The Bile Acid Tauroursodeoxycholic Acid Modulates Phosphorylation and Translocation of Bad via Phosphatidylinositol 3-Kinase in Glutamate-Induced Apoptosis of Rat Cortical Neurons

Bile acids are inherently cytotoxic molecules that can influence cellular function and structure. They act as intracellular signaling agents, which modulate cellular transport, alter Ca²⁺ intracellular levels, and activate cell surface receptors (Rodrigues et al., 2004). In contrast, ursodeoxycholic acid (UDCA) is a hydrophilic bile acid with proven clinical efficacy in the treatment of hepatobiliary disorders (Lazaridis et al., 2001). Once administrated, this bile acid is rapidly conjugated with glycine or taurine, forming glycoursodeoxycolic and tauroursodeoxycholic (TUDCA) acids, respectively. There is now evidence that the cytoprotective mechanism of UDCA and its conjugates results from their ability, in part, to inhibit apoptosis. Interestingly, the antiapoptotic properties of these bile acids are independent of the cell type and death signal, suggesting a common antiapoptotic mechanism. We have shown that TUDCA inhibits apoptosis by modulating mitochondrial membrane perturbation, cytochrome c release, caspase activation, and associated nuclear instability in several cell types, including neuronal cells (Rodrigues et al., 2000). In addition, bile acids can also block apoptosis through survival signals such as the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase pathways (Rust et al., 2000; Qiao et al., 2002; Solá et al., 2003).

The regulation of mitochondrial membrane function and the release of apoptogenic factors from mitochondria are key components of the apoptotic repertoire, tightly controlled by the Bcl-2 family of proteins (Kroemer and Reed, 2000). The function of Bcl-2 members as cell death agonists or antagonists is modulated by transcriptional and post-transcriptional modifications. For example, pro-apoptotic Bad forms heterodimers with the antiapoptotic proteins Bcl-x_L or Bcl-2 (del Peso et al., 1997), antagonizing their antiapoptotic activity. Loss of survival signals reduces levels of Bad phosphorylation and promotes its translocation to mitochondria (Wang et al., 1999). In contrast, survival signals induce Bad phosphorylation, thereby sequestering it from mitochondrial targets. In this regard, the PI3K signaling cascade has been implicated in the phosphorylation of pro-apoptotic Bad (Datta et al., 1997).

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system, whose activities result in excitatory neurotransmission, regulation of neuronal plasticity, and induction of cell death, among others. A number of parameters change dramatically during central nervous system stress and can lead to high levels of glutamate. These include the direct release of glutamate from cells, enzymatic conversion of high extracellular glutamine to glutamate, and shutdown of nerve and glial glutamate uptake systems by pro-oxidant conditions (Schubert and Piasecki, 2001). Thus, cell death induced by glutamate may be involved in neuronal injury associated with several acute and chronic neurodegenerative disorders, such as hypoxia-reperfusion or Huntington's and Alzheimer's diseases. Both apoptotic and necrotic pathways have been implicated in the death process, depending on the severity of the insult (Bonfoco et al., 1995; Ayata et al., 1997; Du et al., 1997). The exacerbated rise in intracellular Ca²⁺ is thought to trigger excitotoxicity through protease activation, mitochondrial toxicity, and nitric oxide production, among others, that ultimately lead to cell death.

The potential role of apoptosis in glutamate-induced toxicity suggests that its regulation may slow acute and chronic neurodegenerative processes. We have recently shown that TUDCA is neuroprotective in a transgenic mouse model of Huntington's disease (Keene et al., 2002) and in rat models of ischemic and hemorrhagic stroke (Rodrigues et al., 2002, 2003). In this study, we tested the hypothesis that TUDCA can reduce the apoptotic threshold induced by glutamate in rat cortical neurons and examined potential transduction pathways involved in both apoptotic signaling and neuroprotection by TUDCA. Our data confirm that glutamate induces apoptosis in rat cortical neurons and demonstrate that TUDCA reduces the apoptotic threshold induced by glutamate. In addition, our results indicate that glutamate modulates expression of Bcl-2 family proteins, thus inducing cytochrome c release, caspase activation, and nuclear fragmentation. TUDCA markedly inhibits apoptosis, in part, by promoting the phosphorylation and translocation of pro-apoptotic Bad. Moreover, the phosphorylation of Bad by TUDCA occurs through a PI3K-dependent mechanism. Inhibition of PI3K prevents both the phosphorylation of Bad by TUDCA and its translocation from the mitochondria to the cytosol, thus abrogating the protective effect of the bile acid.

Materials and Methods

Isolation and Culture of Rat Cortical Neurons. Primary cultures of rat cortical neurons were prepared from 17- to 18-day-old fetuses of Wistar rats as previously described (Brewer et al., 1993) with minor modifications. In short, pregnant rats were ether-anesthetized and decapitated. The fetuses were collected in Hanks' balanced salt solution (HBSS-1; Invitrogen, Carlsbad, CA) and rapidly decapitated. After removal of meninges and white matter, the brain cortex was collected in HBSS without Ca²⁺ and Mg²⁺ (HBSS-2; Invitrogen). The cortex was then mechanically fragmented, transferred to an HBSS-2 solution containing 0.025% trypsin, and incubated for 15 min at 37°C. Following trypsinization, cells were washed twice in HBSS-2 with 10% fetal bovine serum and resuspended in Neurobasal medium (Invitrogen), supplemented with 0.5 mM l-glutamine, 25 μM l-glutamic acid, and 2% B-27 supplement (Invitrogen), and 12 mg/ml gentamicin. Aliquots of 2 × 10⁶ cells/ml were plated on tissue culture plates precoated with poly-d-lysine and maintained at 37°C in a humidified atmosphere of 5% CO₂. At 5 days, one-half of the medium was removed and replaced by the same volume of fresh medium without glutamic acid and B-27 supplement for the duration of the experiments. All assays were performed on cells cultured for 5 days. Cells were characterized by phase contrast microscopy and indirect immunocytochemistry for neurofilaments and glial fibrillary acidic protein. Neuronal cultures were >95% pure. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the national Academy of Sciences and published by the National Institutes of Health (National Institutes of Health publication 86-23 revised 1985).

Induction of Apoptosis. Isolated rat neurons cultured for 5 days were incubated with either 125 μM glutamate (Sigma-Aldrich, St. Louis, MO) for 5, 15, and 30 min and 1, 8, 24, and 48 h, with or without 100 μM TUDCA (Sigma-Aldrich) or no addition (control). In coincubation experiments, TUDCA was added to neurons 12 h prior to incubation with glutamate. Wortmannin (Calbiochem, San Diego, CA), an inhibitor of PI3K phosphorylation, was added to cells 1 h prior to TUDCA treatment at a final concentration of 200 nM. The medium was gently removed at the indicated times, and attached cells were fixed for morphologic assessment of apoptosis or processed for viability assays. In addition, total, cytosolic and mitochondrial protein fractions were extracted for immunoblotting and caspase activity assays.

Measurement of Cell Death. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously (Mosmann, 1983). In brief, the culture medium was replaced with 0.5 ml of the MTT (Sigma-Aldrich) solution at 0.5 mg/ml, and the cells were incubated for 1 h at 37°C. One milliliter of the solubilization solution (isopropanol:0.04 M HCl; 1:1, v/v) was then added to cells, and the absorption was measured at 570 nm after 15 min of incubation. In addition, apoptosis was assessed by analysis of nuclear morphology. Briefly, neurons were fixed with 4% formaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 10 min at room temperature, incubated with Hoechst dye 33258 (Sigma-Aldrich) at 5 μg/ml in PBS for 5 min, washed with PBS, and mounted using PBS/glycerol (3:1, v/v). Fluorescence was visualized using an Axioskop fluorescence microscope (Carl Zeiss GmbH, Jena, Germany). Fluorescent nuclei were scored and categorized according to the condensation and staining characteristics of chromatin. Normal nuclei showed noncondensed chromatin dispersed over the entire nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous to the nuclear membrane, as well as nuclear fragmentation and apoptotic bodies. Three random microscopic fields per sample of approximately 250 nuclei were counted and mean values expressed as the percentage of apoptotic nuclei.

Cytochrome c Release and Bad Translocation. Cellular distribution of cytochrome c and phosphorylated Bad (p-Bad) in neuronal cells was determined using mitochondrial and cytosolic protein extracts. Cells were harvested and centrifuged at 600g for 5 min at 4°C. The pellets were washed once in ice-cold PBS and resuspended with 3 volumes of isolation buffer containing 20 mM HEPES/KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and supplemented with protease inhibitor cocktail tablets (Complete; Roche Diagnostics, Indianapolis, IN), in 250 mM sucrose. After chilling on ice for 15 min, cells were disrupted by 40 strokes of a glass homogenizer, and homogenates were centrifuged twice at 2500g for 10 min at 4°C to remove unbroken cells and nuclei. The mitochondrial fraction was then centrifuged at 12,000g for 30 min at 4°C, and the pellet was resuspended in isolation buffer and frozen at -80°C. For cytosolic proteins, the 12,000g supernatants were removed, filtered sequentially through 0.2- and 0.1-μm Ultra-free MC filters (Millipore Corporation, Bedford, MA) to remove other cellular organelles, and frozen at -80°C. Typically, 25 and 100 μg of mitochondrial and cytosolic proteins, respectively, was separated by 15% SDS-polyacrylamide gel electrophoresis (PAGE) and processed cytochrome c and p-Bad detection.

Following electrophoretic transfer onto nitrocellulose membranes, the immunoblots were incubated with 15% H₂O₂ for 15 min at room temperature. Blots were then sequentially incubated with 5% milk blocking solution, primary monoclonal antibody to cytochrome c (BD Biosciences PharMingen, San Diego, CA) at a dilution of 1:5000 overnight at 4°C, and finally with secondary goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA) for 3 h at room temperature. In addition, blots were probed with primary polyclonal antibody to p-Bad (Ser-136; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:500 and then with secondary anti-rabbit antibody conjugated with horseradish peroxidase. The membranes were processed for detection of cytochrome c and p-Bad using the SuperSignal substrate (Pierce Chemical, Rockford, IL). Mitochondrial contamination of the cytosolic protein extracts was determined by western blot analysis of cytochrome c oxidase (subunit II). Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad) according to the manufacturer's specifications.

N-Acetyl-Asp-Glu-Val-Asp-Specific Caspase Activity. Caspase activation was determined in cytosolic protein extracts. Cells were harvested and homogenized in isolation buffer containing 10 mM Tris-HCl, pH 7.6, 5 mM MgCl₂, 1.5 mM KAc, 2 mM dithiothreitol, and protease inhibitor cocktail tablets (Roche Diagnostics). General caspase-3-like activity was evaluated by enzymatic cleavage of the chromophore p-nitroanilide (pNA) from the substrate N-acetyl-Asp-Glu-Val-Asp (DEVD) pNA (Sigma-Aldrich). The proteolytic reaction was carried out in isolation buffer containing 50 μg of cytosolic protein and 50 μM DEVD-pNA. The reaction mixtures were incubated at 37°C for 1 h, and the formation of pNA was measured at 405 nm using a 96-well plate reader.

Immunoblotting Analysis of Total Protein Expression. Bcl-2 family, caspase-3, p-Akt1, and p-Bad protein levels were determined from total protein homogenates separated by 12% SDS-PAGE. Blots were probed with either primary mouse monoclonal antibodies reactive with Bcl-2 or with primary rabbit polyclonal antibodies to caspase-3, Bcl-x_L, Bax, p-Akt1 (Ser-473), and p-Bad (Ser-136) at a dilution of 1:500 (Santa Cruz Biotechnology, Inc.) overnight at 4°C. Subsequently, blots were incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with horseradish peroxidase. Finally, membranes were processed for specific protein detection using the SuperSignal substrate. β-actin, total Bad, and total Akt1/2 were used for lane loading standards.

Densitometry and Statistical Analysis. Densitometry was accomplished using a personal computer coupled to an HP scan jet 2200c scanner (Hewlett Packard, Palo Alto, CA). The relative intensities of the protein bands were analyzed using the ImageMaster 1D Elite densitometric analysis program (Amersham Biosciences AB, Uppsala, Sweden). All values were normalized to their respective lane loading controls. All data were expressed as mean ± S.E.M. from at least three separate experiments. Statistical analysis was performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software Inc., San Diego, CA) for the ANOVA and Bonferroni multiple comparison tests. Values of p < 0.05 were considered statistically significant.

Results

Glutamate Induces and TUDCA Prevents Apoptosis in Rat Cortical Neurons. Glutamate toxicity has been extensively studied in several experimental paradigms of cell death. However, less is known about the role of apoptosis in glutamate-induced neurotoxicity. Primary cultures of rat cortical neurons were incubated with 125 μM glutamate for 24 h and examined for the characteristic morphological nuclear changes associated with apoptosis (Fig. 1A). After 24 h of incubation, almost 20% of cells exhibited condensed chromatin and nuclear fragmentation with formation of apoptotic bodies at a >3-fold increase over control values (p < 0.01). Incubation with 50 μM glutamate resulted in significantly less apoptosis, whereas 250 or 500 μM glutamate induced accelerated cell death. Incubations with 125 μM glutamate for 48 h induced ∼ 22% apoptotic cells (data not shown).

We next evaluated cytochrome c release and caspase-3 activity in isolated cortical neurons (Fig. 1, B and C). In fact, it has been suggested that glutamate induces free radical formation and disrupts mitochondrial membrane potential (Sastry and Rao, 2000), which in turn may promote cytochrome c release. Control cells exhibited almost undetectable levels of cytosolic cytochrome c. In contrast, glutamate induced a >5-fold accumulation of cytochrome c in the cytosol, concomitant with a significant reduction in mitochondrial levels (p < 0.05). Using Western blot analysis, we also show a marked increase of the 20-kDa active caspase-3 fragment in neurons incubated with glutamate compared with controls (p < 0.05).

TUDCA inhibits cytochrome c release, caspase activation, and ultimately cell death in several models of neuronal apoptosis (Rodrigues et al., 2000; Solá et al., 2003; Ramalho et al., 2004). Therefore, we next determined whether glutamate toxicity was reduced with TUDCA. Our results indicated that TUDCA significantly decreased the observed nuclear changes in rat cortical neurons by >75% (p < 0.01) (Fig. 2A) and inhibited mitochondrial efflux of cytochrome c (p < 0.05) (Fig. 2B). Finally, pretreatment with TUDCA reduced glutamate-induced caspase-3-like activation to almost control values (p < 0.05) (Fig. 2C). Taken together, these findings suggested that glutamate-mediated apoptosis in rat cortical neurons is markedly inhibited by TUDCA. However, preincubations with lower TUDCA concentrations and/or shorter periods were not as protective. Indeed, pretreatment of neurons with 100 μM TUDCA for 1 h prior to incubation with glutamate resulted in ∼ 20% inhibition of apoptosis (data not shown).

Glutamate-Induced Apoptosis Is Associated with Down-Regulation of Antiapoptotic Bcl-2 Family Proteins. The Bcl-2 family of proteins plays a key role in the apoptotic process. In this specific model, we sought to investigate the effects of glutamate on antiapoptotic Bcl-2 and Bcl-x_L and pro-apoptotic Bax. By Western blot analysis of total protein extracts, incubation of neurons with glutamate significantly altered Bcl-2 and Bcl-x_L protein levels as compared with controls (Fig. 3). Glutamate reduced Bcl-2 protein by ∼60% after 30 min of incubation (p < 0.01), which remained below control values for at least 48 h. Moreover, Bcl-x_L was also down-regulated, especially at 1 h (p < 0.01). In contrast, steady-state levels of Bax protein did not significantly change throughout the incubation period (data not shown). Pretreatment with TUDCA only slightly inhibited the decrease in Bcl-2 and Bcl-x_L induced by glutamate (Fig. 3), suggesting that TUDCA cytoprotection results from mechanisms other than modulation of Bcl-2 and Bcl-x_L protein levels.

TUDCA Induces Phosphorylation and Translocation of Bad through a PI3K-Dependent Pathway. The proapoptotic protein Bad is another member of the Bcl-2 family that is known to be involved in the regulation of apoptosis by binding to and inactivating Bcl-2 and Bcl-x_L (Yang et al., 1995). However, under nonapoptotic conditions, Bad is phosphorylated by serine-threonine kinases such as Akt (Datta et al., 1997; del Peso et al., 1997), protein kinase A (Harada et al., 1999), and Raf-1 (Wang et al., 1996) and remains inactive in the cytosol. Therefore, to examine the mechanism(s) by which TUDCA prevents glutamate-induced apoptosis, we examined whether TUDCA alters the phosphorylation state and cellular distribution of Bad.

Neurons treated with glutamate only exhibited marginal and transient changes in p-Bad levels (Fig. 4A). Similarly, TUDCA alone induced slight increases in Bad phosphorylation. However, p-Bad was increased by >75% after incubation of cells with glutamate plus TUDCA (p < 0.05), suggesting that Bad is an important regulatory target for this bile acid in the presence of a toxic stimulus. Also, glutamate alone induced a rapid and reversible phosphorylation of Akt at 5 min (p < 0.05) (Fig. 4B). After 30 min of incubation, p-Akt decreased ∼ 50% (p < 0.01) and remained below control values throughout the time course. However, pretreatment with TUDCA only slightly increased Akt phosphorylation, suggesting that TUDCA modulation of Bad phosphorylation is at least partially independent of Akt.

Bad phosphorylation appears to be an important factor in PI3K survival signaling (Datta et al., 1999). To address the role of the PI3K pathway in TUDCA protection, neurons were preincubated with the PI3K inhibitor wortmannin. As assessed by MTT metabolism and Hoechst staining, control and glutamate-treated cells were less viable after exposure to wortmannin alone (Fig. 5A). Not surprisingly, wortmannin increased apoptosis by TUDCA, which has also been observed previously with inhibition of the PI3K pathway in several cell types (Qiao et al., 2002; Solá et al., 2003). More importantly, wortmannin reduced the protective effect of TUDCA in modulating glutamate-induced cell death (Fig. 5A) and appeared to do so by inhibiting Bad phosphorylation (Fig. 5B). We next measured the translocation of Bad from mitochondria to cytosol in intact neurons after cell fractionation. The results revealed a >2-fold increase in cytosolic levels of p-Bad in cells treated with glutamate plus TUDCA compared with glutamate alone (p < 0.05) (Fig. 5C). However, Bad translocation with TUDCA plus glutamate was absent with wortmannin inhibition of PI3K signaling. These data suggest that TUDCA-induced PI3K activation is required for phosphorylation and translocation of Bad and subsequent protection of neuronal death from glutamate.

Discussion

Our results indicate that glutamate induces cytochrome c release, caspase activation, and changes in nuclear morphology consistent with apoptosis in rat cortical neurons. The changes are associated with down-regulation of antiapoptotic Bcl-2 and Bcl-x_L, and although TUDCA reduces the apoptotic threshold, it does so without significantly altering their steady-state levels. However, Bad phosphorylation on Ser-136 is in response to the treatment of cortical neurons with glutamate plus TUDCA. This phosphorylation is accompanied by the translocation of Bad from mitochondria to the cytosol. Furthermore, inhibition of PI3K with wortmannin prevented the phosphorylation and translocation of Bad and abrogated the antiapoptotic effect of TUDCA. Phosphorylation of Bad is thought to prevent its interaction with anti-apoptotic protein Bcl-2 or Bcl-x_L, thereby allowing them to promote cell survival.

Overstimulation of glutamate receptors may contribute to neuronal cell death that occurs in a variety of physiological and pathological settings. Exposure of hippocampal and cortical neurons to glutamate can induce apoptosis, which is mediated by Ca²⁺ influx, mitochondrial perturbation, and caspase activation (Ankarcrona et al., 1995; Du et al., 1997; Larm et al., 1997; Cheung et al., 1998). In addition, glutamate receptor activation is thought to contribute to cell death in Alzheimer's and Parkinson's diseases, whereas pharmacological inhibition of glutamate receptors prevents neuronal death in experimental models of stroke (Mattson, 1997; Dirnagl et al., 1999) and Huntington's disease (Petersen et al., 1999).

The present study confirms that apoptosis clearly plays a role in glutamate-induced neurotoxicity in rat cortical neurons. Cytochrome c was significantly depleted from mitochondria, and caspase-3 was activated in response to incubation with glutamate. It is generally accepted that mitochondria make substantial contribution to the effector phase of apoptosis in various cell types. Following a death signal, the release of cytochrome c activates the apoptosis-activating factor-1 to form a protein complex, which in turn activates caspase-3 (Kroemer and Reed, 2000). As expected, cytochrome c efflux and caspase-3 processing were accompanied by the morphologic changes of apoptosis in neurons exposed to glutamate.

One possible explanation for cytochrome c release from mitochondria relates to the channel forming activity of the pro-apoptotic protein Bax. It has been demonstrated that the involvement of Bax on glutamate-induced apoptosis is dependent on the nature of the insult and the experimental conditions (Dargusch et al., 2001). Our results show that glutamate-induced apoptosis occurs independently of changes in Bax expression. Nevertheless, glutamate may activate signal transduction pathways that stabilize Bax protein, alter its intracellular distribution, or change the ratio to other Bcl-2 family antagonists. In fact, antiapoptotic Bcl-2 and Bcl-x_L were rapidly down-regulated after glutamate incubation. In this regard, Bcl-2 overexpression in the central nervous system has been shown to protect cells from glutamate toxicity (Behl et al., 1993; Lawrence et al., 1996; Howard et al., 2002).

UDCA and TUDCA are nontoxic, endogenous bile acids that can act as antiapoptotic agents. They directly inhibit reactive oxygen species production, collapse of the transmembrane potential, and disruption of the outer mitochondrial membrane. Additionally, both bile acids mitigate mitochondrial insufficiency and toxicity by inhibiting Bax translocation from cytosol to mitochondria (Rodrigues et al., 2000). Membrane stability inhibits cytochrome c release, thereby reducing downstream events, such as caspase activation and substrate cleavage. In addition to inhibiting mitochondrial membrane depolarization and channel formation, TUDCA may also play key regulatory roles in signal transduction by modulating Ca²⁺ levels and gene expression (Ozes et al., 1999). Finally, we have recently shown that TUDCA can protect neurons against cell death in experimental models of acute neurodegenerative conditions such as ischemic and hemorrhagic stroke (Rodrigues et al., 2002, 2003), as well as in chronic neurodegenerative disorders such as Huntington's disease (Keene et al., 2002).

The present study now shows a neuroprotective effect of TUDCA in rat cortical neurons exposed to glutamate. TUDCA significantly reduced cytochrome c release, caspase activation, and changes in nuclear morphology. In addition, coincubation with TUDCA and glutamate markedly increased Bad phosphorylation and translocation to the cytosol, suggesting an important target for the antiapoptotic function of this bile acid. Interestingly, neither glutamate nor TUDCA alone increased phosphorylation of Bad significantly, suggesting that a toxic stimulus may be required for TUDCA to trigger the survival pathway. The phosphorylation of Bad prevents its interaction with Bcl-2 or Bcl-x_L, thereby allowing these proteins to complex with Bax. Although TUDCA does not significantly alter Bcl-2 and Bcl-x_L protein levels, it may increase their antiapoptotic activity by phosphorylating, thus neutralizing the repressor Bad.

Inhibition of PI3K with wortmannin prevented Bad phosphorylation and translocation to the cytosol associated with TUDCA, an effect that was accompanied by increased apoptosis of the cortical neurons. This suggests that TUDCA-induced Bad phosphorylation was triggered by a PI3K-mediated signal that was partially independent of Akt activation. Other studies have demonstrated that PI3K stimulates Akt-independent Bad-phosphorylation and induces antiapoptotic signals (Hinton and Welham, 1999; Wolf et al., 2001). PI3K may activate the small GTP-binding protein Rac (Hawkins et al., 1995), as well as a number of other kinases, including an atypical protein kinase C, PKCζ. In this regard, the bile acid taurochenodeoxycholate is thought to activate PI3K-dependent cell survival pathways through PKCζ and independently of Akt (Rust et al., 2000). However, the mechanism(s) by which TUDCA activates PI3K is still unclear. Some studies could not identify a direct effect of bile acids on PI3K activity (Misra et al., 1998), suggesting an indirect mechanism for activation. Bile acids likely stimulate PI3K activity by facilitating its association with receptor complexes known to activate this lipid kinase.

Our results provide further insight into how TUDCA and glutamate mediate their effects in cortical neurons. This may have implications for treatment and prevention of human neurodegenerative diseases. In fact, excessive glutamate release is thought to play a pivotal role in several neurological injuries, in part by activating apoptosis. Its toxicity appears to be mediated through mitochondrial reactive oxygen production, energy depletion, and apoptogenic factor release or activation. We show here that glutamate reduced Bcl-2 and Bcl-x_L expression, and this was associated with cytochrome c release, caspase activation, and morphologic changes of apoptosis. TUDCA proved to be a potent inhibitor of cell death by glutamate, in part, by promoting the phosphorylation and translocation of pro-apoptotic Bad and thus preserving mitochondrial membrane stability. Moreover, the phosphorylation of Bad by TUDCA occurs through a PI3K-dependent mechanism. It remains to be determined whether the activation of PI3K is modulated directly by TUDCA or the result of a downstream effect.