Calmodulin Mediates Brain-derived Neurotrophic Factor Cell Survival Signaling Upstream of Akt Kinase in Embryonic Neocortical Neurons*

Aiwu ChengDagger , Shuqin WangDagger , Dongmei Yang§, Ruiping Xiao§, and Mark P. MattsonDagger ||**

From the Laboratories of Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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
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INTRODUCTION
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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 beta -lactone, KN-92, KN-93, and AIP were from Calbiochem-Novabiochem. LY294002 and PD98059 were from Cell Signaling Technology. Nicardipine was from Sigma.

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 beta -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 beta -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.

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.

beta -Galactosidase Staining-- After 3 days of infection with adeno-beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.


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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 beta -galactosidase activity in uninfected control cells (Con) and in cells 3 days after infection with adeno-beta -galactosidase (Adeno-beta -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).

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-beta -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 beta -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).

The cells were cultured for 5 days in Neurobasal medium with B27 supplements and then infected with adeno-beta -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-beta -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-beta -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 beta -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-beta -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-beta -galactosidase (Adeno-beta -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-beta -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-beta -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).

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).


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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 (beta -galactosidase (beta -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.

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.


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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-beta -galactosidase (beta -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).

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).


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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-beta -galactosidase (beta -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 beta -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 beta -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 beta -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).

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-beta -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 beta -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 beta -lactone for 6 h, followed by stimulation with BDNF (25 ng/ml) for 10 min. We found that treatment with clasto-lactacystin beta -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-beta -galactosidase (beta -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.

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.


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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 beta -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

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).

    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-beta -D-galactopyranoside; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling; ANOVA, analysis of variance; ERK, extracellular signal-regulated kinase.

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