SEARCH

SEARCH BY CITATION

Keywords:

  • Alzheimer;
  • Bcl-2;
  • excitotoxicity;
  • hippocampus;
  • laminin;
  • wortmanin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Integrins are integral membrane proteins that mediate adhesive interactions of cells with the extracellular matrix and with other cells. Integrin engagement results in activation of intracellular signaling cascades that effect several different cellular responses including motility, proliferation and survival. Although integrins are known to provide cell survival signaling in various types of non-neuronal cells, the possibility that integrins modulate neuron survival has not been explored. We now report data demonstrating a neuroprotective function of integrins in embryonic hippocampal neurons. Neurons grown on laminin, an integrin ligand, exhibit increased resistance to glutamate-induced apoptosis compared with neurons grown on polylysine. Neurons expressed integrin β1 and treatment of cultures with an antibody against integrin β1 abolished the protective effect of laminin. Neurons maintained on laminin exhibited a sustained activation of the Akt signaling pathway demonstrated in immunoblot analyses using an antibody that selectively recognizes phosphorylated Akt. The neuroprotective effect of integrin engagement by laminin was mimicked by an IKLLI-containing integrin-binding peptide and was abolished by treatment of neurons with the PI3 kinase inhibitor wortmanin. Levels of the anti-apoptotic protein Bcl-2 were increased in neurons grown on laminin and decreased by wortmanin, suggesting a mechanism for the neuroprotective effect of integrin-mediated signaling. The ability of integrin-mediated signaling to prevent glutamate-induced apoptosis suggests a mechanism whereby neuron–substrate interactions can promote neuron survival under conditions of glutamate receptor overactivation.

Abbreviations used
BDNF

brain-derived neurotrophic factor

ECM

extracellular matrix

FAK

focal adhesion kinase

IGF

insulin-like growth factor

MAP

mitogen-activated protein

NF-κB

nuclear factor kappa of B cells

PI3

phosphatidyl inositol-3

TNF

tumor necrosis factor

Integrins are expressed in neurons throughout the nervous system, wherein they are concentrated in membranes of growth cones and synaptic terminals. Accordingly, integrins control certain aspects of neurite outgrowth and pathfinding during development (Weaver et al. 1995; Jones 1996; Ivins et al. 1998), and may play roles in modulating synaptic plasticity in the hippocampus (Grotewiel et al. 1998; Staubli et al. 1998). Integrin receptors are heterodimeric and consist of α and β subunits, and several isoforms of each subunit are expressed in the brain (Grooms et al. 1993; Jones 1996; Zhang and Galileo 1998). Proteins of the extracellular matrix (ECM) interact with and activate integrins and several of these proteins including laminin are found in the CNS (Jucker et al. 1996). Laminin immunoreactivity has been demonstrated in association with astrocytes and neurons in several regions of the developing and adult brain including the hippocampus (Hagg et al. 1989; Chen and Strickland 1997). In non-neuronal cells integrin receptors promote cell survival through anti-apoptotic signaling upon their activation by ECM proteins (Boudreau et al. 1995; Giancotti and Ruoslahti 1999). Examples include induction of Bcl-2 expression in Chinese hamster ovary cells via integrin α5/β1–fibronectin interactions (Zhang et al. 1995), suppression of p53-mediated apoptosis in fibroblasts attatched to fibronectin (Ilic et al. 1998), and protection of lung cancer cells in contact with fibronectin, laminin and collagen IV from death induced by chemotheropuetic agents (Sethi et al. 1999). It was recently reported that short peptides containing the sequence IKLLI, which may represent the integrin-activating site of laminin, can activate integrin signaling in PC12 cells (Tashiro et al. 1999). The possibility that integrin engagement by ECM modulates neuronal survival has not been explored.

Neuronal apoptosis occurs in a variety of physiological and pathological settings, and overactivation of glutamate receptors may contribute to such neuronal cell deaths. Exposure of cultured rat hippocampal and cortical neurons to glutamate can induce apoptosis which is mediated by calcium influx, mitochondrial dysfunction and caspase activation (Ankarcrona et al. 1995; Du et al. 1997; Larm et al. 1997; Leist et al. 1997; Cheung et al. 1998). Pharmacological blockade of glutamate receptors prevents neuronal death in vivo in experimental models of epilepsy (Rogawski and Donevan 1999), stroke (Mattson 1997a; Dirnagl et al. 1999) and Huntington's disease (Petersen et al. 1999). Glutamate receptor activation is also believed to contribute to the death of hippocampal and cortical neurons in Alzheimer's disease (Mattson 1997b) and substantia nigra dopaminergic neurons in Parkinson's disease (Doble 1999). Several different neurotrophic factors and cytokines can protect neurons against glutamate-induced cell death including basic fibroblast growth factor (Mattson et al. 1989), insulin-like growth factors (Cheng and Mattson 1992), tumor necrosis factor (Cheng et al. 1994) and transforming growth factor-β (Prehn et al. 1993). The signal transduction pathways mediating such anti-apoptotic actions of neurotrophic factors may include those involving MAP kinase, PI3 kinase-Akt, and NF-kB (Mattson et al. 1997, 2000b). Recent studies suggest that binding of integrins to extracellular matrix molecules results in activation of PI3 kinase and Akt in tumor cells and that this pathway can promote cell survival (Frisch and Ruoslahti 1997). We therefore designed a set of experiments to test the hypothesis that a similar integrin-mediated signaling pathway can be activated in neurons, and that activation of this ECM-dependent pathway can modify neuronal vulnerability to glutamate-induced death.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Hippocampal cell cultures and experimental treatments

Primary hippocampal cell cultures were established from E18 rat embryos using methods described previously (Mattson et al. 1995). Dissociated cells were seeded onto plastic dishes or 22-mm2 glass coverslips that had been coated overnight with either 10 µg/mL poly-l-lysine (prepared in borate buffer, pH 8.4) or 10 µg/mL laminin [prepared in phosphate-buffered saline (PBS), pH 7.4]. Dishes and coverslips were then washed thoroughly with sterile water, allowed to dry, and exposed to UV light for 5 min. Cells were plated at a density of 70–100 neurons/mm2 of culture surface. Cultures were maintained in medium consisting of Neurobasal medium containing B-27 supplements, 2 mm l-glutamine, 1 mm HEPES and 0.001% gentamicin sulfate. All experiments were performed using 6–8-day-old cultures, at which time they contain approximately 90–95% neurons and 5–10% astrocytes. The neurons in these cultures express both NMDA and non-NMDA glutamate receptors, and are vulerable to glutamate-induced apoptosis (Mattson et al. 1991, 1993; Glazner and Mattson 2000). Glutamate was prepared as a 200X stock in Locke's buffer (pH 7.2), and wortmanin (Sigma) and zVAD-fmk (Calbiochem, Inc.) were prepared as 500X stocks in ethanol and dimethylsulfoxide respectively. The β1 integrin blocking antibody (hamster polyclonal antibody from Pharmingen International, San Diego, CA) and synthetic peptides (the integrin-activating peptide EIKLLIS and a scrambled control peptide ILEKSLI; Tashiro et al. 1999) were diluted directly into the culture medium.

Quantification of neuron survival and apoptosis

Neuron survival was quantified using methods described previously (Mattson et al. 1995). Briefly, for each culture four microscope fields (10X objective) were photographed prior to experimental treatment, and the same microscope fields were photographed at designated time points after treatment. The photographic negatives were used to count undamaged neurons in each microscope field at each time point. Neurons with fragmented neurites and crenated cell bodies were considered non-viable. To quantify apoptosis, cells were fixed in 4% paraformaldehyde and stained with the fluorescent DNA-binding dye Hoechst 33342 as described previously (Kruman et al. 1997). Hoechst-stained cells were visualized and photographed under epifluorescence illumination (340 nm excitation and 510 nm barrier filter) using a 40X oil immersion objective (150 cells/culture were counted, and counts were made in at least four separate cultures/treatment condition). Analyses were performed without knowledge of the treatment history of the cultures. The percentage of ‘apoptotic’ cells (cells with condensed and fragmented DNA were considered apoptotic) in each culture was determined.

Immunoblot and immnunocytochemical procedures

For immunoblot analysis, proteins in hippocampal cell homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel) and transferred to a nitrocellulose membrane. The membrane was incubated for 2 h in 5% non-fat milk at room temperature and then overnight at 4°C in the presence of primary antibody. The membrane was then exposed for 1 h to horseradish peroxidase-conjugated secondary antibody (1 : 20 000; Jackson ImmunoResearch Labs, West Grove, PA, USA) and immunoreactive protein was visualized using a chemiluminescence-based detection kit (Amersham Corp., Arlington Heights, IL, USA). The primary antibodies employed included a rabbit polyclonal antibody that selectively recognizes Akt only when phosphorylated at serine 473 (1 : 1000 dilution, New England Biolabs, Beverly, MA, USA), a rabbit polyclonal antibody against Akt (phosphorylation insensitive; 1 : 1000 dilution), a rabbit polyclonal antibody that selectively recognizes focal adhesion kinase (FAK) only when phosphorylated at tyrosine 397 (Biosource International, Camarillo, CA, USA), and a mouse monoclonal antibody against FAK (phosphorylation insensitive; 1 : 1000, Transduction Laboratories, Lexington, KY, USA). The methods for immunostaining cultured cells were similar to those described previously (Mattson et al. 1997; Guo et al. 1998). Cells were fixed in 2% paraformaldehyde, membranes were permeabilized by exposure for 5 min to 0.2% Triton X-100 in PBS, and cells were placed in blocking serum (5% goat serum in PBS) for 30 min. Cells were then exposed to rabbit polyclonal antibody against integrin β1 (1 : 500 dilution, Chemicon) overnight at 4°C, followed by an incubation for 1 h with biotinylated goat anti-rabbit secondary antibody (1 : 200), and 30 min in the presence of fluorescein isothiocyanate–avidin (Vector Labs, Burlingame, CA, USA). Images were acquired using a confocal laser scanning microscope with a 60× oil immersion objective (488 nm excitation and 510 nm emission). All images were acquired using the same laser intensity and photodetector gain to allow quantitative comparisons of relative levels of immunoreactivity between cultures; the average pixel intensity within neuronal cell bodies was determined using the Imagespace software provided by the manufacturer (Molecular Probes, Eugene, OR, USA). All images were analyzed without knowledge of culture substrate or treatment history.

Assessment of mitochondrial function

The dye rhodamine 123 was employed to assess relative levels of mitochondrial function (transmembrane potential) using methods described previously (Guo et al. 1998; Mattson et al. 1998). Briefly, cultures were incubated for 30 min in medium containing 5 µm rhodamine 123 and were then washed with Locke's buffer (154 mm NaCl; 5.6 mm KCl; 2.3 mm CaCl2; 1 mm MgCl2; 3.6 mm NaHCO3; 5 mm glucose; 5 mm HEPES; pH 7.2). Confocal images of cellular fluorescence were acquired and the average pixel intensity/cell was quantified using ImageSpace software (Molecular Dynamics); measurements were made in at least 20 cells/culture. Analyses were performed without knowledge of culture substrate or treatment history.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Hippocampal neurons maintained on a laminin substrate exhibit increased resistance to glutamate-induced death

Laminin is a well-established ligand for integrins that is widely expressed in the brain (Jucker et al. 1996) and is known to promote neurite outgrowth in cultured neurons (Jones 1996). In order to determine whether integrin signaling might affect neuronal vulnerability to apoptosis, we employed a paradigm of neuronal apoptosis in which neurons are exposed to a moderately high concentration of the excitatory amino acid glutamate (Ankarcrona et al. 1995; Duan et al. 1999). Dissociated embryonic hippocampal neurons were plated in culture dishes containing either a laminin or polylysine substrate, and 6 days later cultures were exposed to saline or 15 µm glutamate, and neuronal death was quantified 24 h later. Basal levels of cell death were similar in neurons grown on polylysine and laminin (Fig. 1a). Glutamate induced death of 35% of the neurons in cultures grown on polylysine, whereas neurons grown on laminin were resistant to glutamate-induced cell death (Figs 1a and b). β1 integrin is the predominant β subunit comprising the laminin binding integrin receptor, and antibodies against the β1 subunit have been shown to block laminin-induced integrin signaling (Albelda and Buck 1990). When neurons grown on laminin were treated with an antibody against β1-integrin, their vulnerability to glutamate was increased to a level similar to that of neurons grown on polylysine (Fig. 1c). Treatment of cultures with IgG from normal rabbit serum did not affect vulnerability of neurons grown on laminin. As expected, cultured hippocampal neurons contained relatively high levels of β1 integrin which was present in the cell body and in neurites (Fig. 1d).

image

Figure 1. Laminin protects hippocampal neurons against glutamate-induced death via an integrin-dependent mechanism. (a) Hippocampal cultures that had been maintained for 6 days on either a polylysine (P) or laminin (L) substrate were exposed for 24 h to 15 µm glutamate and cell survival was quantified. Values are the mean and SE of determinations made in at least four cultures. *p < 0.01 compared with each of the other values. (b) Phase-contrast micrographs of neurons grown for 6 days on either polysine or laminin, prior to (upper panels) and 24 h after exposure to 15 µm glutamate (lower panels). Note that several neurons on polylysine had degenerated after exposure to glutamate (e.g. arrow), while neurons on laminin remained intact. (c) Cultures on the indicated substrates were pretreated for 48 h with antibody against β1 integrin (aB1; 40 µg/mL) or IgG from non-immune rabbit serum (40 µg/mL). Cultures were then exposed for 24 h to 15 µm gltuamate or saline (P, L) and neuronal survival was quantified. Values are the mean and SD of determinations made in at least four cultures. *p < 0.01 compared with values for conditions, P, L and L + IgG + Glut (anova with Scheffe post hoc tests). (d) Confocal laser scanning microscope images showing immunoreactivity of neurons with an antibody against β1 integrin (lower panel) and neurons reacted without primary antibody (upper panel).

Download figure to PowerPoint

In additional experiments, apoptosis was assessed by staining cells with the fluorescent DNA-binding dye Hoechst 33342 and the percentage of neurons exhibiting nuclear DNA condensation and fragmentation in each culture was determined. Glutamate induced a threefold increase in the number of neurons with apoptotic nuclei in cultures grown on polylysine (Figs 2a and b). In contrast, glutamate did not cause a significant increase in the number of apoptotic neurons in cultures grown on laminin. Apoptosis induced by glutamate in neurons grown on polylysine was completely abolished by ptreatreatment of cultures with the caspase inhibitor zVAD-fmk (Fig. 2b), confirming an apoptotic mode of cell death. In addition, neurons grown on polylysine exhibited mitochondrial dysfunction after exposure to glutamate, as indicated by a decrease in mitochondrial rhodamine 123 fluorescence (Fig. 3). Mitochondrial function was maintained after exposure to glutamate in neurons grown on laminin, suggesting that the signaling pathway activated by integrin engagement interrupted the cell death process at a premitochondrial step (Fig. 3). Neurons exposed to a higher concentration of glutamate (200 µm) that induced necrosis exhibited similar levels of death (80–90%) whether grown on polylysine or laminin, indicating that activation of the integrin signaling pathway can prevent apoptosis but not excitotoxic necrosis (data not shown).

image

Figure 2. Neurons maintained on a laminin substrate exhibit increased resistance to glutamate-induced apoptosis. (a) Images show nuclear DNA-associated fluorescence in neurons stained with Hoechst dye. PC, neurons on polylysine that had been exposed for 24 h to saline. PG, neurons on polylysine that had been exposed for 24 h to 15 µm glutamate. LG, neurons on laminin that had been exposed for 24 h to 15 µm glutamate. Note nuclear DNA condensation and fragmentation in glutamate-treated neurons on the polylysine substrate. (b) Cultures on polylysine or laminin substrata were pretreated for 48 h with β1-integrin antibody or not; or for 2 h with zVAD-fmk (50 µm). Cultures were then exposed to 15 µm glutamate or saline for 24 h. Cells were then stained with Hoechst dye and the percentage of neurons with apoptotic nuclei in each cultre was determined. Values are the mean and SE of determinations made in at least four cultures. *p < 0.01 compared with values for conditions, P, Glut + zVAD and L + Glut (anova with Scheffe post hoc tests).

Download figure to PowerPoint

image

Figure 3. Preservation of mitochondrial function after exposure to glutamate in neurons maintained on a laminin substrate. (a) Confocal laser scanning microscope images of rhodamine 123 fluorescence in hippocampal neurons in cultures that had been maintained for 6 days on either a polylysine (P) or laminin (L) substrate, and then exposed for 8 h to either 15 µm glutamate (G) or saline (C). Note that the level of rhodamine 123 fluorescence is decreased after exposure to glutamate in neurons maintained on a polylysine substrate, but not in neurons maintained on a laminin substrate. (b) Cultures maintained on either polylysine or laminin substrata were exposed for 8 h to either 15 µm glutamate or saline and relative levels of rhodamine 123 fluorescence were quantified (see Methods). Values are the mean and SE of determinations made in at least 4 cultures (measurements made in at least 20 neurons/culture). *p < 0.01 compared with each of the other values (anova with Scheffe post hoc tests).

Download figure to PowerPoint

The neuroprotective effect of laminin is mediated by the PI3 kinase – Akt signaling pathway

Previous studies of non-neuronal cells have shown that engagement of integrins by extracellular matrix proteins can activate a signal transduction pathway involving FAK, PI3 kinase and Akt (Giancotti and Ruoslahti 1999). We therefore assessed activity of FAK and Akt in our paradigm in order to determine their role in signaling in neurons cultured on laminin. The activity of Akt is controlled by phosphorylation of threonine residue 308 and serine residue 473, with both residues being phosphorylated by phosphatidyl-dependent kinase(s) (Coffer et al. 1998). We used serine 473 phosphorylation as an indirect measure of Akt activation to investigate the regulation of Akt activity in response to integrin stimulation. Immunoblot analysis showed that levels of Akt phosphorylation were higher in neurons grown on laminin compared with neurons grown on polylysine (Fig. 4). Levels of Akt phosphorylation decreased between days 4 and 6 in culture in neurons grown on polylysine and laminin, with the decrease being greater in neurons grown on polylysine (Fig. 4). Treatment of cultures with wortmanin, a selective inhibitor of PI3 kinase (Ui et al. 1995), resulted in a marked decrease in the level of Akt phosphorylation in neurons grown on either laminin or polylysine (Fig. 4).

image

Figure 4. Levels of phosphorylated Akt are increased in hippocampal neurons maintained on a laminin substrate. (a) Cultures were maintained on either polylysine (P) or laminin (L) substrata for 2, 4 or 6 days. Proteins in cell homogenates (50 µg/lane) were then subjected to immunoblot analysis using antibodies against phophorylated Akt (Akt-p) or total Akt (Akt; an antibody that binds to Akt in a phosphorylation-independent manner). (b) Cultures were maintained on either polylysine or laminin substrata for the indicated number of days in either the absence or presence of 50 nm wortmaninin (W; added to cultures daily). Proteins in cell homogenates (50 µg/lane) were then subjected to immunonblot analysis using an antibody against phosphorylated Akt. (c) Relative levels of Akt-p were measured by densitometric analysis of immunoblots and the ratio of Akt-p levels in neurons maintained on laminin compared with polylysine was determined for the indicated time periods in culture. Values are the mean and SE from three separate experiments. *p < 0.05, **p < 0.01. (d) Relative level of Akt-p in neurons maintained for the indicated time periods in culture on either polylysine or laminin. The ratio represents the levels of p-Akt in each group on the indicated day compared with the level on day 1 for that group.

Download figure to PowerPoint

In order to assess FAK activation, we used an antibody which recognizes FAK when it is phosphorylated on tyrosine residue 397; phosphorylation on this residue is essential for activation of FAK, although other residues can also contribute to FAK activation under certain conditions (Maa and Leu 1998). Immunoblot analyses using this antibody revealed no apparent differences in the phosphorylation of FAK in neurons grown on laminin compared with neurons grown on polylysine (Fig. 5). Levels of FAK phosphorylation decreased over the course of 6 days in culture to a similar extent in neurons grown on laminin and polylysine (data not shown).

image

Figure 5. Levels of phosphorylated FAK are not different in neurons maintained on laminin versus polylysine. (a) Cultures were maintained on either a polylysine or laminin substrate for the indicated number of days. Proteins in cell homogenates (50 µg/lane) were then subjected to immunoblot analysis using either an antibody that recognizes the tyrosine-397 phosphorylated species of FAK (upper) or an antibody insensitive to FAK phosphorylation. (b) Relative levels of FAK-p were measured by densitometric analysis of immunoblots and the ratio of FAK-p levels in neurons maintained on laminin compared with polylysine was determined for the indicated time periods in culture. Values are the mean and SE from three separate experiments.

Download figure to PowerPoint

In order to determine whether activation of the PI3 kinase pathway mediated the neuroprotective effects of laminin, we used wortmanin in cell survival assays. Cultures were pretreated with 50 nm wortmanin or vehicle for three days at which time cultures were exposed to glutamate or saline. Wortmanin alone induced considerable neuronal death in neurons grown on polylysine and potentiated glutamate-induced neuronal death (Fig. 6). Neurons grown on laminin were less sensitive to wortmnanin alone than neurons grown on polylysine, and wortmanin abolished the neuroprotective effect of the laminin substrate against glutamate toxicity (Fig. 6).

image

Figure 6. Evidence that the PI3 kinase pathway mediates the neuroprotective effect of laminin in neurons. Cultures were maintained on either polylysine (P) or laminin (L) substrata for 6 days and treated on days 4–6 with 50 nm wortmannin (W) or vehicle. Cultures were then exposed to either saline or 15 µm glutamate, and neuronal survival was quantified 24 h later. Values are the mean and SD of determinations made in at least four cultures. *p < 0.05 and **p < 0.01.

Download figure to PowerPoint

An integrin-activating peptide protects mature cultured hippocampal neurons against glutamate toxicity

Because the results to this point were obtained in cultures in which neurons had been maintained on a laminin substrate during the entire process of differentiation into mature neurons, it was unclear whether the increased resistance of these neurons to glutamate toxicity was the result of an integrin-mediated effect of the laminin substrate on the process of neuronal differentiation. To address this issue we allowed neurons to mature during a one week period of culture on a polylysine substrate, and then exposed the neurons for 24 h to a peptide (EIKLLIS) recently shown to activate integrin receptors (Tashiro et al. 1999). Glutamate-induced neuronal death was significantly attenuated in cultures treated with EIKLLIS compared with control cultures treated with a control peptide with a scrambled amino acid sequence (Fig. 7). The neuroprotective effect of EIKLLIS was completely abolished in cultures cotreated with wortmanin, suggesting a requirement for PI3 kinase. As expected, exposure of mature neurons to EIKLLIS resulted in an increase in the level of Akt phosphorylation (data not shown). These findings suggest that the neuroprotective effect of integrin signaling can occur in mature neurons.

image

Figure 7. An integrin-activating peptide protects mature hippocampal neurons in culture against glutamate-induced cell death. Cultures were maintained for one week on a polylysine substrate and then were exposed for 24 h to either the integrin-activating peptide EIKLLIS (P) or a control peptide with a scrambled amino acid sequence (S) (100 µg/mL for each peptide). Additional cultures were cotreated with 50 nm wortmanin (W). Cultures were then exposed for 24 h to 15 µm glutamate and neuronal survival was quantified. Values are the mean and SE of determinations made in a least four cultures. *p < 0.05 compared with values for G + S, W, W + P and G + W + P (anova with Scheffe post hoc tests).

Download figure to PowerPoint

Levels of the anti-apoptotic protein Bcl-2 are increased upon activation of the integrin signaling pathway

Previous studies have shown that activation of the PI3 kinase–Akt pathway can induce expression of Bcl-2 (Matsuzaki et al. 1999; Pugazhenthi et al. 2000), a protein known to protect hippocampal neurons against apoptosis (Kruman et al. 1997) and excitotoxicity (Lawrence et al. 1996). In order to determine if up-regulation of Bcl-2 might mediate the neuroprotective effect of integrin signaling, we performed immunoblot analysis of cell homogenates from neurons that had been grown on laminin or polylysine. Levels of Bcl-2 protein were increase approximately two- to fourfold in neurons that had been maintained on laminin for 4–6 days (Fig. 8). The increase in Bcl-2 levels in neurons on laminin was suppressed by treatment of the neurons with wortmanin, indicating the involvement of PI3 kinase in the upregulation of Bcl-2 protein levels. Collectively, our findings suggest that activation of an integrin signaling pathway in neurons results in upregulation of anti-apoptotic pathways that can suppress neuronal death at an early step prior to mitochondrial dysfunction.

image

Figure 8. Levels of Bcl-2 are increased in neurons grown on laminin, and this effect is blocked by wortmanin. (a) Immunoblot showing relative levels of Bcl-2 in cultures that had been maintained for 4 or 6 days on either polylysine (P) or laminin (L) substrates. Some cultures were also exposed to 50 nm wortmanin (W). Note that levels of Bcl-2 are increased in neurons grown on laminin and that this increase is suppressed in neurons treated with wortmanin. (b) Relative levels of Bcl-2 were measured by densitometric analysis of immunoblots and the ratio of Bcl-2 levels in neurons maintained on laminin compared with polylysine was determined for the indicated time periods in culture. Values are the mean and SE from three separate experiments. *p < 0.05 compared with the basal level.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our data suggest that engagement of integrins by laminin in hippocampal neurons activates a neuroprotective signaling pathway involving PI3 kinase and Akt. Previous studies of non-neuronal cells have shown that interactions with extracellular matrix proteins can promote cell survival (Clark and Brugge 1995; Zhang et al. 1995; Bachelder et al. 1999; Sethi et al. 1999). A signaling mechanism similar to that identified in hippocampal neurons in the present study may also mediate the anti-apoptotic effects of laminin in non-neuronal cells as suggested by recent data showing that epithelial cells grown on laminin are resistant to apoptosis and exhibit increased PI3 kinase activity (Farrelly et al. 1999). Studies of normal epithelial cells and tumor cells have shown that integrins mediate cell survival effects of cell–extracellular matrix interactions (Howlett et al. 1995; Sethi et al. 1999). A prior study showed that laminin increases long-term survival of mesencephalic dopamine-producing neurons in culture (Dong et al. 1994). We found that neurons grown on laminin exhibit increased tonic activation of Akt as demonstrated by increased Akt phosphorylation. The ability of a laminin substrate to protect neurons against glutamate-induced death was abolished when neurons were treated with antibodies against β1 integrin. The PI3 kinase inhibitor wortmanin, blocked the protective effects of laminin, as well as the integrin-activating peptide EIKLLIS, demonstrating a requirement for activation of the PI3 kinase pathway. These findings are consistent with previous reports that demonstrated increased activation of the PI3 kinase/Akt pathway initiated by integrin signaling (King et al. 1997; Guilherme and Czech 1998; Tan et al. 1999). Moreover, previous studies have shown that exposure of neurons to glutamate results in a decrease in the level of Akt phosphorylation (Chalecka-Franaszek and Chuang 1999), suggesting that suppression of this survival-promoting pathway may contribute to the neurotoxic effects of glutamate.

The PI3 kinase–Akt signaling pathway has been shown to be an important pathway that promotes survival of many different cell types (Philpott et al. 1997; Crowder and Freeman 1998; Morita et al. 1999; Sabbatini and McCormick 1999). PI3 kinase activity can be induced via activation of receptors for growth factors and cytokines including those for brain-derived neurotrophic factor (BDNF; Hetman et al. 1999), insulin-like growth factors (IGF; Ryu et al. 1999) and tumor necrosis factor (TNF; Kim et al. 1999). BDNF (Cheng and Mattson 1994; Takei et al. 1999), IGFs (Kermer et al. 2000) and TNF (Barger et al. 1995; Mattson et al. 1997) have each been shown to protect neurons against death induced by glutamate, and oxidative and metabolic insults. Signaling through the PI3 kinase pathway leads to activation of Akt which, in turn, has multiple targets for its anti-apoptotic actions (Marte and Downward 1997; Coffer et al. 1998). Akt has been shown to phosphorylate Bad, thereby decreasing the interaction of Bad with BclxL and increasing the BclxL/Bad ratio (Khwaja 1999). Akt has also been shown to phosphorylate human caspase 9, thereby inactivating it and promoting cell survival (Cardone et al. 1998). Akt phosphorylates and inactivates GSK-3β, a protein recently shown to play a role in promotoing apoptotic death of cultured cortical neurons (Hetman et al. 2000). In addition, Akt has can prevent the release of cytochrome c from mitochondria in a Bcl-2/Bad independent manner (Kennedy et al. 1999), and participates in the activation of the anti-apoptotic transcription factor NF-κB in a variety of cell types including neurons (Matsuzaki et al. 1999; Ozes et al. 1999; Romashkova and Makarov 1999; Mattson 2000).

Akt can stimulate expression of Bcl-2 in several different cell types including hippocampal neurons and PC12 cells (de la Fuente et al. 1999; Matsuzaki et al. 1999; Pugazhenthi et al. 2000). In our model of integrin stimulation, such mechanisms may play a role in the protection of neurons against excitotoxic death. Indeed, we found that levels of Bcl-2 are increased in hippocampal neurons grown on a laminin substrate, and that wortmanin can suppress the increased production of Bcl-2, suggesting that PI3 kinase–Akt signaling is necessary for the upregulation of Bcl-2 in neurons in which integrins are activated. It is known that interactions with extracellular matrix can upregulate expression of anti-apoptotic proteins. For example, lymphocytic leukemia cells in contact with fibronectin exhibit increased levels of Bcl-2 (de la Fuente et al. 1999). Neurons grown on laminin exhibited preserved mitochondrial function after glutamate exposure compared with neurons grown on polylysine. Bcl-2 is known to protect cells from apoptotic death by preservation of mitochondrial function (Kluck et al. 1997; Kroemer 1997; Yang et al. 1997). Since Bcl-2 has previously been shown to protect neurons against apoptosis induced by glutamate and other insults, increased production of Bcl-2 may account for the increased resistance of neurons grown on laminin to glutamate-induced apoptosis.

Activation of integrin receptors by proteins of the extracellular matrix such as fibronectin and laminin can activate FAK during the formation of focal adhesions in non-neuronal cells (Burridge et al. 1992) and recent findings implicate FAK in the PI3 kinase signaling pathway (Milani et al. 1998; Sonoda et al. 1999; Tamura et al. 1999). However, we found no differences in levels of phosphorylated FAK in neurons grown on laminin versus polylysine. Nevertheless, our data do not completely rule out the participation of FAK in the neuroprotective signaling pathway activated by laminin binding to integrins. In addition, it is possible that other signaling pathways such as MAP kinase and NF-κB may be stimulated in neurons on laminin, and might affect neuronal vulnerability to glutamate. It would be particularly interesting to investigate the possibility that NF-κB mediates the protective effect of integrin signaling in light of previous studies showing that Akt can induce NF-κB activation (Ozes et al. 1999), and that activation of NF-κB can induce Bcl-2 production (Camandola and Mattson 2000) and protect neurons against excitotoxicity (Yu et al. 1999).

Although the role of integrin-mediated signaling in the survival of neurons in the developing and adult brain is unknown, emerging data are consistent with its involvment. For example, it was recently shown that blocking integrin binding at the developing neuromuscular junction results in motoneuron death (Wong et al. 1999). Apoptosis involving overactivaton of glutamate receptors is thought to contribute to neuronal death in acute neurodegenerative conditions such as stroke (Dirnagl et al. 1999; Mattson et al. 2000a) and traumatic brain injury (Obrenovitch and Urenjak 1997), as well as in chronic neurodegenerative disorders such as Alzheimer's (Cotman 1998; Mattson 2000), Parkinson's (Jenner and Olanow 1998; Doble 1999) and Huntington's (Petersen et al. 1999) diseases. The ability of laminin-induced integrin signaling to protect neurons against glutamate-induced death therefore has important implications for such disorders. Although the present findings are the first to demonstrate that integrin signaling can prevent excitotoxic apoptosis, prior studies have suggested that interactions of extracellular matrix with neural cells can modify injury. For example, soluble laminin up-regulates Bcl-2 and inhibits apoptotic death in non-adherent neuroblastoma cells (Bozzo et al. 1997). In addition, neurotrophic factors known to activate the PI3 kinase–Akt pathway can protect neurons against excitotoxic injury. For example, pretreatment of cultured rat hippocampal neurons with IGFs (Cheng and Mattson 1992) and BDNF (Cheng and Mattson 1994) protects them against glutamate-induced death, stimuli that activate PI3 kinase and Akt can protect cultured hippocampal neurons against amyloid β-peptide toxicity (Guo et al. 1999; Miho et al. 1999), and BDNF protects cultured cerebellar granule neurons against glutamate-induced apoptosis (Skaper et al. 1998). Agents that activate integrin signaling, such as IKLLI-containing peptides, may therefore prove useful as therapeutic agents in a variety of neurological disorders in which apoptosis and excitotoxicity occur.

In several models of brain injury, extracellular proteases are up-regulated and may act primarily to cleave extracellular matrix proteins (Heo et al. 1998; Matsuoka et al. 1998; Fujimura et al. 1999). In the hippocampus levels of laminin decrease following seizures prior to death of pyramidal neurons (Chen and Strickland 1997). Under conditions in which laminin degradation does not occur (in mice lacking tissue plasminogen activator and in mice administered a plasminogen inhibitor), seizure-induced damage to neurons is greatly decreased suggesting that disruption of extracellular matrix–neuron interactions promotes neuronal death. Levels of active extracellular proteases are increased in brain tissue from Alzheimer's patients compared with age-matched controls (Lukes et al. 1999), making it tempting to speculate that excessive degradation of proteins such as laminin contributes to neuronal death by suppressing anti-apoptotic signaling. Although it remains to be established whether alterations in integrin-mediated signaling contributes to the pathogenesis of neurodegenerative disorders, our data demonstrating a neuroprotective role for activation of this pathway are consistent with such a possibility.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Staci Lyvers and Simonetta Camandola for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Albelda S. M. & Buck C. A. (1990) Integrins and other cell adhesion molecules. FASEB J. 4, 28682880.
  • Ankarcrona M., Dypbukt J. M., Bonfoco E., Zhivotovsky B., Orrenius S., Lipton S. A. & Nicotera P. (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961973.
  • Bachelder R. E., Ribick M. J., Marchetti A., Falcioni R., Soddu S., Davis K. R. & Mercurio A. M. (1999) p53 inhibits α6β4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J. Cell Biol. 147, 10631072.
  • Barger S. W., Horster D., Furukawa K., Goodman Y., Krieglstein J. & Mattson M. P. (1995) Tumour necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc. Natl. Acad. Sci. USA 92, 93289332.
  • Bozzo C., Bellomo G., Silengo L., Tarone G. & Altruda F. (1997) Soluble integrin ligands and growth factors independently rescue neuroblastoma cells from apoptosis under non-adherent conditions. Exp. Cell Res. 237, 326337.DOI: 10.1006/excr.1997.3777
  • Burridge K., Turner C. E. & Romer L. H. (1992) Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to exrtacellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119, 893903.
  • Camandola S. & Mattson M. P. (2000) Pro-apoptotic action of Par-4 involves inhibition of NF-kappaB activity and suppression of BCL-2 expression. J. Neurosci. Res. 61, 134139.DOI: 10.1002/1097-4547(20000715)61:2<134::aid-jnr3>3.0.co;2-p
  • Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbridge E., Frisch S. & Reed J. C. (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 13181321.
  • Chalecka-Franaszek E. & Chuang D. M. (1999) Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc. Natl. Acad. Sci. USA 96, 87458750.DOI: 10.1073/pnas.96.15.8745
  • Chen Z. L. & Strickland S. (1997) Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91, 917925.
  • Cheng B. & Mattson M. P. (1992) IGF-I and IGF-II protect cultured hippocampal and septal neurons against calcium-mediated hypoglycemic damage. J. Neurosci. 12, 15581566.
  • Cheng B. & Mattson M. P. (1994) NT-3 and BDNF protect CNS neurons against metabolic/excitotoxic insults. Brain Res. 640, 5667.
  • Cheng B., Christakos S. & Mattson M. P. (1994) Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron 12, 139153.
  • Cheung N. S., Pascoe C. J., Giardina S. F., John C. A. & Beart P. M. (1998) Micromolar l-glutamate induces extensive apoptosis in an apoptotic- necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones. Neuropharmacology 37, 14191429.DOI: 10.1016/s0028-3908(98)00123-3
  • Clark E. A. & Brugge J. S. (1995) Integrins and signal transduction pathways: the road taken. Science 268, 233239.
  • Coffer P. J., Jin J. & Woodgett J. R. (1998) Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem. J. 335, 113.
  • Cotman C. W. (1998) Apoptosis decision cascades and neuronal degeneration in Alzheimer's disease. Neurobiol. Aging 19, S29S32.DOI: 10.1016/s0197-4580(98)00042-6
  • Crowder R. J. & Freeman. R. S. (1998) Phosphatidyl 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J. Neurosci. 18, 29332943.
  • Dirnagl U., Iadecola C. & Moskowitz M. A. (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 22, 391397.DOI: 10.1016/s0166-2236(99)01401-0
  • Doble A. (1999) The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacol. Ther. 81, 163221.DOI: 10.1016/s0163-7258(98)00042-4
  • Dong J. F., Detta A. & Hitchcock E. R. (1994) Enhanced in vitro survival and growth of foetal human mesencephalic dopaminergic neurones on laminin and collagen: implications for cell banking. Neurosci. Lett. 178, 2731.
  • Du Y., Bales K. R., Dodel R. C., Hamilton-Byrd E., Horn J. W., Czilli D. L., Simmons L. K., Ni B. & Paul S. M. (1997) Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons. Proc. Natl. Acad. Sci. USA 94, 1165711662.DOI: 10.1073/pnas.94.21.11657
  • Duan W., Rangnekar V. M. & Mattson M. P. (1999) Prostate apoptosis response-4 production in synaptic compartments following apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration. J. Neurochem. 72, 23122322.
  • Farrelly N., Lee Y. J., Oliver J., Dive C. & Streuli C. H. (1999Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling. J. Cell Biol. 144, 13371348.
  • Frisch S. M. & Ruoslahti E. (1997) Integrins and anoikis. Curr. Opin. Cell Biol. 9, 701706.
  • De La Fuente M. T., Casanova B., Garcia-Gila M., Silva A. & Garcia-Pardo A. (1999) Fibronectin interaction with a4b1 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: correlation with Bcl-2 and Bax. Leukemia 13, 266274.
  • Fujimura M., Gasche Y., Morita-Fujimura Y., Massengale J., Kawase M. & Chan P. H. (1999) Early appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion. J. Cereb. Blood Flow Metab. 19, 624633.
  • Giancotti F. G. & Ruoslahti E. (1999) Integrin signaling. Science 285, 10281032.DOI: 10.1126/science.285.5430.1028
  • Glazner G. W. & Mattson M. P. (2000) Differential effects of BDNF, ADNF9, and TNFα on levels of NMDA receptor subunits, calcium homeostasis, and neuronal vulnerability to excitotoxicity. Exp. Neurol. 161, 442452.DOI: 10.1006/exnr.1999.7242
  • Grooms S. Y., Terracio L. & Jones L. S. (1993) Anatomical localization of b1 integrin-like immunoreactivity in rat brain. Exp. Neurol. 122, 253259.DOI: 10.1006/exnr.1993.1125
  • Grotewiel M. S., Beck C. D., Wu K. H., Zhu X. R. & Davis R. L. (1998) Integrin-mediated short-term memory in Drosophila. Nature 391, 455460.DOI: 10.1038/35079
  • Guilherme A. & Czech M. P. (1998) Stimulation of IRS1-associated phosphatidylinositol 3-kinase and Akt/protein kinase B, but not glucose transport, by β1-integrin signaling in rat adipocytes. J. Biol. Chem. 273, 3311933122.
  • Guo Q., Fu W., Xie J., Luo H., Sells S. F., Geddes J. W., Bondada V., Rangnekar V. & Mattson M. P. (1998) Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease. Nature Med. 4, 957962.
  • Guo Q., Sebastian L., Sopher B. L., Miller M. W., Glazner G. W., Ware C. B., Martin G. M. & Mattson M. P. (1999) Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation. Proc. Natl. Acad. Sci. USA 96, 41254130.DOI: 10.1073/pnas.96.7.4125
  • Hagg T., Muir D., Engvall E., Varon S. & Manthorpe M. (1989) Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment. Neuron 3, 71732.
  • Heo J. H., Lucero J., Abumiya T., Koziol J. A., Copeland B. R. & Del Zoppo G. J. (1998) Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. Neurosci. Lett. 252, 119122.DOI: 10.1016/s0304-3940(98)00562-x
  • Hetman M., Kanning K., Cavanaugh J. E. & Xia Z. (1999) Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J. Biol. Chem. 274, 2256922580.
  • Hetman M., Cavanaugh J. E., Kimelman D. & Xia Z. (2000) Role of glycogen synthase kinase-3β in neuronal apoptosis induced by trophic factor withdrawal. J. Neurosci. 20, 25672574.
  • Howlett A. R., Bailey N., Damsky C., Petersen O. W. & Bissell M. J. (1995) Cellular growth and survival are mediated by β1 integrins in normal human breast epithelium but not in breast carcinoma. J. Cell Sci. 108, 19451957.
  • Ilic D., Almeida E. A., Schlaepfer D. D., Dazin P., Aizawa S. & Damsky C. H. (1998) Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J. Cell Biol. 143, 547560.
  • Ivins J. K., Colognato H., Kreidberg J. A., Yurchenco P. D. & Lander A. D. (1998) Neronal receptors mediating responses to antibody activated laminin-1. J. Neurosci. 18, 97039715.
  • Jenner P. & Olanow C. W. (1998) Understanding cell death in Parkinson's disease. Ann. Neurol. 44, S72S84.
  • Jones L. S. (1996) Integrins: possible functions in the adult CNS. Trends Neurosci. 19, 6872.
  • Jucker M., Tian M. & Ingram D. K. (1996) Laminins in the adult and aged brain. Mol. Chem. Neuropathol. 28, 209218.
  • Kennedy S. G., Kandel E. S., Cross T. K. & Hay N. (1999) Akt/protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol. Cell Biol. 19, 58005810.
  • Khwaja A. (1999) Akt is more than just a Bad kinase. Nature 401, 3334.DOI: 10.1038/43354
  • Kim B. C., Lee M. N., Kim J. Y., Lee S. S., Chang J. D., Kim S. S., Lee S. Y. & Kim J. H. (1999) Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts. J. Biol. Chem. 274, 2437224377.
  • King W. G., Mattaliano M. D., Chan T. O., Tsichlis P. N. & Brugge J. S. (1997) Phosphatidylinositol-3-kinase is required for integrin-stimulated Akt and Raf-2/mitogen-activated protein kinase pathway activation. Mol. Cell. Biol. 17, 44064418.
  • Kluck R. M., Bossy-Wetzel E., Green D. R. & Newmeyer D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 11321136.DOI: 10.1126/science.275.5303.1132
  • Kermer P., Klocker N., Labes M. & Bahr M. (2000) Insulin-like growth factors-I protects axotomized rat retinal ganglion cells from secondary death via PI3-k-dependent. Akt. J. Neurosci. 20, 28.
  • Kroemer G. (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Med. 3, 614620.
  • Kruman I., Bruce-Keller A. J., Bredesen D., Waeg G. & Mattson M. P. (1997) Evidence that 4 hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 50895100.
  • Larm J. A., Cheung N. S. & Beart P. M. (1997) Apoptosis induced via AMPA-selective glutamate receptors in cultured murine cortical neurons. J. Neurochem. 69, 617622.
  • Lawrence M. S., Ho D. Y., Sun G. H., Steinberg G. K. & Sapolsky R. M. (1996) Overexpression of Bcl-2 with herpes simplex virus vectors protects CNS neurons against neurological insults in vitro and in vivo. J. Neurosci. 16, 486496.
  • Leist M., Volbracht C., Kuhnle S., Fava E., Ferrando-May E. & Nicotera P. (1997) Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide. Mol. Med. 3, 750764.
  • Lukes A., Mun-Bryce S., Lukes M. & Rosenberg G. A. (1999) Extracellular matrix degradation by metalloproteinases and central nervous system diseases. Mol. Neurobiol. 19, 267284.
  • Maa M. C. & Leu T. H. (1998) Vanadate-dependent FAK activation is accomplished by the sustained FAK Tyr-576/577 phosphorylation. Biochem. Biophys. Res. Commun. 251, 344349.DOI: 10.1006/bbrc.1998.9464
  • Marte B. M. & Downward J. (1997) PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem. Sci. 22, 355358.DOI: 10.1016/s0968-0004(97)01097-9
  • Matsuoka Y., Kitamura Y. & Taniguchi T. (1998) Induction of plasminogen in rat hippocampal pyramidal neurons by kainic acid. Curr. Biol. 8, 1925.
  • Matsuzaki H., Tamatani M., Mitsuda N., Namikawa K., Kiyama H., Miyake S. & Tohyama M. (1999) Activation of Akt kinase inhibits apoptosis and changes in Bcl-2 and Bax expression induced by nitric oxide in primary hippocampal neurons. J. Neurochem. 73, 20372046.
  • Mattson M. P. (1997a) Neuroprotective signal transduction: relevance to stroke. Neurosci. Biobehav. Rev. 21, 193206.DOI: 10.1016/s0149-7634(96)00010-3
  • Mattson M. P. (1997b) Cellular actions of β-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol. Rev. 77, 10811132.
  • Mattson M. P. (2000) Apoptosis in neurodegenerative disorders. Nature Rev. Mol. Cell Biol. 1, in press.
  • Mattson M. P., Murrain M., Guthrie P. B. & Kater S. B. (1989) Fibroblast growth factor and glutamate: opposing actions in the generation and degeneration of hippocampal neuroarchitecture. J. Neurosci. 9, 37283740.
  • Mattson M. P., Wang H. & Michaelis E. K. (1991) Developmental expression, compartmentalization, and possible role in excitotoxicity of a putative NMDA receptor protein in cultured hippocampal neurons. Brain Res. 565, 94108.
  • Mattson M. P., Kumar K., Cheng B., Wang H. & Michaelis E. (1993) Basic FGF regulates the expression of a functional 71 kDa NMDA receptor protein that mediates calcium influx and neurotoxicity in cultured hippocampal neurons. J. Neurosci. 13, 45754588.
  • Mattson M. P., Lovell M., Furukawa K. & Markesbery W. R. (1995) Neurotrophic factors attenuate glutamate-induced accumulation of peroxides, elevation of [Ca2+]i and neurotoxicity, and increase anti-oxidant enzyme activities in hippocampal neurons. J. Neurochem. 65, 17401751.
  • Mattson M. P., Goodman Y., Luo H., Fu W. & Furukawa K. (1997) Activation of NF-κB protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of Mn-SOD and suppression of peroxynitrite production and protein tyrosine nitration. J. Neurosci. Res. 49, 681697.DOI: 10.1002/(sici)1097-4547(19970915)49:6<681::aid-jnr3>3.0.co;2-3
  • Mattson M. P., Keller J. N. & Begley J. G. (1998) Evidence for synaptic apoptosis. Exp. Neurol. 53, 3548.
  • Mattson M. P., Culmsee C. & Yu Z. F. (2000a) Apoptotic and anti-apoptotic mechanisms in stroke. Cell Tiss. Res. 301, 173187.
  • Mattson M. P., Culmsee C., Yu Z. & Camandola S. (2000b) Roles of NF-κB in neuronal survival and plasticity. J. Neurochem. 74, 443456.
  • Miho Y., Kouroku Y., Fujita E., Mukasa T., Urase K., Kasahara T., Isoai A., Momoi M. Y. & Momoi T. (1999) bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation. Cell Death Differ. 6, 43470.
  • Milani D., Mazzoni M., Zauli G., Mischiati C., Gibellini D., Giacca M. & Capitani S. (1998) HIV-1 Tat induces tyrosine phophorylation of p125FAK and its association with phosphoinositide 3-kinase in PC12 cells. AIDS 12, 12751284.
  • Morita Y., Manganaro T. F., Tao X. J., Martimbeau S., Donahoe P. K. & Tilly J. L. (1999) Requirement of phosphatidyl-3′-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse. Endocrinology 140, 941949.
  • Obrenovitch T. P. & Urenjak J. (1997) Is high extracellular glutamate the key to excitotoxicity in traumatic brain injury? J. Neurotrauma 14, 677698.
  • Ozes O. N., Mayo L. D., Gustin J. A., Pfeffer S. R., Pfeffer L. M. & Donner B. D. (1999) NF-κB activation by tumor necrosis factor requires the Akt serine-threonine kinase. Nature 401, 8285.DOI: 10.1038/43466
  • Petersen A., Mani K. & Brundin P. (1999) Recent advances in the pathogenesis of Huntington's disease. Exp. Neurol. 157, 118.DOI: 10.1006/exnr.1998.7006
  • Philpott K. L., McCarthy M. J., Klippel A. & Rubin L. L. (1997) Activated phophatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons. J. Cell Biol. 139, 809815.
  • Prehn J. H., Backhauss C. & Krieglstein J. (1993) Transforming growth factor-beta 1 prevents glutamate neurotoxicity in rat neocortical cultures and protects mouse neocortex from ischemic injury in vivo. J. Cereb. Blood Flow Metab. 13, 521525.
  • Pugazhenthi S., Nesterova A., Sable C., Heidenreich D. A., Boxer L. M., Heasley L. E. & Reusch J. E. (2000) Akt/protein kinase B upregulates Bcl-2 expression through cAMP response element-binding proein. J. Biol. Chem. 275, 1076110766.
  • Rogawski M. A. & Donevan S. D. (1999) AMPA receptors in epilepsy and as targets for anti-epileptic drugs. Adv. Neurol. 79, 947963.
  • Romashkova J. A. & Makatov S. S. (1999) NF-κB is a target of Akt in anti-apoptotic PDGF signaling. Nature 401, 8690.DOI: 10.1038/43474
  • Ryu B. R., Ko H. W., Jou I., Hoh J. S. & Bwag B. J. (1999) Phosphatidylinositol 3-kinase-mediated regulation of neuronal apoptosis and necrosis by insultin and IGF-1. J. Neurobiol. 39, 536546.DOI: 10.1002/(sici)1097-4695(19990615)39:4<536::aid-neu7>3.3.co;2-a
  • Sabbatini P. & McCormick F. (1999) Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis. J. Biol. Chem. 274, 2426324269.
  • Sethi T., Rintoul R. C., Moore S. M., MacKinnon A. C., Salter D., Choo C., Chilvers E. R., Dransfield I., Donnelly S. C., Strieter R. & Haslett C. (1999) Extracellular matrix proteins protect small lung cancer cells against apoptosis: a mechanism from small cell lung cancer growth and drug resistance. Nature Med. 5, 662668.
  • Skaper S. D., Florani M., Negro A., Facci L. & Giusti P. (1998) Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway. J. Neurochem. 70, 18591868.
  • Sonoda Y., Watanabe S., Matsumoto Y., Aizu-Yokota E. & Kasahara T. (1999) FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line. J. Biol. Chem. 274, 1056610570.
  • Staubli U., Chun D. & Lynch G. (1998) Time-dependent reversal of long-term potentiation by an integrin antagonist. J. Neurosci. 18, 34603469.
  • Takei N., Tanaka O., Endo Y., Lindholm D. & Hatanaka H. (1999) BDNF and NT-3 but not CNTF counteract calcium ionophore-induced apoptosis of cultured cortical neurons: involvement of dual pathways. Neuropharmacology 38, 283288.DOI: 10.1016/s0028-3908(98)00189-0
  • Tamura M., Gu J., Danen E. H., Takino T., Miyamoto S. & Yamada K. M. (1999) PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem. 274, 2069320703.
  • Tan K., Nie D., Cai Y. & Honn K. V. (1999) The β4 integrin subunit rescues A431 cells from apoptosis through a PI3K/Akt kinase signaling pathway. Biochem. Biophys. Res. Commun. 264, 127132.DOI: 10.1006/bbrc.1999.1496
  • Tashiro K., Monji A., Yoshida I., Hayashi Y., Matsuda K., Tashiro N. & Mitsuyama Y. (1999) An IKLLI-containing peptide derived from the laminin alpha1 chain mediating heparin-binding, cell adhesion, neurite outgrowth and proliferation, represents a binding site for integrin alpha3beta1 and heparan sulphate proteoglycan. Biochem. J. 340, 119126.DOI: 10.1042/0264-6021:3400119
  • Ui M., Okada T., Hazeki K. & Hazeki O. (1995) Wortmannin as a unique probe for an intracellular signaling protein, phosphoinositide 3-kinase. Trends Biochem. Sci. 20, 303307.
  • Weaver C. D., Yoshida C. K., De Curtis I. & Reichardt L. F. (1995) Expression and in vitro function of β1-integrin laminin receptors in the developing ciliar ganglion. J. Neurosci. 15, 52755285.
  • Wong K. C., Meyer T., Harding D. I., Dick J. R., Vrbova G. & Greensmith L. (1999) Integrins at the neuromuscular junction are important for motorneuron survival. Eur. J. Neurosci. 11, 32873292.DOI: 10.1046/j.1460-9568.1999.00749.x
  • Yang J., Liu X., Bhalla K., Kim C. N., Ibrado A. M., Cai J., Peng T. I., Jones D. P. & Wang X. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 11291132.DOI: 10.1126/science.275.5303.1129
  • Yu Z., Zhou D., Bruce-Keller A. J., Kindy M. S. & Mattson M. P. (1999) Lack of the p50 subunit of nuclear factor-kappaB increases the vulnerability of hippocampal neurons to excitotoxic injury. J. Neurosci. 19, 88568865.
  • Zhang Z. & Galileo D. S. (1998) Retroviral transfer of antisense integrin α6 or α8 sequences results in laminar redistribution or clonal cell death in developing brain. J. Neurosci. 18, 69286938.
  • Zhang Z., Vuori K., Reed J. C. & Ruoslahti E. (1995) The a5b1 integrin supports survival of cells on fibronectin and upregulates Bcl-2 expression. Proc. Natl Acad. Sci. USA 92, 61616165.