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The Journal of Neuroscience, July 1, 1998, 18(13):4973-4984
cAMP-Mediated Regulation of Neurotrophin-Induced Collapse of
Nerve Growth Cones
Qun
Wang and
James Q.
Zheng
Department of Neuroscience and Cell Biology, University of Medicine
and Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854
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ABSTRACT |
Neurotrophins are known to promote the survival, differentiation,
and neurite outgrowth of developing neurons. Here we report that
acutely applied brain-derived neurotrophic factor (BDNF) induces rapid
growth cone collapse and neurite retraction of embryonic Xenopus spinal neurons in culture. The collapsing effect
of BDNF depends on the activation of Trk receptor tyrosine kinase,
requires an influx of extracellular Ca2+, and is
regulated by cAMP-dependent activity. Elevation of intracellular cAMP
levels ([cAMP]i) by forskolin or
(Sp)-cAMP completely blocked the
collapsing effect, whereas inhibition of protein kinase A (PKA) by
(Rp)-cAMP potentiated the collapsing
action. BDNF-induced growth cone collapse was only observed in 6 hr
cultures but not in 24 hr cultures. However, inhibition of PKA by
(Rp)-cAMP restored the collapsing
response of these "old" neurons in 24 hr cultures, suggesting that
embryonic Xenopus spinal neurons may upregulate their
endogenous cAMP-dependent activity during development in culture,
leading to the blockade of their collapsing response to BDNF. Taken
together, our results suggest the presence of cross-talk between
Ca2+- and cAMP-signaling pathways involved in the
collapsing action of neurotrophins, in which the cAMP-pathway regulates
the Ca2+-mediated signal transduction required for
BDNF-induced collapse. By modulating the cAMP-dependent activity
through the intrinsic programming or interaction with other factors
present in the environment, a neuron thus could respond to the same
extracellular factors with different morphological and cellular changes
at different stages during development.
Key words:
growth cone; collapse; neurotrophin; calcium; cAMP; guidance; repulsion
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INTRODUCTION |
Formation of neuronal circuitry
during development requires the growth and guidance of developing axons
to their correct target cells. This process depends on the response of
the growing tip of an axon, the growth cone, to a variety of
extracellular factors present in developing embryos, including
surface-bound as well as diffusible molecules (Bray and Hollenbeck,
1988 ; Keynes and Cook, 1995a; Tessier-Lavigne and Goodman, 1996).
Guidance by positive cues that promote axonal extension and attract
growth cones has been considered the main mechanism in vivo.
However, recent studies have established an equally important role for
inhibitory or repulsive cues in axonal guidance (Luo and Raper, 1994;
Pini, 1994; Dodd and Schuchardt, 1995; Keynes and Cook, 1995b;
Tessier-Lavigne and Goodman, 1996). By inhibiting axonal growth in
"wrong" directions and by collapsing the growth cones entering
inappropriate regions, these molecules can repel elongating axons and
thus help them to reach the correct destination (Pini, 1993; Fan and
Raper, 1995; Messersmith et al., 1995; Püschel et al., 1995).
Neurotrophins are a family of neurotrophic factors with profound
influence on neuronal proliferation, survival, and differentiation (for
review, see Barde, 1990; Thoenen, 1991; Davies, 1994b; Klein, 1994;
Snider, 1994; Lindsay, 1996). Recent studies have revealed a number of
novel biological actions of neurotrophins on various aspects of
developing nervous systems, including axonal (Diamond et al., 1992;
Cohen-Cory and Fraser, 1995) and dendritic (Cohen-Cory et al., 1991;
McAllister et al., 1995, 1996) morphology, synaptic activity (Lohof et
al., 1993; Kim et al., 1994; Kang and Schuman, 1995; Levine et al.,
1995; Figurov et al., 1996) and maturation (Wang et al., 1995), and
synaptic patterning (Cabelli et al., 1995). A role for neurotrophins in
the regulation of axonal growth is supported by the remarkable ability
of neurotrophins to promote neurite outgrowth of sensitive populations
of neurons (Snider and Johnson, 1989; Kuffler, 1994; Lundborg et al.,
1994). Furthermore, the chemoattractive effect of nerve growth factor
(NGF) was demonstrated previously in vitro (Letourneau,
1978; Gundersen and Barrett, 1980) as well as in vivo
(Menesini-Chen et al., 1978), suggesting a role for neurotrophins in
axonal guidance. Despite subsequent evidence that NGF is not
responsible for long-range growth cone guidance (Lumsden and Davies,
1983; Davies et al., 1987), a role for NGF in local regulation of
axonal growth has been proposed (Gallo et al., 1997). Recent findings
that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)
are expressed relatively early during development (Maisonpierre et al.,
1990; Hallböök et al., 1993) further raise the possibility
for neurotrophins to play a role in the early development of the
nervous system. The demonstration of chemoattractive effects of BDNF
and NT-3 in culture (Ming et al., 1997; Song et al., 1997) suggests
that they could be candidates involved in axonal growth and
guidance.
We now report a collapsing effect of neurotrophins on growth cones of
embryonic Xenopus spinal neurons in early stages in culture.
Acute application of BDNF induces rapid growth cone collapse followed
by neurite retraction of Xenopus neurons in 6 hr cultures. The collapsing effect of BDNF depends on the activation of Trk receptor
tyrosine kinase and requires an influx of extracellular Ca2+. Furthermore, the
Ca2+-mediated BDNF-induced collapse is regulated by
cytosolic cAMP-dependent activity; elevation or inhibition of
cAMP-dependent activity blocked or enhanced the collapsing effect,
respectively. Such cAMP-dependent regulation is found to be responsible
for the disappearance of the collapsing effect of BDNF on neurons in 24 hr cultures. Our results suggest that neurotrophins may play important
roles in the regulation of growth cone motility and extension; such
regulation may well depend on the developmental stages of the neurons
and could be influenced by other extracellular factors that modulate the cytosolic cAMP activity, leading to differential responses of the
neuron to the same factor.
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MATERIALS AND METHODS |
Cell culture. Cultures of embryonic
Xenopus spinal neurons were prepared according to procedures
reported previously (Spitzer and Lamborghini, 1976; Tabti and Poo,
1990). The neural tube tissue from developing embryos at stage 20-22
(Nieuwkoop and Faber, 1967) was dissociated in a
Ca2+- and Mg2+-free Ringer's
solution supplemented with EDTA (in mM: 115 NaCl, 2.5 KCl,
10 HEPES, and 0.5 EDTA, pH 7.6), plated on clean glass coverslips, and
incubated at room temperature (20-22°C). The culture medium
consisted of 50% (vol/vol) Leibovitz medium (Life Technologies, Gaithersburg, MD), 1% (v/v) fetal bovine serum (Life Technologies), and 49% (v/v) Ringer's solution (in mM: 115 NaCl, 2 CaCl2, 2.5 KCl, and 10 HEPES, pH 7.6).
Neurotrophins and chemicals. Human recombinant BDNF and NGF
were generously provided by Regeneron Pharmaceuticals, (Tarrytown, NY).
All neurotrophins were aliquoted at 10 mg/ml and stored at  85°C.
Working stock solutions of neurotrophins at 100 µg/ml were prepared
and used within 1 week. Neurotrophins at working concentrations were
prepared in culture medium before each experiment. A water-soluble derivative of forskolin
(7-deacetyl-7-(O-N-methylpiperazino)- -butyryl-dihydrochloride), K252a, (Rp)-cAMP,
(Sp)-cAMP, and staurosporine were all
purchased from CalBiochem (La Jolla, CA). Cytochalasin D was purchased
from Sigma (St. Louis, MO).
Collapsing experiments. Most of the experiments were
performed on a Nikon TMS inverted microscope equipped with
phase-contrast optics and a 20× objective (Nikon, Tokyo, Japan). The
images of individual neurons were acquired through a  inch
CCD video camera (Coordinated Systems, East Hartford, CT) and digitized
by a SNAPPY video digitizer (Play, Rancho Cordova, CA). For
high-resolution imaging, cells grown on a glass coverslip were mounted
on a microscopy chamber using silicon vacuum grease (Dow Corning,
Midland, MI) and examined on a Nikon Diaphot 300 inverted microscope
equipped with differential interference contrast (DIC) optics and a
40×, numerical aperture 1.3 oil-immersion objective. A 1/2 inch
CCD video camera (C2400-75i; Hamamatsu Photonics System, Bridgewater,
NJ) was used for video imaging in conjunction with an Argus-20 image
processor (Hamamatsu) for image enhancement. The video images were
background-subtracted, averaged over four video frames, and
contrast-enhanced in real time using Argus-20. The enhanced video
images were digitized and acquired by a personal computer at a standard
rate of one frame every 2 min, although faster rates were sometimes
used to examine the dynamic changes of growth cone morphology. For all
the experiments, a control period of 5 min observation was performed on
each neuron to assess the normal neurite extension before the
neurotrophin addition. Neurotrophins at their final working
concentrations were applied to the culture by rapid perfusion of the
culture medium. Immediately after the perfusion, 10 min of time-lapse
recording at the standard rate was performed. For each pharmacological
treatment, the cells were pretreated for 20 min with various chemicals
before the addition of neurotrophins, and the effect of these chemicals
alone on growth cone extension was monitored during the 20 min
pretreatment. Only those chemicals that did not significantly affect
growth cone extension were used for further experiments.
Image analysis was performed using the ImageTool program (developed at
the University of Texas Health Science Center, San Antonio, TX, and
available from the Internet by anonymous file transfer protocol from
ftp://maxrad6.uthscsa.edu). The lengths of neurite processes were
measured by tracing the entire trajectory of neurite extension,
including all major branches. Because of the dynamic nature of
filopodia and lamellipodia, they were not included in the length
measurement. Therefore, the length measurement for each neurite process
ends at the center of the phase-dark "palm" of the growth cone.
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RESULTS |
Neurotrophin collapsing effects
Isolated spinal neurons in 6 hr Xenopus cultures were
used for these experiments. Active neurite extension at a rate of ~15 µm/hr is observed in these cultures (Zheng et al., 1996b). Time-lapse video imaging was performed to monitor the changes in growth cone motility and neurite lengths before and after the addition of BDNF. In
a typical experiment, a cell was monitored for 5 min before and 10 min
after BDNF application. BDNF was applied to the culture by rapid
perfusion with the culture medium containing the final concentration of
BDNF. Exposure of cultured Xenopus neurons to 50 ng/ml BDNF
caused rapid collapse of growth cones and subsequent withdrawal of
neurite processes (Fig. 1). The
collapsing response was observed as early as 1 min after BDNF addition,
and the maximal neurite retraction was observed within the next 4 min.
Whereas some neurons responded to BDNF by drastically withdrawing their
processes (Fig. 1A), others displayed moderate
neurite retraction and were able to resume extension after the washout
of BDNF (Fig. 1B). Growth cones undergoing drastic
collapse appeared to maintain some adhesion to the substratum, as
evidenced by the thin membrane traces left behind (Fig.
1A, double arrowheads), suggesting that the collapse
did not result from a complete loss of cell adhesion. The growth cone
collapse induced by BDNF is accompanied by a number of morphological
changes at the growth cone, most notably including the transient loss
of filopodia (Fig. 1, arrows) and the later appearance of
lamellipodial protrusion (arrowheads), an effect of BDNF
that was also observed in 24 hr cultured Xenopus spinal neurons (Ming et al., 1997).
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Figure 1.
BDNF-induced collapse of growth cones of cultured
embryonic Xenopus spinal neurons. Time-lapse DIC
sequences showing growth cone collapse followed by dramatic
(A) or moderate (B) neurite
retraction induced by BDNF. Numbers represent minutes at
various times before (negative numbers) and after
(positive numbers) the application of 50 ng/ml
BDNF (at time 0). In B, BDNF was washed out at 10 min
after the application, and neurite extension was observed thereafter.
Note the transient loss of filopodia (arrows) after BDNF
application and the appearance of lamellipodial protrusion at later
times (arrowheads). In A, also note that
some adhesion remained during growth cone collapse (double
arrowheads). Dashed lines depict the positions
of the growth cones at various times. Scale bar, 15 µm.
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To quantify the collapsing effect, we directly measured the length of
each neurite process at the beginning and at various times after the
addition of BDNF. A growth cone is considered exhibiting collapsing
response when it displays the morphological changes described above and
withdraws at least 10 µm (approximately the average size of
Xenopus growth cones) at any time point during the 10 min
observation after exposure to BDNF. The percentage of growth cones
exhibiting collapse was determined and used to assess the overall
responsiveness of Xenopus growth cones to neurotrophins (Fig. 2A). To further
quantitatively analyze the collapsing response of growth cones from a
population of neurons, the length of each neurite process at various
times after BDNF addition was measured and normalized against the
length before BDNF application and presented as the percentage of
original length (POL). POL did not exhibit a normal distribution;
therefore, results from populations of neurons are presented as box
whisker plots to illustrate the overall response from a population of
cells (Fig. 2B). The boxes enclose the 25th and 75th
percentiles, the horizontal lines mark the median, and the error bars
denote the 10th and 90th percentiles of the distributions. The mean POL
at various times after BDNF application is also presented on the same
graph as symbol line plots. The majority of neurons responded to
acutely applied BDNF by collapsing their growth cones followed by
neurite withdrawal within 4 min after BDNF addition; the neurite
retraction induced by BDNF was attenuated thereafter. Although some
neurons largely withdrew their neurite processes within the 10 min
period (indicated by the small POL at the 25th percentile: 41% at 4 min and 49% at 10 min after BDNF addition), most neurons only showed
growth cone collapse with a small amount of neurite retraction (median POL, 88% at 4 min and 80% at 10 min), indicating selective
responsiveness by different neurons. The collapsing effect seems to be
specific for BDNF. The related factor, NGF, at the same concentration
was ineffective in inducing collapse of the majority of growth cones (35% of growth cones showing collapse, compared with 60% for BDNF; see Fig. 2A). This is further evidenced by the near
100% median POL after NGF addition (Fig. 2C; median POL,
97% at 4 min and 99% at 10 min), which is significantly higher than
that of BDNF alone (*p < 0.05 when compared at
corresponding time points, Mann-Whitney test). The collapsing effect
of BDNF appears to be reversible. Although regrowth of these retracted
processes was not observed during the 10 min period, retracted neurite
processes did reextend after BDNF washout (Fig.
2D).
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Figure 2.
Quantitative analysis of growth cone collapse
induced by neurotrophins. A, The percentage of growth
cones collapsed in response to various neurotrophins is determined by
measuring the lengths of each neurite at various times after the
addition of neurotrophins. A growth cone is considered to exhibit
collapse when it withdraws at least 10 µm, in addition to the
morphological changes described in Results. B-F, The
growth cone collapse was quantified by measuring the lengths of each
neurite at various times after the addition of neurotrophins,
normalized against the lengths before the neurotrophin addition, and
presented as the percentage of original length (POL). POL did not
follow a normal distribution; therefore, the data are presented as the
box whisker plots. The boxes enclose the 25th and 75th
percentiles; the horizontal lines mark the median; and
the error bars denote the 10th and 90th percentiles of
the distributions. Circles represent the mean POL.
B, Acute application of 50 ng/ml BDNF induced rapid
growth cone collapse and neurite retraction, but the related factor NGF
had little effect on the neurite length
(C). When compared with the POL of BDNF
(B) at corresponding times after the neurotrophin
addition, NGF caused significantly less collapse than BDNF
(*p < 0.05, Mann-Whitney test). D,
BDNF-induced growth cone collapse is reversible. When BDNF was washed
out at the end of 10 min application, growth cones recovered and
resumed extension. E, F, BDNF collapsing effect seems to
be mediated by Trk receptor tyrosine kinases. Incubation of cells with
200 nM K252a (E) or 100 nM staurosporine (F) completely
blocked BDNF-induced collapse [*p < 0.0001, Mann-Whitney test, compared with the POL of BDNF alone
(B) at corresponding times after BDNF addition].
Numbers indicate the numbers of cells (growth cones)
examined.
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Involvement of Trk receptor tyrosine kinase
Most of the biological effects of neurotrophins are believed to be
mediated by the high-affinity Trk receptor tyrosine kinases: NGF
activates TrkA, BDNF and NT-4 activate TrkB, and NT-3 activates TrkC
(for review, see Chao, 1992; Barbacid, 1994; Dechant et al., 1994).
Binding of neurotrophins to Trk receptors induces dimerization of the
receptors followed by autophosphorylation, leading to cascades of
signaling events (Kaplan and Stephens, 1994; Barbacid, 1995). The
growth cone collapsing effect of BDNF appeared to be mediated by Trk
receptors, because it was completely blocked by 200 nM K252a (Fig. 2A,E) or 100 nM staurosporine
(Fig. 2A,F). These two potent, structurally
related inhibitors for a broad range of serine and threonine
protein kinases have been shown to effectively block the activity of
Trk receptor tyrosine kinases (Knusel and Hefti, 1992; Nye et al.,
1992; Tapley et al., 1992). All neurotrophins also bind to a
low-affinity receptor p75, which could modulate Trk activity (Chao and
Hempstead, 1995). The potential contribution of p75 receptors in the
BDNF-induced growth cone collapse remains to be elucidated.
Ca2+ mediates the collapsing effect
Activation of Trk receptor tyrosine kinases by neurotrophins
is known to elicit a range of second messenger responses, including increases in intracellular Ca2+ (Nikodijevic and
Guroff, 1991; Berninger et al., 1993; De Bernardi et al., 1996; Stoop
and Poo, 1996), cAMP (Knipper et al., 1993a,b), cGMP (Laasberg et al.,
1988), and phosphoinositide turnover (Contreras and Guroff, 1987), as
well as to activate the protein kinases Src, Raf, and the GTP-binding
protein Ras (D'Arcangelo and Halegoua, 1993; Heumann, 1994; for
review, see Kaplan and Stephens, 1994; Greene and Kaplan, 1995; Segal
and Greenberg, 1996). Ca2+ and cAMP are two
important second messengers that are known to affect growth cone
motility and behavior (Kater and Mills, 1991; Kim and Wu, 1996). To
test whether Ca2+ is involved in BDNF-induced growth
cone collapse, we removed extracellular Ca2+ by
using a Ca2+-free medium (in mM: 115 NaCl, 2.5 KCl, 2 MgCl2, 1 EGTA, and 10 HEPES, pH
7.4). BDNF-induced growth cone collapse was completely blocked in the
Ca2+-free medium (Fig.
3A-C). Results from a
population of neurons show that growth cones actually extended in the
Ca2+-free medium during the 10 min exposure to BDNF
(Fig. 3C). The possibility that the removal of extracellular
Ca2+ may have interfered with the activation of
Trk receptors by BDNF is argued against by the finding that
BDNF-induced lamellipodial protrusion was still observed in the
Ca2+-free medium (Fig. 3A,B; also see
Ming et al., 1997), suggesting that the action of
Ca2+-free medium on collapse is likely downstream of
the receptor activation. To further test whether an influx of
Ca2+ is involved in BDNF-induced growth cone
collapse, we loaded the cells with BAPTA (10 µM BAPTA AM
for 20 min), a rapid Ca2+ chelator (Tsien, 1980),
before the application of BDNF. Loading neurons with BAPTA appeared to
slow down the growth cone extension but did not cause growth cone
collapse or retraction. However, the BDNF collapsing effect was
diminished by BAPTA (Fig. 3D). Taken together, our results
suggest that an influx of extracellular Ca2+ is
required for the collapse, and that the Ca2+
signaling pathway mediates the collapsing effect of BDNF.
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Figure 3.
Ca2+ mediates the collapsing
effect of BDNF. A, Representative images showing the
changes induced by BDNF (50 ng/ml) in Ca2+-free
medium. No growth cone collapse and neurite retraction were observed
after BDNF addition. However, BDNF-induced lamellipodial protrusion was
still observed (arrows). Numbers
represent minutes after BDNF addition. Dashed lines
indicate corresponding positions along the neurite. B,
BDNF-induced lamellipodial protrusion was observed not only at the
growth cone (arrow) but also along the neurite shaft
(arrowhead). The image was taken 10 min after BDNF
application. C, Data from populations of cells in the
Ca2+-free medium are presented as a
box whisker plot. Apparently no collapse was observed after BDNF
application. D, Loading cells with 10 µM
BAPTA AM for 20 min also blocked the collapsing effect of BDNF. No
substantial changes in neurite length were observed. *Significantly
different from the POL of BDNF in culture medium (Fig.
2B) at corresponding times after BDNF addition
(p < 0.001, Mann-Whitney test).
Numbers in C and D
indicate the numbers of cells (growth cones) examined. Scale bars, 10 µm.
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cAMP regulates the collapsing effect
We further tested the role of cAMP in BDNF-induced growth cone
collapse. Application of 100 µM
(Rp)-cAMP, a competitive inhibitor of
protein kinase A (Rothermel and Parker Botelho, 1988; Dostmann et al.,
1990), did not affect normal neurite extension, nor did it inhibit
growth cone collapse induced by 50 ng/ml BDNF (Fig. 4B). Approximately 66%
of (Rp)-cAMP-treated growth cones were collapsed by the addition of BDNF, a percentage not significantly different from that observed for growth cones exposed to BDNF alone
(60%; see Fig. 2A). However, the collapsing effect
of BDNF was completely blocked by 10 µM water-soluble
derivative of forskolin (Fig. 4C, *p < 0.05, Mann-Whitney test), an agent known to stimulate membrane-bound
adenylate cyclase, resulting in an elevation of [cAMP]i
(Laurenza et al., 1987). To further confirm that forskolin blockade of
the collapse is directly associated with an elevation of
[cAMP]i, we applied
(Sp)-cAMP, a membrane-permeant cAMP
analog that is resistant to hydrolysis (Rothermel and Parker Botelho, 1988; Dostmann et al., 1990). Although not as effective as forskolin, application of 500 µM
(Sp)-cAMP blocked BDNF-induced growth
cone collapse (Fig. 4D, *p < 0.05, Mann-Whitney test). The blockade of the BDNF collapsing effect by the
elevation of [cAMP]i is further supported by the small
percentage of growth cones collapsed after exposure to 50 ng/ml BDNF
(14.5 and 19.4% for forskolin- and
(Sp)-cAMP-treated growth cones,
respectively, compared with 60% for growth cones exposed to BDNF
only).
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Figure 4.
cAMP regulation of BDNF-induced growth cone
collapse. A, Growth cone collapse induced by BDNF alone
(from Fig. 2B). B, The culture was
preincubated with 100 µM
(Rp)-cAMP for 20 min before BDNF
application, and (Rp)-cAMP was
present throughout the experiment. No significant difference from
A was detected (*p > 0.5, Mann-Whitney test). C, D, Application of 10 µM forskolin (C) or 500 µM (Sp)-cAMP
(D) blocked the collapsing effect. Both chemicals
were preincubated with the culture for 20 min before the application of
BDNF and present throughout the experiment. Numbers
represent numbers of cells (growth cones) examined. *Significantly
different from BDNF alone (A) at corresponding
times (p < 0.05, Mann-Whitney
tests).
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Although inhibition of protein kinase A did not significantly affect
the collapse induced by 50 ng/ml BDNF,
(Rp)-cAMP did exert influence on the
collapse induced by BDNF at lower concentrations. For example, BDNF at
5 ng/ml was ineffective to induce growth cone collapse. The percentage
of growth cones collapsed (25%) and the extent of neurite retraction
induced by 5 ng/ml BDNF were small (Fig.
5A). When 100 µM
(Rp)-cAMP was applied to the culture, the
collapsing effect was potentiated (Fig. 5B). The percentage of growth cones collapsed in response to 5 ng/ml BDNF was also increased to 46% when growth cones were treated with
(Rp)-cAMP. To better illustrate the
potentiation of the BDNF collapsing effect by
(Rp)-cAMP, the distribution of POL from
populations of growth cones at 10 min after BDNF addition was plotted
as a histogram. The presence of (Rp)-cAMP
caused more growth cones to respond to BDNF by collapse, as indicated
by an increased number of growth cones with smaller POL values when
compared with that of 5 ng/ml BDNF alone (Fig. 5C, arrow).
Statistical analysis further confirms the significance of the
potentiation effect on BDNF-induced collapse by the inhibition of PKA
(Fig. 5B, *p < 0.05, Mann-Whitney
test).
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Figure 5.
Potentiation of BDNF-induced growth cone collapse
by (Rp)-cAMP. A,
Growth cone collapse induced by 5 ng/ml BDNF alone. B,
One hundred micromolar (Rp)-cAMP was
added to the culture 20 min before the application of 5 ng/ml BDNF.
Increased collapse was observed [*p < 0.05, Mann-Whitney test, compared with the POL of BDNF alone
(A) at the corresponding time].
Numbers in A and B
represent numbers of cells (growth cones) examined. C,
Histogram showing the difference in the distribution of POL between
BDNF alone (top panel) and BDNF with
(Rp)-cAMP treatment (bottom
panel) at 10 min after BDNF application (5 ng/ml).
Presence of (Rp)-cAMP in the medium
caused more growth cones to respond to BDNF with collapse, as evidenced
by the appearance of a peak of the POL distribution at smaller POL
(arrow).
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Loss of collapsing response of old neurons to BDNF in 24 hr cultures
The collapsing effect of BDNF was only observed on neurons in 6-8
hr cultures in which motile growth cones and active neurite extension
are observed. In 24 hr cultures, although active neurite extension was
still observed but at a slightly reduced rate (Ming et al., 1997), no
apparent collapsing effect of BDNF was observed (Fig.
6A). Conversely, BDNF
was found to elicit extensive lamellipodial protrusion along the
neurite shaft and to induce chemoattractive turning of the growth cone
(Ming et al., 1997; Song et al., 1997). These results suggest that
neurons may respond to the same tropic factors differently at different
developmental stages. Because a clear cAMP regulation of BDNF-induced
collapse was observed in 6 hr cultures, it is possible that neurons in
24 hr cultures may have higher resting levels of endogenous
cAMP-dependent activity, which could block the collapsing effect of
BDNF. To test this hypothesis, we applied
(Rp)-cAMP (100 µM) to 24 hr
cultures to inhibit the activity of PKA. When 50 ng/ml BDNF was applied
to these (Rp)-cAMP-treated neurons,
significant collapse of growth cones was observed in these 24 hr
cultures (Fig. 6B, *p < 0.001, Mann-Whitney test). It appears that
(Rp)-cAMP restored the collapsing response of 24 hr cultures to a level similar to that of 6 hr cultures.
The percentage of growth cones collapsed by 50 ng/ml BDNF was also
increased from 25 to 81% after growth cones were treated with
(Rp)-cAMP in 24 hr cultures. These
results suggest that the loss of the collapsing response to BDNF by
neurons in 24 hr cultures is tightly associated with the endogenous
cAMP-dependent activity, which may be elevated during the
development.
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Figure 6.
Endogenous cAMP-dependent activity plays a key
role in the disappearance of the BDNF collapsing effect on neurons in
24 hr cultures. A, BDNF was apparently ineffective in
inducing growth cone collapse in 24 hr cultures. However, when
endogenous cAMP-dependent activity was inhibited by 100 µM (Rp)-cAMP, the
collapsing effect of BDNF was clearly observed
(B). *Significantly different from the data in
A at corresponding times (p < 0.001, Mann-Whitney test).
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Involvement of the actin cytoskeleton in BDNF-induced growth
cone collapse
Although the signaling events downstream of second messengers are
unknown, the actin cytoskeleton seems to be one of the final targets in
BDNF-induced growth cone collapse. Cytochalasins are fungal metabolites
that bind to the barbed ends of actin filaments to prevent further
polymerization, leading to the disassembly of actin filaments over
time. Application of cytochalasin D at 1 µM did not cause
neurite retraction but effectively abolished BDNF-induced growth cone
collapse (Fig. 7) as well as
lamellipodial protrusion (Ming et al., 1997). Dependence on actin
polymerization has been observed previously in NGF-induced rapid
neurite retraction of cultured sensory neurons (Griffin and Letourneau,
1980). It was suggested that extensive membrane ruffling induced at
such thin neurite processes by growth factors may directly cause
neurite retraction (Griffin and Letourneau, 1980), mainly as the result of cytoskeletal reorganization. However, the presence of lamellipodial protrusion and the blockade of collapsing effect in the
Ca2+-free medium (Fig. 3) argue strongly against
this possibility. Other actin-based mechanism(s) must be involved in
the collapse.
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Figure 7.
Cytochalasin D blocks growth cone collapse induced
by BDNF. The mean POL from growth cones exposed to BDNF alone (Fig.
2B) is presented as the reference
(squares, dashed line). Cytochalasin D at 1 µM did not reduce the neurite length but blocked the
collapsing effect of BDNF. *Significantly different from that of BDNF
alone (Fig. 2B) at corresponding times
(p < 0.05, Mann-Whitney test).
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DISCUSSION |
Acute, inhibitory effects of neurotrophins on growth
cone motility
An increasing number of studies have established a "positive"
role for neurotrophins in axonal and dendritic growth. In addition to
the key activity of neurotrophins in promoting neurite outgrowth of
responsive neuronal populations, neurotrophins have been shown to
increase axonal sprouting (Diamond et al., 1992; Cohen-Cory and Fraser,
1995) and to regulate morphological development of dendrites
(Cohen-Cory et al., 1991; McAllister et al., 1995, 1996). In our
Xenopus cultures, addition of neurotrophins enhances
neuronal survival and promotes neurite outgrowth of embryonic spinal
neurons; when applied as concentration gradients, BDNF and NT-3 also
exert chemotropic effects on growth cones in 18-24 hr cultures (Ming et al., 1997; Song et al., 1997). In this study, we provide evidence on
a "negative" effect of BDNF on growth cone extension of embryonic Xenopus neurons in 6 hr cultures: acute application of BDNF
induces rapid growth cone collapse and neurite retraction. The
effective neurotrophin concentrations for growth cone collapse are
similar to those known to promote neuronal survival as well as to
elicit a number of acute effects on these neurons (Ming et al., 1997). The collapsing effect appears to be relatively specific for BDNF, because NGF was ineffective in inducing growth cone collapse. Only a
small portion of growth cones collapsed in response to NGF (35% of
total growth cones examined, compared with 60% for BDNF). The lack of
the collapsing effect of NGF is further demonstrated by the
significantly smaller extent of neurite retraction than that of BDNF
(Fig. 2C). Given the heterogeneity of Xenopus
cultures, it is not surprising for the existence of subpopulations of
neurons that may respond to different neurotrophins differently (Lohof et al., 1993; Ming et al., 1997). Nevertheless, the significant difference between BDNF and NGF in the ability of inducing growth cone
collapse further supports the conclusion that the high-affinity Trk
receptor (TrkB for BDNF), rather than the low-affinity p75 receptor,
mediates the collapsing effect; otherwise, similar extents of growth
cone collapse would have been observed for BDNF and NGF, because both
can activate p75 effectively. However, our results do not exclude the
possibility that p75 may be involved, but not required, in the
collapsing action of neurotrophins. The possible contribution from the
p75 receptor activation in BDNF-induced growth cone collapse remains to
be determined.
Ca2+ is an important second messenger involved in
regulation of a wide range of cellular activities, including growth
cone motility (Kater and Mills, 1991; Zheng et al., 1996a). In
BDNF-induced growth cone collapse, an influx of Ca2+
is required for the collapse, suggesting that the
Ca2+-dependent signaling pathway mediates the
collapse. Furthermore, the collapsing response is regulated by
cAMP-dependent activity, because elevation or inhibition of cytosolic
cAMP-dependent activity attenuated or potentiated the collapse,
respectively. This cAMP-mediated regulation appears to play a key role
in the loss of the collapsing response to BDNF by old neurons in 24 hr
cultures. The endogenous cAMP-dependent activity in these neurons may
be elevated to a higher level than that in neurons from 6 hr cultures,
therefore leading to the blockade of the collapsing effect of BDNF. Our finding that inhibition of PKA by
(Rp)-cAMP restored the collapsing response of these old neurons confirms such a notion. The cellular mechanism underlying the elevated levels of cAMP-dependent activity in
these old neurons remains to be studied. Nevertheless, these results
thus resolve the apparent contradiction between the negative collapsing
effect and the positive effects of neurotrophins on neurite growth
observed on same Xenopus cultures. By modulating its
cytosolic levels of cAMP-dependent activity at different stages during
development, a neuron could thus exhibit different responses to the
same extracellular signals.
Although the cellular mechanism underlying BDNF-induced growth cone
collapse is largely unknown at this moment, the actin cytoskeleton
seems to play an important role. The result that cytochalasin D
completely blocked the collapse induced by BDNF suggests that actin
polymerization may be required for the collapse. However, in growth
cone collapse induced by collapsin-1, actin depolymerization was
observed (Fan et al., 1993), indicating that the collapse induced by
BDNF and collapsin-1 may involve different cellular processes. On the
other hand, cytochalasins are known not only to prevent actin
polymerization but also to disrupt the existing actin cytoskeletal
network. Therefore, the blockade of BDNF-induced collapse by
cytochalasins could also suggest that an intact actin network is
required for BDNF collapsing action, either for signal transduction or
for force generating. In neurite retraction induced by
lysophosphatidade (LPA) and thrombin, a similar dependence on the actin
cytoskeleton was observed, and an actomyosin contractile mechanism was
suggested to mediate the neurite retraction (Jalink and Moolenaar,
1992; Jalink et al., 1993). However, whether the same mechanism
operates in BDNF-induced growth cone collapse and neurite retraction
remains to be determined.
Mechanism(s) underlying the cAMP regulation of BDNF-induced
collapse: a tentative model
The cAMP pathway has been suggested to regulate the activity of
other signal transduction pathways (for review, see Iyengar, 1996). In
smooth muscle, contractility is regulated by myosin light-chain kinase
(MLCK), which can be phosphorylated by PKA, cGMP-dependent protein
kinases, and Ca2+-/calmodulin-dependent protein
kinase II (for review, see Burridge and Chrzanowska-Wodnicka, 1996).
Phosphorylation of MLCK decreases its affinity for
Ca2+ and calmodulin, inhibiting MLCK activity and
contractility (Nishikawa et al., 1984; Tansey et al., 1994). It was
suggested that LPA-induced neurite retraction is mediated by the
contraction of cortical actin cytoskeleton, which may also be subjected
to this kind of regulation (Tigyi et al., 1996). We hypothesize a
similar cAMP-dependent regulation in BDNF-induced growth cone collapse
(see Fig. 8). Elevation of
[cAMP]i in these cultured Xenopus neurons
activates protein kinase A, which in turn phosphorylates the unknown
protein "X" that might be involved in the main signaling pathway
(Ca2+-mediated) in BDNF-induced collapse. The
phosphorylation inactivates X and blocks the collapse;
dephosphorylation of X by phosphatases could reactivate the protein.
The resting levels of PKA and phosphatase activity in the cell are
likely to reach a balance, leading to an equilibrium between active and
inactive forms of X. Inhibition of PKA thus can change the equilibrium
and increase the level of active X, leading to the potentiation of the
collapsing effect induced by low concentrations of BDNF, which was
observed in this study. At high concentrations of BDNF, the collapsing
effect may already be saturated, and further enhancement by PKA
inhibitors cannot be produced. Although our hypothesized model places
cAMP-mediated regulation downstream of Ca2+, it is
possible that such cAMP regulation is upstream of
Ca2+; that is, X mediates BDNF-induced
Ca2+ influx to induce collapse that can be regulated
by cAMP-dependent activity in a similar matter.
View larger version (20K):
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|
Figure 8.
Schematic diagram showing the proposed model
involved in the cAMP regulation of BDNF-induced collapse. Activation of
TrkB receptors by BDNF induces multiple intracellular signaling
cascades including a Ca2+ influx through
Ca2+ channels, and the Ca2+
pathway mediates the collapsing effect (double lines
with arrows). Although the unknown protein X is involved
in the main Ca2+-signaling pathway for collapse, its
activity can be modulated by phosphorylation and dephosphorylation by
cAMP-dependent activity (single lines with
arrows). PKA phosphorylates and inactivates () X,
whereas phosphotase(s) dephosphorylate(s) and activate(s) (+) X. An
equilibrium between the active and inactive X is likely reached in the
cell. Experimental manipulation of PKA activity by
(Rp)-cAMP or
(Sp)-cAMP (dashed
lines with arrows) can change the equilibrium
and thus regulate the collapsing effect of BDNF. Activation of PKA by
(Sp)-cAMP causes more X to become
inactive, leading to the blockade of the collapsing effect; inhibition
of the resting level of PKA activity by
(Rp)-cAMP results in more active X,
leading to the potentiation of the collapsing effect induced by the low
concentrations of BDNF. Alternatively, the cAMP-mediated regulation can
be upstream of Ca2+. In this case, the protein X
mediates BDNF-induced Ca2+ influx required for
growth cone collapse (dotted double lines with
arrows), and its activity can be regulated by
cAMP-dependent activity in a similar way.
|
|
Collapsing effect and growth cone guidance: the in
vivo relevance
Recent studies have identified an increasing number of receptor
tyrosine kinases involved in axonal guidance (Tessier-Lavigne and
Goodman, 1996). Although no in vivo evidence is available for a role of neurotrophic factors in axonal guidance, the
chemoattractive effects of neurotrophins in culture suggest such a
potential (Letourneau, 1978; Menesini-Chen et al., 1978; Gundersen and
Barrett, 1980; Ming et al., 1997; Song et al., 1997). The collapsing
effect observed here, together with previous observations of similar
inhibitory effects of neurotrophins in vivo and in cell
culture (Griffin and Letourneau, 1980; Zhang et al., 1994; Paves and
Saarma, 1997), suggests that neurotrophins may exert acute, negative
regulation on neuronal development. The observation that BDNF
administered through a micropipette could also produce a similar
inhibitory effect on Xenopus growth cones in 6 hr cultures
(Q. Wang and J. Q. Zheng, unpublished observation) suggests that
the negative regulation of axonal growth could be achieved by
neurotrophins derived from point sources, such as those
neurotrophin-expressing cells along developing axonal pathways, as well
as target tissues (Elkabes et al., 1994; Hallböök et al.,
1995). Such negative regulation of axonal growth, if occurring in the
early stages during neuronal development in vivo, could be
used to temporarily halt axonal extension to prevent developing axons
from entering incorrect intermediate regions and targeting prematurely;
by switching the dependence on specific neurotrophins (Davies, 1994a)
or by modulating its endogenous cAMP activity in the later stages of development, a growth cone could then change its responsiveness to
accommodate the positive influence of neurotrophins (O'Connor et al.,
1990; Kuhn et al., 1995; Gallo et al., 1997).
The cAMP-dependent activity has recently been shown to regulate the
turning direction of Xenopus growth cones exposed to BDNF gradients in 18-24 hr cultures (Song et al., 1997). A gradient of BDNF
normally induces a chemoattractive response in these neurons (Ming et
al., 1997; Song et al., 1997). It was proposed that a cytoplasmic
Ca2+ gradient induced by the BDNF gradient produces
the repulsive turning of the growth cone, which is normally overridden
by a Ca2+-induced cAMP gradient that attracts growth
cones. Inhibition of cAMP-dependent activity thus results in an
opposite turning response. Because the repulsive turning of growth
cones is thought to result from a local, asymmetric collapse of the
growth cone (Fan and Raper, 1995), our finding of the collapsing effect
of BDNF provides the direct evidence to support the repulsive action of
BDNF on embryonic Xenopus neurons. However, our data show
that such a collapsing and repulsive effect of BDNF is mainly blocked by the change of endogenous cAMP-dependent activity in these 18-24 hr
neurons; the Ca2+-induced cAMP gradient, on the
other hand, might be responsible for inducing attractive turning of the
growth cone in BDNF gradients after the repulsive effect is blocked.
When endogenous cAMP-dependent activity is inhibited in these 24 hr
cultures, the collapsing (this study) and chemorepulsive (Song et
al., 1997) effect of BDNF is observed. The cAMP-mediated regulation
observed in this study might also play a role in repulsive guidance of
growth cones by other molecules. Our preliminary study has shown that
elevation of [cAMP]i by forskolin completely blocked the
repulsive turning of Xenopus growth cones induced by
extracellular gradients of semaphorin III in culture (Q. Wang and
J. Q. Zheng, unpublished observations), suggesting that the
cAMP-dependent activity could regulate the repulsive action of
semaphorins. Developing neurons in vivo do encounter many
extracellular factors that act through second messenger-dependent
mechanisms, including the cAMP-dependent pathway. Concurrent presence
or absence of these factors may thus regulate the different responses
of growth cones to the guidance cues. Furthermore, a neuron may switch
on or off the pathways responsible for different cellular responses to
the same guidance cues by its intrinsic programming, allowing different
responses at different stages during development.
| |
FOOTNOTES |
Received Feb. 12, 1998; revised March 30, 1998; accepted April 10, 1998.
This work was supported by a University of Medicine and Dentistry of
New Jersey Foundation research seed grant and a startup fund from the
Department of Neuroscience and Cell Biology at University of Medicine
and Dentistry of New Jersey-Robert Wood Johnson Medical School. We
thank Dr. Ann M. Lohof (Laboratoire de Neurobiologie, Ecole Normale
Supérieure, Paris, France) for valuable input during manuscript
preparation, Dr. Ira B. Black for review and comments on this paper,
and Ms. Jean Gibney for technical assistance.
Correspondence should be addressed to Dr. James Q. Zheng, Department of
Neuroscience and Cell Biology, University of Medicine and Dentistry of
New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane,
Piscataway, NJ 08854.
| |
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Abstract
Introduction
