Bax Involvement in p53-Mediated Neuronal Cell Death

Neurons lacking Bax are resistant to excitotoxicity and DNA damage

We recently have postulated a direct role for p53 in initiating neuronal degeneration on the basis of the observation that wild-type (p53 +/+) neurons were extremely susceptible to glutamate- and kainate-mediated cell death, whereas p53-deficient (−/−) neurons were resistant (H. Xiang et al., 1996). We reasoned that if Bax activity were essential to a cell death pathway activated by p53 in response to excitotoxic challenge, then Bax-deficient neurons should display a degree of resistance similar to that of the p53-deficient neurons. Glutamate (Glu) or kainate (KA) administration induced cell death in cortical neuronal cultures derived from newborn Bax (+/+) mice (Fig.1), with both a time and a dose dependence (data not shown). A significant difference in the percentage of surviving neurons was observed between control and Glu- or KA-treated neurons as early as 24 hr, with this difference becoming more pronounced with time. Approximately 70–80% of Bax (+/+) neurons present at the time of treatment had died 3 d after adding Glu (50 μm) or KA (40 μm). The extent of neuronal death induced by Glu or KA did not differ significantly between Bax (+/+) and Bax (+/−) neuronal cultures (p > 0.55) (Fig. 1). The morphological changes induced by Glu treatment were striking. Nontreated wild-type cultures consisted primarily of intact cell bodies, which were demonstrable by phase-contrast optics or by detection of the viability dye, calcein (Fig.2A, Control, green fluorescence). Nontreated wild-type neurons displayed an abundant outgrowth of arborizing neurites that were visualized clearly by the calcein fluorescence. However, even at 1 d after Glu treatment (50 μm) there was significant loss of viable cell bodies, as visualized by phase-contrast optics and demonstrated by the reduced number of elements exhibiting calcein fluorescence (Fig.2A, Glutamate). Many cell bodies that remained after Glu treatment were damaged severely, as demonstrated by their uptake of ethidium homodimer (red fluorescence).

The loss of viability induced by KA or Glu treatment was determined not only by direct cell counting but also by evaluating mitochondrial integrity as assessed by conversion of the tetrazolium salt, MTT, to formazan by the respiratory chain enzyme succinate–tetrazolium reductase (Slater et al., 1963; Altman, 1976). Treatment of Bax (+/+) cortical neurons with Glu (50 μm) or KA (40 μm) for 5 d produced a significant reduction in MTT conversion (Table 1; glutamate, 29% of control; kainate, 77% of control) relative to nontreated control cultures.

Neurons deficient in Bax (−/−) were resistant to Glu- and KA-induced cell death relative to Bax wild-type neurons (Fig. 1). However, in contrast to p53-deficient neurons (H. Xiang et al., 1996), there was a small but significant decline in the percentage (20%) of Bax (−/−) neurons that survived 3 d after exposure to Glu (50 μm) or KA (40 μm), as compared with control cultures (i.e., nontreated) (Glu, p < 0.01; KA,p < 0.001). A decline in viability of Bax (−/−) neurons exposed to Glu for 5 d was confirmed by the reduction in MTT conversion (Table 1; glutamate, 65% of control). Thus, the protection afforded by Bax deletion was relative to controls, but not absolute. The protection conferred by the absence of Bax was consistent with the lack of substantial morphological damage observed in the cultures; whereas Bax (+/+) neurons displayed obvious signs of damage, most Bax (−/−) neurons retained viable cell bodies and neurites after excitotoxic challenge (Fig. 2B, compareControl and Glutamate). The viability of Bax (−/−) neurons also was reflected in the retention of calcein fluorescence.

The resistance displayed by Bax-deficient cortical neurons to excitotoxic injury was not restricted to this single form of damage. In the present study Bax- and p53-deficient cortical neurons also were protected from DNA damage induced by the addition of the topoisomerase I inhibitor, camptothecin. The cellular toxicity of camptothecin is associated with the generation of DNA strand breaks (Ryan et al., 1991). Camptothecin treatment, which leads to cell cycle arrest or apoptosis, has been shown to elevate p53 protein levels (X. Chen et al., 1996; M. Johnson and R. Morrison, unpublished data). In cultures of p53 (Fig. 3A, +/+) and Bax (Figs. 2A, 3B, +/+) wild-type cortical neurons, camptothecin exposure produced significant cell death. Camptothecin-induced cell death was dose-dependent and proceeded more rapidly (2 d) than cell death induced by Glu (50 μm) treatment (3–4 d). Morphological evidence of neuronal damage was observed clearly at 24 hr after treatment and was associated with significant loss of viable cell bodies, as seen under phase-contrast optics and by the paucity of calcein fluorescence (Fig.2A, Camptothecin). The majority of cell bodies that remained after camptothecin treatment was severely damaged, as demonstrated by their uptake of ethidium homodimer (red fluorescence).

The significant degree of damage produced by camptothecin treatment in Bax (+/+) cultures was reflected in the absence of detectable MTT conversion (Table 1). Interestingly, the damage induced by camptothecin and glutamate was not associated with DNA laddering (data not shown), consistent with previous studies (Ankarcrona et al., 1995; Finiels et al., 1995). In marked contrast, camptothecin treatment did not significantly alter the survival of p53- and Bax-deficient cortical neurons when compared with nontreated or DMSO-treated control neurons (Figs. 2A,B, 3A,B). Bax- and p53-deficient neurons appeared remarkably healthy 2 d after camptothecin treatment and showed no morphological evidence of damage. Cell bodies failed to exhibit condensation, and neurites did not display the blebbing and disintegration (Fig. 2B) that were prominent in camptothecin-treated wild-type cultures (Fig.2A). The enhanced viability of Bax (−/−) neurons relative to wild-type neurons after camptothecin treatment was consistent with the high level of MTT conversion present in the Bax (−/−) cultures (Table 1).

The Bax protein in cultured cortical neurons is selectively elevated by excitotoxic injury

The resistance displayed by p53- and Bax-deficient neurons to Glu- and camptothecin-mediated cell death suggested that these forms of toxicity must be capable of altering the levels, activity, or intracellular distribution of the Bax protein. Because the Bax promoter previously has been shown to contain a p53-responsive binding element (Miyashita et al., 1994), we therefore examined cortical neurons for alterations in the levels of Bax protein after Glu and camptothecin treatment. Glutamate treatment (50 μm) produced a significant increase in the steady-state levels of the Bax protein in neurons containing the p53 gene (Fig. 4, Table 2). The levels of Bax protein increased ∼18-fold in Glu-treated cultures relative to saline-treated control cultures (n = 3; p < 0.05) and were evident 24 hr after treatment. In contrast to the dramatic change induced by Glu, camptothecin induced a small change in the steady-state levels of Bax protein (Fig. 4, Table 2). The Glu-induced increase in Bax protein was not observed when this treatment was imposed on p53-deficient neurons (Fig. 4, p53 −/−, n = 4), suggesting that p53 was needed to mediate the rise in Bax protein. Neurons containing a full complement of p53 alleles (p53 +/+) were derived from both the p53 and Bax strains of mice. Therefore, the p53-dependent glutamate-induced increase in Bax protein was a general phenomenon observed in neurons derived from at least two distinct strains of mice. However, increased p53 expression resulting from adenovirus-mediated gene transfer was not sufficient, by itself, to elevate Bax protein levels (Fig. 4, p53_ADV). This result suggests that other pathways must be required, in addition to p53 expression, for Glu to promote an increase in the steady-state levels of the Bax protein. These results suggest that neuronal viability may be compromised by complex p53-mediated effects on the Bax protein.

p53 induces neuronal cell death via a complex signaling cascade

Previous studies from this laboratory demonstrated that increased p53 expression was sufficient to induce neuronal cell death, even in the absence of a cytotoxic stimulus (H. Xiang et al., 1996). The same study showed that, although p53-deficient neurons were resistant to excitotoxic injury, they were still competent to respond to a p53-initiated cell death pathway after introduction of the p53 gene via adenovirus-mediated transduction. In the present study the same paradigm was used to determine whether Bax is essential to a p53-regulated cell death pathway in neurons. A control virus containing the β-galactosidase gene (LacZ) was used previously to determine the efficiency of transduction as well as the effect of viral infection on neuronal viability (H. Xiang et al., 1996). These results demonstrated that neurons could be infected with a high degree of efficiency by using adenovirus and that at the appropriate titer the infection process by itself does not alter neuronal survival.

Adenoviral infection at a multiplicity of infection (MOI) of 250 was shown previously to produce significant neuronal cell death in p53-deficient neurons (5% survival 4 d postinfection; H. Xiang et al., 1996). In the present study the p53 adenovirus did not affect the viability of Bax-deficient neurons when used at a titer of 250 (Fig.5, p53, MOI-250). However, this resistance to introduction of p53 was not absolute. When the p53 adenovirus titer was increased to an MOI of 500, Bax-deficient (Bax −/−) neurons developed pathological morphology (Fig.6A, p53-Adenovirus) and showed a dramatic decline in survival (Fig. 5, p53, MOI-500). At 4 d after infection only 25% of the Bax (−/−) neurons present at the time of infection survived (MOI-500). This decline in viability was confirmed by the loss of calcein fluorescence (Fig. 6C, green fluorescence) and by the increased uptake of ethidium homodimer (Fig. 6C, red fluorescence). The morphological appearance of neurons in the p53 adenovirus-infected cultures was consistent with their declining viability. Most noticeable was the blebbing and dissolution of neuritic processes, followed by shrinkage of the soma and condensation of the nucleus. Because infection with an adenovirus containing the β-galactosidase gene at the higher titer of MOI-500 had only a modest effect on Bax (−/−) neuronal survival on the basis of cell counts of morphologically viable cells (Fig. 5, LacZ, MOI-500) and the continued presence of calcein fluorescence (Fig.6D, green fluorescence), cell death observed with p53 adenovirus infection was attributed specifically to increased p53 expression.

Despite the different outcomes observed with p53- and Bax-deficient neurons in response to p53 adenovirus infection at an MOI of 250, the amount of p53 protein expressed in both types of neurons was similar (Fig. 7). Further, the extent of p53 expressed after adenovirus infection greatly exceeded the p53 levels induced under physiological conditions of injury. p53-Immunoreactive protein could not be detected by Western blot analysis after adenovirus infection with the β-galactosidase gene (Fig. 7, LacZ). We conclude from this study that, under the appropriate circumstances, p53 can induce neuronal cell death in the absence of Bax, suggesting that p53 activates multiple cell death signaling pathways in neurons.

Bax–p53 coupling in cell death pathways

Our results demonstrate that Bax plays a critical role in regulating neuronal sensitivity to excitotoxic and genotoxic damage. Previous results from our laboratory demonstrated that p53-deficient neurons were resistant to glutamate-mediated excitotoxicity. It recently has been shown that excitotoxicity may be associated with the accumulation of single-strand DNA breaks (Didier et al., 1996). Therefore, it is especially noteworthy that Bax- and p53-deficient neurons were resistant to the toxic effects of camptothecin, which, at the concentrations used in this study, directly promotes DNA strand breaks (Ryan et al., 1991). DNA strand breaks, but not other DNA lesions, are capable of inducing p53 accumulation (Nelson and Kastan, 1994; Jayaraman and Prives, 1995; Lee et al., 1995). These observations suggest that excitotoxicity and genotoxic damage share a common pathway for activating neuronal cell death–DNA damage-induced p53 activation. The protection conferred by the absence of either the p53or Bax gene suggests that these proteins are coupled within a common cell death pathway that is activated in response to some, but not all, death-inducing stimuli.

In contrast to our major results, Bax involvement in programmed cell death that occurs during the development of the nervous system may be independent of p53 activation. For example, the declining viability of neonatal sympathetic neurons after trophic factor deprivation recently has been shown to be dependent on the Bax protein (Deckwerth et al., 1996). However, the absence of p53 does not confer protection on embryonic sensory and sympathetic neurons after the withdrawal of neurotrophins (Davies and Rosenthal, 1994; Sadoul et al., 1996). These results suggest that neurotrophin-deprived peripheral neurons die via a pathway that is dependent on Bax expression but independent of p53. Thus, Bax may function in multiple pathways to regulate neuronal viability during development and in response to injury.

Regulation of Bax expression/activity

The involvement of Bax in multiple cell death pathways suggests that Bax expression and/or activity is subject to complex patterns of regulation. Our results demonstrated that Bax is expressed constitutively in cultured cortical neurons in the absence of a cytotoxic stimulus and independently of p53. Although camptothecin-induced DNA damage had little effect on Bax expression, excitotoxic injury significantly upregulated the Bax protein. The glutamate-mediated increase in Bax protein was dependent on the presence of the p53 gene, providing additional evidence for p53 activation after excitotoxic injury. Interestingly, increased p53 levels alone, resulting from adenovirus infection, were not sufficient to elevate Bax. Therefore, excitotoxic injury must stimulate other signal transduction pathways that augment Bax transcription, stabilize the Bax protein, or alter its intracellular localization (Hsu et al., 1997). Because Bax-deficient neurons are resistant to both glutamate- and camptothecin-induced cell death, we conclude that Bax must be activated and incorporated into a cell death pathway independently of changes in its expression. Thus, the glutamate-mediated increase in Bax protein observed in the present study may not contribute directly to neuronal cell death but may be secondary to (or associated with) other modifications in the Bax protein.

Bax activity could be integrated into a proapoptotic pathway as a result of distinct changes in the ratio of the Bax protein to cellular antagonists. For example, it is conceivable that p53 indirectly may augment Bax activity by activating additional proapoptotic proteins. Because multiple Bcl-2 family members are expressed in the nervous system, there may be several pathways for regulating Bax that are ultimately contingent on the identity of the injured neuron and the context of the injury.

Results from several different model systems have demonstrated either that Bax is required for apoptosis (McCurrach et al., 1997; Yin et al., 1997) or that an increase in Bax expression precedes apoptosis (Zhan et al., 1994; Bargou et al., 1995; Brady et al., 1996). The results of the present study are consistent with several reports demonstrating that upregulation of the Bax protein occurs in neurons after cerebral ischemia (Krajewski et al., 1995; J. Chen et al., 1996) and after exposure to the amyloid-β peptide (Paradis et al., 1996). Trophic factor deprivation-induced cell death in sympathetic and motor neurons was abated in the absence of Bax; however, it was not actually determined whether trophic factor withdrawal elevated Bax expression in wild-type neurons (Deckwerth et al., 1996). In addition, several mutant p53 proteins have been identified that have lost the ability to transactivate some, but not all, cellular p53-responsive promoters (Friedlander et al., 1996; Ludwig et al., 1996). In particular, one mutant p53 protein retained an ability to activate expression of the cyclin-dependent kinase inhibitor p21^cip1/waf1, and this activity correlated with the ability to induce cell cycle arrest. However, this particular p53 mutant was defective in the activation of p53-responsive sequences derived from the Bax and the insulin-like growth factor-binding protein-3 gene promoters. Failure to activate Bax correlated with the impaired apoptotic activity of this mutant p53 gene.

In contrast, several studies have failed to observe detectable increases in Bax expression after p53 activation (Allday et al., 1995;Canman et al., 1995; Knudson et al., 1995; Rowan et al., 1996). These results suggest that p53-induced cell death may require the activation of additional signaling pathways and may be only partially dependent on the presence of Bax. For example, thymocytes derived from Bax-deficient mice exhibit an apparently normal p53-mediated apoptotic response, suggesting that Bax expression is not necessarily coupled to p53-mediated apoptotic activity under all circumstances. The present demonstration that high levels of p53 can induce neuronal cell death in the absence of Bax expression is consistent with this possibility.

Mechanisms of Bax-mediated cell death

Although the present study demonstrated that Bax activity is often involved in p53-mediated cell death, the mechanism by which Bax promotes CNS cell death is not understood. One intriguing possibility is that p53-induced changes in neuronal viability may stem from declining mitochondrial function initiated by alterations in Bax activity. This hypothesis is consistent with the demonstration that mitochondrial dysfunction, including the loss of mitochondrial membrane potential and increased production of reactive oxygen species, plays an obligate role in excitotoxic damage (Ankarcrona et al., 1995; Dugan et al., 1995; Reynolds and Hastings, 1995; Schinder et al., 1996). Disruption of the mitochondrial membrane potential and increased production of reactive oxygen species have been defined as early events in the process of apoptosis (Deckwerth and Johnson, 1993; Vayssiere et al., 1994; Petit et al., 1995; Zamzami et al., 1995; Marchetti et al., 1996) A relationship between Bax activity and alterations in mitochondrial function also would be consistent with the mitochondrial localization of the Bax protein (Zha et al., 1996). Indeed, increased Bax expression has been shown to reduce mitochondrial membrane potential and to activate ICE-like proteases in a lymphoid cell line (J. Xiang et al., 1996) and in sympathetic neurons (Vekrellis et al., 1997). These effects of Bax expression are prevented by Bcl-2 and Bcl-x_L (Chinnaiyan et al., 1996; Monney et al., 1996;Shimizu et al., 1996). Interestingly, Bcl-2 recently has been shown to prevent the release of cytochrome c from mitochondria (Kharbanda et al., 1997; Kluck et al., 1997; Yang et al., 1997); cytochrome c promotes caspase activation and apoptotic changes in nuclei in a cell free system (Liu et al., 1996). In addition, recent studies have shown that the crystal structure of Bcl-x_L is related to certain bacterial pore-forming proteins (Sattler et al., 1997) consistent with the demonstration that the Bcl-x_L, Bcl-2, and Bax proteins are capable of forming an ion-conducting channel in synthetic lipid membranes (Antonsson et al., 1997; Minn et al., 1997; Schendel et al., 1997;Schlesinger et al., 1997). Although not yet proven, these channels may play a role in regulating mitochondrial permeability transition. Although the precise relationship between Bax and other Bcl-2 family members remains to be elucidated, it appears that they are tied intimately to the integrity of mitochondrial function.

Recent investigations have demonstrated a perhaps unexpected richness and complexity of cell death pathways. Key elements of the signaling sequence undoubtedly depend not only on the cell type involved but also on the nature of the insult and the developmental stage. Results of the present study demonstrate that, in CNS cortical neurons, excitotoxicity and genotoxic damage induce neuronal cell death by activating a p53-mediated Bax-dependent pathway. Thus, suppressing p53-mediated pathways, including alterations in Bax activity, may provide a means for maintaining neuronal viability and metabolic competence after cytopathic insults.