From the Laboratories of Neurosciences and
§ Cardiovascular Science, Gerontology Research Center, NIA,
National Institutes of Health, Baltimore, Maryland 21224, the
¶ National Laboratory of Biomembrane and Membrane Biotechnology,
College of Life Sciences, Peking University, Beijing, 100871 China, and the
Department of Neuroscience, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
Received for publication, July 18, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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As a calcium-sensing protein, calmodulin acts as
a transducer of the intracellular calcium signal for a variety of
cellular responses. Although calcium is an important regulator of
neuronal survival during development of the nervous system and is also implicated in the pathogenesis of neurodegenerative disorders, it is
not known if calmodulin mediates these actions of calcium. To determine
the role of calmodulin in regulating neuronal survival and death, we
overexpressed calmodulin with mutations in all four Ca2+-binding sites (CaM(1-4)) or with disabled
C-terminal Ca2+-binding sites (CaM(3,4)) in cultured
neocortical neurons by adenoviral gene transfer. Long-term neuronal
survival was decreased in neurons overexpressing CaM(1-4) and
CaM(3,4), which could not be rescued by brain-derived neurotrophic
factor (BDNF). The basal level of Akt kinase activation was decreased,
and the ability of BDNF to activate Akt was completely abolished in
neurons overexpressing CaM(1-4) or CaM(3,4). In contrast, BDNF-induced
activation of p42/44 MAPKs was unaffected by calmodulin
mutations. Treatment of neurons with calmodulin antagonists and a
phosphatidylinositol 3-kinase inhibitor blocked the ability of BDNF to
prevent neuronal death, whereas inhibitors of calcium/
calmodulin-dependent protein kinase II did not. Our
findings demonstrate a pivotal role for calmodulin in survival
signaling by BDNF in developing neocortical neurons by activating a
transduction pathway involving phosphatidylinositol 3-kinase and Akt.
In addition, our findings show that the C-terminal Ca2+-binding sites are critical for calmodulin-mediated
cell survival signaling.
Neurotrophins are a family of neurotrophic factors involved in the
development, maintenance, and repair of the nervous system (1, 2). The
neurotrophin brain-derived neurotrophic factor (BDNF)1 and its high-affinity
receptor TrkB are widely expressed in neurons throughout the central
nervous system (2-4). Binding of BDNF to TrkB results in
phosphorylation of tyrosine residues in the cytoplasmic domain of the
receptor (5) and subsequent recruitment and activation of various
signaling proteins, including Shc, Gab1, FRS-2, and SH2-B (6-9). Such
signaling proteins may link Trk activation to Ras and the downstream
activation of mitogen-activated protein kinases (MAPKs) and the
phosphatidylinositol 3-kinase (PI3K)-Akt kinase pathway (10-13). MAPK
and Akt kinase have been reported to promote the survival of neurons in
various physiological and pathological settings (14-20).
Calcium is a second messenger that mediates a variety of physiological
responses of neurons to neurotransmitters and neurotrophic factors,
including cell survival responses (14, 21-26). An increase in
cytoplasmic calcium levels can activate Ras, resulting in the activation of Raf, MEK, and MAPKs (27, 28); and calcium can also
activate the PI3K-Akt pathway (14). BDNF has several effects on
cellular calcium homeostasis, including enhancement of calcium oscillations in cultured hippocampal neurons (29) and stimulation of
calcium release from inositol 1,4,5-trisphosphate-sensitive stores in myocytes (30). In addition, studies have suggested roles for
calcium/calmodulin-dependent protein kinase (CaMK) IV in
the effects of BDNF on gene expression (31). However, it is not known
if and how calcium mediates survival signaling by BDNF.
Calmodulin is a loop-helix-loop Ca2+-binding protein that
is evolutionarily conserved and is expressed in all mammalian cells (32). Calmodulin transduces Ca2+ signals by interacting
with specific target proteins; examples include CaMKII, CaMKIV,
calcineurin, spectrin A2, p21, and neuronal nitric-oxide synthase (33,
34). Calmodulin has four Ca2+-binding sites, with two in
the globular N-terminal domain and two in the globular C-terminal
domain; the Ca2+-binding sites are separated by flexible
Neocortical Cell Cultures and Experimental
Treatments--
Cultures of embryonic neocortex were prepared from
embryonic day 18 Sprague-Dawley rats as described previously (36).
Cells were plated at a density of 50,000 cells/cm2 on 12-mm
glass coverslips photoetched with a grid (Eppendorf) to allow
relocation of the same microscope field at different time points. The
coverslips were coated with poly-L-lysine (Sigma). The
culture medium consisted of Neurobasal medium with B27
supplements (Invitrogen); cultures were maintained in a 95% room air
and 5% CO2 humidified atmosphere at 37 °C. BAPTA-AM and
EGTA were from Molecular Probes, Inc. BDNF was from Invitrogen. W-12,
W-13, clasto-lactacystin Viral Vectors--
Site-directed mutagenesis was performed by
PCR using the overlap extension method (37). A synthetic gene encoding
wild-type bovine calmodulin (38) was used as template for the PCRs. In mutant CaM(1,2), Asp21 and Asp57 were changed
to Ala. In mutant CaM(3,4), Asp94 and Asp130
were mutated to Ala, whereas in mutant CaM(1-4), Asp21,
Asp57, Asp94, and Asp130 were all
changed to Ala. A 5'-flanking BamHI restriction site and a
3'-flanking EcoRI restriction site were introduced into the
wild-type and mutant calmodulin cDNAs. DNA fragments
(BamHI-EcoRI) containing the entire coding region
of wild-type or mutant calmodulin were first subcloned into the pGEX-4T
plasmid. DNA fragments (HindIII-XbaI) containing
the wild-type or mutant calmodulin cDNA were then removed from the
pGEX-4T plasmid and subcloned into the adenoviral shuttle plasmid
pAdv/RSV (39). A portion of each construct (15 µg) and an equal
amount of the adenoviral package plasmid pJM17 were cotransfected into
the E1 trans-complementing cell line 293 using
calcium phosphate. The recombinant adenoviruses, generated by
homologous recombination, were isolated by plaque formation in 293 cells. Viral DNA was isolated from at least five separate plaques for
each construct, and the insertion of calmodulin or its mutants or
Immunohistochemistry--
At 8 days in culture, the cells were
fixed in 4% paraformaldehyde in PBS for 30 min at room temperature and
then washed with PBS. The fixed cells were permeabilized with 0.2%
Triton X-100 for 10 min, followed by a 2-h incubation at room
temperature in blocking solution (5% normal goat serum and 0.2%
Triton X-100 in PBS, pH 7.4) containing primary monoclonal antibody
against MAP2, a microtubule-associated protein used as neuronal marker. After washing with PBS, cells were incubated for 2 h in PBS
containing fluorescein-conjugated goat anti-mouse IgG (1:200;
ImmunoResearch Laboratory). After washing with PBS, some cultures were
counterstained with propidium iodide (10 µg/ml), a DNA-binding dye,
to label nuclei.
Evaluation of Neuronal Survival--
At 5 days in
vitro, live neurons in each of four 175-µm2 regions
of the grid on each coverslip were counted from images captured using a
digital camera; at the beginning of each experiment, the average number
of neurons in a 175-µm2 region was in the range of
30-50. Four separate coverslips were used for each condition; and
thus, ~800 neurons were quantified for each experiment. Four separate
experiments were performed. Neurons that were in the process of
degenerating (i.e. that displayed nuclear condensation,
membrane blebbing, and extensive neurite fragmentation) were excluded.
On each subsequent day, the same areas were located and recounted; the
number of live neurons remaining at each day was expressed as a
percentage of the initial number. To identify and quantify apoptotic
neurons, cells were fixed in 4% paraformaldehyde in PBS and then
stained with the DNA-binding dye Hoechst 33258. Coverslips were mounted
onto glass slides and examined under epifluorescence illumination using
a ×40 objective lens. Cells were considered "apoptotic" if their
nuclear chromatin was condensed or fragmented, whereas cells were
considered viable if their chromatin was diffusely and evenly
distributed throughout the nucleus.
To further confirm and detect apoptotic DNA fragmentation, a TUNEL kit
(R&D Systems) was used. Briefly, the cells were fixed with 4%
paraformaldehyde in PBS for 30 min and washed with PBS, pH 7.4. After
blocking of endogenous peroxidases, the cells were incubated at
37 °C for 1 h in a reaction mixture containing terminal transferase, biotinylated nucleotide (dNTP), or PBS as a control. Cells
were then incubated in the presence of streptavidin-conjugated horseradish peroxidase for 20 min at room temperature. After rinsing in
PBS, DNA strand breakage was visualized in the presence of horseradish
peroxidase substrate. The apoptotic cells exhibited dark blue nuclear
staining. Approximately 500 cells on each coverslip were scored; three
separate coverslips were assessed for each condition in each
experiment; and three separate experiments were performed for both
Hoechst 33258 staining and TUNEL staining.
Immunoblot Analysis--
After infection and experimental
treatment, the cells were solubilized in SDS-PAGE sample buffer, and
the protein concentration in each sample was determined using a Bio-Rad
protein assay kit with bovine serum albumin as the standard. Proteins
(50 µg of protein/lane) were then resolved on 7.5-12%
SDS-polyacrylamide gels and electrophoretically transferred to a
nitrocellulose membrane. Membranes were blocked with 4% nonfat milk in
Tris-HCl-based buffer with 0.2% Tween 20, pH 7.5, and then incubated
overnight at 4 °C in the presence of primary antibody. Cells were
incubated for 1 h in the presence of a 1:5000 dilution of
secondary antibody (IgG) conjugated to horseradish peroxidase. Reaction
product was visualized using an enhanced chemiluminescence Western blot
detection kit (ECL, Amersham Biosciences). The primary antibodies
included anti-tubulin (mouse, 1:5000; Sigma), anti-phospho-p42/44 MAPK (mouse, 1:1000; New England Biolabs Inc.), anti-p42/44 MAPK (rabbit, 1:1000; New England Biolabs Inc.), anti-phospho-Akt (rabbit, 1:1000; Cell Signaling Technology), anti-Akt (rabbit, 1:1000; Cell Signaling Technology), and anti-calmodulin (mouse, 1:1000; Upstate Biotechnology, Inc.).
Statistics--
All data are presented as means ± S.E.
Comparisons between controls and treatments were performed using
Student's unpaired t test or ANOVA when appropriate. A
value of p < 0.05 was considered to be statistically significant.
Calcium-binding Domains 3 and 4 of Calmodulin Are Required for the
Long-term Survival of Neocortical Neurons--
To investigate how
calmodulin, a calcium-sensing protein, can influence neuronal
survival, we used primary neocortical neurons that were isolated and
cultured from embryonic day 18 Sprague-Dawley rats in serum-free
medium. The cultures consisted of two populations of cells (based on
morphology and expression of cell type-specific proteins). Cells with
round cell bodies and long thin neurites were immunoreactive with an
antibody against MAP2, whereas flat cells with no neurites were
immunoreactive with an antibody against glial fibrillary acidic protein
(Fig. 1, A-C; and data not
shown). At 8 days in culture, 91 ± 3.3% (n = 3)
of the cells were MAP2-positive and 9 ± 4.5% (n = 3) were MAP2-negative, whereas at 14 days in culture, the percentage
of MAP2-negative cells was increased to ~25%. The distinct
morphologies of neurons and astrocytes allowed us to unambiguously
identify neurons for the various analyses performed in this study.
To study the role of calmodulin in neuronal survival, we performed
site-directed mutagenesis to generate forms of calmodulin in which
either Ca2+-binding domains 1 and 2 (CaM(1,2)) or 3 and 4 (CaM(3,4)) or all four Ca2+-binding domains (CaM(1-4))
were mutated at Asp residues known to be critical for Ca2+
binding (Asp21, Asp57, Asp94, and
Asp130); in each case, the mutation changed the Asp residue
to an Ala residue. Adenoviral constructs were used to deliver the
calmodulin cDNAs into primary neocortical neurons in culture. To
control for nonspecific effects of infection and overexpression of any exogenous protein, we used neurons infected with
adeno-
The cells were cultured for 5 days in Neurobasal medium with B27
supplements and then infected with adeno- Evidence That Calmodulin with Functional Calcium-binding Domains 3 and 4 Is Required for the Neuronal Survival-promoting Activity of
BDNF--
The initial results suggested a role for calmodulin in cell
survival signaling in developing neocortical neurons. To link calmodulin to a specific trophic factor signaling pathway, we employed
BDNF, a neurotrophin previously shown to promote the survival of
cultured embryonic hippocampal and neocortical neurons (42-44). When
cultures that had been infected with the control adenoviral construct
or adeno-CaM(1,2) were treated with BDNF after withdrawal of trophic
supplements, long-term neuronal survival was significantly increased
(Fig. 3A). In contrast, the
ability of BDNF to promote neuronal survival was completely abolished in neurons expressing either the CaM(1-4) or CaM(3,4) mutant (Fig. 3,
B and C), demonstrating that Ca2+
binding in the C-terminal EF-hands of calmodulin is required for BDNF
cell survival signaling. To confirm a role for calmodulin in BDNF cell
survival signaling, we employed W-13, a calmodulin antagonist, and
W-12, a less active structural analog of W-13 (W-13 IC50 = 68 µM versus W-12 IC50 = 260 µM) (45). In these experiments, neuronal death was
assessed 2 days following withdrawal of trophic supplements by counting
Hoechst-stained neurons with condensed nuclei or TUNEL-positive
cells (Fig. 4A). In cultures subjected to withdrawal of trophic supplements, ~20% of the cells died during a 2-day period, whereas ~10% of the cells died when they
were treated with BDNF. When cells were treated with 80 µM W-13, a concentration previously shown to inhibit
calmodulin maximally (14), the neuronal survival-promoting effect of
BDNF was completely abolished (Fig. 4, A and B).
At this concentration, the effect of W-13 was specific because the same
concentration of W-12 did not alter the ability of BDNF to prevent
apoptotic neuronal death (Fig. 4B). As was the case when
neuronal survival was evaluated in neurons expressing a calmodulin
mutant by morphological criteria (Fig. 3), W-13 completely abolished
the ability of BDNF to prevent neuronal death as assessed by counting
apoptotic nuclei (Fig. 4, A and B).
Two pathways for BDNF cell survival signaling in neurons have been
identified, one involving PI3K and Akt kinase and the other involving
MAPKs p42 and p44 (also known as ERK1 and ERK2) (12, 13). To determine
which of these pathways is involved in BDNF cell survival signaling in
our neocortical cultures, we pretreated cultures with either LY294002,
an inhibitor of PI3K (46), or PD98059, an inhibitor of MEK1 (47).
LY294002 completely abolished the neuronal survival-promoting effect of
BDNF, whereas PD98059 did not (Fig. 4B). In other studies,
we found that the same batch of PD98059 could block the activation of
ERKs by BDNF (data not shown), suggesting that failure of PD98059 to
abolish the neuronal survival-promoting effect of BDNF is not due to
instability of PD98059. These results suggest that activation of
PI3K is required for BDNF cell survival signaling, whereas activation
of ERKs is not.
Calmodulin Activity and Intact Calcium-binding Domains 3 and 4 Are
Essential for Activation of Akt Kinase, but Not p42/44
MAPKs, by BDNF--
To determine whether calmodulin acts upstream of
one or both of the kinase cascades activated by BDNF, we exposed
neocortical cultures to BDNF in the absence or presence of the
calmodulin antagonist W-13 and then evaluated the activation levels of
Akt and ERKs by performing immunoblot analyses of cell lysates using antibodies against the phosphorylated (active) forms of Akt and ERK1/2.
The levels of phospho-Akt and phospho-ERK1/2 were greatly increased
during a 10-min exposure to BDNF (Fig.
5A). The levels of Akt and ERK
proteins were unchanged in neurons exposed to BDNF, thus indicating
that the increased levels of phospho-Akt and phospho-ERKs are the
result of increased phosphorylation of a constant level of proteins.
Treatment of cultures with W-13 largely abolished the ability of BDNF
to activate Akt, whereas W-12 had no effect on BDNF-induced activation
of this kinase (Fig. 5A). In contrast, W-13 did not
compromise the ability of BDNF to activate ERK1 or ERK2. These results
suggest that calmodulin is a pivotal upstream mediator of BDNF-induced
Akt activation and cell survival signaling in embryonic neocortical
neurons.
We next sought to determine the Ca2+-binding requirements
for activation of Akt and ERKs by calmodulin. The basal levels of Akt
phosphorylation were greatly decreased in neurons overexpressing the
CaM(1-4) and CaM(3,4) mutants compared with uninfected neurons or
neurons infected with control virus or with adeno-CaM(1,2) (Fig. 5,
B and C). Exposure of control cultures to BDNF
resulted in a large increase in phospho-Akt levels in neurons infected with control virus or virus containing CaM(1,2); Akt phosphorylation was increased within 10 min of exposure to BDNF, was further increased at 30 min, and remained elevated through 240 min of exposure to BDNF
(Fig. 6, A and B).
In contrast, BDNF did not cause a significant increase in the levels of
phospho-Akt in neurons expressing the CaM(1-4) or CaM(3,4) mutant
(Fig. 6, A and B).
We did not observe any significant differences in the levels of
calmodulin in neurons expressing adeno-wild-type calmodulin (data not
shown), adeno-CaM(1,2), and adeno- Akt Activation by BDNF Requires Intracellular Free Calcium and
Activation of PI3K, but Is Independent of CaMKs--
To determine the
events upstream of Akt activation in the calmodulin-mediated BDNF
signaling pathway, we employed several pharmacological tools.
Incubation of neurons in the presence of the intracellular
Ca2+ chelator BAPTA-AM significantly reduced activation
of Akt by BDNF (Fig. 8A). In
contrast, the extracellular Ca2+ chelator EGTA, switching
the culture medium to calcium-free medium prior to BDNF stimulation, or
treatment with the L-type calcium channel blocker nicardipine did not
prevent activation of Akt by BDNF. These findings demonstrate a
requirement for intracellular free Ca2+, but not for
extracellular Ca2+, in Akt activation. Our data are
consistent with a recent study showing that CaMKII can promote cell
survival by directly phosphorylating and activating Akt (48). To
determine the involvement of CaMKs in Akt activation by BDNF, we
employed KN-93 (a CaMKII inhibitor that selectively binds to the
calmodulin-binding site of the enzyme and prevents the association of
calmodulin with CaMKII), KN-92 (an inactive analog of KN-93 as a
control), and AIP (a cell-permeable competitive peptide inhibitor of
CaMKII). Cultures were pretreated with KN-93 and AIP at concentrations
previously shown to inhibit CaMKII (49) and then stimulated with BDNF.
KN-93 and AIP did not prevent activation of Akt by BDNF (Fig.
8B), indicating that CaMKII is not required for BDNF-induced
Akt activation. Because PI3K is an upstream activator of Akt in several
cell survival signaling pathways, including a pathway activated by
BDNF, we determined whether PI3K activation is required for Akt
activation by BDNF. When cultures were pretreated with the PI3K
inhibitor LY29002, the ability of BDNF to activate Akt was completely
abolished (Fig. 8B). Collectively, these findings suggest
a cell survival signaling pathway in which BDNF activates Akt by
a signaling pathway involving activation of calmodulin by calcium.
Our findings establish a pivotal role for calmodulin as a mediator
of the cell survival-promoting action of BDNF in embryonic neocortical
neurons. An antagonist of calmodulin and calmodulin mutants with
disabled Ca2+-binding domains significantly decreased
long-term neuronal survival and completely abolished the ability of
BDNF to support neuronal survival. BDNF activated two signal
transduction pathways previously linked to cell survival signaling in
neurons: one pathway involves PI3K and Akt, and the other involves
ERK1/2. However, we found that inhibition of calmodulin prevented
activation of Akt by BDNF, but did not prevent activation of ERK1/2.
These findings suggest that calmodulin acts upstream of Akt in the cell
survival pathway activated by BDNF. They further suggest that ERKs do
not play an essential role in cell survival signaling by BDNF; and
indeed, inhibition of ERKs with PD98059 did not compromise the ability of BDNF to support neuronal survival. A similar dependence of BDNF cell
survival signaling on PI3K, but not on ERKs, was recently reported in a
study of motor neurons (20). The survival of sympathetic neurons
induced by depolarization and calcium influx requires activation of a
pathway involving Ras, PI3K, and Akt (50). Our data are consistent with
calcium/calmodulin activating a similar cell survival pathway in
neocortical neurons. Based upon the prominent involvement of CaMKII and
other CaMKs as mediators of biological actions of calmodulin in neurons
(34), we attempted to block the neuronal survival-promoting action of
BDNF with inhibitors of CaMKII. Surprisingly, inhibition of CaMKII did
not block activation of Akt induced by BDNF, suggesting that calmodulin
acts on other proteins upstream of Akt. Although our data clearly show
that calmodulin acts upstream of Akt, the protein(s) with which
calmodulin interacts in this pathway remains to be established. Based
upon recent studies of the involvement of calmodulin in insulin
signaling, a site of action of calmodulin in cell survival signaling by
BDNF downstream of receptor activation, but upstream of PI3K
activation, seems likely. Thus, it was shown that calmodulin
antagonists have no effect on insulin receptor autophosphorylation or
tyrosine phosphorylation of insulin receptor substrate-1, but
completely abolish insulin-induced activation of PI3K (51). Further
analyses in the latter study showed that calmodulin is essential for
the formation of phosphatidylinositol 3,4,5-triphosphate. A candidate target protein for activation of PI3K by calmodulin is Ras because it
has been reported that calmodulin can bind directly to Ras and modulate
its downstream signaling (52).
p42/44 MAPKs can be activated by membrane depolarization and
Ca2+ influx through voltage-dependent L-type
calcium channels in PC12 cells through initial activation of Ras (27,
28). This Ras-MAPK pathway can also be activated by neurotrophins such
as BDNF by ligand binding to a specific receptor tyrosine kinase,
resulting in tyrosine autophosphorylation of the receptor and tyrosine
phosphorylation of Shc (12, 53). Previous studies suggested that
Ca2+ flux and activation of calmodulin is necessary
for activation of the Ras-MAPK pathway in response to membrane
depolarization (54, 55); however, whether receptor tyrosine kinase
phosphorylation and the subsequent Shc-Ras-MAPK activation are
calmodulin-modulated is neurotrophin- and cell
type-dependent. Nerve growth factor activation of the ERK
pathway in PC12 cells is modulated by Ca2+ and calmodulin
(56), whereas activation of ERKs by epidermal growth factor in the same
cell type is not modulated by calmodulin (54). In our studies of
primary cortical neurons, we found that Ca2+-insensitive
calmodulin mutants specifically blocked BDNF-induced Akt activation
without affecting ERK activity. Our data demonstrate a novel
calmodulin-mediated, but CaMK- and ERK-independent, neuronal survival
signaling pathway in neocortical neurons.
The Ca2+-binding requirements for the biological activities
of calmodulin in neurons had not been studied previously, although it
had been shown that occupancy of all four Ca2+-binding
domains is required for full activity of calmodulin in other cell types
(14, 51, 56, 57). To determine the roles of Ca2+ binding in
the neuronal survival-promoting function of calmodulin, we mutated
critical asparagine residues in each of the Ca2+-binding
domains of calmodulin. However, we observed that neurons overexpressing
mutant calmodulin also expressed lower levels of wild-type calmodulin,
suggesting that the forms of calmodulin with mutated calcium-binding
domains interfere with the ability of endogenous calmodulin to activate
Akt and to promote neuronal survival in response to BDNF. Previous
studies employed co-immunoprecipitation and affinity chromatography to
demonstrate that calmodulin associates with SH2 domains in the 85-kDa
regulatory subunits of PI3K, thereby enhancing PI3K activity in
vitro and in intact cells in the presence of calcium (57). We
found that overexpression of calmodulin with mutations in all four
Ca2+-binding domains completely abolished the activation of
Akt and the neuronal survival-promoting action of BDNF. It is therefore reasonable to consider a model in which calmodulin associates with the
SH2 domains in the 85-kDa regulatory subunits of PI3K in unstimulated
cells. In response to a stimulus such as BDNF, calcium binds to
calmodulin, resulting in a conformational change required to activate
PI3K. When mutant (calcium-unresponsive) forms of calmodulin are
expressed in neurons, they therefore act in a dominant-negative manner
by competing with wild-type calmodulin for binding to PI3K. Expression
of the mutant forms of calmodulin therefore renders neurons
unresponsive to BDNF. It is also possible that calcium-insensitive
mutants of calmodulin compete, in a dominant-negative manner, with
other calmodulin-regulated proteins such as Ras (52).
Although disabling all four Ca2+-binding domains was
expected to completely abolish the biological activities of calmodulin, we also found that disabling only the two C-terminal
Ca2+-binding domains was sufficient to render calmodulin
completely dysfunctional. Previous studies have shown that the
C-terminal pair of EF-hands has a 3-5-fold higher affinity for
Ca2+ than the N-terminal pair of sites (32). It is
therefore likely that disabling the two C-terminal
Ca2+-binding domains of calmodulin (CaM(3,4)) creates a
low-affinity Ca2+-binding protein that can no longer act as
a Ca2+ sensor and therefore fails to transduce the calcium
signal to downstream effector proteins such as Akt. Our observations
that overexpression of the CaM(3,4) mutant, but not the CaM(1,2)
mutant, could mimic the effects of the CaM(1-4) mutant by blocking the activation of Akt (Fig. 6) support the latter mechanism. Collectively, our data suggest that, to activate the PI3K-Akt pathway,
calcium-binding domains 3 and 4 of calmodulin are essential.
It has been reported previously that activation of TrkA leads to a
small and rapid increase in [Ca2+]i (58), and
also we observed that intracellular Ca2+ was required for
activation of Akt by BDNF in neocortical neurons (Fig. 8). Because
BDNF-induced activation of Akt was abolished by the intracellular
Ca2+ chelator BAPTA-AM, but not by the extracellular
Ca2+ chelator EGTA, calcium-free medium, or an L-type
calcium channel blocker, it appears that BDNF signaling mobilizes an
intracellular pool of Ca2+ that is required for activation
of calmodulin. Indeed, it was recently reported that BDNF stimulates a
rapid release of Ca2+ from endoplasmic reticulum stores in
cerebellar granule neurons (59).
The downstream targets of Akt that promote neuronal survival have begun
to be elucidated. Activation of the PI3K-Akt pathway can increase the
expression of anti-apoptotic members of the Bcl-2 family of proteins
(60), can phosphorylate and thereby inactivate the pro-apoptotic
protein Bad (61), and may also act at a post-mitochondrial step to
prevent caspase activation (62). Consistent with the latter mechanisms
in the neuronal survival-promoting actions of BDNF, it has been shown
that BDNF induces the expression of Bcl-2 in sensory and
parasympathetic neurons (63) and that BDNF blocks ischemia-induced
activation of caspase-3 in cortical neurons of neonatal rats (64). BDNF
has also been shown to increase the production of antioxidant enzymes
in cultured rat hippocampal neurons (65). BDNF can also stabilize
cellular calcium homeostasis, an action that may contribute to its
ability to protect neurons against apoptosis (44, 65). The latter
action of BDNF is interesting in light of the present findings
demonstrating a pivotal role for the Ca2+-binding protein
calmodulin as a mediator of neuronal cell survival signaling. Thus, the
calcium/calmodulin signal appears to activate genes that encode
proteins that help neurons cope with more severe and potentially lethal
stimuli. Based upon previous studies of the neuroprotective effects of
BDNF, such proteins may include the calcium-binding protein calbindin
(66) and glutamate receptor subunits (36).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical domains. When bound to Ca2+, calmodulin
undergoes a conformational change that allows it to bind to target
effector proteins and to stimulate or inhibit their activities.
Calmodulin plays important roles in regulating synaptic plasticity
(35), but its possible roles in mediating actions of neurotrophic
factors are unknown. Because BDNF has been shown to increase the levels
of intracellular Ca2+ in neurons on the one hand and also
activates PI3K-Akt and Ras-MEK-MAPK pathways on the other hand, we
sought to establish a role for calmodulin in one or both of these BDNF
cell survival signaling pathways.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactone, KN-92, KN-93, and AIP
were from Calbiochem-Novabiochem. LY294002 and PD98059 were from Cell
Signaling Technology. Nicardipine was from Sigma.
-galactosidase in the viral genome was confirmed by restriction
endonuclease digestion. High-titer stocks of recombinant adenoviruses
were grown in 293 cells and purified by density gradient
ultracentrifugation. The titers of viral stocks were ~2.8 × 109 pfu/ml for wild-type calmodulin, 6 × 109 pfu/ml for CaM(1,2), 4.4 × 109 pfu/ml
for CaM(3,4), 3 × 109 pfu/ml for CaM(1-4), and
4.8 × 109 pfu/ml for
-galactosidase. The methods
for delivery of the adenovirus to the cultures were described
previously (40). The cultured neurons were infected at a multiplicity
of infection of 50 pfu/cell.
-Galactosidase Staining--
After 3 days of infection with
adeno-
-galactosidase, the cells were rinsed briefly in ice-cold PBS
and incubated for 1 h in fixative solution (2% formaldehyde,
0.2% glutaraldehyde, and 0.1 M NaHPO4, pH
7.3). Cells were washed three times with rinse solution (0.01% sodium
deoxycholate, 0.02% Nonidet P-40, 2 mM MgCl2,
and 0.1 M NaHPO4, pH 7.3) and then placed in
X-gal stain solution containing 2 mM MgCl2, 5 mM K3Fe(CN)6, and 5 mM
K4Fe(CN)6 plus 0.1 M
NaHPO4, pH 7.3, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and 8 mg/ml X-gal for 12-16 h at 37 °C. Stained cells were rinsed and equilibrated in sucrose solution (20% sucrose, 0.05% NaN3, and 0.1 M NaHPO4, pH 7.3) at
4 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of primary cultured neurons
expressing the CaM(1-4) and CaM(3,4) mutants.
A, immunostaining of cells with MAP2
(green), a neuron-specific marker, showing that the vast
majority (91 ± 3.3%, n = 3) of the cells in the
cultures are neurons at 8 days in culture. PI, propidium
iodide, a fluorescent DNA-binding dye that labels nuclei. B,
the process-bearing cells (arrows) are exclusively
MAP2-positive, indicating that they are neurons; a small percentage
(9 ± 4.5%, n = 3) of the cells are MAP2-negative
flat glial cells (arrowhead). In this photomicrograph, we
enhanced the background staining to show the morphology of the flat
cells. C, representative photomicrographs of neocortical
neurons in culture stained for -galactosidase activity in
uninfected control cells (Con) and in cells 3 days
after infection with adeno-
-galactosidase (Adeno-
-gal)
using a multiplicity of infection of 50. The infection efficiency was
essentially 100% as indicated by the blue color in all
neurons. D, Western blot analysis showing the levels of
calmodulin proteins after 24, 48, or 72 h of infection with
adeno-wild-type calmodulin (wt), adeno-CaM(1,2),
adeno-CaM(1-4), and adeno-CaM(3,4).
-galactosidase as the control in this study. Using a
multiplicity of infection of 50, we found that nearly 100% of the
neurons were infected as demonstrated by
-galactosidase staining
(Fig. 1C). Immunoblot analysis of cell lysates showed that
the CaM(3,4) and CaM(1-4) proteins were overexpressed in infected
cells and that, as expected for adenoviral vectors, expression was
sustained at a high level (Fig. 1D). The mutant forms of
calmodulin exhibited a slightly lower apparent molecular mass than
endogenous calmodulin (Fig. 1B), consistent with a previous
finding that calmodulin displays a
Ca2+-dependent shift in electrophoretic
mobility (41). In a previous study, we found that when cardiac myocytes
were infected with adenoviral vector containing wild-type calmodulin or
mutant CaM(1,2), the levels of calmodulin were tightly regulated such
that little or no overexpression of calmodulin
occurred.2 We obtained a
similar result in primary neocortical neurons infected with adenovirus
containing wild-type calmodulin or mutant CaM(1,2) (Fig.
1D).
-galactosidase (control),
adeno-CaM(1,2), adeno-CaM(1-4), or adeno-CaM(3,4). At 5 days in
culture, cells were infected with viral vectors. Three days after
infection, the culture medium was changed to Neurobasal medium lacking
B27 trophic supplements (withdrawal of trophic support). Fig.
2A shows representative
phase-contrast micrographs of cultured cortical neurons that had been
infected with adeno-
-galactosidase and adeno-CaM(1-4) immediately
prior to withdrawal of trophic supplements and 4 days later. Many more neurons had degenerated 4 days after withdrawal of trophic support in
cultures infected with CaM(1-4) compared with cultures infected with
control virus. To quantitatively analyze the time course of neuronal
survival, the total number of cells identifiable as live neurons by
phase-contrast microscopy in each grid square was counted daily for 6 days. The results show that neuronal survival was significantly
decreased in neurons expressing the CaM(3,4) and CaM(1-4) mutants 2, 4, and 6 days after withdrawal of trophic support compared with neurons
infected with control virus or virus containing the CaM(1,2) mutant
(Fig. 2B). In an additional experiment, we performed TUNEL
staining analysis of adeno-
-galactosidase-, adeno-CaM(1,2)-,
adeno-CaM(1-4)-, or adeno-CaM(3,4)-infected neurons 6 days after
withdrawal of trophic support. Essentially all of the neurons
expressing CaM(3,4) or CaM(1-4) were TUNEL-positive, whereas ~70%
of the neurons expressing
-galactosidase (control) or infected with
adeno-CaM(1,2) were TUNEL-positive (Fig. 2, C and
D). When trophic support was not removed from the cultures, adeno-CaM(3,4)- and adeno-CaM(1-4)-infected neurons died
significantly faster compared with adeno-
-galactosidase- and
CaM(1,2)-infected neurons (Fig. 2, C and
D). Thus, the CaM(3,4) and adeno-CaM(1-4) mutations promote
neuronal death in the absence or presence of trophic support,
indicating that the effect of the mutations is not the result of an
altered response to one or more components of the B27
supplements.
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Fig. 2.
Overexpression of the CaM(1-4) and CaM(3,4)
mutants accelerates neocortical neuronal death following withdrawal of
trophic support. A, representative phase-contrast
photomicrographs showing adeno- -galactosidase
(Adeno-
-gal)- and adeno-CaM(1-4)-infected cortical
neuronal cultures immediately prior to withdrawal of B27 trophic
supplements and 4 days later. The upper and lower
panels show the same microscope field at the two time points.
B, survival curves of adeno-
-galactosidase-,
adeno-CaM(1,2)-, adeno-CaM(1-4)-, and adeno-CaM(3,4)-infected neurons
prior to and following withdrawal of trophic support (WTS).
Live neurons were counted daily by phase-contrast microscopy (see
"Materials and Methods"), and survival is expressed as a percentage
of the initial number of neurons present on day 0. Data are the
means ± S.D. of four separate experiments. *, p < 0.05, and **, p < 0.001 compared with the
corresponding values for cells infected with adeno-CaM(1-4) or
adeno-CaM(3,4) (ANOVA with Scheffe post-hoc tests).
C, TUNEL staining of adeno-
-galactosidase- and
adeno-CaM(1-4)-infected neurons with or without B27 supplements 6 days
post-infection. Arrows show the represented TUNEL-positive
cells. D, quantification data of TUNEL-positive cells. The
TUNEL-positive cells were counted and expressed as a percentage of
total cells at 6 days post-infection. Data are the means ± S.D.
of six separate coverslips in two separate experiments. **,
p < 0.001 compared with the corresponding values for
cells infected with adeno-CaM(1-4) or adeno-CaM(3,4) with or without
B27 supplements (ANOVA with Scheffe post-hoc tests).
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Fig. 3.
Calmodulins with mutated calcium-binding
domains 1-4 or 3 and 4 abolish the cell survival-promoting action of
BDNF. Shown are survival curves for neurons infected with control
virus ( -galactosidase (
-gal)) (A) or with
virus containing CaM(1,2) (B), CaM(1-4) (C), or
CaM(3,4) (D) in cultures maintained in medium lacking B27
trophic supplements in the absence or presence of 25 ng/ml BDNF as
indicated. Values are the means ± S.D. of three separate
experiments. BDNF significantly increased the survival of neurons
infected with control virus and virus containing CaM(1,2) (*,
p < 0.05; **, p < 0.01), but failed
to protect neurons expressing CaM(1-4) or CaM(3,4).
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Fig. 4.
Calmodulin and Akt activation is essential
for the neuronal survival-promoting effect of BDNF, whereas ERK
activation is not required. Primary cultured neurons were
incubated in medium lacking B27 trophic supplements in the absence or
presence of 25 ng/ml BDNF as indicated. Cultures were treated with the
indicated agents, which were added at the time of removal of trophic
supplements: 80 µM W-12, 80 µM W-13, 20 µM LY294002, and 10 µM PD98059. After
48 h, cells were fixed and stained with Hoechst 33258 or
processed to TUNEL staining. A, shown are representative
photomicrographs illustrating the morphology of the nuclei of the cells
or TUNEL-positive nuclei subjected to the indicated treatments.
B, the percentage of cells displaying nuclear apoptotic
morphology was determined for each culture; values are the means ± S.D. of three independent experiments. **, p < 0.01 (ANOVA with Scheffe post-hoc tests). Con, control.
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Fig. 5.
Calmodulin is essential for activation of Akt
by BDNF and contributes to the basal level of Akt phosphorylation.
A, neurons were pretreated for 30 min with W-13 (80 µM) or W-12 (80 µM) and then stimulated
with BDNF (25 ng/ml) for 10 min. The relative levels of activation of
Akt kinase and p42/44 MAPKs were assessed by Western blot analysis
using antibodies that selectively recognize phospho-Akt
(p-Akt) and phospho-ERKs (p-Erks). The same
membranes were then stripped and reprobed with antibodies against total
Akt kinase and p42/44 MAPKs (phosphorylation-independent antibodies).
B, 3 days after infection with adeno- -galactosidase
(
-gal), adeno-CaM(1,2) (1, 2), adeno-CaM(1-4)
(1-4), or adeno-CaM(3,4) (3, 4), protein
extracts were prepared to assess the phosphorylation level of Akt.
C, the immunoreactive bands were quantified by densitometric
analysis, and the Akt phosphorylation level was expressed as -fold of
control (Con). Values are the means ± S.D. of
determinations made in three separate experiments. **,
p < 0.01 (unpaired t test).
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Fig. 6.
Activation of Akt by BDNF is abolished by
expression of CaM(1-4) and CaM(3,4), but not CaM(1,2).
A, after 3 days of infection with adeno- -galactosidase
(
-gal), adeno-CaM(1,2), adeno-CaM(1-4), or
adeno-CaM(3,4), the cultures were stimulated with BDNF (25 ng/ml)
for the indicated time periods. Cell extracts were analyzed by Western
blotting with antibodies against phosphorylated Akt (P-Akt)
and total Akt. B, data were obtained by densitometric
analysis of blots from three separate experiments; values are expressed
as -fold change compared with the time 0 level. **, p < 0.01 compared with
-galactosidase; with the time 0 value; and
with each value for cells infected with CaM(1,2), CaM(1-4), or
CaM(3,4) (ANOVA with Scheffe post-hoc tests). C, Western
blot analysis showed that treatment with clasto-lactacystin
-lactone (CLL; 5 µM) in control cultures
(Con) or cultures infected with adeno-CaM(1,2) for 48 or
72 h resulted in an increase in the levels of calmodulin and
CaM(1,2) (arrows). After 3 days of infection with
adeno-CaM(1,2), the cultures were treated with
clasto-lactacystin
-lactone (5 µM) for
6 h and then stimulated with BDNF (25 ng/ml) for 10 min. Cell
extracts were analyzed by Western blotting with antibodies against
phosphorylated Akt and total Akt. D, shown are the results
from densitometric analysis of total Akt and phospho-Akt levels. Values
are the means ± S.D. of determinations made in three separate
experiments and are expressed as -fold of control (no BDNF
stimulation). **, p < 0.01 compared with the control
and with each value for cells infected with CaM(1,2) in the absence or
presence of CLL with or without BDNF stimulation (ANOVA with Scheffe
post-hoc tests).
-galactosidase, apparently because
of the tight regulation of the endogenous calmodulin expression levels
(Fig. 1D). We therefore designed experiments to rule out the
possibility that the effects of the CaM(3,4) and CaM(1-4) mutants were
the result of nonspecific effects of overexpression of the mutant
proteins. When the cultures were treated with
clasto-lactacystin
-lactone (an irreversible 20 S
proteasome inhibitor) 48 or 72 h after infection with wild-type
calmodulin or mutant CaM(1,2), both wild-type calmodulin (data not
shown) and mutant CaM(1,2) accumulated in the neurons to greater
amounts than in untransfected cells treated with the proteasome
inhibitor (Fig. 6C). These results demonstrate that the
proteasome system is involved in tightly regulating the levels of
calmodulin in neurons. Immunoblot analysis of cell lysates showed that
mutant CaM(1,2) also exhibited a slightly lower apparent molecular mass
compared with endogenous calmodulin (Fig. 6C). To determine
whether overexpression of mutant CaM(1,2) can block the phosphorylation
of Akt, we treated adeno-CaM(1,2)-infected cultures (72-h infection)
with clasto-lactacystin
-lactone for 6 h, followed
by stimulation with BDNF (25 ng/ml) for 10 min. We found that treatment
with clasto-lactacystin
-lactone did not change the
amount of Akt protein in the neurons and that Akt phosphorylation was
increased to a level similar to that in control neurons stimulated by
BDNF (Fig. 6, C and D). In contrast to a requirement for functional Ca2+-binding domains 3 and 4 in
BDNF-induced activation of Akt, the ability of BDNF to activate ERK1/2
was not compromised in neurons overexpressing the CaM(3,4) or CaM(1-4)
mutant (Fig. 7).
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Fig. 7.
Activation of ERKs by BDNF is not affected by
expression of CaM(1-4) and CaM(3,4). After 3 days of infection
with adeno- -galactosidase (
-gal), adeno-CaM(1,2),
adeno-CaM(1-4), or adeno-CaM(3,4), the cultures were stimulated with
BDNF (25 ng/ml) for the indicated time periods. Cell extracts were
analyzed by Western blotting with antibodies against phosphorylated
ERK1/2 (p-Erk1, 2) and total ERK1/2.
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Fig. 8.
Activation of Akt by BDNF requires
intracellular Ca2+, but not extracellular Ca2+,
and is independent of CaMKII. A, neuronal cultures were
pretreated with BAPTA-AM (50 µM), nicardipine (15 µM), EGTA (5 mM), KN-92 (1 µM),
KN-93 (1 µM), AIP (10 µM), or
LY294002 (20 µM) for 2 h or switched to
calcium-free medium for 10 min and then stimulated with BDNF (25 ng/ml)
for 10 min. Phosphorylation of Akt was analyzed by Western blot
analysis. B, the data were obtained by densitometric
analysis of blots from three separate experiments; values are expressed
as -fold change compared with the -galactosidase time 0 level. **,
p < 0.01 compared with control (Con),
BAPTA-AM + BDNF, and LY29002 + BDNF values (ANOVA with Scheffe post-hoc
tests). p-Akt, phospho-Akt.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Lab. of Neurosciences, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8463; Fax: 410-558-8465; E-mail: mattsonm@grc.nia.nih.gov.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M207232200
2 D. Yang, A. Cheng, and R. Xiao, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BDNF, brain-derived
neurotrophic factor;
SH2, Src homology 2;
MAPK, mitogen-activated
protein kinase;
PI3K, phosphatidylinositol 3-kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
CaM, calmodulin;
CaMK, calcium/calmodulin-dependent
protein kinase;
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester;
AIP, myristolated
autocamtide-2-related inhibitory peptide;
pfu, plaque-forming unit;
PBS, phosphate-buffered saline;
MAP2, microtubule-associated protein-2;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick
end labeling;
ANOVA, analysis of variance;
ERK, extracellular
signal-regulated kinase.
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REFERENCES |
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