Article |
Address correspondence to Ruth S. Slack, Ottawa Health Research Institute, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5. Tel.: (613) 562-5800 ext. 8458. Fax.: (613) 562-5403. E-mail: rslack{at}uottawa.ca
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: neurodegeneration; neurons; apoptosis; p53; Bax
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of death regulatory molecules have been implicated in neuronal injury induced by ischemia, including p53, PARP, c-jun, and plasma membrane death receptor ligand systems (Eliasson et al., 1997; Endres et al., 1997; Herdegen et al., 1998; Morrison and Kinoshita, 2000; Martin-Villalba et al., 2001). Importantly, several lines of evidence suggest that p53 is a key upstream initiator of the cell death process after neuronal injury. P53 expression has been reported to be upregulated in response to excitotoxins, hypoxia, and ischemia (Xiang et al., 1996; Banasiak and Haddad, 1998; McGahan et al., 1998). Accordingly, we and others have shown that enforced expression of p53 alone is sufficient to trigger apoptosis in postmitotic neurons (Slack et al., 1996; Xiang et al., 1998; Cregan et al., 1999). In addition, it has been demonstrated that brain damage induced by ischemia or kainic acid excitotoxicity is significantly reduced in mice carrying a null mutation for the p53 gene (Crumrine et al., 1994; Morrison et al., 1996). Furthermore, cultured neurons derived from p53-deficient mice have been shown to be resistant to excitotoxins (Xiang et al., 1996, 1998), DNA damaging agents (Johnson et al., 1998; Xiang et al., 1998; Morris et al., 2001), and hypoxia (Halterman et al., 1999).
Caspases are a family of cysteine proteases that have been implicated as key effector molecules in the execution of apoptotic cell death (Cryns and Yuan, 1998). Recent studies have demonstrated the involvement of caspases in the execution of neuronal cell death both during development and after injury. Mouse embryos deficient for apoptotic activating factor-1 (Apaf1),* caspase-9, or caspase-3 display severe craniofacial malformations and dramatically enhanced neuronal cell numbers (Kuida et al., 1996, 1998; Cecconi et al., 1998). These gross developmental defects were attributed to failed apoptosis in the neuroepithelium. The importance of the caspase signaling cascade has also been demonstrated in many models of neuronal injury, including traumatic brain injury and ischemia (Hara et al., 1997; Yakovlev et al., 1997; Cheng et al., 1998).
Although caspases have been recognized as important mediators of apoptosis, there is accumulating evidence indicating the existence of caspase-independent mechanisms of neuronal cell death (Rideout and Stefanis, 2001). For example, several groups have indicated that in excitotoxic cell death, caspases are not activated and peptide-based caspase inhibitors do not invoke neuroprotection (Johnson et al., 1999; Lankiewicz et al., 2000). Similarly, in experimental models of stroke, caspase inhibition affords protection in certain neuronal populations, but not in others (Rideout and Stefanis, 2001; Zhan et al., 2001). Furthermore, in a number of neuronal cell death paradigms in which caspases are normally activated, inhibition of caspase activity delays, but does not prevent cell death from occurring (Miller et al., 1997; Stefanis et al., 1999; D'Mello et al., 2000; Keramaris et al., 2000; Selznick et al., 2000). Thus it appears that, at least in certain neuronal death paradigms, caspase inhibition simply results in the activation or recruitment of compensatory cell death processes. Although there has been extensive investigation on caspase-mediated cell death processes, much less is known about the molecular mechanisms involved in the regulation of caspase-independent cell death.
Apoptosis-inducing factor (AIF) is a putative caspase-independent effector of cell death that has recently been cloned and characterized (Susin et al., 1999). AIF is a mitochondrial intermembrane flavoprotein that has been reported to be released from the mitochondria and to translocate to the nucleus in response to specific death signals (Daugas et al., 2000). Furthermore, this apoptotic factor has been shown to cause high molecular weight DNA fragmentation and chromatin condensation in cells and isolated nuclei in a caspase-independent manner (Susin et al., 1999, 2000; Daugas et al., 2000).
In the present study, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process in the presence of Apaf1 and via a delayed onset caspase-independent mechanism in the absence of Apaf1. More importantly, we demonstrate that AIF is an important factor involved in the regulation of caspase-independent cell death induced by p53-mediated neuronal injury.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we examined long-term survival in Apaf1-deficient neurons to determine whether caspase-independent cell death occurred. We treated wild-type or Apaf1-deficient neurons with the DNA damaging agent camptothecin, which has previously been shown to induce neuronal cell death through a p53-dependent mechanism (Xiang et al., 1998; Morris et al., 2001). Camptothecin treatments were administered in the presence or absence of a broad spectrum caspase inhibitor, Boc-aspartyl (OMe)-fluoromethylketone (BAF), to account for the possible involvement of Apaf1-independent pathways of caspase activation. In wild-type neurons, treatment with camptothecin resulted in a rapid loss of neuronal survival beginning at 12 h, and within 24 h, survival had decreased to <20% (Fig. 1 A). In contrast, Apaf1-deficient neurons remained largely viable during the first 24 h of treatment. However, after 24 h, cell survival began to decline and by 48 h, <30% of Apaf1-/- neurons remained viable. These results demonstrate that p53 induces neuronal cell death through a rapid Apaf1-dependent pathway in wild-type neurons, and through a delayed pathway in Apaf1-deficient neurons.
|
Next, to determine whether p53 could directly induce caspase-independent cell death in the absence of Apaf1, we infected Apaf1+/+ and Apaf1-/- cortical neurons with a recombinant adenoviral vector carrying an expression cassette for either p53 or the control gene LacZ. Adenoviral-mediated expression of p53 resulted in a significant induction of TUNEL-positive cell death in both wild-type (61.4 vs. 16.8%) and Apaf1-deficient neurons (44.5 vs. 14.9%) relative to Ad-LacZinfected controls (Fig. 2 A). Cell death induced by direct p53 expression was associated with a significant induction of caspase-3 activity in wild-type neurons (4.7-fold increase), but not in Apaf1-deficient neurons (Fig. 2 B). Furthermore, Ad-p53induced TUNEL labeling was not prevented in the presence of a pan-caspase inhibitor (unpublished data). This indicated that, similar to the delayed cell death induced by camptothecin, DNA fragmentation triggered by direct p53 expression in Apaf1-deficient neurons was induced in a caspase-independent manner. Furthermore, whereas wild-type neurons undergoing cell death exhibited classical apoptotic nuclear morphology, Apaf1-deficient neurons again displayed only peripheral chromatin condensation and intermediate pyknosis (unpublished data). These results indicate that the p53 pathway can trigger a caspase-mediated cell death process in the presence of Apaf1 and a slower caspase-independent death process in the absence of Apaf1.
|
To determine whether AIF was released from the mitochondria during p53-mediated neuronal injury, we examined the cellular localization of AIF by immunofluorescence in neurons infected with Ad-p53 or Ad-LacZ. Our results demonstrate that the majority of neurons infected with the control vector displayed a punctate, cytoplasmic staining pattern that colocalized with a mitochondrial-specific marker (Fig. 3 A). In contrast, AIF staining was dramatically diminished and did not colocalize with the mitochondrial marker in Ad-p53infected neurons exhibiting morphological features of apoptotic cell death (Fig. 3 A, arrow). Despite the significant decrease in immunostaining intensity, Western blot analysis demonstrated that the dying cells retained AIF and cytochrome-c (Fig. 3 B), suggesting that these proteins may diffuse from the cells during the fixation and permeabilization procedure. Furthermore, many Apaf1-deficient neurons infected with Ad-p53 exhibited diffuse AIF staining in the nucleus (Fig. 3 A, arrowhead). These results suggest that AIF is released from the mitochondria and can translocate to the nucleus during p53-induced neuronal cell death. It is unclear why AIF fails to translocate to the nucleus in Apaf1+/+ neurons; however, it is possible that the early activation of caspases in wild-type cells results in the inactivation of the process responsible for AIF nuclear translocation.
|
We then assessed whether AIF release was associated with other mitochondrial events involved in DNA damageinduced neuronal cell death, such as mitochondrial depolarization and cytochrome-c release (Stefanis et al., 1999). Neurons treated with camptothecin were monitored for the mitochondrial release of cytochrome-c or AIF by immunofluorescence staining. In parallel cultures, mitochondrial depolarization was assessed in live cells by CMX-Ros labeling. This fluorescent dye is selectively incorporated into mitochondria with an intact transmembrane potential and therefore serves as an indicator of mitochondrial depolarization. Our results demonstrate that camptothecin induced a time-dependent decrease in the fraction of cells retaining mitochondrial transmembrane potential as well as mitochondrial cytochrome-c and AIF (Fig. 4 A). Interestingly, release of cytochrome-c appeared to precede the loss of AIF and mitochondrial membrane potential such that within 12 h of camptothecin treatment, there was already a significant decrease in the fraction of cells maintaining mitochondrial cytochrome-c staining (45%), but only a modest decrease in the fraction of cells exhibiting mitochondrial AIF (
5%) and CMX-Ros (
15%) staining (Fig. 4, A and B). These results suggest that AIF and cytochrome-c release occur by different mechanisms during p53-induced neuronal cell death.
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
P53 has been recognized as a key regulator of cell death after neuronal injury (Morrison and Kinoshita, 2000). In this paper, we have demonstrated that p53 triggers neuronal cell death via a caspase-mediated process in the presence of Apaf1, and via a caspase-independent process in the absence of Apaf1. More importantly, we provide several lines of evidence supporting a role for AIF in the regulation of caspase-independent cell death triggered by neuronal injury. We have demonstrated that (a) AIF redistributes from the mitochondria to the nucleus after p53-mediated neuronal injury, that (b) enforced expression of AIF can induce neuronal cell death in the absence of caspase activity with morphological features characteristic of caspase-independent cell death, and that (c) microinjection of neutralizing antibodies against AIF significantly decreases caspase-independent cell death induced by DNA damage. It was noted, however, that microinjected AIF antisera was moderately more effective at preventing AIF translocation (at least as detectable by immunostaining) than neuronal cell death. It is unclear whether this is due to incomplete inhibition of AIF translocation or rather to the possible involvement of other mediators of caspase-independent cell death. Indeed, the mitochondrial-derived proteins endonucleaseG and HtrA2 have recently been identified as potential caspase-independent mediators of cell death (Li et al., 2001; Suzuki et al., 2001; van Loo et al., 2001). Unfortunately, more definitive studies on the role of AIF in neuronal injury have been precluded by the lack of an AIF-null mouse model. Interestingly, AIF appears to be essential for the programmed cell death that occurs during cavitation of embryoid bodies (Joza et al., 2001). Attempts to generate chimeric mice from AIF-deficient ES cells have likely failed as a result of this defect in the early stages of embryogenesis (Joza et al., 2001). Thus, future studies of AIF function in neuronal injury in vivo will rely on the development of a conditional or tissue-specific knockout mouse model.
Two distinct stages of nuclear apoptosis have been identified during the apoptotic process (Daugas et al., 2000; Susin et al., 2000). In stage I, nuclei exhibit a wrinkled pattern of peripheral chromatin condensation, which is typically associated with high molecular weight DNA fragmentation (50 kb). As cell death progresses, nuclei adopt a stage II morphology, which is characterized by marked chromatin condensation and the formation of nuclear bodies. Furthermore, it has been shown that nuclear apoptosis is restricted to a stage I morphology when caspases are inhibited (Daugas et al., 2000; Susin et al., 2000). When recombinant AIF is added to isolated nuclei or injected into mouse embryo fibroblasts, nuclei adopt a stage Itype apoptotic morphology and exhibit high molecular weight DNA fragmentation, both of which occur independently of caspase activation (Susin et al., 1999, 2000; Daugas et al., 2000). In contrast, injection of active caspase-3 or caspase-activated DNase (CAD/DFF) caused a stage IItype nuclear morphology and resulted in the cleavage of DNA into oligonucleosomal fragments (Susin et al., 2000). In this paper, we have shown that in wild-type neurons, p53 initiates a caspase-mediated cell death process that manifests in a stage IItype nuclear apoptotic morphology. In contrast, p53-mediated cell death processes in Apaf1-deficient neurons resulted in a stage Ilike apoptotic nuclear morphology, consistent with a caspase-independent apoptotic mechanism. Interestingly, wild-type neurons undergoing p53-mediated cell death in the presence of a caspase inhibitor also exhibited stage I apoptotic nuclei. Furthermore, we have shown that enforced expression of AIF induces stage I apoptotic morphology and DNA fragmentation in neurons in a caspase-independent manner.
The Bcl-2 protein family consists of proapoptotic and antiapoptotic members that are thought to regulate cell death through their opposing effects on mitochondrial-mediated death processes (Adams and Cory, 1998; Gross et al., 1999). Accordingly, the proapoptotic member Bax has been reported to translocate from the cytoplasm to the mitochondria in response to certain death signals (Goping et al., 1998), where it causes permeabilization of the mitochondrial membrane and the release of apoptogenic factors like cytochrome-c (Finucane et al., 1999). Upon release into the cytosol, cytochrome-c forms a complex with dATP and Apaf1, which then recruits and activates caspase-9 and initiates the caspase cascade. We and others have shown that Bax deficiency provides long-term protection against p53-induced cell death, suggesting that Bax is required for both caspase-dependent and -independent modes of neuronal cell death (Xiang et al., 1998; Cregan et al., 1999). Similarly, we report here that Bcl-2 can block both Apaf1/caspase-dependent and -independent neuronal death pathways induced by p53. We have previously demonstrated that Bax is required for p53-induced cytochrome-c release (Keramaris et al., 2000) and caspase activation in postmitotic neurons (Cregan et al., 1999). The results presented here indicate that Bax is also required for p53-induced mitochondrial depolarization and the release of AIF. Interestingly, our studies on the relative kinetics of p53-induced mitochondrial events indicated that cytochrome-c release preceded the release of AIF, at least in the absence of caspase activation. Furthermore, similar to a previous report (Stefanis et al., 1999), our results suggest that cytochrome-c is released before the collapse of mitochondrial membrane potential. In contrast, the release of AIF appears to occur after, and possibly as a consequence of, mitochondrial depolarization during p53-mediated cell death. It has previously been reported that AIF can be released from the mitochondria simultaneously with, or even before, cytochrome-c during staurosporine-induced cell death of Rat-1 fibroblasts (Daugas et al., 2000; Loeffler et al., 2001). One may hypothesize that the sequence of these events may depend upon the nature of the processes leading to mitochondrial permeabilization. Because we have shown that Bax is required for the release of both cytochrome-c and AIF, the different kinetics of their release suggests that Bax mediates the release of these factors through either distinct mechanisms or the progressive action of a common process.
The precise mechanism by which Bax mediates mitochondrial membrane permeabilization and the release of apoptogenic factors remains highly controversial. However, two main hypothetical models have been proposed. In the first, Bax is proposed to oligomerize upon insertion into the mitochondria and to directly form pores within the outer membrane. This hypothesis is supported by the finding that Bcl-2 family proteins share some structural homology with the transmembrane domain of diptheria toxin and the colicins, and that Bcl-2 family proteins can form pores in artificial membranes (Muchmore et al., 1996; Antonsson et al., 2000; Saito et al., 2000). In the other model, Bax is proposed to interact with existing membrane channels and to modulate their conductivity. Accordingly, Bax has been reported to interact with the voltage-dependent anion channel and studies in yeast cells and isolated mitochondria have suggested that this interaction is required for Bax-mediated mitochondrial effects (Shimizu et al., 1999, 2000). On the other hand, other research groups have indicated that the inner mitochondrial membrane protein, adenine nucleotide translocator (ANT), is the critical Bax target (Marzo et al., 1998). It is possible that more than one of these models is correct and Bax can form different types of channels. In this case, it is conceivable that the different apoptogenic factors could be released through distinct channels. Alternatively, Bax could alter membrane permeability either by forming pores itself or by modulating existing channels, allowing the selective release of smaller molecules like cytochrome-c. This increase in membrane permeability could lead to swelling of the mitochondrial matrix and eventual lysis of the outer mitochondrial membrane (Vander Heiden et al., 1997), resulting in the release of larger apoptogenic molecules like AIF.
In summary, we have shown that p53 induces neuronal cell death through a caspase-mediated process in the presence of Apaf1, and through a caspase-independent process in the absence of Apaf1. Furthermore, we have shown that AIF is an important regulator of the caspase-independent cell death pathway and functions downstream of Bax. The fact that blocking AIF function with neutralizing antibodies provides significant protection against cell death suggests that AIF may represent an important therapeutic target for neuroprotection after acute injury.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant adenovirus infection
Total RNA was extracted from postnatal day eight mouse cortices using Trizol reagent (Invitrogen). 1 µg of total RNA was used for first strand cDNA synthesis and targeted gene amplification using Superscript One-Step RT-PCR kit (Invitrogen). cDNA synthesis was performed at 50°C for 45 min followed by a 2-min initial denaturation step at 94°C. This was followed by 37 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min using mouse-specific AIF primers: AIF forward (CCCGGGATGTTCCGGTGTGGAGG) and AIF reverse (CCCGGGTCAATCTTCATGAATG) containing Xma1 restriction sites. The resulting product was sequenced and confirmed to be AIF. Recombinant adenoviral vectors carrying human p53, Bcl-2, AIF, or LacZ expression cassettes were constructed, purified, and titered as previously described (Cregan et al., 2000). Recombinant adenoviral vectors were added to cell suspensions immediately before plating.
Cell viability assays
Neurons were infected with adenovirus at the time of plating or treated with camptothecin with or without BAF (Enzyme System Products) after 2 d in culture. Cell survival was measured by three different methods: live/dead staining, MTT assay (Cell Proliferation Kit; Promega), or TUNEL. At the times indicated, neuronal viability was determined using the Live/Dead Cytotoxicity Kit (Molecular Probes Inc.) according to the manufacturer's instructions. Representative samples were photographed using ZEISS Axiovert 100 with a Northern Eclipse Sony Power HAD 3CCD color video camera. Survival was determined as the fraction of total cells exhibiting positive staining for calcein-AM. TUNEL labeling was used to visualize cells with fragmented DNA. At the indicated times, cells were fixed in 4% paraformaldehyde for 20 min, washed in three changes of PBS, and then labeled by TUNEL, as previously described (Cregan et al., 1999), and counterstained with Hoechst 33258 (1 µg/µl) for 5 min. The fraction of TUNEL-positive cells as a percentage of total cell number was determined. For both live/dead and TUNEL assays, a minimum of 500 cells was scored for each treatment and the data represent the mean and standard deviation from three independent experiments. In certain experiments, survival was measured by colorimetric MTT assay as previously described (Cregan et al., 1999).
Antibodies, immunofluorescence staining, and cell counts
The rabbit antiserum for AIF was produced by immunizing rabbits with a mixture of synthetic peptides (coupled to keyhole limpet hemocyanin) corresponding to amino acid residues 151170 and 181200. The polyclonal antibody was purified through peptide-conjugated affinity chromatography and was found to specifically recognize both forms of AIF (67 and 57 kD), but predominately recognized the 57-kD truncated form of AIF. No other immunoreactive bands were detected with the AIF antibody. The specificities of anti-AIF antiserum and purified antibody were confirmed by the absence of 57- and 67-kD bands after preadsorption with peptides corresponding to amino acid residues 151170 and 181200 of AIF (unpublished data). Cytochrome-c and AIF immunostaining was performed as previously described (Keramaris et al., 2000). Representative fields were photographed and images were captured using a ZEISS Axioskop-II or Axiovert 100 microscope equipped with a Northern Eclipse Sony power HAD 3CCD color video camera. The fraction of cytochrome-c or AIF-positive cells was determined as the proportion of total cells exhibiting a punctate cytoplasmic staining pattern. A minimum of 400 cells was scored per treatment and data represent the mean and standard deviation from three independent experiments.
Mitochondrial membrane potential
Loss of mitochondrial membrane potential was monitored in unfixed cells using the membrane potentialdependent dye Mitotracker CMX-Ros (Molecular Probes Inc.). This fluorescent dye is selectively incorporated into mitochondria with an intact transmembrane potential and therefore serves as an indicator of mitochondrial depolarization. Cells were incubated with CMX-Ros at 0.25 µM for 30 min at 37°C, washed in fresh media, and images were captured as described above. The fraction of cells maintaining mitochondrial transmembrane potential was determined by counting CMX-Rospositive cells relative to total cell number in corresponding phase images. A minimum of 400 cells was scored per treatment and data represent the mean and standard deviation from three independent experiments.
Western blot analysis
Cells were lysed in RIPA buffer for 20 min on ice and the soluble extract was recovered by centrifugation. Extracts containing 30 µg of protein were separated on a 10% acrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked for 2 h in 5% skim milk and then incubated for 1 h with monoclonal antibodies directed against Bcl-2 (BD Transduction Labs)/cytochrome-c (BD Biosciences), or polyclonal antibodies to AIF (Dawson) and actin (Santa Cruz Biotechnology, Inc.) for standardization. Membranes were washed in TPBS (25 mM Na2HPO4, 5 mM NaH2PO4, 0.9% NaCl, 0.1% Tween-20) and then incubated for 1 h with appropriate secondary antibodies. Membranes were again washed and then developed by an enhanced chemiluminescence system according to the manufacturer's instructions (PerkinElmer).
Caspase activity assay
Cells were harvested and extracted for 15 min on ice in caspase lysis buffer, and 10 µg of protein was used for caspase activity assay as previously described (Cregan et al., 1999). Caspase activity is reported as the ratio of fluorescence output in treated samples relative to corresponding untreated controls.
Microinjection and cell death quantitation
Microinjection solution containing 3 mg/ml Alexa®488-dextran (Molecular Probes Inc.) and either AIF antiserum (Susin et al., 1999) or preimmune rabbit serum diluted in PBS was injected at 150 hPa (0.5 s) into neurons in 35-mm dishes using Femtotip needles (Eppendorf Inc.). Neurons were then treated with camptothecin (or vehicle control) and after 36 h, cells were fixed in 4% paraformaldehyde, washed in PBS, and stained with Hoechst 33258 (1 µg/ml). The extent of cell death was determined as the fraction of Alexa®488positive cells exhibiting pyknotic nuclei. Where indicated, the cells were immunostained for AIF and the fraction of microinjection-positive cells exhibiting nuclear AIF staining was determined. A minimum of 200 cells were scored per treatment and data represent the mean and standard deviation from three independent experiments.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Canadian Stroke Network (CSN) to R.S. Slack, a special grant from the Ligue Contre le Cancer to G. Kroemer, and grant NS43691 to T.M. Dawson. R.S. Slack is a CIHR Scholar, S.P. Cregan is supported by a CIHR fellowship, and A. Fortin is supported by a graduate scholarship from the CSN. F. Cecconi is an Assistant Telethon Scientist (grant 38/CP).
Submitted: 27 February 2002
Revised: 22 May 2002
Accepted: 24 June 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J.M., and S. Cory. 1998. The Bcl-2 protein family: arbiters of cell survival. Science. 281:13221326.
Banasiak, K.J., and G.G. Haddad. 1998. Hypoxia-induced apoptosis: effect of hypoxic severity and role of p53 in neuronal cell death. Brain Res. 797:295304.[CrossRef][Medline]
Cheng, Y., M. Deshmukh, A. D'Costa, J.A. Demaro, J.M. Gidday, A. Shah, Y. Sun, M.F. Jacquin, E.M. Johnson, and D.M. Holtzman. 1998. Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J. Clin. Invest. 101:19921999.
Cregan, S.P., J.G. MacLaurin, C.G. Craig, G.S. Robertson, D.W. Nicholson, D.S. Park, and R.S. Slack. 1999. Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons. J. Neurosci. 19:78607869.
Crumrine, R.C., A.L. Thomas, and P.F. Morgan. 1994. Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J. Cereb. Blood Flow Metab. 14:887891.[Medline]
Cryns, V., and J. Yuan. 1998. Proteases to die for. Genes Dev. 12:15511570.
Daugas, E., S.A. Susin, N. Zamzami, K.F. Ferri, T. Irinopoulou, N. Larochette, M.C. Prevost, B. Leber, D. Andrews, J. Penninger, and G. Kroemer. 2000. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J. 14:729739.
Eldadah, B.A., and A.I. Faden. 2000. Caspase pathways, neuronal apoptosis, and CNS injury. J. Neurotrauma. 17:811829.[Medline]
Endres, M., Z.Q. Wang, S. Namura, C. Waeber, and M.A. Moskowitz. 1997. Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase. J. Cereb. Blood Flow Metab. 17:11431151.[Medline]
Finucane, D.M., E. Bossy-Wetzel, N.J. Waterhouse, T.G. Cotter, and D.R. Green. 1999. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274:22252233.
Fortin, A., S.P. Cregan, J.G. MacLaurin, N. Kushwaha, E.S. Hickman, C.S. Thompson, A. Hakim, P.R. Albert, F. Cecconi, K. Helin, et al. 2001. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death. J. Cell Biol. 155:207216.
Goping, I.S., A. Gross, J.N. Lavoie, M. Nguyen, R. Jemmerson, K. Roth, S.J. Korsmeyer, and G.C. Shore. 1998. Regulated targeting of BAX to mitochondria. J. Cell Biol. 143:207215.
Gross, A., J.M. McDonnell, and S.J. Korsmeyer. 1999. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13:18991911.
Halterman, M.W., C.C. Miller, and H.J. Federoff. 1999. Hypoxia-inducible factor-1alpha mediates hypoxia-induced delayed neuronal death that involves p53. J. Neurosci. 19:68186824.
Hara, H., R.M. Friedlander, V. Gagliardini, C. Ayata, K. Fink, Z. Huang, M. Shimizu-Sasamata, J. Yuan, and M.A. Moskowitz. 1997. Inhibition of interleukin 1ß converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. USA. 94:20072012.
Herdegen, T., F.X. Claret, T. Kallunki, A. Martin-Villalba, C. Winter, T. Hunter, and M. Karin. 1998. Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J. Neurosci. 18:51245135.
Hu, Y., M.A. Benedict, L. Ding, and G. Nunez. 1999. Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. EMBO J. 18:35863595.
Johnson, M.D., Y. Kinoshita, H. Xiang, S. Ghatan, and R.S. Morrison. 1999. Contribution of p53-dependent caspase activation to neuronal cell death declines with neuronal maturation. J. Neurosci. 19:29963006.
Keramaris, E., L. Stefanis, J. MacLaurin, N. Harada, K. Takaku, T. Ishikawa, M.M. Taketo, G.S. Robertson, D.W. Nicholson, R.S. Slack, and D.S. Park. 2000. Involvement of caspase 3 in apoptotic death of cortical neurons evoked by DNA damage. Mol. Cell. Neurosci. 15:368379.[CrossRef][Medline]
Kuida, K., T.F. Haydar, C.Y. Kuan, Y. Gu, C. Taya, H. Karasuyama, M.S. Su, P. Rakic, and R.A. Flavell. 1998. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell. 94:325337.[Medline]
Lankiewicz, S., C. Marc Luetjens, N. Truc Bui, A.J. Krohn, M. Poppe, G.M. Cole, T.C. Saido, and J.H. Prehn. 2000. Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death. J. Biol. Chem. 275:1706417071.
Li, P., D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, and X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479489.[Medline]
Loeffler, M., E. Daugas, S.A. Susin, N. Zamzami, D. Metivier, A.L. Nieminen, G. Brothers, J.M. Penninger, and G. Kroemer. 2001. Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J. 15:758767.
Marzo, I., C. Brenner, N. Zamzami, J.M. Jurgensmeier, S.A. Susin, H.L. Vieira, M.C. Prevost, Z. Xie, S. Matsuyama, J.C. Reed, and G. Kroemer. 1998. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science. 281:20272031.
Miller, T.M., K.L. Moulder, C.M. Knudson, D.J. Creedon, M. Deshmukh, S.J. Korsmeyer, and E.M. Johnson, Jr. 1997. Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death. J. Cell Biol. 139:205217.
Morris, E.J., E. Keramaris, H.J. Rideout, R.S. Slack, N.J. Dyson, L. Stefanis, and D.S. Park. 2001. Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage. J. Neurosci. 21:50175026.
Morrison, R.S., H.J. Wenzel, Y. Kinoshita, C.A. Robbins, L.A. Donehower, and P.A. Schwartzkroin. 1996. Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death. J. Neurosci. 16:13371345.[Abstract]
Nitatori, T., N. Sato, S. Waguri, Y. Karasawa, H. Araki, K. Shibanai, E. Kominami, and Y. Uchiyama. 1995. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J. Neurosci. 15:10011011.[Abstract]
Portera-Cailliau, C., J.C. Hedreen, D.L. Price, and V.E. Koliatsos. 1995. Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci. 15:37753787.[Abstract]
Saito, M., S.J. Korsmeyer, and P.H. Schlesinger. 2000. BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat. Cell Biol. 2:553555.[CrossRef][Medline]
Shimizu, S., M. Narita, and Y. Tsujimoto. 1999. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 399:483487.[CrossRef][Medline]
Shimizu, S., T. Ide, T. Yanagida, and Y. Tsujimoto. 2000. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J. Biol. Chem. 275:1232112325.
Slack, R.S., D.J. Belliveau, M. Rosenberg, J. Atwal, H. Lochmuller, R. Aloyz, A. Haghighi, B. Lach, P. Seth, E. Cooper, and F.D. Miller. 1996. Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons. J. Cell Biol. 135:10851096.[Abstract]
Stefanis, L., D.S. Park, W.J. Friedman, and L.A. Greene. 1999. Caspase-dependent and -independent death of camptothecin-treated embryonic cortical neurons. J. Neurosci. 19:62356247.
Susin, S.A., E. Daugas, L. Ravagnan, K. Samejima, N. Zamzami, M. Loeffler, P. Costantini, K.F. Ferri, T. Irinopoulou, M.C. Prevost, et al. 2000. Two distinct pathways leading to nuclear apoptosis. J. Exp. Med. 192:571580.
Vander Heiden, M.G., N.S. Chandel, E.K. Williamson, P.T. Schumacker, and C.B. Thompson. 1997. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 91:627637.[CrossRef][Medline]
Xiang, H., D.W. Hochman, H. Saya, T. Fujiwara, P.A. Schwartzkroin, and R.S. Morrison. 1996. Evidence for p53-mediated modulation of neuronal viability. J. Neurosci. 16:67536765.
Xiang, H., Y. Kinoshita, C.M. Knudson, S.J. Korsmeyer, P.A. Schwartzkroin, and R.S. Morrison. 1998. Bax involvement in p53-mediated neuronal cell death. J. Neurosci. 18:13631373.
Yakovlev, A.G., S.M. Knoblach, L. Fan, G.B. Fox, R. Goodnight, and A.I. Faden. 1997. Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J. Neurosci. 17:74157424.