(Received for publication, July 5, 1995)
From the
Neurotrophins activate the Trk tyrosine kinase receptors, which
subsequently initiate signaling pathways that have yet to be fully
resolved, resulting in neuronal survival and differentiation. The
ability of nerve growth factor (NGF) and brain-derived neurotrophic
factor (BDNF) to activate GTP binding to p21 was
investigated using cultured embryonic chick neurons. In both
sympathetic and sensory neurons, the addition of NGF markedly increased
the formation of Ras-GTP. The magnitude of the effect was found to
depend upon the developmental stage, peaking at embryonic day 11 in
sympathetic neurons and at embryonic day 9 in sensory neurons, times
when large numbers of neurons depend on NGF for survival. Surprisingly,
following the addition of BDNF, no formation of Ras-GTP could be
observed in neurons cultured with BDNF. When sensory neurons were
cultured with NGF alone, both NGF and BDNF stimulated GTP binding to
Ras. In rat cerebellar granule cells, while the acute exposure of these
cells to BDNF resulted in the formation Ras-GTP, no response was
observed following previous exposure of the cells to BDNF, as was
observed with sensory neurons. However, this desensitization was not
observed in a transformed cell line expressing TrkB. In neurons, the
mechanism underlying the loss of the BDNF response appeared to involve
a dramatic loss of binding to cell-surface receptors, as determined by
cross-linking with radiolabeled BDNF. Receptor degradation could not
account for the desensitization since cell lysates from neurons
pretreated with BDNF revealed that the levels of TrkB were comparable
to those in untreated cells. These results indicate that in neurons,
the pathways activated by NGF and BDNF are differentially regulated and
that prolonged exposure to BDNF results in the inability of TrkB to
bind its ligand.
The formation of the vertebrate nervous system is regulated by
the availability of a variety of soluble factors promoting the survival
and differentiation of neurons. Among these factors is a family of
related proteins referred to as neurotrophins that includes nerve
growth factor (NGF), ()brain-derived neurotrophic factor
(BDNF), and neurotrophins 3-6 (NT-3, NT-4/5, and NT-6). Gene
knockout and antibody-mediated deprivation experiments have established
the crucial role played by several of these proteins in the formation
of the nervous system (for review, see (1) ). The involvement
of the Trk subgroup of receptor tyrosine kinases in mediating the
biological function of the neurotrophins has been demonstrated by the
observation that loss-of-function mutations introduced in the genes
coding for each of the three Trk receptors cause defects in the nervous
system that are analogous to those observed in mice lacking the
corresponding receptor ligand (for review, see (2) ). While
these results do not indicate that the Trk proteins are the only
receptor components necessary for mediating a neurotrophin response in
neurons, they do show that this subgroup of tyrosine kinases plays a
necessary role in mediating neuronal responses to neurotrophins.
Binding of neurotrophins to their respective Trk receptors results
in the stimulation of tyrosine kinase activity, which initiates a
signaling cascade involving the downstream targets of many growth
factor receptors, including mitogen-activated protein kinase,
phosphatidylinositol 3-kinase, phospholipase C, and, in particular,
p21 (for review, see (3) ). The
importance of the Ras pathway in neurotrophin signaling was first
revealed using the rat pheochromocytoma PC12 cell line. The
introduction of Ras antibodies or of dominant negative Ras mutants into
these tumor cells suppressed NGF-induced fiber outgrowth(4) .
While much less work has been done using post-mitotic neurons, similar
results were obtained with embryonic neurons: the introduction of a
constitutively active mutant of Ras into chick sensory neurons led to
neurotrophin-independent survival and fiber outgrowth(5) .
Similarly, Fab fragments blocking the activity of Ras prevented
NGF-induced survival in these neurons(6) . However,
neurotrophin signaling appears to be differentially regulated depending
on the neuronal context: in this same study, the introduction of
anti-Ras Fab fragments failed to block the survival effects of NGF in
chick (unlike rat(7) ) sympathetic neurons, and constitutively
active Ras was unable to support survival of these neurons. Taken
together, these findings indicate that although neurotrophins act like
many other growth factors, there appears to be differential regulation
of the signal transduction pathways in different neurons.
We were interested in investigating the involvement and regulation of the Ras pathway by measuring the formation of Ras-GTP following stimulation by NGF and BDNF of different populations of neurons at various embryonic ages. In confirmation of a recent report, we found that BDNF and NGF were both able to stimulate GTP binding to Ras in chick sensory neurons(8) . NGF was also found to activate Ras in sympathetic neurons. Surprisingly, in contrast to the results obtained with NGF, cultured neurons became completely desensitized to BDNF when exposed for prolonged periods of time to BDNF.
Sympathetic neuronal cultures from embryos at E7, E11, or E15, as indicated, were prepared as described(11) . Briefly, lumbosacral chains were isolated, dissociated, and preplated for 2 h and plated on 6-cm dishes coated as described above.
Cerebellar granule neurons were prepared from P6
rats as described (12) . Cerebella were dissected and incubated
for 20 min at 37 °C in phosphate-buffered saline containing 10
mM glucose, 1 mg/ml albumin, 10 mg/ml DNase, and 12 units/ml
papain. The cells were dissociated by trituration through a plastic
pipette, pelleted at 900 g for 5 min, and resuspended
in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum. The cells were plated on 6-cm polyornithine-coated dishes,
and the medium was changed after 12 h to a serum-free medium with or
without BDNF(12) .
Analysis of the
dose-response curve for NGF in sympathetic cultures was done by
nonlinear regression using the software package Graphpad.
All data were simultaneously fit to a sigmoidal curve describing
dose-response functions according to the following equation: Y = A + (B - A)/(1
+
(10
/10
)
),
where Y is the percent of GTP binding, X is the
logarithm of the ligand concentration, A is the minimum and B is the maximum of the curve, C is the
EC
, and D is the slope factor.
To prepare membranes for cross-linking experiments,
granule cells were cultured and washed as described above, removed by
scraping in phosphate-buffered saline, and pelleted at 10,000 g for 5 min. The cells were then disrupted in
phosphate-buffered saline with the addition of 1 mM PMSF and 4
µg/ml leupeptin using a glass-Teflon tissue homogenizer for 1 min
at 1500 rpm (B. Braun Biotech International), followed by sonication
for 10 s on ice (Bronson sonifier). After removal of the nuclei by
centrifugation at 1000
g for 5 min, the membranes
remaining in solution were pelleted at 100,000
g for 1
h. These membranes were then resuspended in KRH buffer, and
radiolabeled BDNF was added and cross-linked as described for intact
cells. The membranes were then repelleted at 100,000
g for 1 h to separate them from the free ligand, and TrkB was
immunoprecipitated as described above.
Figure 1:
Time and concentration dependence of
p21 stimulation by NGF in neurons from the
sympathetic ganglion. Cultured sympathetic neurons from E11 embryos
were treated with NGF (1 nM) for various times (upperpanel) or at various concentrations for 2 min (lowerpanel) at 37 °C. The cells were lysed, and the
percent of GTP bound to p21
was assessed as
described under ``Experimental Procedures.'' The half-maximal
point of the curve depicted in the lowerpanel is 60
± 1.2 pM, determined by nonlinear regression analysis
as described under ``Experimental Procedures.'' All points
depict the means ± S.E. of at least three
experiments.
After 2 days in culture, the medium containing the factor(s) was removed, and acute stimulation of Ras was measured. A maximally effective concentration of NGF (1 nM or 26 ng/ml) was able to stimulate GTP binding to Ras in both sensory and sympathetic neurons at all stages examined. The maximal response to NGF varied during development with a peak at E9 in DRG and at E11 in sympathetic neurons and a decreased response in both populations of cells in late development. However, even at the earliest time point, significant stimulation by this neurotrophin was observed (Fig. 2). With sympathetic neurons at E7, NT-3 weakly activated Ras at a stage when these neurons are known to show a survival response to this factor(18) . Marginal but significant activation was also observed at E11, a time point at which NT-3, at low concentrations, no longer supports these cells in culture(18) . However, by E15, there was no significant effect of NT-3 on Ras (Fig. 2).
Figure 2:
Developmental changes in neurotrophin
efficacy in activating p21 in DRG and
sympathetic ganglia. Cultured neurons from sympathetic ganglia at
embryonic day 7, 11, or 15 (upperpanel) and from DRG
at embryonic day 6, 9, or 12 (lowerpanel) were
acutely treated with 1 nM NGF (solidbars),
BDNF (dottedbars), NT-3 (stripedbars), or buffer (openbars) for 2 min
at 37 °C and lysed, and the percent of GTP bound to p21
was assessed as described under ``Experimental
Procedures.'' Shown are the means ± S.E. of three or more
experiments. The asterisks indicate the degree of significance
relative to that in the absence of factor based on Student's t test (**, p < 0.01; *, p < 0.05).
The insets depict representative images from the TLC plates
for sympathetic neurons at E15 (upperpanel) and for
DRG at E12 (lowerpanel). con,
control.
Unlike with NGF and NT-3, BDNF was found not to stimulate the formation of Ras-GTP above control levels in sensory neurons at any stage examined. The reasons for this unexpected observation were then explored.
Figure 3:
Neurotrophin stimulation of
p21 in DRG neurons cultured with a heterologous
or homologous factor. DRG neurons from E9 embryos were selected by
culturing with either 5 ng/ml BDNF (leftpanel) or
0.1 ng/ml NGF (rightpanel). After removing the
factor used for culturing, the cells were then acutely exposed to NGF (solidbars), BDNF (dottedbars),
or no factor (openbars) and lysed, and the percent
of GTP bound to p21
was assessed as described
under ``Experimental Procedures.'' Shown are the means
± S.E. of three or more experiments. The asterisks indicate the degree of significance relative to that in the
absence of factor based on Student's t test (p
0.01). CON, control.
Figure 4: Expression of TrkB mRNA in DRG cells cultured with NGF or BDNF. DRG neurons from E9 embryos were cultured for the indicated time with 5 ng/ml NGF or BDNF, and RNA was collected, reverse-transcribed (Alanes) or used directly (B lanes), and then amplified by PCR using oligonucleotides from the extracellular domain (EC) and the tyrosine kinase domain (TK) of TrkB. The cDNA was then transferred to nylon membrane and hybridized with a radiolabeled oligonucleotide from the juxtamembrane region of the intracellular domain (*) as described under ``Experimental Procedures.'' TM, transmembrane domain.
When BDNF was added to granule cells that had not been cultured with BDNF, the formation of Ras-GTP could readily be observed (Fig. 5). However, exposure of the cells to BDNF for 24 h led to the disappearance of the response to acutely added BDNF (Fig. 5). This result indicates that the desensitization to BDNF observed with chick sensory neurons can also be observed with other TrkB-expressing neurons. Interestingly, this loss of responsiveness is not observed when TrkB is expressed in a non-neuronal context. Incubation of the cell line A293 expressing chick TrkB (14) for 24 h with 20 ng/ml BDNF did not lead to any measurable desensitization when compared with cells acutely exposed to BDNF (Fig. 6).
Figure 5:
Desensitization of p21 stimulation by BDNF in rat cerebellar granule neurons.
Cerebellar granule cells from postnatal day 6 rats were cultured for 24
h in the absence (leftpanel) or presence (rightpanel) of 20 ng/ml BDNF. The neurons were then thoroughly
rinsed to remove the culture medium, acutely treated with BDNF (dottedbars) or buffer (openbars), and lysed, and the percent of GTP bound to
p21
was assessed as described under
``Experimental Procedures.'' Shown are the means ±
S.E. of three or more experiments. The asterisk indicates the
degree of significance relative to that in the absence of factor based
on Student's t test (p
0.01). CON, control.
Figure 6:
Desensitization of p21 stimulation by BDNF does not occur in TrkB-transfected
fibroblasts. A293 fibroblasts expressing chicken TrkB were cultured for
24 h in the absence (leftpanel) or presence (rightpanel) of 20 ng/ml BDNF. The cells were then
thoroughly rinsed to remove the culture medium, acutely treated with
BDNF (dottedbars) or buffer (openbars), and lysed, and the percent of GTP bound to
p21
was assessed as described under
``Experimental Procedures.'' Shown are the means ±
S.E. of three or more experiments. CON,
control.
To further investigate the mechanisms underlying
the loss of BDNF signaling through Ras, changes in cell-surface
receptor availability were assessed in granule cells using radiolabeled
BDNF. After cross-linking and immunoprecipitation with a Trk-specific
antibody, a radioactive band at 150 kDa was detected (Fig. 7, lanes A and B), which was absent when
incubation was performed in the presence of 100-fold excess unlabeled
BDNF (Fig. 7, laneD). Immunoprecipitates from
cells treated for 24 h with BDNF revealed a dramatic decrease in
receptor availability, suggesting a loss of receptor binding capability
in BDNF-pretreated neurons (Fig. 7, laneC).
Figure 7: Inactivation of TrkB binding capability following chronic BDNF treatment. Cerebellar granule cells from postnatal day 6 rats were cultured for 24 h without factor (lanesA, B, D, and E) or in the presence of 20 ng/ml BDNF (lanes C and F). The neurons were then thoroughly rinsed to remove the culture medium, and intact cells (lanesA-D) or isolated membranes (lanesE and F) were incubated with 1 nM iodinated BDNF alone (lanesA-C, E, and F) or in the presence of 100 nM unlabeled BDNF (laneD). Following cross-linking, the neurons were lysed or the membranes were pelleted, TrkB was immunoprecipitated, and the immune complex was separated by 6.5% SDS-PAGE as described under ``Experimental Procedures.'' Shown are representative images from a Fuji PhosphorImager.
The cross-linking experiments were repeated using cell membranes
isolated by high speed centrifugation to probe for receptors no longer
at the cell surface. No detectable levels of I-BDNF could
be cross-linked to the receptor in membranes isolated from cells
pretreated with BDNF (Fig. 7, lanesE and F).
To determine if the loss of binding was a result of receptor degradation, cell lysates obtained from either naive or BDNF-treated granule cells were compared for their TrkB receptor content by immunoprecipitation and Western blotting using an antiserum specific for the extracellular domain of TrkB. The levels of full-length TrkB observed in neurons incubated for 24 h with BDNF compared with untreated cells were similar (Fig. 8). This result was confirmed using a TrkB antiserum specific for the intracellular domain (data not shown). This finding suggests that although the ability of TrkB to bind BDNF is lost following prolonged exposure of the cells to BDNF, TrkB protein levels in the cell remain similar.
Figure 8: Western blot of TrkB protein in granule cell lysates following chronic BDNF treatment. Cerebellar granule cells from postnatal day 6 rats were cultured for 24 h without factor (control (CON)) or in the presence of 20 ng/ml BDNF. Lysates were prepared from the cells, and equal amounts of protein were separated by 6.5% SDS-PAGE and transferred to Immobilon-P membranes, and TrkB was detected with an antibody raised to a specific extracellular sequence of rat TrkB as described under ``Experimental Procedures.''
To be certain that BDNF from the culture medium was completely removed by the standard washing procedure preceding acute exposure of neurons to the neurotrophins, the naive cells were exposed for 5 min to 20 ng/ml BDNF. This period of time is sufficient for receptor activation, as indicated by the Ras stimulation (Fig. 1, upper panel). After washing, cross-linking with radioiodinated BDNF, and immunoprecipitation, the cross-linked protein band was of the same intensity as for the control cells (Fig. 7, laneB). Thus, following a 5-min exposure of the cells to BDNF at 37 °C, the washing procedure is sufficient to render the BDNF receptors fully accessible to binding of the ligand.
This study shows that cultured sensory and cerebellar neurons respond to the addition of neurotrophins by the formation of Ras-GTP. This response is also observed in sympathetic neurons, where the activation of Ras has been previously shown to be neither sufficient nor necessary for neuronal survival. Unexpectedly, the formation of Ras-GTP in response to BDNF can only be seen when neurons are acutely exposed to BDNF: culturing them in the presence of BDNF leads to the complete disappearance of the response. This phenomenon is not observed with NGF, nor is it observed when the receptor tyrosine kinase for BDNF, TrkB, is expressed in non-neuronal cells.
Coexpression of neurotrophin receptors in at least some neurons is also likely in view of the results obtained with sensory neurons demonstrating that many NGF neurons first depend on NT-3 or BDNF for survival(25) . A shift from NT-3 to NGF dependence has also been suggested by results obtained in vivo in antibody deprivation experiments(26) .
The formation of Ras-GTP in response to BDNF was not only observed
in sensory neurons, but also in granule cells isolated from the
postnatal rat cerebellum. Just like with sensory neurons, a striking
feature of this response is that it was completely abolished by
preincubation of the neurons with BDNF. Unlike with peripheral neurons,
cultured neurons from the central nervous system are much less
dependent on the addition of exogenous neurotrophin for their
survival(19) . This, together with the availability of large
numbers of cells displaying 10 times more BDNF receptors than
sensory neurons, made them the ideal object for studying the unexpected
phenomenon of desensitization.
To investigate the mechanisms involved in the loss of response to BDNF, we looked for changes at the level of the receptor. The experiments with the granule cells indicate that in cells treated with BDNF, receptor availability decreases dramatically, as indicated by cross-linking experiments. This is not likely due to a massive receptor degradation since cell lysates obtained from treated and untreated cells seemed to contain comparable levels of full-length TrkB protein in Western blot experiments, in contrast with the drastic loss in binding capacity. Internalized intact receptors are also unlikely to account for the loss in binding since BDNF could not be cross-linked to membranes isolated from BDNF-treated cells. What biochemical mechanism underlies the receptor inactivation is currently under investigation.
Interestingly, the phenomenon of
desensitization is not observed when similar experiments are performed
with TrkB expressed in a cell line able to internalize BDNF at a rate
such that 30% of surface-bound BDNF is internalized within 30
min(27) . The conclusion that neuronal receptors for BDNF,
while not degraded, become incapable of binding BDNF might also explain
recent observations made with embryonic rat cortical and hippocampal
neurons(28) . A loss of phosphatidylinositol signaling was
observed after pre-exposure to BDNF or NT-3. While the physiological
significance of BDNF desensitization and receptor inactivation in these
neurons is not clear, it is possible that such mechanisms are also
observed in vivo. Indeed, in a recent study, it was observed
that during rat development, the phosphorylation of TrkB through BDNF
in isolated brain tissues becomes increasingly attenuated to become
only marginal in adult animals(29) . This could reflect
prolonged exposure of neurons to BDNF in vivo since during
development, BDNF levels increase from very low levels in early
embryonic tissue to substantial levels in the adult brain (see, for
example, (30) and (31) ). Interestingly, in the study
by Knüsel et al.(29) , the
phosphorylation of TrkA by NGF was shown to still be possible with
tissue from adult brain, suggesting that also in vivo, the
phenomenon of desensitization to NGF does not occur.
It is interesting to consider the ability of the BDNF receptor to undergo desensitization in the context of recent evidence indicating that BDNF is able to rapidly enhance spontaneous synaptic activity (32) . Receptor inactivation provides an efficient means of protecting neurons exposed for prolonged periods to BDNF. In addition, it has been reported that cells expressing truncated TrkB receptors can internalize BDNF(27) , thus preventing accumulation of BDNF and avoiding desensitization. Of note in this context is the observation that no truncated variants of the NGF receptor TrkA have been found. Finding the mechanisms by which BDNF receptors lose the ability to bind their ligand will be important in order to understand the conditions under which BDNF could be best applied to adult neurons in vivo.