To test this hypothesis, hippocampal
neurons were cultured in the presence or absence (control cultures) of 50 ng
ml-1 BDNF and then labeled with antiserum to ßgc.
The control cells developed several minor processes within 24-36 hours DIV and
a relatively short axon (Fig.
8A,C) with a weakly ßgc-positive growth cone
(Fig. 8B,D). Cells cultured for
24 hours in the presence of BDNF (Fig.
8E,F) were similar to controls. After 36 hours in culture with
BDNF, however, a significant increment in ßgc immunostaining
was evident in the distal axons and growth cones
(Fig. 8D,H).
ßgc immunoreactivity in distal axons was measured in 36-hour
cultures and the data are shown in Table
1. The results demonstrate that BDNF increased the fluorescence
intensity of ßgc more than fivefold in the distal third of the
axon, including the growth cone.
Effects of BDNF and KIF2 expression
The increase in ßgc in the distal axon could be the result
of enhanced ßgc production and/or redistribution by export
from the perikaryon into the axon. Surprisingly, western blots failed to
reveal an increase in ßgc immunoreactivity in hippocampal
neurons cultured with BDNF (Fig.
9). By contrast, BDNF greatly enhanced the expression of the
central-domain microtubule motor KIF2 (Fig.
9). Thus, KIF2 might be responsible for ßgc
redistribution during axonal differentiation. To test this hypothesis, we
cultured hippocampal pyramidal cells in the presence of both BDNF and an
antisense oligonucleotide to KIF2 (asKIF2). Western blots of
total extracts of such cultures demonstrated significantly diminished KIF2
production compared with controls. However, asKIF2 did not affect
cell survival and/or the expression of other proteins such as tyrosinated
-tubulin, ßgc, kinesin heavy chain and synaptophysin
(data not shown) (see Morfini et al.,
1997
). Immunofluorescence shows that asKIF2 substantially
reduced axon outgrowth (Fig.
8G,I) and caused the absence of ßgc in the distal
axon (Fig. 8J,H). The effects
are shown quantitatively in Table
1. In the presence of BDNF and asKIF2, both axonal length
and ßgc fluorescence intensity in the distal third of the axon
were attenuated significantly compared with those in cultures treated with
BDNF alone. Thus, ßgc distribution to the axonal growth cone
is KIF2-dependent.
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Discussion |
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Regulation of plasmalemmal expansion by trophic factors
The present results extend our earlier observations on the role of
depolarization and Ca2+ influx in plasmalemmal expansion to the
action of growth factors in both intact neurons and isolated growth cones. As
expected, BODIPY-ceramide incorporation into sphingomyelin and its
glycosylation to glucosylceramide (Pagano
et al., 1991) result in strong, stable labeling of the Golgi
complex. Intense red fluorescence indicates a high concentration of the label
in this organelle. Labeled smaller compartments, presumably vesicles and their
clusters, emerge from the Golgi and begin to appear at the growth cones within
2 hours. Excimer formation by the BODIPY label and recording in the red
channel enable the selective imaging of these Golgi-derived membranes.
Although overlap of these compartments with specific phase-dense or
phase-bright structures was not evident, their overall distribution is
consistent with that of PPV clusters labeled with another lipid precursor,
glycerol (Pfenninger and Friedman,
1993
), as expected. The time-lapse observations shown here reveal
that IGF-1, but not BDNF, accelerates the disappearance of Golgi-derived,
BODIPY-labeled structures from the growth cones. This is consistent with the
coalescence into the plasmalemma of vesicles or vesicle clusters visible
initially as bright red spots owing to their high BODIPY content. Upon
plasmalemmal insertion, the BODIPY label becomes diluted into a much larger
lipid pool and so the fluorescent signal in the red channel disappears and
shifts to green.
These fluorescence microscopic observations document the regulated
`disappearance' of BODIPY-labeled structures at the growth cone but do not
provide direct evidence about membrane insertion. Therefore, we complemented
these experiments with an assay of membrane externalization using GCPs. We
have characterized this cell-free assay in detail
(Lockerbie et al., 1991) and
shown that changes in WGA binding (e.g. induced by Ca2+ influx)
reflect changes in Bmax (maximum lipid bound) not in
KD. We have shown, furthermore, that
depolarization-induced externalization of WGA binding sites is paralleled by
an ultrastructurally measurable and comparable decrease in the membrane area
of internal precursor vesicles (operationally defined as vesicles <180 nm
in diameter). This earlier characterization of the assay readily applies to
the experiments presented here. BDNF was used over a wide concentration range
(0.01-100 nM) in order to test whether it triggered externalization of WGA
binding sites. However, WGA binding was not affected in these assays, even
though western blots had demonstrated the presence and enrichment of intact
TrkB, as well as its activation, in the same GCP preparation. By contrast,
IGF-1 stimulated the externalization of WGA binding sites in a dose-, time-
and temperature-dependent manner. A maximal effect was observed at
1 nM
(the KD of the IGF-1 receptor was 0.75 nM) and amounted to
40% externalization of the internal pool. This value was comparable to,
or higher than, the numbers obtained for K+ depolarization and
Ca2+ ionophore treatment
(Lockerbie et al., 1991
). It
follows from both sets of experiments that insertion into growth-cone
plasmalemma of PPVs is a regulated phenomenon and stimulated by the growth
factor IGF-1, but not by BDNF.
BDNF and redistribution of ßgc
BDNF was discovered as a neuronal survival, differentiation and
axonal-elongation factor (Davies et al.,
1986; Hofer and Barde,
1988
). Our data confirm in our experimental system that BDNF
increases the axonal growth of hippocampal pyramidal neurons in culture almost
twofold. However, unlike IGF-1, BDNF does not promote outgrowth by stimulating
plasmalemmal assembly at the growth cone. Instead, our data show that it
affects IGF-1 receptor distribution. Without exogenous BDNF,
ßgc appears in our cultures only transiently. BDNF is required
for, and greatly stimulates, the sustained distal axonal enrichment of
ßgc. These results parallel our previous finding that
enrichment of ßgc in the distal neurites of PC12 cells is
tightly regulated by nerve-growth factor (NGF)
(Mascotti et al., 1997
).
Together these observations suggest that neurotrophins of the NGF family
regulate IGF-1-receptor distribution in the growing neuron.
Experiments performed in whole cells or with cell extracts indicate that
KIF2 is a plus-end-directed microtubule-based motor protein (Noda et al.,
1994; Morfini et al., 1997;
Santama et al., 1998
). BDNF
causes a striking increase in KIF2 protein level in hippocampal pyramidal
cells, together with the distal axonal enrichment of ßgc.
Conversely, transfection of neurons with asKIF2 blocks distal
accumulation of ßgc and reduces axonal growth. These results
suggest that ßgc reaches the axonal growth cone by
KIF-dependent anterograde transport that is regulated by BDNF.
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Conclusions |
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The growth cone receives BDNF signals, as indicated by the presence of
full-length, activatable TrkB. Interestingly, the distribution of TrkB at the
growth cone is different to that of the ßgc-containing IGF-1
receptor. TrkB is present throughout the growth-cone periphery, together with
filamentous actin. By contrast, ßgc is located primarily in
the proximal growth cone, together with microtubules
(Mascotti et al., 1997). The
significance of this different receptor distribution is not known, but the
distinct patterns are likely to be relevant to the receptors' different
biological effects. Neurotrophins have been established as target-derived
polypeptides that promote neuron survival via retrograde transport from the
nerve terminal to the perikaryon (Reynolds
et al., 2000
). However, it has been suggested that the temporal
and spatial characteristics of many effects of BDNF in the central nervous
system are more consistent with local actions than with signaling mediated by
long-distance retrograde transport (e.g.
Altar et al., 1997
). Indeed,
neurotrophins might act as chemoattractants and have been implicated in the
modulation of growth-cone responses to guidance molecules
(Campenot, 1977
;
Paves and Saarma, 1997
;
Tuttle and O'Leary, 1998
;
Ming et al., 1999
;
Gallo and Letourneau, 2000
).
Yet, at least in the two assays used here, BDNF does not directly influence
membrane addition at the growth cone. Our findings suggest instead an indirect
effect. By controlling the distribution of ßgc, BDNF probably
influences distal growth regulation via retrograde signaling to the
perikaryon, the synthesis of KIF2 (and probably other proteins) and increased
transport of ßgc to the axonal growth cone.
Overall, our studies support the hypothesis that the actions of BDNF and IGF-1 on the growth cone are different but interdependent. They seem to occur in sequence, with BDNF regulating the distribution of ßgc-containing IGF-1 receptors. Once the IGF-1 receptor is present in the distal axon, IGF-1 directly regulates membrane assembly and outgrowth at the growth cone.
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Acknowledgments |
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