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The Journal of Neuroscience, October 1, 2000, 20(19):7228-7237
Phosphatidylinositol 3-Kinase Is Required for the Trophic, But
Not the Survival-Promoting, Actions of NGF on Sympathetic Neurons
Brian A.
Tsui-Pierchala,
Girish V.
Putcha, and
Eugene M.
Johnson Jr
Department of Neurology and Department of Molecular Biology and
Pharmacology, Washington University School of Medicine, St. Louis,
Missouri 63110
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ABSTRACT |
Nerve growth factor (NGF) supports target-dependent survival of
sympathetic and other neurons during development; however, the
NGF-regulated signaling pathways required for survival are not fully
understood. Sympathetic neurons are able to abort acutely the cell
death pathway initiated by NGF deprivation at early, as well as late,
time points after readdition of NGF. We found that NGF-dependent
phosphatidylinositol 3-kinase (PI-3-K) activity inhibited an early cell
death event proximal to c-Jun phosphorylation. However, PI-3-K activity
was not required for NGF to inhibit the translocation of Bax from the
cytoplasm to the mitochondria, nor was it required for NGF to inhibit
the subsequent release of mitochondrial cytochrome c,
two events required for NGF deprivation-induced apoptosis.
MEK/MAPK activity did not account for any of these NGF-dependent
events. When subjected to long-term PI-3-K inhibition in the presence
of NGF, the majority of sympathetic neurons did not die. Those that did
die exhibited significant differences in the characteristics of death
caused by PI-3-K inhibition as compared with NGF deprivation.
Additionally, PI-3-K inhibition in the presence of NGF did not induce
release of mitochondrial cytochrome c, indicating that
these neurons were unable to complete the apoptotic program. In
contrast to its modest effects on survival, inhibition of PI-3-K
induced marked decreases in somal diameter and metabolic function, as
measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-yltetrazolium bromide (MTT) reduction, suggesting that PI-3-K is required for the
trophic effects of NGF. Therefore, although PI-3-K is important for the
trophic effects of NGF, it is not required for survival. Other, or at
least additional, signaling pathways contribute to NGF-mediated
survival of sympathetic neurons.
Key words:
phosphatidylinositol 3-kinase; sympathetic neurons; NGF; Bax; cytochrome c; apoptosis
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INTRODUCTION |
Target-dependent survival of
sympathetic neurons is regulated during development by nerve growth
factor (NGF) through activation of its receptor tyrosine kinase, TrkA
(Levi-Montalcini, 1987 ; Korsching, 1993; Bothwell, 1995). A signaling
molecule important for NGF-dependent survival of sympathetic neurons is
phosphatidylinositol 3-kinase (PI-3-K). A mechanism by which PI-3-K
promotes survival of neurons is via activation of the serine/threonine
kinase Akt/protein kinase B (PKB) (Franke et al., 1997; Datta et
al., 1999). Akt/PKB inhibits apoptosis through phosphorylation of cell
death regulatory molecules such as BAD (Datta et al., 1997), caspase-9
(Cardone et al., 1998), and Forkhead transcription factors (Biggs et
al., 1999; Brunet et al., 1999). Although PI-3-K-dependent survival of
cerebellar granule neurons is well established (D'Mello et al., 1997;
Dudek et al., 1997; Miller et al., 1997), the exact role of PI-3-K in
sympathetic neuron survival is less clear. Constitutively active or
overexpressed PI-3-K and Akt/PKB support survival of sympathetic
neurons after NGF withdrawal, demonstrating that either is sufficient
for survival (Philpott et al., 1997; Crowder and Freeman, 1998).
Whether PI-3-K is necessary for the survival-promoting activity of NGF,
however, is controversial. Although some have observed that inhibition
of PI-3-K activity results in death of sympathetic neurons (Crowder and
Freeman, 1998; Mazzoni et al., 1999; Vaillant et al., 1999), others
have not (Philpott et al., 1997; Virdee et al., 1999).
NGF deprivation of sympathetic neurons initiates a sequence of
molecular events that culminates in caspase activation and death
(Deshmukh and Johnson, 1997). An early event is the phosphorylation and
activation of c-Jun that may be responsible in part for the expression
of death-promoting genes (Estus et al., 1994; Ham et al., 1995).
Consistent with c-Jun activation, several genes are induced after NGF
deprivation, such as c-jun, cyclin D1, and SM-20, that may be important
for NGF deprivation-induced cell death (Estus et al., 1994; Freeman et
al., 1994; Lipscomb et al., 1999). The expression of proapoptotic genes
is thought to mediate translocation of Bax from the cytoplasm to the
mitochondria, an event that promotes cytochrome c release
from the mitochondria (Putcha et al., 1999). Cytoplasmic cytochrome
c is required, but not sufficient, for apoptosis of
sympathetic neurons, which also must develop "competence-to-die," the molecular nature of which is unclear (Deshmukh and Johnson, 1998;
Neame et al., 1998).
NGF can abort the cell death pathway at multiple points after NGF
deprivation. Sympathetic neurons can be deprived of NGF for 22 hr, a
time when 50% of neurons have released cytochrome c from
the mitochondria; readdition of NGF at this time halts any further
cytochrome c release and death (Deckwerth and Johnson, 1993;
Deshmukh and Johnson, 1998). Similarly, NGF can inhibit acutely any
further Bax translocation after 18 hr of NGF deprivation, a time when
50% of rat neurons have already translocated Bax (Putcha et al.,
1999). We examined which signaling pathways account for NGF-dependent
inhibition of c-Jun phosphorylation, Bax translocation to mitochondria,
and mitochondrial cytochrome c release. These experiments
helped determine whether TrkA used multiple signaling pathways to
promote survival of sympathetic neurons or relied solely on PI-3-K activity.
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MATERIALS AND METHODS |
Materials. LY294002, PD98059, SB203580, H89, and
Wortmannin were obtained from BIOMOL">Biomol (Plymouth Meeting, PA) and diluted
in DMSO to the concentrations recommended. Boc-aspartyl
fluoromethyl ketone (BAF) was obtained from Enzyme Systems Products
(Livermore, CA) and diluted in DMSO as well. All other pharmacological
agents were reagent grade and diluted into medium just before use.
Anti- -tubulin (Sigma, St. Louis, MO), anti-phosphoMAPK (New England
Biolabs, Beverly, MA), anti-phosphoAKT (Ser472, New England Biolabs),
anti-phosphoAKT (Thr308, Upstate Biotechnology, Inc., Lake Placid, NY),
anti-LDH (Rockland Immunochemicals, Gilbertsville, PA), anti-COX
IV (Molecular Probes, Eugene, OR), and anti-Bax (Upstate Biotechnology,
Inc.) were used for Western blotting at the dilutions described below. Anti-phospho-c-Jun (New England Biolabs), anti-cytochrome c
(PharMingen, San Diego, CA), and CM1 (IDUN, San Diego, CA) were used
for immunocytochemistry.
Sympathetic neuronal cultures and treatments. The superior
cervical ganglia from postnatal day 1 Sprague Dawley rats were dissected, dissociated, and seeded onto collagen-coated 35 mm culture
dishes, 24-well culture plates, or two-well glass chamber slides (Nunc,
Naperville, IL) as described previously (Martin et al., 1992). Cultures
were maintained for 5 d in vitro (DIV) in medium (90%
Minimum Essential medium, 10% fetal bovine serum, 2 mM glutamine, 20 µM
uridine, 20 µM fluorodeoxyuridine, 100 U/ml penicillin, and 100 µg/ml streptomycin) containing NGF (50 ng/ml), after which time cultures were washed twice with medium without NGF
(AM0). AM0 medium containing anti-NGF (goat polyclonal, 1:10,000 dilution) was then added. The neurons were maintained in this medium
for the amount of time described in each experiment, washed twice with
AM0, and next treated with AM0 containing NGF (300 ng/ml) in the
presence or absence of the described pharmacological agent. For
experiments examining the survival and metabolism of sympathetic
neurons after PI-3-K blockade, LY294002 (50 µM)
was added in the presence of NGF, and control cultures were treated with NGF and vehicle alone (0.1% DMSO). In these experiments the medium was replaced every 2 d. For c-Jun phosphorylation studies, neurons were maintained in anti-NGF for 4 hr, and the described pharmacological agent was added for an additional 0.5 hr before NGF
readdition in the continued presence of the drug or vehicle alone
(DMSO). For Bax translocation experiments, neurons were maintained in
anti-NGF for 18 hr before drug pretreatment and NGF readdition. For
cytochrome c translocation experiments, sympathetic neurons
were maintained in anti-NGF for 22 hr before drug and NGF additions.
For Bax, cytochrome c, and in some survival experiments, BAF
(50 µM) was added to the cultures during the
entire treatment regimen to prevent the neurons from lifting off the
collagen substrate after caspase activation and apoptotic death.
Immunocytochemistry. All sympathetic neuronal cultures
processed for immunofluorescence were grown on glass two-well chamber slides. After the described treatments, cultures were washed once with
ice-cold PBS and fixed with fresh 4% paraformaldehyde in PBS for 30 min at 4°C. Cultures were next washed three times with Tris-buffered
saline (TBS) and incubated in blocking solution [5% normal goat serum
(NGS) in TBS that contained 0.3% Triton X-100] for 30 60 min at room
temperature. The neurons were then incubated with primary antibodies in
blocking solution overnight at 4°C. The phospho-c-Jun (P-Jun)
antibody was used at a dilution of 1:400, and the cytochrome
c antibody was used at a dilution of 1:1000 (0.5 µg/ml).
The cultures were next washed three times with TBS and incubated in
blocking solution containing fluorescently labeled secondary antibodies
for 24 hr at 4°C (1.5 µg/ml anti-rabbit Cy-3 and 2 µg/ml
anti-mouse Alexa 488). The cultures were then washed twice with TBS,
incubated for 20 min with Hoechst 33258 (1 µg/ml) in TBS, washed a
final two times in TBS, and mounted for fluorescence microscopy. All
counts of positively or negatively stained neurons were obtained in a
blinded manner.
Survival assays. Toluidine blue staining was performed on
sympathetic neuronal cultures grown in two-well chamber slides. After
the indicated treatments, cultures were washed with ice-cold PBS and
next fixed for 12 d with 4% paraformaldehyde. Cultures were then
washed with deionized water, stained for 4560 sec in toluidine blue-O
(1 gm/l), and incubated for an additional 60 sec in deionized water.
The cells were dehydrated by using successive 2 min washes in deionized
water containing increasing concentrations of ethanol to reach 100%,
after which the slides were washed in toluene, and coverslips mounted
with Permount (Sigma). Neurons displaying smooth cell bodies that were
Nissl-stained were considered alive and were counted. All survival
counts were obtained in a blinded manner.
Subcellular fractionation. After the described treatments,
sympathetic neurons (106) were harvested
into 10 ml of isotonic fractionation buffer (250 mM
sucrose, 0.5 mM EDTA, 20 mM HEPES, 500 µM Na3VO4, pH
7.2) supplemented with protease inhibitors (Inhibitor cocktail
complete, Roche Molecular Biochemicals, Indianapolis, IN) and
centrifuged at 900 × g for 5 min. All subsequent steps
were conducted at 4°C. The pellet was resuspended into 200 µl of
fractionation buffer, homogenized with a ball-bearing homogenizer, and
centrifuged at 900 × g for 5 min to remove nuclei and
intact cells. The postnuclear supernatant was transferred to a
microfuge tube (Beckman, Palo Alto, CA) and centrifuged at 25,000 × g for 10 min to collect the heavy membrane (HM) fraction
that is enriched in mitochondria, followed by centrifugation of the
post-HM supernatant at 100,000 × g for 1020 min to
obtain the microsomal and cytosolic fractions. All pellets were
resuspended in a volume of fractionation buffer equivalent to the
cytosolic volume. These fractions were then resuspended to equivalent
volumes with 2× sample buffer and evaluated by Western blotting.
Western blotting. Sympathetic neurons maintained in 35 mm
culture dishes were washed twice with ice-cold TBS and incubated with
lysis buffer (1% Nonidet P-40, 10% glycerol, 500 µM
sodium orthovanadate, and protease inhibitors in TBS) for 20 min at
4°C with gentle agitation. The cell extracts were centrifuged at
maximum speed in a microfuge to remove collagen and nuclei from the
detergent extracts. The supernatants were next diluted into 2× sample
buffer to arrive at 1×, and boiled for 510 min. SDS-PAGE was
performed on these denatured extracts using 412% gradient gels
(Novex, San Diego, CA). Resolved proteins were transferred to
polyvinylidene difluoride membranes (Millipore, Burlington, MA) and
washed with TBS containing 0.1% Tween-20 (TBST). All immunoblots were
incubated with a blocking solution consisting of TBST containing 4%
heat-inactivated horse serum (HS) for 1 hr at room temperature, and
further incubated for 2 hr in TBST containing the primary antibody, 4%
BSA, and 2% normal donkey serum. Immunoblots were washed three times
with TBST containing 1% HS, incubated with the secondary antibody
(1:10,000 dilution) in blocking solution for 1 hr, washed twice with
TBST containing 1% HS, and finally washed once with TBST alone.
Proteins were visualized by using the supersignal chemiluminescent
detection system (Pierce, Rockford, IL). The tubulin antibody was
used at a 1:50,000 dilution, whereas all other antibodies were used at
a 1:1000 dilution. Immunoblots were reprobed by using the Western analysis protocol described above after stripping residual antibodies from the blots by incubation in 100 mM glycine, pH 2.75, for 1 hr at room temperature followed by two washes with TBST.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assays. Sympathetic neurons maintained in 24-well culture plates (20,000 neurons per well) were treated as described in the text. After
2 d the plates were washed with PBS once and incubated for 10 min
in L15 medium containing 10% fetal bovine serum and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (0.4 mg/ml, Sigma). The medium was then removed, and DMSO (250 µl) was added to each well, mixed, and transferred to a translucent
microtiter plate. The absorbance at 550 nm was measured by using a
microplate reader and subtracted from the background absorbance
measured at 650 nm. The background level of metabolic activity of
contaminating glial cells was subtracted from all conditions by
performing the above analysis on sympathetic neuronal cultures
maintained in anti-NGF for 7 d beginning on the first day of
plating, which killed all neurons leaving only glial cells behind.
Lipid kinase assays. Sympathetic neurons (5 DIV) were
deprived of NGF for 8 hr and then treated with medium alone or NGF (100 ng/ml) in the presence or absence of LY294002 (50 µM) or
Wortmannin (1-10 µM) for 3 min. Both LY294002 and
Wortmannin were added to the culture medium 30 min before NGF
treatment. Because LY294002 is a reversible inhibitor, it had to be
added to all subsequent buffers and washes, whereas Wortmannin, which
is an irreversible inhibitor, was only added to the culture medium
immediately before and during the NGF treatments. The sympathetic
neurons were next washed twice with TBS, and detergent extracts were
produced using the NP40-containing lysis buffer described above. The
extracts were subjected to immunoprecipitation using
anti-phosphotyrosine antibody conjugated to agarose (50 µl)
(Calbiochem, San Diego, CA) overnight at 4°C. The immunoprecipitates
were then subjected to lipid kinase analysis essentially as described
previously (Auger et al., 1990). Briefly, the immunoprecipitates were
washed at 4°C three times with PBS containing NP40 (1%), three times
with PBS containing lithium chloride (0.5 M), and finally
three times with TBS (20 mM Tris, 137 mM NaCl).
The immunoprecipitates were then incubated with phosphatidylinositol
(20 µg, Sigma), cold ATP (40 mM), and
[ 32P]ATP (10 µCi) (ICN
Radiochemicals, Irvine, CA) in TBS containing magnesium chloride (15 mM) for 15 min at room temperature under constant
agitation. The reaction was stopped by adding HCl (6 M, 20 µl), and the lipids was extracted with addition of
chloroform/methanol (1:1, 160 µl total). The lower organic phase,
obtained after centrifugation, was subjected to TLC using
oxalate-coated silica gel chromatography plates (Whatman Inc., Clifton,
NJ) and developed in chloroform/methanol/water/ammonium hydroxide
(60:47:11.3:2). The plate was then dried and subjected to
phosphorimaging analysis.
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RESULTS |
PI-3-K inhibits a cell death event proximal to
c-Jun phosphorylation
A critical early event in the cell death pathway initiated by NGF
deprivation of sympathetic neurons is the Ser-63 phosphorylation and
transcriptional activation of c-Jun (Estus et al., 1994; Ham et al.,
1995). After NGF deprivation c-Jun was rapidly phosphorylated, with
30% of neurons displaying nuclear P-Jun staining after 4 hr, and
almost 60% were P-Jun positive after 9.5 hr (Fig.
1C, D). The number
of P-Jun-positive neurons increased more slowly thereafter, reaching a
maximum of 80% after 2 d (see Fig. 6B). To
determine whether NGF could inhibit acutely c-Jun phosphorylation, sympathetic neurons were deprived of NGF for 4 hr to reach a
half-maximal number of P-Jun-positive neurons. This medium was replaced
with medium containing NGF or medium without NGF, and the cultures were
incubated for an additional 5.5 hr (Fig. 1B).
After treatment, the sympathetic neurons were fixed, and
immunocytochemical detection of Ser-63-phosphorylated c-Jun was
performed as before. Acute NGF readdition to cultured sympathetic
neurons not only halted the number of P-Jun positive neurons (Fig.
1C), it also reversed the number to basal levels . As
expected, readdition of medium alone did not alter the number of
neurons that became P-Jun positive (Fig. 1C). Therefore, NGF
can reverse the phosphorylation of c-Jun after NGF deprivation.
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Figure 1.
PI-3-K inhibits a cell death event proximal to
c-Jun phosphorylation. A, Cultures of sympathetic
neurons maintained in NGF (50 ng/ml) were treated with medium
containing LY294002 (50 µM, left
panel) or PD98059 (25 µM, right
panel) to inhibit PI-3-K or MEK activities,
respectively, or medium containing vehicle alone (DMSO, 0.1%) for 30 min. The cultures were then treated with medium alone, medium
containing NGF (50 ng/ml), or medium containing NGF (50 ng/ml) in the
continued presence of LY294002 or PD98059 for an additional 30 min.
Detergent extracts were prepared, and equal amounts of the extracts
were subjected to phospho-Akt (P-Akt) or phospho-MAPK
(P-MAPK) immunoblotting. Reprobing the same blot with
anti--tubulin after stripping demonstrates equal amounts of protein
in the extracts. B, This schematic diagram depicts the
experimental paradigm for the experiment described in C.
Sympathetic neurons (5 DIV) were deprived of NGF for 4 hr, preincubated
with medium containing LY294003 (50 µM), PD98059 (25 µM), or vehicle alone for 30 min, and next treated with
medium containing NGF (300 ng/nl) in the continued presence of LY294002
or PD98059 for an additional 5 hr. C, Sympathetic
neurons treated as in B were fixed after the treatments,
and P-Jun was detected immunocytochemically. The number of neurons
showing nuclear P-Jun staining is shown as a percentage of total cells.
This experiment was performed in duplicate in three independent cultures.
D, Sympathetic neurons were deprived of NGF
(anti-NGF) or treated with LY294003 (50 µM) in the continued presence of NGF (NGF
+LY29400). The cultures were fixed 2, 4, 6, or 9 hr after
treatment and subjected to P-Jun immunocytochemistry. This experiment
was performed in duplicate in three independent cultures. Error bars
represent SEM.
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We next determined whether the ability of NGF to reverse c-Jun
phosphorylation required PI-3-K or MEK activity. Sympathetic neurons
were deprived of NGF for 4 hr. The neurons were then treated with
LY294002 or PD98059 for 30 min to inhibit the activity of PI-3-K or
MEK, respectively; controls were treated with vehicle (0.10.2%
DMSO). NGF was next added in the continued presence of LY294002 or
PD98059, and these cultures incubated for an additional 5 hr, after
which time they were processed for P-Jun immunocytochemistry (Fig.
1B). PD98059 did not inhibit NGF-mediated
reversal of c-Jun phosphorylation (Fig. 1C). PD98059
inhibited NGF-dependent MAPK activation, as determined by phospho-MAPK
Western analysis of extracts made from PD98059-treated sympathetic
neurons (Fig. 1A), confirming that an effective dose
of PD98059 was used. Treatment of sympathetic neurons with LY294002
completely inhibited NGF-dependent Akt phosphorylation, as determined
by P-Akt (S473) Western analysis (Fig. 1A). In
contrast to inhibition of MEK activity, inhibition of PI-3-K activity
completely blocked NGF-mediated reversal of c-Jun phosphorylation (Fig.
1C).
Because P-Jun staining was often intense when neurons were treated with
LY294002 in either the presence or absence of NGF, we determined
whether PI-3-K inhibition in the presence of NGF was sufficient to
induce c-Jun phosphorylation in sympathetic neurons. Neurons were
treated with LY294002 in the presence of NGF for 29 hr and fixed, and
P-Jun immunocytochemistry was performed. To compare PI-3-K inhibition
with NGF withdrawal, sympathetic neurons were deprived of NGF for the
same amounts of time, and P-Jun immunocytochemistry was performed as
well. PI-3-K inhibition was sufficient to induce rapid and sustained
c-Jun phosphorylation in the presence of NGF that reached a maximum
within 4 hr (Fig. 1D). NGF deprivation, in contrast,
did not induce maximal c-Jun phosphorylation until at least 9 hr after
NGF withdrawal (Fig. 1D). Therefore, PI-3-K activity
inhibits an event in the cell death pathway proximal to c-Jun
phosphorylation, whereas MEK/MAPK activity is not required for
NGF-dependent inhibition of c-Jun phosphorylation initiated by NGF
withdrawal. These results are consistent with reports demonstrating
that PI-3-K inhibition induces c-Jun kinase activity in cerebellar
granule neurons as well as c-Jun phosphorylation in DRG neurons
(Vogelbaum et al., 1998; Shimoke et al., 1999).
Neither PI-3-K nor MEK activity is required for NGF-dependent
inhibition of Bax translocation from cytoplasm to mitochondria
Bax, a proapoptotic Bcl-2 family member, is required for NGF
deprivation-induced cell death of sympathetic neurons (Deckwerth et
al., 1996). The translocation of Bax from the cytoplasm to the
mitochondria is an event that occurs after NGF deprivation of
sympathetic neurons and is required for the release of cytochrome c from the mitochondria and subsequent activation of
caspases (Putcha et al., 1999, 2000). Because NGF can
acutely inhibit the translocation of Bax to the mitochondria, we
determined whether PI-3-K or MEK activity is required for the ability
of NGF to regulate this event. Sympathetic neurons were deprived of NGF
for 18 hr, after which time they were treated with LY294002, PD98059,
or vehicle alone (0.10.2% DMSO) for 30 min. These cultures were then
treated with NGF for an additional 12 hr in the continued presence of
LY294002 or PD98059 (Fig.
2A). Cultures were then lysed, and subcellular fractionation was performed to separate cytoplasmic proteins from lysosomal and mitochondrial proteins. Cytoplasmic and HM fractions were subjected to SDS-PAGE, and Western analysis was performed with antibodies directed against Bax and cytochrome c. NGF deprivation resulted in the loss of Bax
from the cytoplasmic fraction and the coincident accumulation of Bax in
the HM fraction (containing mitochondria), consistent with its
translocation from the cytoplasm to the mitochondria (Fig. 2B). If NGF was replaced after 18 hr of deprivation,
Bax redistribution was halted as compared with replacement of medium
alone (Fig. 2B). Inhibition of PI-3-K or MEK activity
did not alter the ability of NGF to inhibit redistribution of Bax (Fig.
2B), although these pharmacological agents did block
downstream Akt and MAPK phosphorylation, respectively (Fig.
1A). Therefore, neither the PI-3-K nor MEK/MAPK pathways are required for NGF-dependent inhibition of Bax translocation from the cytoplasm to mitochondria.
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Figure 2.
Neither PI-3-K nor MEK/MAPK is required for
NGF-dependent inhibition of Bax translocation from cytoplasm to
mitochondria. A, This schematic diagram depicts the
treatment paradigm used in B. Sympathetic neurons were
deprived of NGF for 18 hr before a 30 min incubation with LY294002 (50 µM), PD98059 (25 µM), or vehicle alone
(DMSO, 0.1%). Neurons were next washed and treated with NGF (300 ng/ml) in the continued presence of LY294002, PD98059, or DMSO for an
additional 12 hr. B, Sympathetic neurons treated as in
A were homogenized in an isotonic buffer and subcellular
fractions produced by differential centrifugation. Equal volumes of the
cytosolic (Cyto) fractions and heavy membrane
(HM) fractions containing mitochondria were
subjected to Western analysis by using antibodies directed against Bax,
cytochrome c, lactate dehydrogenase (LDH,
a cytosolic protein), and cytochrome oxidase IV (COX IV,
a mitochondrial protein). LDH and COX IV immunoblots demonstrate not
only that the cytoplasmic and heavy membrane fractions do not contain
significant amounts of contamination, but also that equal amounts of
protein from each of these fractions were analyzed. This experiment was
performed on two independent cultures with similar results.
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Neither PI-3-K nor MEK activity is required for NGF-dependent
inhibition of cytochrome c release in sympathetic
neurons
NGF can act late in the cell death pathway to inhibit the release
of cytochrome c from the mitochondria, thus inhibiting
activation of caspases (Deshmukh and Johnson, 1998; Putcha et al.,
1999). Because the signaling pathway that NGF uses to inhibit release of mitochondrial cytochrome c is unknown, we determined
whether NGF-dependent inhibition of cytochrome c release
required PI-3-K or MEK activity. Sympathetic neurons were deprived of
NGF for 22 hr, after which time LY294002, PD98059, or vehicle alone
(DMSO) was added for an additional 30 min. The neurons were then
subjected to NGF readdition, or to readdition of medium alone, in the
continued presence of LY294002 or PD98059 for an additional 12 hr (Fig. 3A). The sympathetic neurons
were then fixed, and cytochrome c immunocytochemistry was
performed. As observed previously, acute NGF readdition after 22 hr of
NGF deprivation inhibited any further release of cytochrome
c from the mitochondria (Fig. 3B). When sympathetic neurons were treated with levels of LY294002 that completely inhibited Akt phosphorylation (data not shown), PI-3-K inhibition did not alter NGF-dependent inhibition of cytochrome c release (Fig. 3B). NGF readdition after 22 hr
of deprivation induced Akt phosphorylation (data not shown), indicating
that signaling downstream of TrkA is still intact after 22 hr of NGF deprivation. NGF-mediated inhibition of cytochrome c release
was also not blocked by concentrations of PD98059 that completely blocked MEK activity (Fig. 3B). Therefore, neither PI-3-K
activity nor the MEK/MAPK cascade was required for NGF-dependent
inhibition of cytochrome c release from the mitochondria. In
fact, inhibition of both PI-3-K and MEK activities simultaneously by
treatment with both LY294002 and PD98059 failed to alter NGF-dependent
inhibition of cytochrome c release (data not shown). These
findings are consistent with the data from subcellular fractionations
demonstrating that inhibition of PI-3-K activity or MEK activity did
not affect NGF-dependent inhibition of cytochrome c
release (Fig. 2B).
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Figure 3.
Neither PI-3-K nor MEK/MAPK mediates NGF-dependent
inhibition of mitochondrial cytochrome c release.
A, This schematic diagram represents the experimental
paradigm used in B and C. Sympathetic
neurons were deprived of NGF for 22 hr and next treated with vehicle
alone (DMSO, 0.1%), LY294002 (50 µM), or PD98059 (25 µM) for 30 min. The cultures were then treated with
medium alone or medium containing NGF (300 ng/ml) in the continued
presence of LY294002 or PD98059 for an additional 12 hr before
fixation. B, Cultures treated as in A
were immunostained for cytochrome c, and the number of
neurons displaying punctate cytochrome c are shown as
the percentage of total cells. This experiment was performed in
duplicate in three independent cultures. Error bars represent SEM.
Open symbols indicate cultures that were treated
with medium alone in the presence of the listed pharmacological agent;
closed symbols represent cultures treated with NGF.
C, Sympathetic neuronal cultures were treated as in
A, but the pharmacological agents added were the p38MAPK
inhibitor SB203580 (30 µM), the PKA inhibitor H89 (5 µM), the L-type calcium channel antagonist nifedipine
(200 nM), or vehicle alone, before NGF readdition. The
number of neurons displaying punctate cytochrome c is
displayed as a vertical bar chart depicting only the
percentage of cytochrome c-positive cells remaining at
the end of the treatments. This experiment was performed in duplicate
in three independent cultures, with error bars representing SEM.
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Because neuroprotectants such as cAMP and potassium depolarization are
able to inhibit cytochrome c release after BAX translocation to the mitochondria (Putcha et al., 1999), we determined whether NGF-dependent inhibition of cytochrome c release might use
similar signaling pathways. Sympathetic neurons were deprived of NGF
for 22 hr and treated with nifedipine (200 nM) to
block L-type channel-dependent calcium influx or with H-89 (5 µM) to block protein kinase A (PKA) activity,
both of which are downstream effectors of potassium depolarization and
cAMP, respectively. The neurons were next treated with NGF in the
continued presence of nifedipine or H-89 for an additional 12 hr, and
the number of mitochondrial cytochrome c-positive neurons
was determined. Neither calcium influx nor PKA activity altered the
ability of NGF to inhibit cytochrome c release from mitochondria (Fig. 3C). Nifedipine and H-89, however, were
able to inhibit >50% of KCl-dependent and cAMP-dependent inhibition of cytochrome c release, respectively, thus demonstrating
the effectiveness of these compounds (data not shown).
We also determined whether p38MAPK, another NGF-regulated kinase, might
mediate the ability of NGF to block cytochrome c release. Treatment of sympathetic neurons with a specific inhibitor of p38MAPK
activation, SB203580, with concentrations that completely inhibit
p38MAPK activity did not alter NGF-dependent inhibition of cytochrome
c release (Fig. 3C). Therefore, p38MAPK did not mediate NGF inhibition of mitochondrial cytochrome c
release. In summary, although acute application of NGF can inhibit the release of mitochondrial cytochrome c, activation of PI-3-K,
MEK/MAPK, or p38MAPK was not required. Furthermore, neither PKA
nor calcium influx through L-type channels was responsible for
NGF-dependent inhibition of cytochrome c release.
PI-3-K activity contributes to NGF-dependent metabolism of
sympathetic neurons
Although the above experiments demonstrated that PI-3-K was not
required for the later anti-apoptotic actions of NGF, sympathetic neurons treated with LY294002 atrophied, even in the presence of NGF.
Therefore, the metabolic effects of PI-3-K were examined. To determine
the minimum concentration of LY294002 that is effective in inhibiting
PI-3-K activity over prolonged periods of time, a dose-response
analysis of LY294002 was performed. As a first step, sympathetic
neurons were stimulated with NGF (100 ng/ml) or medium alone, in the
presence of either LY294002 (50 µM) or Wortmannin (1-10
µM), and the activity of PI-3-K was measured directly
using lipid kinase assays. NGF treatment lead to a ~2.5-fold induction of PI-3-K activity (Fig.
4A), similar to what
has been reported previously for sympathetic neurons (Virdee et al.,
1999). LY294002 completely inhibited NGF-dependent PI-3-K activity
(Fig. 4A), but because LY294002 is a reversible
inhibitor, its inhibitory effect could be washed out of the immune
complexes if LY294002 was not included in subsequent
immunoprecipitation and kinase buffers (data not shown). An
irreversible PI-3-K inhibitor, Wortmannin, when added only to the
culture medium immediately before and during the NGF treatment, also
blocked PI-3-K activity at concentrations known to be maximal in intact
cells (10 µM), with a lower concentration (1 µM) leading to a ~60% inhibition (Fig.
4A). In conjunction with the PI-3-K assays, Western
analysis was performed on the supernatants from the lipid kinase assays
performed above with antibodies specific for phosphorylated threonine
308 (T308) or phosphorylated serine 473 (S473) of Akt. Importantly, the
phosphorylation of both residues, and thus the kinase activity,
correlated with the level of catalytic activity of PI-3-K (Fig.
4A). Therefore, Akt phosphorylation and activity were
completely dependent on PI-3-K activity in 5 DIV sympathetic neurons,
and thus monitoring the phosphorylation state of Akt was a reliable
measure of the activity of PI-3-K in these neurons. To perform a
dose-response analysis of LY294002, sympathetic neurons were treated
with increasing concentrations of LY294002 in the presence of NGF for
2 d, after which detergent extracts were prepared from the
cultures that were then subjected to P-Akt (S473) Western analysis
(Fig. 4B). LY294002 (25 µM
and 50 µM) completely inhibited Akt
phosphorylation in the presence of NGF. Similar results were obtained
with P-Akt (T308) immunoblotting as well (data not shown). Therefore,
as in previous experiments, a concentration of 50 µM LY294002 was used for all subsequent
treatments to inhibit PI-3-K activity completely in sympathetic neurons
maintained in NGF.
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Figure 4.
PI-3-K regulates NGF-dependent
metabolism of sympathetic neurons. A, Sympathetic
neurons were treated for 3 min with medium alone, medium containing NGF
(100 ng/ml), or pretreated with LY294002 (50 µM)
or Wortmannin (1-10 µM) for 30 min and then treated with
NGF in the continued presence of the respective PI-3-K inhibitor.
Detergent extracts were produced, and lipid kinase assays
(top panel) were performed on phosphotyrosine
immunoprecipitates. P-Akt (S473 or T308) immunoblotting was performed
on the supernatants from the lipid kinase assays (middle
panels), and -tubulin immunoblotting (bottom
panel) was also performed to confirm equal protein
loading. This experiment was performed twice with identical results,
and the bar graph represents the
means. B, Cultures of sympathetic neurons were treated
with NGF (50 ng/ml) in the presence of increasing concentrations of
LY294002 for 2 d. After this time, detergent extracts produced
from these cultures were subjected to P-Akt (S473) immunoblotting
(top panel). Equal protein loading was confirmed
by reprobing the same immunoblot with antibodies directed against
-tubulin (bottom panel). C,
Sympathetic neurons were treated with NGF (50 ng/ml), vehicle (DMSO,
0.1%), anti-NGF in the presence of BAF (50 µM), or NGF
(50 ng/ml) with LY294002 (50 µM) and BAF (50 µM) for 2 d. The neurons were fixed and subjected to
toluidine blue staining as described in Materials and Methods. Somal
diameters of 100120 neurons were determined from each condition from
three independent cultures. Error bars represent SEM. The somal
diameter of neurons in NGF medium was smaller than that normally
observed because of the fixation, staining, and dehydration of
the neurons. D, Sympathetic neurons were treated with
NGF containing vehicle alone (DMSO, 0.1%), NGF with LY294002 (50 µM), NGF with LY294002 and BAF (50 µM), or anti-NGF with BAF for 2 d. Cultures were
then subjected to MTT analysis as described in Materials and Methods.
The data are presented as the percentage of MTT reduction compared with
NGF-maintained neurons, and each condition was performed in
quadruplicate in four independent cultures. Error bars represent
SEM.
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To determine whether PI-3-K activity regulates NGF-dependent growth of
the cell body, the somal diameter of sympathetic neurons was measured
after PI-3-K inhibition or NGF deprivation. BAF was included in all
conditions to inhibit neuronal death. Neurons maintained in NGF for
7 d had an average somal diameter of 15 µm, whereas neurons
maintained in NGF for 5 d and then deprived of NGF for 2 d
had an average somal diameter of 8 µm (Fig. 4C). PI-3-K
inhibition of 5 DIV, NGF-maintained sympathetic neurons caused a
marked atrophy of the cell bodies to 10 µm (Fig. 4C). PI-3-K inhibition induced less somal atrophy than NGF deprivation, suggesting that although PI-3-K is required, it did not completely account for NGF-dependent growth of the neuronal cell body.
To examine whether PI-3-K also accounts for the metabolic effects of
NGF, we performed MTT reduction assays, which measure the level of
mitochondrial oxidative phosphorylation, on NGF-maintained sympathetic
neurons subjected to PI-3-K inhibition. Two days of NGF deprivation in
the presence of BAF resulted in a 70% loss of metabolic activity (Fig.
4D). PI-3-K inhibition for the same amount of time in
the presence of NGF decreased the metabolic activity of sympathetic
neurons by 35% (Fig. 4D). Similar to LY294002 treatment alone, PI-3-K inhibition of NGF-maintained sympathetic neurons in the presence of BAF, which completely prevents LY-dependent death after 2 d (Fig.
5A), displayed a 45% loss of
metabolic activity, demonstrating that LY294002-dependent inhibition of
metabolic function was not the cause of neuronal death (Fig.
4D). Therefore, PI-3-K contributes to the
growth-promoting and anabolic effects of NGF on sympathetic
neurons.
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Figure 5.
PI-3-K is not solely required for the
survival-promoting activity of NGF. A, Sympathetic
neurons were deprived of NGF in the absence or presence of BAF (50 µM) or were treated with LY294002 (50 µM)
in the presence of NGF (50 ng/ml) with and without BAF. The neurons
were fixed after 24 d. The neurons were stained with toluidine blue,
and the number of surviving neurons was counted and graphed as a
percentage (compared with age-matched NGF-maintained neurons). Each
condition was performed in duplicate in four independent cultures.
B, Sympathetic neurons maintained for 5 d in either
50 ng/ml NGF (open symbols) or 10 ng/ml NGF
(closed symbols) were deprived of NGF or maintained in
NGF in the presence of LY294002 (50 µM). In addition,
sympathetic neurons maintained in 10 ng/ml NGF were also deprived of
NGF in the presence of BAF (50 µM) to confirm that they
could be rescued from apoptosis by a caspase inhibitor. Surviving
neurons were counted from duplicate wells of each condition from two
independent cultures. Error bars represent SEM.
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PI-3-K is not required for the survival-promoting effects of NGF on
sympathetic neurons
Because PI-3-K activity is not required for NGF-mediated
inhibition of Bax translocation or cytochrome c release in
sympathetic neurons, we hypothesized that PI-3-K inhibition in the
presence of NGF would not activate caspases or induce apoptosis. To
test this hypothesis, NGF-maintained sympathetic neurons were incubated with LY294002 for 24 d. The number of neurons that survived these treatments was ascertained by counting the number of Nissl-stained cells in each condition. In most experiments, detergent extracts were
produced from parallel cultures treated with NGF or NGF in the presence
of LY294002 for 2 d. These extracts were subjected to P-Akt
Western analysis to confirm that LY294002 blocked PI-3-K activity (data
not shown). We consistently observed that 70% of sympathetic neurons
treated with LY294002 in the presence of NGF survived after 4 d,
whereas <20% of neurons survived only 2 d of NGF deprivation
(Fig. 5A). To determine whether the LY294002-induced death
required activation of caspases, parallel NGF-maintained cultures were
treated with LY294002 and BAF. Caspase inhibition blocked
LY294002-dependent death after 2 d of treatment, but only partially blocked LY294002-dependent death after 4 d, in contrast to BAF treatment of NGF-deprived neurons, which rescued all neurons from death after 4 d. Taken together, these results indicate that PI-3-K did not completely account for NGF-dependent survival of sympathetic neurons in culture. All neurons treated with LY294002 in
the presence of NGF atrophied (Fig. 5B). Therefore, although LY294002 was able to inhibit PI-3-K in all sympathetic neurons, resulting in a loss of somal diameter and metabolic activity, only 30%
died after PI-3-K inhibition.
One explanation for the inability of PI-3-K inhibition to induce
apoptosis is that maintaining sympathetic neurons in high concentrations of NGF (50 ng/ml) results in activation of survival pathways that would not normally be used in vivo. To test
this hypothesis, sympathetic neurons were maintained in 50 ng/ml NGF as
before, or in a lower concentration of NGF (10 ng/ml). Cultures maintained in 10 ng/ml NGF were phase bright and projected complex neuritic arborizations, as did the neurons maintained in 50 ng/ml NGF.
However, the somal diameter of sympathetic neurons maintained in 10 ng/ml NGF was smaller than neurons maintained in 50 ng/ml NGF (data not
shown). In addition, cell counts revealed that 85% of neurons survived
in 10 ng/ml NGF as compared with neurons maintained in 50 ng/ml NGF
(data not shown). When cultures maintained in 50 or 10 ng/ml NGF for
5 d were deprived of NGF, those maintained in 50 ng/ml NGF as well
as those maintained in 10 ng/ml NGF underwent rapid apoptotic death
(Fig. 5B). Sympathetic neurons maintained in 10 ng/ml NGF
were completely rescued from NGF deprivation-induced apoptosis by
caspase inhibition (Fig. 5B), similar to cultures maintained
in 50 ng/ml NGF. To determine whether their susceptibility to PI-3-K
inhibition differed, sympathetic neurons were treated with LY294002, as
before, in the continued presence of either 50 or 10 ng/ml NGF for 24
d. In cultures maintained in 50 ng/ml NGF, only 2530% of neurons
died after 4 d of PI-3-K inhibition (Fig. 5B). A
similar number of neurons died after 4 d of PI-3-K inhibition when
maintained in only 10 ng/ml NGF (Fig. 5B). Therefore, susceptibility to PI-3-K inhibition does not depend on the
concentration of NGF used to maintain the survival of sympathetic
neurons, suggesting that even at a lower concentration of NGF,
sympathetic neurons rely on additional NGF-regulated survival signals.
PI-3-K inhibition in the presence of NGF does not induce cytochrome
c release in sympathetic neurons
Because PI-3-K inhibition did not kill the majority of sympathetic
neurons and because PI-3-K did not account for NGF-dependent inhibition
of Bax translocation or cytochrome c release, we determined whether LY294002-treated sympathetic neurons were arrested in the cell
death pathway after c-Jun phosphorylation, but before cytochrome
c release. Sympathetic neurons were deprived of NGF, maintained in NGF (50 ng/ml), or maintained in NGF with LY294002 for
24 d, all in the presence of BAF. After 24 d the neurons were
fixed, and simultaneous immunocytochemical detection of cytochrome c and P-Jun was assessed. NGF deprivation induced release of
cytochrome c from 85% of sympathetic neurons after 2 d
(Fig. 6A). PI-3-K inhibition in the presence of NGF, on the other hand, induced cytochrome c release in only 35% of neurons after 4 d
(Fig. 6A). Although as many as 80% of sympathetic
neurons were P-Jun positive shortly after LY294002 treatment (Fig.
1D), many neurons lost c-Jun phosphorylation after
several days, leaving only 3540% of sympathetic neurons c-Jun
positive after 4 d (Fig. 6B). In contrast,
sympathetic neurons deprived of NGF maintained c-Jun phosphorylation in
the presence of BAF (Fig. 6B), suggesting that PI-3-K
inhibition, in contrast to NGF deprivation, did not induce sustained
c-Jun phosphorylation in most neurons. Taken together, these data
indicate that most sympathetic neurons do not complete the cell death
pathway after PI-3-K inhibition but arrest, or perhaps abort, the cell
death process before cytochrome c release. This conclusion
is consistent with the observation that PI-3-K inhibition did not
account for the ability of NGF to block Bax translocation or cytochrome
c release.
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Figure 6.
PI-3-K inhibition does not induce cytochrome
c release from most NGF-dependent sympathetic neurons.
Sympathetic neurons were maintained in NGF (50 ng/ml), maintained in
NGF with LY294002 (50 µM), or deprived of NGF for 2-4 d.
The cultures were then fixed and subjected to simultaneous P-Jun and
cytochrome c immunocytochemistry. Hoechst staining was
also performed to identify nuclei. All conditions included BAF (50 µM) to prevent the loss of neurons. The number of
cytochrome c-positive neurons (A)
and the number of P-Jun-positive neurons (B) were
scored in each condition and graphed as a percentage of total neurons.
Note that initially more neurons are P-Jun positive a few hours after
LY treatment (Fig. 1) but plateau to a lower level after 24 d of
LY294002 treatment in the presence of BAF. C,
Representative examples of immunofluorescent detection of cytochrome
c (Cyt-C, Alexa 488, middle
column), P-Jun (Cy-3, right
column), and nuclei (Hoechst, left
column) in sympathetic neurons after 2 d of treatments as
described above, which are listed to the left of each
panel depicting each condition.
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A similar percentage of neurons released cytochrome c after
PI-3-K inhibition that maintained c-Jun phosphorylation for several days (Fig. 6B). In fact, many of the neurons that
maintained c-Jun phosphorylation also released mitochondrial cytochrome
c, suggesting that these neurons completed the cell death
pathway after PI-3-K inhibition (Fig. 6C). Interestingly,
the size of this population of neurons is similar to the number of
neurons that die after long-term PI-3-K inhibition (Fig.
5A), suggesting that this population of sympathetic neurons
may undergo an apoptotic cell death similar to NGF deprivation.
LY294002-induced death, however, differed significantly from NGF
deprivation-induced death in that 50% of the LY294002-susceptible
neurons were not rescued by caspase inhibition (Fig. 5A). To
determine whether protein synthesis inhibition protected sympathetic
neurons from LY294002-induced death, NGF-maintained neurons were
treated with LY294002 in the presence or absence of 1 µg/ml
cycloheximide (CHX). In contrast to NGF deprivation-induced death,
which is protected by protein synthesis inhibitors (Martin et al.,
1988), CHX treatment exacerbated LY-induced death (Fig. 7). In fact, only 40% of neurons
survived concurrent treatment with LY294002 and CHX after 4 d
(Fig. 7). Therefore, although PI-3-K inhibition-induced death displayed
characteristics similar to the cell death pathway initiated by NGF
deprivation, this death clearly differed from NGF deprivation-induced
death in that it was not fully protein synthesis or caspase
dependent.
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Figure 7.
Inhibition of protein synthesis exacerbates
LY294002-induced death. Sympathetic neurons were maintained in NGF with
vehicle alone (0.1% DMSO) or maintained in NGF with LY294002 (50 µM) in the presence or absence of CHX (1 µg/ml). In
addition, neurons were deprived of NGF, and all conditions were
maintained for 4 d. The cultures were then fixed and stained with
toluidine blue, and the number of surviving neurons was counted. Each
condition was performed in duplicate in two independent cultures, and
error bars represent SEM.
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DISCUSSION |
TrkA activation regulates multiple signaling molecules such as
PI-3-K, phospholipase C- (PLC-), and the MEK/MAPK cascade (Kaplan and Stephens, 1994; Greene and Kaplan, 1995; Segal and Greenberg, 1996). Sympathetic neurons are able to abort acutely the
cell death pathway initiated by NGF deprivation at early and late time
points on readdition of NGF. We found that NGF-dependent PI-3-K
activity inhibited an early cell death event proximal to c-Jun
phosphorylation. PI-3-K activity, however, was not required for NGF to
inhibit the translocation of Bax from the cytoplasm to the mitochondria
or the ability of NGF to inhibit the release of mitochondrial
cytochrome c. Furthermore, the MEK/MAPK pathway did not
account for any of these NGF-dependent events, suggesting that the MAPK
pathway may be more important in regulating neuronal phenotype rather
than survival, as suggested earlier (Virdee and Tolkovsky, 1995;
Creedon et al., 1996; Virdee and Tolkovsky, 1996). Consistent with the
observations that PI-3-K is not required for all of the
survival-promoting effects of NGF, PI-3-K inhibition did not kill the
majority of sympathetic neurons in the presence of NGF. In contrast to
its effects on survival, PI-3-K did contribute to the NGF-dependent
trophic status of sympathetic neurons as measured by the loss of somal
diameter and metabolic function in the presence of PI-3-K inhibitors.
The mechanisms by which NGF or PI-3-K promotes growth and metabolism
are not understood. Consistently, 30% of sympathetic neurons died
after PI-3-K inhibition in the presence of NGF, and this death displays
some, but not all, of the properties of NGF withdrawal-mediated apoptosis.
PI-3-K is not required for the survival-promoting effects of NGF in
sympathetic neurons
Three observations reported here support the conclusion that
PI-3-K alone is not responsible for NGF-dependent survival of sympathetic neurons and that additional signaling pathways cooperate to
maintain survival. First, PI-3-K did not mediate NGF-dependent inhibition of late cell death events such as Bax translocation and
cytochrome c release (Fig. 8).
Second, PI-3-K inhibition did not kill the majority of sympathetic
neurons in the presence of NGF. Third, LY294002-induced death did not
mimic apoptosis induced by NGF deprivation. Specifically, the time
course of LY294002-stimulated death was considerably slower (Fig. 5),
as reported previously (Crowder and Freeman, 1998). In addition,
LY294002-induced death was not fully dependent on caspases (Fig. 5) and
in fact was exacerbated by inhibition of protein synthesis (Fig. 7).
This is in contrast to apoptosis of sympathetic neurons induced by NGF
deprivation, which is completely protein synthesis and caspase
dependent (Martin et al., 1988; Deshmukh et al., 1996). The inability
of BAF and CHX to protect against LY294002-mediated death could be
attributable in part to inhibition of other PI-3-K functions such as
cytoskeletal regulation (Virdee et al., 1999) or metabolic activity
(Fig. 4), although these functions should be lost after NGF
deprivation as well. LY294002-stimulated death, however, had many of
the features observed in NGF deprivation-induced apoptosis such as
phosphorylation of c-Jun (Fig. 1), expression of proapoptotic genes
such as cyclin D1 and c-jun (Estus et al., 1994; Freeman et al., 1994;
Crowder and Freeman, 1998), and release of mitochondrial cytochrome
c (Fig. 6). Additionally, in the few neurons that did die,
LY294002 treatment led to caspase activation as measured by CM-1
staining (data not shown), nuclear condensation, and terminal
deoxynucleotidyl transferase-mediated biotinylated dUTP nick end
labeling (TUNEL) positivity (Crowder and Freeman, 1998; Vaillant et
al., 1999).
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Figure 8.
Multiple signaling pathways regulate the cell
death pathway in sympathetic neurons. This schematic diagram depicts
three points at which NGF can inhibit further progression of the cell
death pathway. PI-3-K activity is required for NGF-dependent inhibition
of c-Jun phosphorylation but is not required for inhibition of Bax
translocation to the mitochondria or release of cytochrome
c from the mitochondria. MEK/MAPK does not account for
any of these NGF-dependent events, and the signaling pathways required
for these two events remain unknown.
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Although overexpression of PI-3-K is sufficient to prevent the death of
sympathetic neurons deprived of NGF (Philpott et al., 1997; Crowder and
Freeman, 1998), whether PI-3-K is necessary for NGF-dependent survival
has been controversial (Philpott et al., 1997; Crowder and Freeman,
1998; Mazzoni et al., 1999; Vaillant et al., 1999; Virdee et al.,
1999). Several methodological differences, such as variations in the
culture conditions, complicate direct comparisons between these
studies. Using culture conditions similar to those reported previously
(Crowder and Freeman, 1998; Mazzoni et al., 1999; Vaillant et al.,
1999), we found that PI-3-K inhibition with LY294002 promoted modest
death of sympathetic neurons over 4 d. In all experiments reported
here we confirmed that NGF deprivation in these cultures led to the
rapid death of >80% of neurons after 2 d and that this death was
completely prevented by caspase inhibition (Fig. 5). In addition, we
frequently confirmed that each lot of LY294002 used was able to inhibit
completely downstream Akt activation by P-Akt immunoblotting. The
amount of death that we observed after LY294002 treatment was not
greatly different from that reported by Crowder and Freeman (1998).
However, we are more impressed with the differences than with the
similarities between LY294002 treatment and NGF deprivation, which
suggests that PI-3-K inhibition does not mimic NGF deprivation.
Furthermore, because much more is now known about the terminal events
involved in NGF deprivation-induced apoptosis, we were able to examine
the function of PI-3-K in these events in greater detail, drawing us to
the conclusion that PI-3-K alone is not required for NGF-dependent
survival. Because PI-3-K inhibition decreased metabolic activity even
when death of sympathetic neurons was prevented by caspase inhibitors
(Fig. 6), relying solely on metabolic assays such as MTT reduction
would lead to the erroneous conclusion that many neurons have died,
when in fact they are atrophic but surviving. Furthermore, in
experiments reported here, we used a concentration of 50 µM LY294002, which completely inhibited downstream Akt
phosphorylation but was a lower concentration than that reported in
several other studies. Although higher concentrations of LY294002 (100 µM) are reported to be more effective in causing death of
sympathetic neurons (Crowder and Freeman, 1998), inhibition of PI-3-K
is maximal by 2550 µM LY294002, suggesting that
higher concentrations of LY294002 may inhibit additional kinases that
contribute to survival.
Cerebellar granule neurons require PI-3-K activity for IGF-I-mediated
and insulin-mediated survival (D'Mello et al., 1997; Dudek et al.,
1997; Miller et al., 1997). When treated with PI-3-K inhibitors in the
presence of IGF-I or insulin, granule cells undergo apoptosis with a
time course similar to survival-factor deprivation and exhibit similar
morphological characteristics as well (D'Mello et al., 1997; Dudek et
al., 1997; Miller et al., 1997). Furthermore, PI-3-K inhibition and
survival-factor deprivation kill the same percentage of neurons,
demonstrating that PI-3-K-independent subpopulations of neurons do not
exist. The sensitivity of granule cells to PI-3-K inhibition under the
conditions used in these experiments is in stark contrast to
sympathetic neurons, which die more slowly and at a lower percentage as
compared with NGF deprivation (Figs. 5, 6) (Crowder and Freeman, 1998).
Taken together, the results reported here, in addition to other studies
on NGF-dependent survival of sympathetic neurons, contrasted with
insulin- and IGF-I-dependent survival of granule cells, support the
conclusion that PI-3-K is not solely responsible for NGF-dependent
survival of sympathetic neurons.
What additional signaling pathways cooperate to promote sympathetic
neuron survival?
Inhibition of PI-3-K activity in NGF-maintained sympathetic
neurons resulted in an arrest in the cell death pathway before mitochondrial cytochrome c release. The fact that PI-3-K was
not required for NGF-inhibition of Bax translocation or cytochrome c release suggests that an additional NGF-regulated pathway
inhibits cell death at this point. This function could not be accounted for by L-type channel, p38MAPK, MEK, and PKA activity because inhibition of these molecules did not alter NGF-dependent inhibition of
cytochrome c release. Although the signaling pathways used by TrkA to block these later cell death events are currently unknown, two possibilities include PLC- and Src family kinases (Atkinson et
al., 1996; al-Ramadi et al., 1998; Hiwasa et al., 1998). In addition,
although this study has focused on three NGF-regulated events in the
cell death pathway, additional NGF-dependent signaling pathways exist,
such as cAMP response element-binding protein-dependent transcriptional
pathways (Bonni et al., 1999; Riccio et al., 1999) and the
competence-to-die pathway (Deshmukh and Johnson, 1998). How NGF
inhibits competence, as well as other cell death events, awaits further investigation.
| |
FOOTNOTES |
Received April 13, 2000; revised July 19, 2000; accepted July 25, 2000.
This work was supported by National Institutes of Health Grants AG12947
and NS38651-01 (E.M.J.) and a National Institutes of Health Training
Grant 5T32-NS07129 (B.A.T.-P.). We thank Patricia A. Osborne for expert
technical assistance and critical reading of this manuscript. We also
thank Dr. Mohanish Deshmukh, Krista Moulder, Charles Harris, Louis
Chang, and Dr. Cynthia Tsui-Pierchala for their thoughtful scientific
discussions and critical reading of this manuscript, and Mary Bloomgren
for secretarial assistance.
Correspondence should be addressed to E. M. Johnson Jr, Department
of Molecular Biology and Pharmacology, Washington University School of
Medicine, 4566 Scott Avenue, Box 8103, St. Louis, MO 63110. E-mail:
ejohnson{at}pcg.wustl.edu.
| |
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ABSTRACT
INTRODUCTION
