Mechanisms underlying hypoxia-induced neuronal apoptosis

Progress in Neurobiology 62 (2000) 215±249

www.elsevier.com/locate/pneurobio

Mechanisms underlying hypoxia-induced neuronal apoptosis Kenneth J. Banasiak a,*, Ying Xia b, Gabriel G. Haddad b,c a

Department of Pediatrics, Section of Critical Care, Yale University School of Medicine, New Haven, CT 06520, USA b Respiratory Medicine, Yale University School of Medicine, New Haven, CT 06520, USA c Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA Received 20 December 1999

Abstract In vivo models of cerebral hypoxia-ischemia have shown that neuronal death may occur via necrosis or apoptosis. Necrosis is, in general, a rapidly occurring form of cell death that has been attributed, in part, to alterations in ionic homeostasis. In contrast, apoptosis is a delayed form of cell death that occurs as the result of activation of a genetic program. In the past decade, we have learned considerably about the mechanisms underlying apoptotic neuronal death following cerebral hypoxiaischemia. With this growth in knowledge, we are coming to the realization that apoptosis and necrosis, although morphologically distinct, are likely part of a continuum of cell death with similar operative mechanisms. For example, following hypoxia-ischemia, excitatory amino acid release and alterations in ionic homeostasis contribute to both necrotic and apoptotic neuronal death. However, apoptosis is distinguished from necrosis in that gene activation is the predominant mechanism regulating cell survival. Following hypoxic-ischemic episodes in the brain, genes that promote as well as inhibit apoptosis are activated. It is the balance in the expression of pro- and anti-apoptotic genes that likely determines the fate of neurons exposed to hypoxia. The balance in expression of pro- and anti-apoptotic genes may also account for the regional di€erences in vulnerability to hypoxic insults. In this review, we will examine the known mechanisms underlying apoptosis in neurons exposed to hypoxia and hypoxia-ischemia. 7 2000 Elsevier Science Ltd. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Characteristics of necrosis and apoptosis . 1.2. Morphologic criteria of apoptosis . . . . . . 1.2.1. DNA fragmentation . . . . . . . . . 1.2.2. Annexin V . . . . . . . . . . . . . . . . 1.2.3. Choice of method . . . . . . . . . . .

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Excitatory amino acids and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 2.1. Glutamate and neuronal apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 2.2. Mechanisms of glutamate-induced apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

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Energy-deprivation and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Abbreviations: DNA, deoxyribonucleic acid; TUNEL, terminal deoxynucleotidyl transferase niick end labeling; NMDA, N-methyl-D-aspartate; ATP, adenosine triphosphateAMPA a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; ROS, reactive oxygen species; KCI, potassium chloride; SAP, stress-activated protein; NHE, sodium hydrogen exchanger; BDNF, brain derived growth factor; IEG, immediate early gene. * Corresponding author. Tel.: +1-203-785-4651; fax: +1-203-785-5833. E-mail address: [email protected] (K.J. Banasiak). 0301-0082/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 - 0 0 8 2 ( 0 0 ) 0 0 0 1 1 - 3

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4.

Ionic 4.1. 4.2. 4.3.

mechanisms and Calcium ions. . Potassium ions Sodium ions . .

neuronal apoptosis . ............... ............... ...............

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Neurotrophic factors and neuronal apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Role of neurotrophic factors in naturally-occurring neuronal death . . . . . . . . . . . . . 5.2. Role of neurotrophic factors in hypoxic/ischemic apoptosis . . . . . . . . . . . . . . . . . . 5.3. Mechanism of neuronal apoptosis: neurotrophic factors and receptors of TrkA and p75 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Regulation of hypoxia-induced neuronal apoptosis. . 6.1. Immediate-early genes . . . . . . . . . . . . . . . . . 6.2. Bcl-2 and related proteins . . . . . . . . . . . . . . 6.2.1. Bcl-2 and Bcl-xl . . . . . . . . . . . . . . . 6.2.2. Other Bcl-2 anti-apoptotic genes. . . . 6.2.3. Bax . . . . . . . . . . . . . . . . . . . . . . . . 6.2.4. Other Bcl-2 pro-apoptotic genes . . . . 6.3. p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Gene activation in hypoxia-induced apoptosis

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Caspases and cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Chemical properties of caspases . . . . . . . . . . . . . . . . . . . . . 7.2. Regulation of caspases. . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1. Cytokine-mediated caspase activation . . . . . . . . . . . 7.2.2. Cytotoxic caspase activation . . . . . . . . . . . . . . . . . 7.3. Role of cytokines and caspases in hypoxia-induced apoptosis

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Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

1. Introduction Over the past decade, there has been a vast and growing interest in the importance of the role of cell death in normal biological processes, e.g., development, and in pathologic conditions. In the 1970s, cell death was ®rst classi®ed, based on histologic criteria, as being necrotic or apoptotic. Since this early classi®cation, we have come to learn that these terms represent more than just morphologic descriptions but rather distinct mechanisms leading to the histologic ®ndings observed. Furthermore, we have realized that apoptosis is not necessarily a pathologic process. Thus, whereas necrotic cell death is the consequence of a pathologic event, apoptosis, in contrast, is most often a developmental event that is critical in maintaining organismal homeostasis. However, apoptosis can also be activated in pathologic conditions as well. Apoptosis serves to remove cells from an organism in an orderly, programmed fashion without inducing a major in¯ammatory response. For example, apoptosis plays an important role in the pathogenesis of a number of disease states including hypoxic-ischemic brain injury. The subject of apoptosis has increased in depth

and breadth. We have learned considerably about apoptosis in hypoxic/ischemic cell injury and death. Since some of this work has been done in the central nervous system, we focus in this review on some of the salient mechanisms of apoptosis and some of the important genes that play a role in programmed cell death. In particular, we review the underlying mechanisms that are critical to cell death in conditions of low oxygen or low oxygen and substrate delivery in the central nervous system. 1.1. Characteristics of necrosis and apoptosis Necrosis is, in general, a form of cell death that rapidly occurs in response to severe insults such as anoxia and cell trauma. Typically, necrosis has been associated with alterations in calcium and sodium ion homeostasis. In contrast, apoptosis is a delayed form of cell death that is the result of less severe insults and is associated with activation of a ``genetic program''. To help investigators distinguish necrosis from apoptosis, a number of criteria have been established that will be discussed below. It is important to recognize that this ``classical'' concept of cell death may not be com-

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pletely accurate. Although necrosis and apoptosis are morphologically distinct, it is becoming more evident that necrosis and apoptosis are likely part of a continuum of cell death and similar mechanisms may be operative in both processes. 1.2. Morphologic criteria of apoptosis The earliest morphologic changes in cells undergoing necrosis include cellular and organellar swelling, especially the mitochondria (Trump et al., 1982). Subsequently, there is dissolution of organelles, rupture of the plasma membrane with leakage of cellular contents into the extracellular space, and random DNA degradation following histone proteolysis. Necrosis usually a€ects large groups of adjacent cells and is associated with in¯ammatory reaction. In contrast, cells undergoing apoptosis exhibit shrinkage of the cytoplasm and condensation of nuclear material into ``clumps'' (Wyllie, 1981; Walker et al., 1988). As the process continues, the nucleus undergoes fragmentation and the endoplasmic reticulum fuses with the plasma membrane forming vesicles and convoluting the surface of the plasma membrane. In the ®nal stages of apoptosis, there is cellular fragmentation forming membrane-bound apoptotic bodies that contain intact cytoplasmic organelles and nuclear fragments. There are several essential gross morphologic features distinguishing necrosis from apoptosis. First, apoptotic cells exhibit loss of cytoplasm while necrotic cells have cytoplasmic swelling. Second, nuclear changes precede plasma membrane changes in apoptosis while the reverse is observed during necrosis. Third, apoptotic cells maintain plasma membrane integrity while necrotic cells undergo plasma membrane rupture. Because of the ability to clearly resolve these relatively unequivocal morphologic di€erences, electron microscopy is much preferred over light microscopy for distinguishing apoptotic from necrotic cells. 1.2.1. DNA fragmentation In necrotic cells, DNA undergoes random fragmentation following histone proteolysis. Because histones remain intact in apoptotic cells, DNA is cleaved into multimers of 180±240 base pairs (Wyllie, 1980). In an attempt to use this event as a means of distinguishing necrotic from apoptotic cells, techniques for gel electrophoresis of genomic DNA and in situ labeling of DNA fragments were developed. Typically, genomic DNA from necrotic cells exhibits a single vertical band on electrophoresis (``smear pattern''). In contrast, genomic DNA will exhibit a ``ladder pattern'' on electrophoresis. Because of potential technical diculties, especially with DNA band resol-

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ution, it is generally dicult to di€erentiate these two patterns. In situ DNA fragmentation can be detected by one of two methods. The ®rst is terminal deoxynucleotidyl transferase nick end labeling (TUNEL) that involves binding of dUTP tagged with a chromagen or ¯uorophore to the 3 '-OH ends of single or double-stranded DNA (Gavrieli et al., 1992) The alternative method, in situ end labeling, involves the addition of labeled nucleotides to the 3 ' ends of oligonucleosomes with 5' overhangs that serve as a template for DNA polymerase I. Although these methods are quite sensitive for detection of in situ DNA fragmentation, they cannot di€erentiate necrotic and apoptotic cells.

1.2.2. Annexin V During the early phases of apoptosis, phosphatidylserine is translocated from the inner surface to the outer surface of the plasma membrane (Vermes et al., 1995). It is believed that this translocation is a phylogenetically conserved process that serves as a marker for phagocyte removal of cells (Meers and Mealy, 1993; Vermes et al., 1995). The calcium-dependent phospholipid binding protein, annexin V, can bind to phosphatidylserine after its translocation to the external surface of the cell (Vermes et al., 1995). The use of ¯uorophore-tagged annexin and a membrane-excluded dye, such as propidium iodide, provides a method of detecting apoptotic cells. Apoptotic cells are typically identi®ed by plasma membrane binding of the tagged annexin V and exclusion of propidium iodide. Controversy exists as to the interpretation of cells exhibiting both annexin V plasma membrane binding and DNA binding of propidium iodide. This pattern of staining could either represent loss of plasma membrane integrity in necrotic cells or cells in the latter stages of apoptosis.

1.2.3. Choice of method In situ DNA labeling and annexin V staining are unable to unequivocally distinguish apoptosis from necrosis. Because of its high resolution of the ultrastructure of cells, electron microscopy is the generally accepted ``gold standard'' for determining the presence of apoptosis. However, electron microscopy does not readily lend itself as a useful application for quantitative studies of cell death since investigators face a statistical sampling problem. Based on our experience, we believe that when performing quantitative apoptotic studies, electron microscopy should be used to determine the presence of apoptosis in conjunction with other methods (e.g., TUNEL, Annexin V) to quantitate apoptotic cells.

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2. Excitatory amino acids and apoptosis One of the commonly used models in vivo or in vitro to further our understanding of O2 deprivation is that involving the use of excitatory amino acids such as glutamate and its receptor agonists. The idea is that glutamate and its agonists, such as NMDA agonists, mimic the e€ect of hypoxia or ischemia on neurons. This idea gained popularity since glutamate is well known to be released in high quantity at nerve terminals during hypoxia. However, it should be noted that, on the one hand, hypoxia in¯uences many di€erent cellular processes that may be di€erent from those a€ected by glutamate. Hence, it is of no surprise that when neurons are exposed to hypoxia, their response in terms of intracellular Ca2+ homeostasis and morphology is very di€erent from those that take place after glutamate exposure, as previously illustrated by work from our laboratory and those of others (Chihab et al., 1998; Chow and Haddad, 1998). On the other hand, it should be clear that the importance of the glutamate model is not only related to models of hypoxia and ischemia but also to those that stem from disease processes that are presumably based on genetic pathophysiologies or those that do pertain to redox or oxidative states (Dubinsky, 1995). In this section, we will focus on glutamate-induced cell death and detail some of the other potential model systems for the sake of comparison. 2.1. Glutamate and neuronal apoptosis Some of the important ideas that, in a sense, delayed our understanding of the long term e€ects of glutamate on neuronal cell death and cell survival were related to the highly popular observations that neurons die when exposed acutely to large amounts of glutamate, as neurons are when deprived of O2 and nutrients (Choi, 1987). Clearly, by the nature of these experiments, long-term e€ects could not be easily appreciated. It was not until the acute e€ects of glutamate were blocked that the delayed cell death that could be induced by glutamate was discovered (Gwag et al., 1995; Choi, 1996; Figiel and Kaczmarek, 1997; Portera-Cailliau et al., 1997; Nicotera et al., 1999). It is the ``peeling'' of one set of mechanisms that made it possible to more fully appreciate the extent of glutamate excito-toxicity! In the past 3±4 years, it has become clear that moderate glutamate exposure over prolonged periods can lead to apoptotic cell death in neurons. This has been associated with a recovery in mitochondrial energy function, unlike neurons dying acutely from massive glutamate exposure in which ATP generation is eliminated and mitochondrial function non-existent (Ankarcrona et al., 1995). Of particular interest also are data

showing that nuclear disintegration and DNA single strand-breaks are early events in granular cell apoptosis that precede any recognizable morphologic changes in these neurons (Didier et al., 1996; Ikeda et al., 1996). This has been clearly shown in culture and evidence of apoptotic changes has been observed not only after glutamate exposure but also after selective AMPA receptor stimulation (Larm et al., 1997). NMDA receptor stimulation can also be part of the glutamate-induced apoptosis and the desensitization of NMDA receptors is neuro-protective against apoptosis that can occur with glutamate (Wood and Bristow, 1998). Furthermore, it has been suggested that lithium protects against apoptosis by blocking NMDAmediated Ca2+ ¯ux into neurons (Nonaka et al., 1998). Recently, however, another interesting observation, seemingly opposite to previous observations, was published: blockade of NMDA receptors led to apoptosis and neuro-degeneration in fetal and neonatal rat brain (Ikonomidou et al., 1999). This is suggestive of the concept that the concentration of glutamate or glutamate receptor agonists is crucial in bringing about a survival or an injury signal whether apoptotic or necrotic in nature. Therefore, it is possible that a certain amount of glutamate ``protects'' against apoptosis and its removal is deleterious and large amounts of glutamate may also be injurious to nerve cells, with a delicate balance being optimal for neuronal growth and integrity. Whether this type of injury resulting from excess glutamate is similar to other types of apoptotic cell death in terms of mechanisms (e.g. apoptosis by serum deprivation) is not understood at present (MacManus et al., 1997; Manev et al., 1997; Piantadosi et al., 1997; Ohgoh et al., 1998). 2.2. Mechanisms of glutamate-induced apoptosis There are various mechanisms and biochemical events that have been implicated in the glutamateinduced apoptotic changes. In the past 2±3 years, it has been shown that the mitochondria is central in glutamate-induced cell death (Montal, 1998). For example, calcineurin inhibition, a calcium-calmodulinregulated phosphatase, prevented the collapse of mitochondrial membrane potential and apoptotic cell death (Ankarcrona et al., 1996; Wang et al., 1999a, 1999b). The way calcineurin works is that it dephosphorylates BAD, a pro-apoptotic gene product, which is then translocated to the mitochondria to dimerize with Bclxl, a process that initiates apoptosis (Fig. 1; Wang et al., 1999a, 1999b). It is this dimerization that induces the release of cytochrome c from mitochondria and, along with Apaf-1 and pro-proteases (see below), proteases are formed and lead the way to cell death by apoptosis (Neame et al., 1998). Whereas BAD can initiate apoptosis, it is well recog-

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nized now that the e€ector molecules are proteases, serine and cysteine proteases. The latter are termed caspases and have been extensively studied and found to be important in mediating apoptotic events (Du et al., 1997; Gottron et al., 1997; Leist et al., 1997; Tenneti et al., 1998). More recently, caspase-3, one of the major ones, has been linked to cell death induced by glutamate and its receptors and glucose/O2 deprivation (Du et al., 1997; Gottron et al., 1997; Leist et al., 1997; Tenneti et al., 1998). The transcription factor AP-1 and c-Jun activation by its kinase Jnk, especially Jnk-3, which is selectively expressed in the CNS, is also critical as a Jnk-3 knock-out protected mice from excito-toxic induced apoptosis (Tabuchi et al., 1996; Yang et al., 1997a, 1997b; Cheung et al., 1998). Glutamate-induced apoptosis also can activate another pathway, that of p53, a tumor suppressor gene, that can lead to cell death by activating Bax-related pathway in neurons (Fig. 1; Uberti et al., 1998; Xiang et al., 1998). Hence, glutamate can lead to apoptosis by activating a number of processes that possibly converge into caspase activation. While it is not clear yet as to what are the gene products that constitute all the targets for caspases, it is clear now, from the preceding discussion, that caspases play a very important role in activating cell death by apoptosis when neurons are exposed to increased levels of glutamate or hypoxia/ ischemia. Reactive oxygen species (ROS) have also been well

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known to induce cell death by either necrosis or apoptosis (Tan et al., 1998; Carmody et al., 1999; Davis and Johnson, 1999). Antioxidants that scavenge ROS inhibit apoptosis in a number of diseases and pathophysiological conditions (Tan et al., 1998; Carmody et al., 1999; Davis and Johnson, 1999). Examples abound for neurologic diseases such as in Alzheimer's disease, Parkinson's disease and retinitis pigmentosa and photoreceptor degeneration (Merad-Boudia et al., 1998). One interesting recent study has demonstrated also that, while Ca2+ in¯ux and collapse of the mitochondrial membrane potential are early events (Nicotera and Rossi, 1994; Takei and Endo, 1994; Wei et al., 1998; Mason et al., 1999), preceding caspase activation, ROS formation and lipid peroxidation are likely downstream events of caspase activation and may be targets of caspase action. 3. Energy-deprivation and apoptosis Oxygen deprivation will lead naturally to energy deprivation and the question that arises is whether energy metabolism is linked to apoptotic cell death. While the mitochondrion is certainly central to apoptosis, ATP and energy metabolism is not necessarily and clearly involved. Furthermore, if metabolism is involved, how is it and whether this involvement is direct through ATP or not is not clearly shown in

Fig. 1. Mechanisms of glutamate-induced apoptosis. Activation of calcineurin, a calcium-calmodulin-regulated phosphatase, by glutamate receptor activation, allows calineurin to dephosphorylate bad. The dephosphorylated form of bad is then translocated to the mitochondrial membrane, where it binds to either bcl-xl or bcl-2. Formation of these heterodimers induces release of cytochrome c from the mitochondria, leading to caspase activation. Alternatively, glutamate can activate bax in a p53-mediated process. With increased protein levels, bax homodimers are formed and contribute to protease activation.

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neurons. There are observations in the literature, actually, that show that ATP levels may be at control levels during apoptosis (Davis and Johnson, 1999). Of interest also is that phosphate incorporation into ATP and the Na+-dependent phosphate entry into cells is markedly decreased in cells (Davis and Johnson, 1999). However, phosphorylation of certain proteins can certainly be increased during apoptosis. This generalized decrease in ATP phosphate incorporation and transport in the presence of increased selective phosphorylation is consistent with the concept that we put forward previously regarding the presence of a cellular ``hierarchy'' which determines the ``use'' or distribution of a variety of moieties, including amino acids and phosphate moieties (Ma and Haddad, 1997).

4. Ionic mechanisms and neuronal apoptosis Although intracellular calcium ions, Ca2+ i , have been known to be associated with intracellular signaling, with activation of a variety of enzymes and with gene activation (Nicotera et al., 1994; Santella and Carafoli, 1997), other ions such as Na+ and K+ have not until recently. In addition, more work has been done fairly recently on intranuclear Ca2+ (Santella and Carafoli, 1997; Scotto et al., 1998) and its relation with apoptotic and anti-apoptotic genes. Below, we detail some of the newer studies on the role potentially played by Ca2+, K+ and Na+ ions in activating or inactivating genetic programs. 4.1. Calcium ions has long been known to be involved in neurCa2+ i onal cell injury, mostly due to the discoveries over the past two decades related to glutamate, its e€ects on neuronal cell death and its simultaneous e€ect on Cai2‡ regulation. In the past 4±5 years, however, Cai2‡ has been demonstrated to be associated with apoptotic cell injury. For example, Ca2+ ionophores (e.g., ionomycin) induce ultra-structural changes of cell body shrinkage, compaction of the nucleus with chromatin condensation and DNA fragmentation that are consistent with an apoptotic process in rat cortical neurons (Takei and Endo, 1994; Wei et al., 1998; Mason et al., 1999). Furthermore, in this model, apoptosis is associated with transcription and translation, since RNA and protein synthesis inhibitors eliminate cell death. Presumably, Ca2+ is entering from the outside to inside the cell with ionomycin. This work on cortical neurons was also recently supported by work on cerebellar granular cells in which dihydropyridine Ca2+ channel blockers (e.g. amlodipine) may have a neuroprotective e€ect by the attenuation of Cai2‡ induced by

KCl depolarization (Takei and Endo, 1994; Wei et al., 1998; Mason et al., 1999). The role of Cai2‡ does not have to be related to the in¯ux of Ca2+ from the outside of cells. Indeed, the overexpression of bcl-2, an anti-apoptotic oncogene, protects against the e€ect of thapsigargin-induced apoptosis (Takei and Endo, 1994; Wei et al., 1998; Mason et al., 1999). Although thapsigargin increases cytosolic Ca2+, bcl-2 did not alter Ca2+, thus casting some doubt on the idea that bcl-2 protects neurons by altering Ca2+ regulation. While an increase in Cai2‡ may be involved in inducing an apoptotic process, the events that lead to cell death are far from being understood. A number of possible Ca2+ involvements have been considered. For instance, the release of caspase-9 from the mitochondria and its accumulation in the cell nucleus with subsequent apoptosis, a bcl-2 inhibitable process, can be triggered by extra-mitochondrial Ca2+ (Walker et al., 1994a, 1994b; Juin et al., 1998; Krajewski et al., 1999). Interestingly, bax has a similar e€ect as Ca2+ in this model system and in activating caspase-9. Another cytosolic e€ect of Ca2+ has recently been shown to be linked to the activation of caspase-3 and that the inhibitors of the latter obliterate the e€ect of Ca2+ in a cell-free model (Walker et al., 1994a, 1994b; Juin et al., 1998; Krajewski et al., 1999). Other possible pathways for the role of Ca2+ involve intranuclear levels and the potential interactions of Ca2+ with DNA and genes. Indeed, intranuclear Ca2+ ¯uctuations have been shown to a€ect chromatin organization, induction of genes and the activation of DNA cleavage by the activation of endonucleases (Walker et al., 1994a, 1994b; Juin et al., 1998; Krajewski et al., 1999). These types of studies have highlighted the importance of the regulation of ionic ¯ux through the nuclear envelope since these control mechanisms will have major implications on Ca2+ or other ionic transport to the nucleus. Ca2+ pumps, Ca2+ channels and inositol-triphosphate channels have been found in the nuclear envelope and most likely participate in Ca2+ homeostasis across the nucleus. Although a number of biochemical reactions take place in the nucleus involving Ca2+, some of these are dependent on calmodulin-dependent kinases and phosphatases. Independent of these calmodulin-dependent processes, apoptosis can take place and the role of Ca2+ in the nucleus does not not need to involve calmodulin (Takei and Endo, 1994; Walker et al., 1994a; Walker et al., 1994b; Juin et al., 1998; Wei et al., 1998; Krajewski et al., 1999; Mason et al., 1999). 4.2. Potassium ions A relatively newer area of research is the relation between K+ channels and apoptosis or proliferation of

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cells. One of the earliest works was performed on astrocytoma cell lines (Chin et al., 1997). Blockers (e.g. 4-aminopyridine) of speci®c K+ channels, such as an outward recti®er, stopped the proliferation of this cell line and induced apoptosis. These investigators were not successful with other blockers such as charybdotoxin, which generally blocks the Ca2+-sensitive, large and voltage-dependent K+ channel (Chin et al., 1997). In a di€erent model system, that of apoptosis induced by serum deprivation or by staurosporine, Choi and colleagues also found out that K+ channels are causally linked to apoptosis (Yu et al., 1997), albeit their results were not consonant with those in the astrocytoma lines. In brief, these studies showed an enhancement, rather than an inhibition, of an outward K+ current with serum deprivation, which led to apoptosis. An increase in extracellular K+, which should lower the driving force for K+ e‚ux, or tetraethylammonium, which blocks K+ channels, showed attenuation of apoptosis (Yu et al., 1997). Two points can be made regarding these studies: (a) It is not clear whether the di€erence obtained is related to the models of apoptosis or whether it is related to the nature of the tissue used; and (b) whether multiple K+ channels can participate di€erently in apoptosis depending on the environmental conditions. More recently, the same group of investigators has shown that the outward K+ current that ensues from Nmethyl-D-aspartate receptor activation plays a role in inducing apoptotic changes in hippocampal neurons in culture (Yu et al., 1999). If K+ channels are involved in a cascade of events that lead to apoptosis, do we know about these events in cells undergoing apoptosis? This cascade is poorly understood at present. However, there is some information related to this question. For example, the nonreceptor tyrosine kinases, the Src family, seem to have as a target the activation of the delayed recti®er K+ channels (Sobko et al., 1998), channels that have been incriminated in inducing apoptosis and in curbing proliferation when they are opened in neuronal culture. The importance of the link between the Src family and the delayed recti®er K+ channels is that this work is related to the onset of myelination and proliferation of Schwann cells. Hence, the inhibition of the Src family and the block of these channels may be relevant to the initiation of myelination. Another possible pathway is that of growth factors and cytokines which have K+ channels as one of their important downstream e€ectors. In cultured oligodendrocytes, ceramide, a lipid mediator induced by cytokines and which can by itself lead to apoptosis, depolarizes these cells by blocking inward recti®er K+ channels via a Ras or a raf-1 pathway. Therefore, it is likely that in these cells, upon the release of pro-in¯ammatory cytokines, ceramide activates a phos-

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phorylation process, which blocks these K+ channels and leads to apoptosis. In this scenario, a block in K+ channels leads to apoptosis (Hida et al., 1998). What is not clear from this work is whether the depolarization is needed to reach apoptosis in oligodendrocytes. Other workers in the ®eld have studied this question from a di€erent angle. Since (a) gene products like reaper, grim and hid have been shown to induce apoptosis in Drosophila (Chen et al., 1996a, 1996b; Avdonin et al., 1998; Wing et al., 1998) and (b) these proteins have an N-terminus that has some homology to the Nterminal portion of K+ channels that plays an important role in K+ channels inactivation, these were expressed in oocytes and examined for the inactivation of K+ channels. They found that both reaper and grim do inhibit these particular channels, but hid did not (Chen et al., 1996a, 1996b; Avdonin et al., 1998; Thress et al., 1998; Vucic et al., 1998; Wing et al., 1998). There are several potential problems with these studies. First, if these are pro-apoptotic genes, one could ask as to why is it that one of the three gene products tested (i.e., hid) does not inhibit a process, like the other two products? Second, the rationale for testing these proteins is not very convincing since the homology is poor and this homology is in the ®rst 6 amino-acid region of the DNA which does not confer inactivation of the K+ channels. Third, in this model, these gene products inhibit K+ channels and do not enhance their activity, unlike other intermediates of apoptosis in which K+ channel activation leads to cell death. Furthermore, what is unclear at present is the sequence of events that is induced by these gene products and whether K+ channels are in that sequence. Also, since reaper induces the release of cytochrome c and activates caspases, the relation between K+ channels and caspases or mitochondrial function is not well understood. Of interest are recent reports demonstrating that K+ channel activation is of functional importance in apoptosis of disease states. For example, these K+ channels were shown to be involved in linking beta-amyloid of Alzheimer's disease and the induced apoptosis in neocortical neurons (Colom et al., 1998; Yu et al., 1998). These studies used in-vitro models and showed that the delayed recti®er K+ channel activation was central to the induction of apoptotic cell death. Other K+ channels were not involved in amyloid-induced apoptosis and the delayed recti®er K+ channels were not involved in cell death induced by other means. Furthermore, K+ channel activation seems also to mediate cell death of myeloblastic leukemia cells when exposed to U-V irradiation (Wang et al., 1999a, Wang et al., 1999b). The activation of K+ channels in this latter model were important for the activation of speci®c kinases, such as c-Jun kinase and SAP kinase, which were central to the induction of apoptosis; and the in-

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hibition of K+ channels prevented the activation of these kinases. Also worthy of investigation is the relation of hypoxia-induced K+ channel activation or inactivation and their relation to apoptosis. 4.3. Sodium ions While a number of investigations have focused their attention on the importance of Cai2‡ in cell death in general, little attention has been paid to the role of Na+ in neuronal injury, let alone apoptosis. Several years ago, it became clear to us that intracellular Na+ …Na‡ i † increases during O2 deprivation (Friedman and Haddad, 1994a, 1994b) and played an important role in the acute depolarization that is induced by hypoxia (Haddad and Jiang, 1993). Furthermore, preventing the increase in Cai2‡ during hypoxia had no e€ect on cell injury and cell death (Friedman and Haddad, 1993). Therefore, there were two important questions at that juncture: (a) Does Na‡ i play a role in cell injury during O2 deprivation and (b) more speci®cally, does Na‡ i play a role in inducing apoptosis when the hypoxic stress is prolonged? Both of these questions are not resolved at this time but there is increasing evidence that Na‡ i plays an important role in cell death. We will mostly address the second question in this section. However, there are very few studies on neurons and most investigations in this area are performed on non-neuronal cells. Recent work on apoptosis in lymphocytes has shown that, as has been the case for other cells, apoptosis is associated with cell shrinkage and indeed this is one morphologic criterion often used for separating apoptosis from necrosis (Bortner et al., 1997). What is interesting in this study was that the cell shrinkage seen was associated with a loss of ionic strength and loss of both intracellular Na+ and K+ measured by ¯ow cytometry and optically (Bortner et al., 1997). This study did not address, however, the importance of each individual ion per se and did not consider the role of Na+ separately (Bortner et al., 1997). Very recently, the ratio of Na+/K+ was investigated in vascular smooth muscles (Orlov et al., 1999). In a model of apoptosis induced by serum deprivation or staurosporine, changing the ratio of Na+/K+ from a low one to a higher one (with the use of ouabain) prevented the occurrence of apoptosis. Activation of caspase-3 and the fragmentation of chromatin, which were associated with this model, did not occur when ouabain was used to increase the ratio of Na+/K+ and inhibit apoptosis. Hence, at rest in these smooth muscle cells, the ratio of these ions is such that cells are primed to undergo apoptosis if the appropriate triggers are present. Clearly, there are a number of membrane proteins that control ionic ¯ux at rest and during O2 depri-

vation. In the recent past, there has been some work examining the importance of some of these proteins in cell death. For example, in experiments on smooth muscle lines that are de®cient in Na/H exchanger (NHE), it was found that these cells do not grow and proliferate in HCO3ÿ -free solutions but they do grow when they were transfected back with the exchanger (Takahashi et al., 1995). In addition, inhibition of the exchanger with the HOE694 blocker also inhibited cell growth (Takahashi et al., 1995). It is of interest, proliferation of pulmonary vascular smooth cells during hypoxia is dependent on NHE (Takahashi et al., 1995). Of major relevance to this chapter is work that has been recently performed during ischemia on cardiac myocytes (Chakrabarti et al., 1997). Apoptosis in such cells (assayed by the TUNEL method) resulting from ischemia was inhibited with the use of HOE642, another inhibitor of NHE. These results on cardiac myocytes contrast, therefore, with results of other studies noted above and it is not clear at present what the role of NHE is in cell death at rest and during stress, and in particular during hypoxia and ischemia. Other studies (Perez-Sala et al., 1995; Reynolds et al., 1996; Furlong et al., 1997; Schlesinger et al., 1997; Shimizu et al., 1998; Park et al., 1999) have focused on pHi regulation and the relation between pHi and apoptosis. It seems that a decrease in pHi precedes caspase activation and that an attenuation of this decrease in pHi can reduce apoptosis and caspase activity (Furlong et al., 1997). Although there are a number of mechanisms and transporters that can a€ect pHi regulation, an important one is NHE. How NHE relates to apoptosis is not clear except that the inhibition of NHE by amiloride derivatives can block the protective e€ect of certain cytokines (IL-3, and GMCSF) in cell lines in which early intracellular acidi®cation precedes apoptosis. Similarly, intracellular alkalinization via increased activity of NHE in a leukemic cell line can suppress apoptosis (Perez-Sala et al., 1995). The inhibition of apoptosis has been shown in this model to be related to the inactivation of endonucleases responsible for DNA degradation (Perez-Sala et al., 1995).

5. Neurotrophic factors and neuronal apoptosis Neurotrophic factors include a large family of target-derived proteins that play an important role in the regulation of neuronal life and death. This is true for both the central and peripheral nervous systems, especially during brain development and under pathologic conditions such as hypoxia-ischemia.

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5.1. Role of neurotrophic factors in naturally-occurring neuronal death A previously held view is that naturally occurring neuronal death depends simply on the absence of appropriate survival signals such as neurotrophic factors. In the past decade, numerous neurotrophic factors and their mechanisms of action have been elucidated and this has greatly improved our understanding of the role of neurotrophic factors in neuronal survival and death. Current literature has shown that, on the one hand, neuronal survival depends on neurotrophic factors; insucient amounts of such neurotrophic factors can lead to cell death. For example, nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) are essential for survival of sympathetic neurons during development (Levi-Montalcini and Booker 1960; Li and Bernd, 1999; LeviMontalcini and Angeletti, 1966; Chun and Patterson, 1977; Johnson et al., 1983; Lindsay, 1988; Martin et al., 1988; Ruit et al., 1990; Snider, 1994; Belliveau et al., 1997; Bamji et al., 1998; Sondell et al., 1999). On the other hand, some of these neurotrophic factors such as BDNF (Bamji et al., 1998) can mediate apoptotic signals and thus facilitate neuronal apoptosis. Therefore, naturally occurring neuronal death is determined by the balance of survival and apoptotic signals elicited by a variety of trophic factors (Bamji et al., 1998; Behrens et al., 1999). 5.2. Role of neurotrophic factors in hypoxic/ischemic apoptosis Several lines of evidence have suggested that neurotrophic factors are up-regulated in the nervous system and are involved in hypoxic/ischemic cascades (Kiyota et al., 1991; Boniece and Wagner, 1993; Cheng and Mattson, 1991; Zhang et al., 1993; Mattson and Sche€, 1994; Pechan, 1995; Sza¯arski et al., 1995; Bossenmeyer-Pourie et al., 1999; DeCoster et al., 1999; Rong et al., 1999). In response to hypoxic-ischemic stress, neurotrophic factors may regulate neuronal survival and death in two ways: (a) direct participation in pathophysiological cascades and neuronal apoptosis and (b) activation of inhibitory mechanisms of neuronal apoptosis. For example, there is evidence showing that BDNF expression increases in adult rat forebrain following limbic seizures (Cheng and Mattson, 1994; Mitchell et al., 1999; Nitta et al., 1999). Since BDNF may activate apoptotic signals, it is possible that the increased BDNF expression is one of the mechanisms underlying epileptic neuronal loss. Indeed, a number of neurotrophic factors have shown a protective role in neuronal apoptosis under pathological conditions. For instance, delivery of ciliary neurotrophic factors (CNTF) into the immediate vicinity of damaged neur-

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ons or directly on cut axonal ends can increase neuronal survival (Sendtner et al., 1991; Hagg and Varon, 1993). Other experiments showed that NGF, CNTF, and glial growth factor block apoptosis in neuronal and glial supporting cells of the central and peripheral nervous system (Louis, 1993; Grombacher, 1995; Trachtenberg, 1996). 5.3. Mechanism of neuronal apoptosis: neurotrophic factors and receptors of TrkA and p75 The e€ects of various neurotrophic factors are mainly mediated by two di€erent cell surface receptors: TrkA and p75. The former mediates a neuronal survival signal and the latter, an apoptotic signal. TrkA is a member of the TrK family of receptors. Binding to TrkA receptors is essential in neurotrophic factor-mediated neuronal survival. A number of studies have shown that NGF binding to TrkA alone is sucient to mediate many of the prototypic biological responses elicited by NGF, including neuronal survival and growth (Ibanez et al., 1992). Other experiments have demonstrated that NT-3 and NT-4 activate TrkA receptors to mediate neuronal survival, albeit less e€ectively than NGF (Belliveau et al., 1997). In contrast, BDNF, which does not elicit neuronal survival, does not activate Trk receptors. These data strongly suggest that activation of TrkA receptors is an important cellular process for neurotrophic factors to promote neuronal survival and suppress neuronal apoptosis. The p75 (p75NTR) is a receptor which can interact with most mammalian neurotrophic factors (Johnson et al., 1986; Levi-Montalcini, 1987; Radeke et al., 1987; Barde, 1989; Snider, 1994; Casaccia-Bonne®l et al., 1999; Roux et al., 1999). The p75NTR signal mediates apoptosis. When neurotrophic factors bind to p75NTR, a series of cellular processes are stimulated such as generation of ceramide, activation and translocation of NF-kB to the nucleus and enhancement of Jun kinase (Jnk) activity which lead to neuronal apoptosis. In BDNFÿ/ÿ mice, neuronal number is larger as compared to BDNF+/+ littermates (Bamji et al., 1998). In p75NTRÿ/ÿ mice, the developmental period that normally precedes neuronal death and which occurs between birth and postnatal day 22, does not take place. In addition, cultured p75NTRÿ/ÿ neurons died much more slowly than their wild type counterparts in the absence of NGF. Therefore, p75NTR receptor activation seems to be essential for neuronal apoptosis. It is important to note that (a) most neurotrophic factors can interact with both TrkA and p75NTR receptors and (b) both TrkA and p75NTR receptors are important for the optimal development of the CNS. For example, during neuronal development, target-derived NGF may, in conjunction with other neurotrophic factors, activate TrkA and p75NTR

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receptors to stimulate terminal growth and eliminate neurons that fail to sequester adequate target territory. In summary, neurotrophic factors are actively involved in neuronal survival and apoptosis during brain development and with environment stress. Their survival and apoptotic signals are mediated mainly by TrkA and p75NTR receptors, respectively. The balance of these two signaling pathways dictate neuronal survival or apoptosis. Since most neurotrophic factors can interact with both types of receptors, the net outcome of neurotrophic stimulation depends on multiple factors including neuronal status, the level of neurotrophic agents, the ratio of neurotrophic factors, developmental stage, and environmental conditions (e.g., hypoxic severity and duration). 6. Regulation of hypoxia-induced neuronal apoptosis In this section, we ®rst present some of our understanding of the genes important in regulating apoptotic cell death. We then present a summary of the role of theses genes in hypoxia-induced apoptotic cell death in neurons. In some instances, we will use non-neuronal examples to illustrate the role of some of these genes in order to strengthen the discussion of the issues raised. 6.1. Immediate-early genes The immediate early genes (IEGs) represent a family of over 100 genes that are induced within minutes to hours following a variety of stimuli. These are termed ``immediate-early'' because they are induced in the absence of de novo protein synthesis. The IEGs code for a variety of proteins including secreted proteins (e.g., cytokines), cytoplasmic enzymes (e.g., Cox-2), ligand-dependent transcription factors (e.g., NGFI-B), and inducible transcription factors (e.g., jun, fos) (Herdegen and Leah, 1998). Because most current literature on immediate-early gene expression following hypoxiaischemia focuses on the inducible transcription factors, the following discussion will center on the functions of this particular subfamily of IEGs. The immediate early genes, c-fos, fosB, c-jun, junD, krox-20, krox-24, NGFI-B Nurr77/TIS1, and NGFI-C are oncogenes and related genes that produce nuclear proteins functioning as transcriptional activators of other genes. The exception is junB, which is ine€ective in activating other promoters and likely functions as a negative regulator of DNA binding and transcriptional activation by c-fos, c-jun and junD (Chiu et al., 1988, 1989; Schutte et al., 1989; Ryseck and Bravo, 1991; Baker et al., 1992; Nikolakaki et al., 1993). The transcriptional activity of IEGs is regulated by phosphorylation by a number of protein kinases. For example,

phosphorylation of the C-terminus by either casein kinase II or glycogen synthase kinase-3 inhibits c-jun DNA binding and transcriptional activity (Lin et al., 1992). In contrast, phosphorylation of c-jun and junD by the stress activated protein kinases (SAPKs; also known as jun kinases or JNKs) enhances transcriptional activity forming a unique signal transduction pathway important in the genesis of apoptosis (Hibi et al., 1993; Derijard et al., 1994; Kyriakis et al., 1994; Minden et al., 1994; Herdegen and Leah, 1998). The IEG proteins not only function directly as transcriptional activators but also form heterodimers altering the DNA binding properties and transcriptional activity of a number of proteins. Heterodimerization of c-jun with c-fos and the binding of the resulting dimer to the DNA consensus sequence, TGACTCA, forms the AP-1 transition complex that functions as a transcriptional activator (Angel et al., 1987; Chui et al., 1988; Sassone-Corsi et al., 1988; Sonnenberg et al., 1989). AP-1 DNA-binding is enhanced in response to apoptotic stimuli but its function remains unclear. Cfos and c-jun also enhance transcription of a variety of other transcription factors. C-jun, fra-1, and fra-2 function to repress transcription factor activity through inhibition of DNA binding or inhibition of transcription factor activation (Metz et al., 1994). JunD is believed to enhance transcription factor activity by preventing degradation of its partner protein (Kovary and Bravo, 1991; Herdegen and Leah, 1998). Rapid induction and transcriptional activation of IEGs occur in response to phenomena observed following hypoxic-ischemic brain injury including electrical depolarization, increased intracellular calcium, excitatory amino acid exposure, and oxygen free radical exposure (Greenberg et al., 1985, 1986; Curran and Morgan, 1986; Morgan and Curran, 1986; Sagar, 1988; Sheng et al., 1988). Following an hypoxicischemic insult, immediate early gene expression occurs in two phases. In the ®rst phase, occurring within 24 h of the insult, there is an increase in c-fos, fosB, c-jun, junB, junD, NGFI-A, and krox24 and increased AP-1 binding activity in the infarct and surrounding regions (Dragunow et al., 1994; Hughes et al., 1998; Walton et al., 1999). In the second phase, occurring 24±48 h following the injury, there is a persistent elevation of fosB, c-jun, NGFI-A, and NGFI-C and a decrease in krox24 (Dragunow et al., 1993, 1994; Hughes et al., 1998; Walton et al., 1999). The IEGs induced following hypoxia-ischemia may transcriptionally activate a number of genes encoding structural proteins, enzymes, ion channels, or neurotransmitters resulting in long-lasting changes in neuronal structure and function (Table 1; Hughes and Dragunow, 1995). However, it remains unclear as to whether these changes in IEG expression promote or inhibit apoptosis in neurons. It seems that IEGs likely serve to both inhibit and pro-

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Table 1 Genes transactivated by immediate early genesa Gene

Mechanism(s) of action

Transin Peripherin SCG10 GADD45 NF-L, NF-M 41A, 41C NCAM Prodynorphin Nerve growth factor (NGF) Tyrosine hydroxylase

Encodes ametalloproteinase Encodes a neuronal speci®c ®lament protein Encodes a vesicle associated protein Binds to PCNA; inhibits cell cycle progression; facilitates DNA repair Encode neuro®lament subunits Encode calcium binding proteins Encodes neural cell adhesion molecule Encodes a proenkephalin (opioid precursor) Encodes a neurotrophic factor Encodes a regulatory enzyme

a

References: Sheng and Greenberg, 1990; Morgan and Curran, 1991; Hughes and Dragunow, 1995.

6.2. Bcl-2 and related proteins

on their structural domains: (a) those that are structurally similar to bcl-2 and possess the BH1, BH2, and BH3 domains such as Bax, Bak, and Bok and (b) those that only possess the BH3 domain such as EGL1 (Chittenden et al., 1995a, 1995b; Cosulich, 1997).

The bcl-2 family is a growing group of proteins regulating cell death (Table 2). To date, more than 15 bcl-2 family members have been identi®ed (Chao and Korsmeyer, 1998). Each member of this gene family contains one or more of four conserved regions, known as the bcl-2 homology domains (BH1±BH4), that control the ability of these proteins to dimerize as well as to regulate apoptotic cell death (Sato et al., 1994; Sedlak et al., 1995). Bcl-2 family members that inhibit apoptosis contain at least both the BH1 and BH2 domains (Yin et al., 1994; Chittenden et al., 1995a, 1995b; Muchmore et al., 1996). These bcl-2 proteins have also been divided into two groups based

6.2.1. Bcl-2 and Bcl-xl Bcl-2 is the ®rst of a growing family of regulators of apoptosis that was initially discovered in the t(14:18) chromosomal breakpoint found in B-cell lymphomas. (Bakhshi et al., 1985; Cleary and Sklar, 1985; Tsujimoto and Croce, 1986). In comparison to other oncoproteins, bcl-2 is unique in that it promotes cell survival rather than cell proliferation and this is achieved via inhibition of apoptosis in multiple cell types, including neurons. Bcl-xl represents one of three known splice variants of the bcl-x gene product (Boise et al., 1993; Gonzalez-Garcia et al., 1995; Ban et al., 1998). Bcl-xl is similar in size and structure to bcl-2

mote apoptosis depending on their post-translational modi®cation, such as phosphorylation by jun kinases, or interaction with other proteins.

Table 2 Mechanism(s) of action of bcl-2 family membersa Gene

Function

Mechanism(s) of Action

bcl-2

Anti-apoptotic

bcl-xl

Anti-apoptotic

bag-1 BI-1 b¯-1 boo bcl-w bax bak bad bid bim diva bcl-xs, blk, bod, bok, btf, hara-kiri

Anti-apoptotic Anti-apoptotic Anti-apoptotic Anti-apoptotic Anti-apoptotic Pro-apoptotic Pro-apoptotic Pro-apoptotic Pro-apoptotic Pro-apoptotic Pro-apoptotic Pro-apoptotic

Inhibits mitochondrial permeability transition; heterodimer formation with proapoptotic proteins; inhibition of ROS generation; regulation of Ca2+ ¯ux; regulation of intracellular pH inhibits mitochondrial permeability transition; heterodimer formation with proapoptotic proteins Enhances raf-1 kinase activity; stimulate Hsc70 and Hsp70 ATPase activity Interacts with bcl-2 and bcl-xl to inhibit bax activity Inhibits p53- and TNF-mediated apoptosis; cell cycle regulation Complexes apaf-1 and caspase-9 Unknown Promotes mitochondrial permeability transition Promotes mitochondrial permeability transition; heterodimer formation; Heterodimer formation; ceramide generation Induces structural change in bax Microtubule binding Binds to Apaf-1 Unknown

a

References: see text for references.

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and, like bcl-2, functions as a suppressor of apoptosis (Chao et al., 1995). bcl-2 and bcl-xl are complex proteins localized to the membranes of the endoplasmic reticulum, nuclear envelope and the outer mitochondrial membrane (Hockenbery et al., 1990; Monaghan et al., 1992; Krajewski et al., 1993; Hsu et al., 1997a, 1997b) indicating that these proteins may play a role in ion and/or protein transport across membrane surfaces. Indeed, it has been shown that recombinant bcl-2 and bcl-xl form pores in liposomes and conductance channels in lipid bilayers (Muchmore et al., 1996; Schendel et al., 1997; Schlesinger et al., 1997). The formation of these channels occurs at acid pH and the resulting channels are cation selective at physiological pH (Minn, 1997; Schendel et al., 1997, 1997). The bcl-2 channel is selective for potassium while the bcl-xl channel conducts sodium (Schendel et al., 1997, 1997; Lam et al., 1998).

The ability to form channels in vitro suggests that bcl2 and bcl-xl regulate the mitochondrial permeability transition pore (PT) and release of proteins from the mitochondrion, e.g., cytochrome c, that are critical to the activation of cellular proteases (Boise and Thompson, 1997; Decaudin et al., 1997; Kim et al., 1997; Kitanaka et al., 1997; Kluck et al., 1997; Manon et al., 1997; Vander Heiden et al., 1997; Yang et al., 1997a, 1997b; Bossy-Wetzel et al., 1998; Clem et al., 1998; Zamzami et al., 1998; Finucane et al., 1999; Gross et al., 1999). The mitochondrial PT is a large (02.9 nm) cyclosporin-sensitive channel present in the inner mitochondrial membrane and opens during the process of apoptotic cell death. The opening of this channel has been associated with release of free radicals, calcium and mitochondrial proteins critical to caspase activation into the cytosol (Fig. 2; Ber-

Fig. 2. Regulation of the mitochondrial permeability transition pore (PT). (A) Bcl-2-bax or bcl-xl-bax heterodimers maintain the PT in a closed state, preventing mitochondrial release of cytochrome c. Under conditions that lead to an increase in bax relative to bcl-2 or bcl-xl, bax homodimers are formed (B). The bax homodimer alters the conformation of the PT allowing mitochondrial release of cytochrome c. Cytochrome c then forms a trimeric complex with apaf-1 and procaspase-9. This complex cleaves procaspase-9 producing caspase-9 in the initial step of the mitochondrial-mediated caspase cascade.

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nardi et al., 1994; Zorati and Szabo, 1994). bcl-2 and bcl-xl have been localized to the outer mitochondrial membrane predominantly at sites of contact between the inner and outer mitochondrial membrane (Hockenbery et al., 1990). (bcl-2 and bclxl prevent mitochondrial membrane depolarization via the mitochondrial permeability transition in apoptosis) (Fig. 2; Boise and Thompson, 1997; Decaudin et al., 1997; Kim et al., 1997; Kitanaka et al., 1997; Kluck et al., 1997; Manon et al., 1997; Vander Heiden et al., 1997; Yang et al., 1997a, 1997b; Bossy-Wetzel et al., 1998; Clem et al., 1998; Zamzami et al., 1998; Finucane et al., 1999; Gross et al., 1999). In addition, these proteins inhibit the release of cytochrome c from the mitochondrion and prevent the activation of caspases. The exact mechanisms by which bcl-2 regulates the PT is yet to be elucidated. Regulation of the PT by bcl-xl occurs as a consequence of binding to the voltagedependent anion channel (VDAC) subunit of the PT and subsequent closure of the VDAC (Shimizu et al., 1998). bcl-xl may also interact with the adenine nucleotide translocator (ANT) subunit of the PT demonstrating that mitochondrial adenylate transport is under active regulation. Furthermore, bcl-xl expression allows cells to maintain sucient mitochondrial ATP/ADP exchange to sustain coupled respiration (Heiden et al., 1999), a mechanism that may enable mitochondria to adapt to the changes in metabolic demand during apoptotic cell death. Although the e€ects of bcl-2 and bcl-xl on mitochondrial permeability transition may be critical to its regulation of cell survival during apoptosis, the presence of these proteins within other cell membranes suggests that bcl-2 and bcl-xl have other apoptosis regulatory functions (Hsu et al., 1997a, 1997b). Overexpression of bcl-2 in vitro has been shown not only to protect cells from oxidative agents but also to prevent the generation of reactive oxygen species (Hockenbery et al., 1993; Kane et al., 1993; Satoh et al., 1996; Wiedau-Pazos et al., 1996; Fabisiak et al., 1997) in response to apoptotic stimuli. Bcl-2 has also been shown to prevent apoptotic cell death associated with increases in intracellular calcium (Zhong et al., 1993; Distelhorst and McCormick, 1996; Marin et al., 1996; Zormich, 1996; Ichimiya, 1998; Kruman et al., 1998) by regulating intracellular calcium in several ways. First, bcl-2 prevents depletion of intracellular calcium from the endoplasmic reticulum (Lam et al., 1994; Distelhorst and McCormick, 1996; He et al., 1997; Wei et al., 1998) by enhancing calcium uptake (Kuo et al., 1998). Second, bcl-2 overexpression potentiates the uptake of calcium by mitochondria (Murphy et al., 1996). Third, bcl-2 inhibits calcium entry into the nucleus (Marin et al., 1996). There is also growing evi-

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dence that bcl-2 may participate in intracellular pH regulation. Intracellular acidi®cation is an important event in the activation of endonucleases and cysteine proteases during apoptosis (Barry and Eastman, 1992; Perez-Sala et al., 1995; Morana et al., 1996; Furlong et al., 1998). Intracellular acidi®cation-mediated caspase activation has been shown to be prevented by overexpression of bcl-2 (Reynolds et al., 1996; Furlong et al., 1997; Ishaque and Al-Rubeai, 1998). The speci®c mechanism by which bcl-2 prevents intracellular acidi®cation is yet to be elucidated. The presence of bcl-2 and bcl-xl within membranes allows these proteins to serve as adapter proteins that can sequester other proteins from the cytosol or facilitate protein±protein interactions as an alternate means of regulating apoptotic pathways. Bcl-2 and bcl-xl have been found to bind or co-immunoprecipitate with a number of proteins including: ced-4 and its mammalian equivalents, bax, raf-1, ras proteins, p53 binding protein 2, Pr-1, and several other proteins of unknown function (Sedlak et al., 1995; Sattler et al., 1997; Tao et al., 1997; Gross et al., 1999; Holinger et al., 1999; Weinmann et al., 1999). The C. elegans equivalent of bcl-2, ced-9, has been shown to bind ced-4, thereby inhibiting its action, i.e., caspase activation. Similarly, bcl-2 and bcl-xl can complex with bax to form a heterodimer through binding at key residues within the BH1 and BH2 domains and serving as an important mechanism in regulating caspase activation (Yin et al., 1994; Sedlak et al., 1995). Bcl-2 also associates with the protein kinase, raf-1, pulling this protein from the cytosol to the mitochondrial membrane (Wang et al., 1996a, 1996b, 1996c). Once this association is formed, raf-1 can phosphorylate the pro-apoptotic protein BAD, nullifying its inhibition of the anti-apoptotic actions of bcl-2. Bcl-2 has also been shown to sequester the pro-apoptotic protein, calcineurin from the cytosol, to intracellular membranes blocking apoptosis. Formation of bcl-xl heterodimers with bax, bak, and bid is an important process that prevents apoptotic cell death by preventing activation of cysteine proteases (Sedlak et al., 1995; Sattler et al., 1997; Tao et al., 1997; Gross et al., 1999; Holinger et al., 1999; Weinmann et al., 1999). The presence of bcl-xl in the cytosol may also prevent activation of proteases in other ways (Hsu et al., 1997a, 1997b). Binding of bcl-xl to ced-4, its mammalian homologue, Apaf-1, and to cytochrome c prevents activation of the cysteine protease, caspase-9, an important step in the mitochondrial-mediated protease cascade (Kharbanda et al., 1997; Li et al., 1997a; 1997b; Hu et al., 1998; Huang, 1998; Pan et al., 1998). In addition, bcl-xl inhibits bax-mediated caspase activation by relocating bax from the mitochondrial membrane to the cytosol (Priault et al., 1999). Bcl-xl also interacts with the integral endoplasmic reticulum mem-

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brane protein, Bap31, regulating the activation of procaspase-8 by ced-4, an important step in the cytokinemediated protease cascade (Ng et al., 1997; Ng and Shore, 1998). In addition to apoptosis regulation, there is growing evidence that bcl-2 also participates in regulation of the cell cycle. Bcl-2 and bcl-xl have been shown to maintain cells in a quiescent state (G0 phase of the cell cycle; O'Reilly et al., 1996). The interaction of bcl-2 with calcineurin prevents the interaction of calcineurin with the transcription factor, NF-AT, in its phosphorylated form, and subsequent translocation of NFAT into the nucleus (Linette et al., 1996; Shibasaki, 1997). Bcl-2 also inhibits the activation of cdk2, a kinase responsible for the G0/G1 to S phase transition of the cell cycle (Gil-Gomez et al., 1998). 6.2.2. Other Bcl-2 anti-apoptotic genes Bag-1 is a protein that interacts with bcl-2 to block apoptotic cell death (Wang et al., 1996a, 1996b, 1996c) via several mechanisms. Binding of bag-1 to bcl-2 prevents caspase activation (Schulz et al., 1997) and reactive oxygen species generation. Bag-1 binding facilitates bcl-2 activation of raf-1 kinase activity following the translocation of raf-1 to the mitochondrial membrane (Wang et al., 1996a, 1996b, 1996c, 1999a, 1999b; Wang and Reed, 1998). The increased kinase activity of raf-1 may be an important mechanism in the phosphorylation/ deactivation of Bad or other related pro-apoptotic proteins (Wang et al., 1999a, 1999b). Bag-1 can also enhance protection from apoptosis binding to the platelet-derived growth factor (PDGF) receptor (Bardelli et al., 1996). In addition, Bag-1 stimulates the ATPase activity of the anti-apoptotic chaperonins, Hsc70 and Hsp70 (Hohfeld and Jentsch, 1997; Stuart et al., 1998). Bax inhibitor 1 (BI-1) is found in intracellular membranes and interacts with bcl-2 and bcl-xl to inhibit bax-mediated apoptosis (Xu and Reed, 1998). B¯-1 is a transcriptional target of NF-kappa B that inhibits p53- and TNF-mediated apoptosis (D'Sa-Eipper et al., 1996; Zong et al., 1999). B¯-1 also interacts with multiple oncogenes and, therefore, may be a regulator of cell proliferation (D'Sa-Eipper and Chinnadurai, 1998). Boo inhibits apoptosis by forming a multimeric protein complex with apaf-1 and caspase-9 (Song et al., 1999). Bcl-w also promotes cell survival via unknown mechanisms (Gibson et al., 1996). The roles of bag-1, BI-1, and boo in neuronal apoptosis following hypoxia-ischemia have not yet been established. In other systems, these proteins interact with bcl-2 and bcl-xl to prevent caspase activation. Because caspase activation is a process known to occur in neurons undergoing apoptosis following hypoxia-ischemia (see below), it is therefore reasonable to expect that bag-1, BI-1, and boo function to pre-

serve neuronal viability after such an insult. However, data are lacking in this area. 6.2.3. Bax Bax is one of the ®rst members of the bcl-2 family found to promote apoptosis. To date, there are six known isoforms of bax, all of which have been shown to induce apoptosis (Oltvai et al., 1993; Apte et al., 1995; Zhou et al., 1998; Shi et al., 1999). Bax has a structure similar to that of bcl-2 and bcl-xl. Unique to bax, however, is its ability to form oligomers but the functional signi®cance of this biochemical property is unknown (Tan et al., 1999). Like bcl-2 and bcl-xl, bax forms membrane channels and binds to other bcl-2 family members, properties that are important to its pro-apoptotic functions (Simonen et al., 1997). Bax exists predominantly in the cytosol under unstressed conditions but translocates to mitochondrial and other membranes in response to apoptotic stimuli (Hsu et al., 1997a, 1997b; Goping et al., 1998; Zhang et al., 1998a, 1998b). Processing of bax by one or more caspases appears to be necessary for this translocation (Goping et al., 1998). The membrane-associating property of bax led to the observations that bax, like bcl-2 and bcl-xl, forms ion channels in lipid bilayers (Antonsson et al., 1997). These channels formed by bax are mildly chloride selective, maximally active at acidic pH (Antonsson et al., 1997; Schlesinger et al., 1997), and are antagonized by bcl-2 (Antonsson et al., 1997). Bax also appears to alter the intrinsic properties of lipid bilayers. Introduction of bax into phospholipid bilayers leads to destabilization and rupture of membranes and their subsequent breakdown (Basanez et al., 1999). The translocation of bax to the mitochondrial membrane and its destabilizing e€ect are important factors that in¯uence mitochondrial function during apoptosis. Once bax translocates to the mitochondrial membrane, it cross-links as a homodimer leading to loss of mitochondrial membrane potential, cytochrome c release, and caspase activation (Fig. 2; Eskes et al., 1998; Gross et al., 1998; Jurgensmeier et al., 1998; Narita et al., 1998; Saikumar et al., 1998; Finucane et al., 1999; Priault et al., 1999). These mitochondrial changes appear to occur via two mechanisms. First, cytochrome c loss may occur via opening of the mitochondrial permeability transition pore in a process that is calcium-dependent (Narita et al., 1998) and enhanced by bax binding to the ANT subunit (Marzo et al., 1998; Priault et al., 1999). The release of cytochrome c through the permeability transition pore by bax appears to require ATP and mitochondrial F1/F0 ATPase activity (Matsuyama et al., 1998; Narita et al., 1998; Priault et al., 1999). Second, bax-dependent cytochrome c release may also occur via a magnesiumdependent mechanism (Eskes et al., 1998). The e€ects

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of bax on cytochrome c and caspase activation are inhibited by both bcl-2 and bcl-xl as described above (St. Clair et al., 1997; Zha and Reed, 1997; Jurgensmeier et al., 1998; Otter et al., 1998; Saikumar et al., 1998; Priault et al., 1999). The binding of bax to other members of the bcl-2 family is critical to its regulation of cell survival. The formation of heterodimers versus homodimers is dependent on the relative amounts of bax and bcl-2 and is important in regulation of caspase activity and cell fate (Oltvai, 1997; Zha et al., 1997; Gross et al., 1998; Otter et al., 1998; Perlman et al., 1999; Weinmann et al., 1999). Bax may also function as a link between cell cycle regulation and cell death. Bax inhibits activation of cdk2, which is responsible for G0/G1 to S phase transition in the cell cycle (Gil-Gomez et al., 1998; Perlman et al., 1998). In response to apoptotic stimuli, bax causes actively dividing cells to enter a quiescent state and subsequently undergo apoptosis in a process mediated by the homeodomain protein gax (Perlman et al., 1998). 6.2.4. Other Bcl-2 pro-apoptotic genes Bak is a pro-apoptotic protein (Chittenden et al., 1995a; Kiefer et al., 1995; Moss et al., 1996) whose activity is mediated through its interaction with bcl-2-related proteins (Chittenden et al., 1995b; Sattler et al., 1997). Bak forms heterodimers with both bcl-2 and bcl-xl within mitochondrial membranes leading to cytochrome c release and caspase activation (McCarthy et al., 1997; Jurgensmeier et al., 1997; Martinou et al., 1998; Griths et al., 1999; Holinger et al., 1999). Bad promotes apoptosis via several mechanisms. First, bad displaces bax from bcl-2 or bcl-xl heterodimers, allowing free bax to carry out its death promoting functions (Kelekar et al., 1997; Ottilie et al., 1997; Yang et al., 1997a, 1997b; Zha et al., 1997). Formation of heterodimers by bad may be prevented by phosphorylation (Zha et al., 1996; Scheid and Duronio, 1998; Zundel and Giaccia, 1998). In contrast, calciumactivated protein phosphatase calcineurin dephosphorylates bad, allowing it to translocate from the cytosol to the mitochondrial membrane where it interacts with bcl-xl (Fig. 1; Wang et al., 1999a, 1999b). Bad may also induce apoptosis through mechanisms mediated by ceramide (Basu et al., 1998). Bid promotes apoptosis through its binding to bax (Wang et al., 1996a, 1996b, 1996c). Bid is cleaved by caspase-8 and the cleavage fragment translocates from the cytosol to the mitochondrial membrane where it induces a structural change in bax conformation leading to cytochrome c release (Li et al., 1998; Luo et al., 1998; Gross et al., 1999). Bim is a protein that appears to promote apoptosis

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via binding to dynein light chains within microtubules (O'Connor et al., 1998; Puthalakath et al., 1999). Diva is a bcl-2 homologue that binds to Apaf-1 to induce apoptosis (Inohara et al., 1998a). Bcl-xs is an m-RNA splice variant of the bcl-x gene (Rouayrenc et al., 1995; Grillot et al., 1997). The mechanisms by which bcl-xs promotes cell death are yet to be elucidated. Similarly, bik, blk, bod, bok, btf, harakiri, and mtd, are proteins that interact with other bcl-2 family members to promote apoptosis via unknown mechanisms (Boyd et al., 1995; Elangovan and Chinnadurai, 1997; Hsu et al., 1997a, 1997b; Inohara et al., 1997; Hegde et al., 1998; Inohara et al., 1998b; Hsu et al., 1998; Kasof et al., 1999). The importance of these pro-apoptotic bcl-2 family members in regulating neuronal apoptosis is yet to be elucidated. However, based on their known mechanisms in other systems, bak, bad, bid, and diva are likely to play a role in inducing neuronal death following hypoxia- ischemia. For example, bak functions to activate caspases in a manner similar to bax. Because other members of the bcl-2 family have been shown to have overlapping functions (e.g., bcl-2 and bcl-xl) in regulating neuronal survival following hypoxia-ischemia, it is logical to predict that bak and bax may function similarly in promoting neuronal death. Also, bid is an important mediator in cytokine-mediated apoptosis, a process that is known to contribute to neuronal death following hypoxia-ischemia. 6.3. p53 The p53 gene and its protein product is likely the most intensively studied tumor suppressor gene and is the most commonly mutated gene in human cancers (Hollstein et al., 1994). Under basal conditions, p53 exists at low concentration within cells and has a halflife of 05±20 min (Levine, 1997; Giaccia and Kastan, 1998). The low concentrations of p53 appear to be the consequence of mdm2-regulated ubiquitin-mediated proteolysis (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997; Midgley and Lane, 1997). Furthermore, the transcriptional activity of p53 is suppressed under basal conditions by cytoplasmic sequestration and by alteration of its phosphorylation state (Moll et al., 1992; Moll et al., 1995; Knippschild et al., 1997). In response to DNA damage, cellular levels of p53 increase primarily because of an increase in protein half-life from enhanced translation rather than an increase in transcription (Maltzman and Czyzyk, 1984; Kastan et al., 1991; Price and Calderwood, 1993; Maki and Howley, 1997). In addition, p53 is translocated to the nucleus where it transcriptionally activates a number of target genes (Goldman et al., 1996; Crook et al., 1998). In addition to an increase in cellular concentration

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following DNA damage, p53 undergoes post-translational modi®cation critical for regulating its transcriptional activity. P53 is phosphorylated on the serine residues in both its amino- and carboxy- domains by a number of protein kinases including casein kinases I and II, protein kinase C, cdk2, and cdc2 (Bischo€ et al., 1990; Lees-Miller et al., 1990; Baudier et al., 1992; Milne et al., 1992; Hupp and Lane, 1994; Price et al., 1995; Hall et al., 1996; Shieh et al., 1997). Phosphorylation of p53 enhances site selective binding of DNA and therefore may regulate the di€erences in transcription of di€erent p53 target genes (Wang et al., 1995; Hecker et al., 1996). P53 DNA binding and transcription are also enhanced by acetylation of p53 in its carboxy domain by members of the histone acetylase family, p300/CPB (Avantaggiati et al., 1997; Gu and Roeder, 1997; Lill et al., 1997; Scolnick et al., 1997). There is new evidence demonstrating that the redox state of p53 is also critical for p53 transcriptional activity. First, oxidation of DNA causes loss of sitespeci®c DNA binding and binding of divalent cations critical to p53 function (Hainaut and Milner, 1993a, 1993b; Delphin et al., 1994; Verhaegh et al., 1997). Second, nitric oxide, a free radical generator, alters p53 protein conformation and decreases p53 sequence speci®c DNA binding (Calmels et al., 1997). Third, the nuclear redox/repair protein, ref-1, reduces oxidized p53, thus enhancing p53 DNA binding and its transcriptional activity (Jayamaran et al., 1997). Following its post-translational modi®cation, p53 transcriptionally activates a number of proteins (Table 3) including bax (the bcl-2 family member), insulin growth factor binding protein 3 (IGF-BP-3), p21WAF1/Cip1, cyclin G, GADD45, and mdm2 (Hansen and Oren, 1997; Levine, 1997). These transcriptionally activated proteins serve a number of functions including induction of apoptosis, cell cycle regulation, DNA repair, and autoregulation of p53. Bax is an important but not the sole mediator of p53-dependent apoptosis as, in some cases, overexpression of bax in the absence of p53 fails to induce apoptosis (Myashita and Reed, 1995; Brady et al., 1996; see above). P53 may also induce apoptosis through the actions of IGF-BP3 (Buckbinder et al., 1995; Ludwig et al., 1996; Ryan

and Vousden, 1998) which binds to the IGF receptor, preventing IGF binding (Buckbinder et al., 1995). Transactivation of p21WAF1/Cip1 and GADD45 by p53 leads to inhibition of cell cycle progression at the G1/S transition phase (El-Deiry et al., 1993; Hollander et al., 1993; Carrier et al., 1994). p21 inhibits cell cycle progression by binding to cyclin-cdk complexes inhibiting their kinase activity (Wu and Schonthal, 1997). In contrast, GADD45 inhibits cell cycle progression at the G1/S transition by binding to proliferating cell nuclear antigen (PCNA). GADD45 also induces growth arrest at the G2/M checkpoint through its activity on the G2-speci®c kinase, cyclin B1/p34 (cdc2) (Wang et al., 1999; Zhan et al., 1999). GADD45 binding to PCNA is also important for stimulating DNA excision repair (Kastan et al., 1992; Smith et al., 1993; Hall et al., 1995). GADD45 binding to PCNA is impaired by p21 binding to GADD45 (Kearsey et al., 1995) indicating that these two proteins regulate PCNA in a competitive manner (Chen et al., 1995a, 1995b). GADD45 may also function in DNA excision repair by enhancing DNA relaxation and cleavage activity by topoisomerases, thereby facilitating accessibility of DNase I to damaged DNA (Carrier et al., 1999). Transcriptional activation of mdm2 by p53 forms a p53 autoregulatory feedback system that functions in several ways. First, mdm2 binds to p53 targeting p53 for ubiquitin-mediated degradation (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997; Midgley and Lane, 1997). Second, mdm2 enhances translocation of p53 from the nucleus to the cytoplasm where it is subjected to proteolysis (Roth et al., 1998). Third, mdm2 binding to p53 inhibits transactivation of p53 downstream genes (Momand et al., 1992; Oliner et al., 1993; Thut et al., 1997) in a process that is blocked by phosphorylation of p53 (Shieh et al., 1997; Kamijo et al., 1998; Pomerantz et al., 1998; Zhang et al., 1998a, 1998b; Zindy et al., 1998). Although many of the functions of p53 occur via transactivation of downstream genes, the e€ects of p53 on cellular functions also occurs via interactions with other proteins (Tables 4 and 5). Following DNA damage, for example, c-abl, an oncogene that func-

Table 3 Genes transactivated by p53a Gene

Mechanism(s) of Action

bax IGF-BP-3 p21WAF1/Cip1 GADD45 mdm2

Promotes mitochondrial permeability transition; promotes apoptosis Inhibits cellular mitogenic response; promotes apoptosis Binds to cyclin-cdk complexes; inhibits cell cycle progression Binds to PCNA; inhibits cell cycle progression; facilitates DNA repair Facilitates proteolytic degradation of p53; inhibits p53 transcriptional activity; faclitates p53 transport from nucleus to cytoplasm

a

Hansen and Oren (1997), Levine (1997).

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Table 4 Proteins interacting with p53a Protein

Mechanism(s) of action

c-abl TFIIH WT1

Proto-oncogene; phosphorylates p53 enhancing p53 transcriptional activity Functions in DNA repair and transcription Enhances p53 DNA binding and transcription; inhibits apoptosis

a

Goga et al. (1995), Maheswaran et al. (1995), Levine (1997).

tions as a kinase, binds p53 and enhances its transcriptional activity resulting in growth arrest (Goga et al., 1995). P53 also binds to the RNA polymerase II basal transcription factor, TFIIH, that functions as a helicase and in DNA repair and transcription (Wang et al., 1995). Cells de®cient in TFIIH helicase activity fail to undergo apoptosis indicating that this interaction may be the critical step in p53-mediated apoptosis (Levine, 1997). The Wilms tumor suppressor gene product, WT1, enhances p53 sequence-speci®c DNA binding and transcriptional activation but appears to inhibit p53-mediated apoptosis (Maheswaran et al., 1995) 6.4. Gene activation in hypoxia-induced apoptosis Exposure of neurons to hypoxia induces changes in immediate early genes, bcl-2 family members, p53, and their downstream genes. Although the genetic mechanisms activated in neurons by hypoxic exposure are not known in detail, a number of important observations can be extracted from the current data. First, in re-

sponse to hypoxia, members of both anti-apoptotic and pro-apoptotic genes families are expressed. It is likely that the balance in the expression of these gene families determines whether neurons will survive or undergo apoptotic cell death following an hypoxic insult. Those neurons, which undergo apoptosis in response to hypoxia, tend to have a predominant expression of fosB, c-jun, NGFI-A, NGFI-C, p53, bax, and bcl-xs while bcl-2, bcl-xl, and krox24 may be unchanged, decreased, or increased (Shimazaki et al., 1994; Krajewski et al., 1995; Hara et al., 1996; Dixon et al., 1997; Li et al., 1997a, 1997b; Antonawich et al., 1998; Rosenbaum et al., 1998; Joo et al., 1999). In neurons surviving exposure to hypoxia, bcl-2, bcl-xl, p21, and GADD45 expression predominates and immediate-early gene expression is transient (Chen et al., 1995a, 1995b, 1997; Lawrence, 1996, 1997; van Lookeren-Campagne and Gill, 1998a, 1998b; Guegan et al., 1999). Measures to alter the balance in favor of antiapoptotic gene expression by hypoxic conditioning, by genetic manipulation, or by attenuating pro-apoptotic gene expression, have resulted in improved neuronal

Table 5 Proteins targeted for proteolysis by caspasesa Caspase

Target protein

Target protein function

Caspase-1 Caspase-2 Caspase-3

pro-interleukin 1b procaspase-3/4 PARP PARP DNA protein kinases U1-70K sn ribonuclear proteins Hn ribonuclear protein-C SREBP D4-GDP dissociation inhibitor DFF-45 gelsolin

Cytokine production proteases DNA repair enzyme

Caspase-4 Caspase-5 Caspase-6 Caspase-7

procaspase-1 unknown lamins A, B, C1 PARP Hn-ribonuclear protein-C Procaspases-3, -4, -7, -9 procaspase-3 PARP Procaspases-3 and -7

Caspase-8 Caspase-9 Caspase-10 a

Nicholson and Thornberry (1997), Villa et al. (1997) Thornberry and Lazebnik (1998).

DNA repair enzyme DNA repair pre-mRNA splicing pre-mRNA splicing sterol biosynthesis regulates Rho-ATPase DNA fragmentation actin protease protease nuclear cytoskeletal proteins DNA repair enzyme Pre-mRNA splicing Proteases protease DNA repair Proteases

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tolerance to hypoxic insults (Martinou et al., 1994; Linnik et al., 1995; Meyer et al., 1995; Banasiak and Haddad, 1998; Bossenmeyer-Pourie and Daval, 1998; Kitagawa et al., 1998; Lawrence et al., 1998; Antonawich et al., 1999). In contrast, inhibiting anti-apoptotic gene expression increases neuronal vulnerability to hypoxic injury (Banasiak et al., 1999). Second, the relative expression of anti- and proapoptotic genes may also account, in part, for regional di€erences in vulnerability to hypoxic insults. In brain regions that are more sensitive to hypoxia, such as the CA1 of the hippocampus, the Purkinje cell layer of the cerebellum, amygdala, thalamus, striatum, and cortex, persistent expression of junB, NGFI-A p53, bax, and bcl-xs is predominant. In contrast, bcl-2, bcl-xl, and krox24 expression may be unchanged or decreased (Krajewski et al., 1995; Chen et al., 1996a, 1996b, 1997; Gillardon et al., 1996; Hara et al., 1996; Ferrer et al., 1998; van Lookeren-Campagne and Gill, 1998a). In contrast, those regions of the brain that are relatively tolerant to hypoxic insults, such as CA3 and dentate gyrus of the hippocampus, have preferential expression of the protective genes bcl-2, bcl-xl, and Gadd45 and only transient increases or no change in immediate-early genes, p53 and bax (Chen et al., 1996a, 1996b, 1998a). In addition, in regions more susceptible to hypoxic injury, there is transcription but not translation of IEGs and other survival promoting genes (Hughes et al., 1998). Third, the severity of the hypoxic insult not only in¯uences the form of neuronal cell death that occurs but also the pattern of gene expression. With prolonged ischemic insults or in central regions of the infarct zone, where oxygen levels are exceedingly low, necrotic cell death predominates (Linnik et al., 1993; Beilharz, 1995; Hill et al., 1995; Li et al., 1995). Under these conditions in vivo, bax and p53 levels are increased while bcl-2, bcl-xl, and GADD45 are decreased (Jin et al., 1996; Hou et al., 1997; Chen et al., 1998a; Ferrer et al., 1998; Isenmann et al., 1998; Matsushita et al., 1998; van Lookeren-Campagne and Gill, 1998a; van Lookeren-Campagne and Gill, 1998b). Similarly, in vitro studies of prolonged hypoxic exposure resulting in neuronal death have shown that bax and p53 are increased while bcl-2 may be increased or decreased (Shimazaki et al., 1994; Ferrer et al., 1997; Niwa et al., 1997; Banasiak and Haddad, 1998; Bossenmeyer-Pourie and Daval, 1998; Tamatani et al., 1998; Banasiak et al., 1999). In contrast, in the penumbral region, where oxygen levels may be graded, both apoptotic and necrotic cell death are detectable (Linnik et al., 1993; Beilharz, 1995; Hill et al., 1995; Li et al., 1995). In the penumbral region, bcl-2, bcl-xl, p21, and GADD45 expression is increased while bax and p53 expression is decreased (Chen et al., 1995a, 1995b, 1998a; Hou et

al., 1997; Antonawich et al., 1998; Isenmann et al., 1998; van Lookeren-Campagne and Gill, 1998b). Similarly, exposure to brief periods of hypoxia in vitro has been associated with upregulation of bcl-2 and c-jun resulting in improved neuronal survival to subsequent insults (Shimazaki et al., 1994; Bossenmeyer-Pourie and Daval, 1998; Whit®eld et al., 1999). Fourth, gene expression may be altered in regions of the brain well beyond the boundary of the infarct zone and penumbra. In models on unilateral focal hypoxiaischemia, transient increases in c-fos, fosB, c-jun, Jun B, junD, NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, krox20, krox24, and Nurr1 have been detected in areas of the cortex outside of the infarct zone extending to the contralateral hemisphere (Gubits et al., 1993; Dragunow et al., 1994; Kinouchi et al., 1994; Lin et al., 1996; Honkaniemi et al., 1997). These and other data seem to support the hypothesis that spreading depression, resulting from excitatory amino acid release and other consequences of ischemic injury, may in¯uence gene activation in regions una€ected by the ischemic lesion. The glutamate receptor antagonists, MK801 and dextromethorphan, have been shown to attenuate c-jun, and c-fos expression and improve neuronal survival following hypoxia-ischemia (Gass et al., 1992; Bokesch et al., 1994; Collaco-Moraes et al., 1994). Increased intracellular calcium, resulting from glutamate or inositol triphosphate receptor activation, is a potent inducer of IEG transcription (Graybiel et al., 1990). Neurotrophic factors and cytokines, such as FGF-2, TGF-1, and IL-1, activated during hypoxiaischemia, have been shown to induce c-fos expression. The consequences of immediate-early gene induction in these ``una€ected'' regions is still to be determined. Fifth, p53 activates both pro-apoptotic as well as survival mechanisms in response to hypoxic insults. Whether apoptosis or survival will occur seems to be dependent on the duration of p53 and downstream gene expression. Following an ischemic insult, a transient rise in p53 with persistence of p21 and GADD45 expression correlates with improved survival in CA3 hippocampal neurons (Tomasevic et al., 1999). It is possible that p21 may confer protection by preventing quiescent cells from entering the cell cycle and allowing for DNA repair by GADD45 and other proteins. In contrast, CA1 hippocampal neurons exhibit persistently elevated levels of p53 and more widespread apoptotic death (van Lookeren-Campagne and Gill, 1998a). Despite the growing knowledge of gene activation, the exact trigger for induction of p53 and other genes during hypoxia is unclear. There is equivocal evidence that DNA strand breaks lead to increased cellular p53 (Stoler et al., 1992; Graeber et al., 1994; Banasiak and Haddad, 1998). There is also no evidence to support the idea that p53 and bcl-2 accumulate during hypoxia as a result of a stress-induced increase in protein syn-

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thesis (McCormick and Penman, 1969; Heacock and Sutherland, 1986; Petterson et al., 1986; Banasiak and Haddad, 1998; Banasiak et al., 1999). However, there is now evidence to suggest that hypoxia induces phosphorylation and consequent stabilization of p53 (Giaccia and Kastan, 1998). Also, redox sensitive proteins, such as HIF 1, contribute to accumulation and subsequent stabilization of p53 and reduction of bcl-2 during hypoxic exposure (An et al., 1998; Carmeliet et al., 1998). The protein products of the downstream genes, GADD 45 and p21, are also increased in response to hypoxia via mechanisms that are HIF 1independent and dependent (Price and Calderwood, 1992; Long et al., 1997; Carmeliet et al., 1998). One must consider that hypoxia may not directly alter gene activity but rather biochemical events consequent to hypoxic exposure, such as altered ionic homeostasis or energy depletion, may alter gene expression during apoptosis.

tively substrate speci®c manner. The substrate speci®city and catalytic eciency of caspases stems from recognition of at least four amino acids NH2-terminal to the cleavage site (Thornberry et al., 1997). The substrates targeted by the caspases are proteins that are vital to cell viability (Cryns and Yuan, 1998). These include: DNA repair proteins (e.g., DNA-protein kinase), negative regulators of apoptosis (e.g., bcl-2), and structural proteins (e.g., nuclear lamina, cytoskeletal proteins) (Orth et al., 1996; Takahashi et al., 1996; Cheng et al., 1997; Kothakota et al., 1997; Rheaume et al., 1997; Rudel and Bokoch, 1997; Wen et al., 1997; Xu and Reed, 1998; Adams and Cory, 1998). The proteolytic activities of the caspases on these substrates provide mechanism for the morphologic changes observed in cells during apoptotic cell death. Regulation of caspase activity by bcl-2 family proteins is discussed above.

7. Caspases and cytokines

7.2.1. Cytokine-mediated caspase activation There is a large body of evidence supporting the existence of a cysteine protease cascade with initiator and e€ector caspases. To date, caspase-3 can be activated via two mechanisms: (a) via cytokine-mediated receptor activation and (b) via alteration of mitochondrial membrane potential (Fig. 3). Currently, four cascades leading to caspase activation in response to apoptotic stimuli are known. The ®rst involves death receptor mediated activation of caspase-8 (Fig. 5; Ashkenazi and Dixit, 1998). The second cascade is characterized by activation of caspase-9 by cytotoxic agents

During the process of apoptosis, there are a number of biochemical events that are critical to the ultimate degradation of the cell. One of these events is the activation of proteolytic enzymes, the cysteine proteases or caspases. The role of caspases in apoptotic cell death was realized with the discovery of Ced-3, a requisite cell death gene in Caenorhabditic elegans homologous to mammalian interleukin-1-b-converting enzyme (ICE; caspase 1) (Thornberry et al., 1992; Yuan et al., 1993). Although, caspase-1 has no clear role in mammals, its discovery led to the identi®cation of a cysteine protease cascade integral to apoptotic cell death. In this section, these proteolytic cascades and their regulation will be discussed.

7.2. Regulation of caspases

7.1. Chemical properties of caspases Caspases are all expressed as proenzymes (30±50 kD) containing three subunits: an NH2-terminal domain, a large subunit (20 kD), and a small subunit (10 kD) (Nicholson and Thornberry, 1997). Caspases are activated following proteolytic processing and association of the large and small subunits (Walker et al., 1994a, 1994b; Wilson et al., 1994; Rotonda et al., 1995). The proenzyme structure has inherent properties critical to the activation of these molecules. First, the NH2-terminal domain acts as a recognition site for activation (Thornberry and Lazebnik, 1998). Second, cleavage of the proenzyme occurs at consensus sites indicating that caspase activation may occur autocatalytically or in a cascade (Thornberry and Lazebnik, 1998). Once activated, caspases cleave proteins in a rela-

Fig. 3. Mechanisms of activation of caspase-3. Caspase-3 can be activated by either of two mechanisms: (a) via cytokine-mediated receptor activation or (b) mitochondrial release of cytochrome c through the permeability transition pore. Binding of fas or TNF-a leads to activation of caspase-8, which, in turn, cleaves procaspase-3, yielding activated caspase-3. Alternatively, mitochondrial release of cytochrome c leads to activation of caspase-9 that, in turn, activates caspase-3.

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(Li et al., 1997a, 1997b; Hakem et al., 1998; Kuida et al., 1998). The other two are less well characterized and involve binding of Apo3 ligand (TWEAK) and Apo2 ligand (TRAIL) to the tumor necrosis factor receptor (TNFR) family members DR3, DR4, and DR5 (Wiley et al., 1995; Marsters et al., 1996; Chicheportiche et al., 1997; McFarlane et al., 1997; Pan et al., 1997a, 1997b; Screaton et al., 1997; Schneider et al., 1997; Sheridan et al., 1997; Walczak et al., 1997; Marsters et al., 1998). Activation of caspase-8 may occur through binding to Fas (also known as CD95 or Apo1), TNFR1, or DR3 (Nagata, 1997). The Fas receptor is a homotrimeric molecule with an adapter protein termed the Fas-associated death domain (FADD) (Fig. 4; Chinnaiyan et al., 1995). Within FADD, a death e€ector domain (DED) binds to an analogous domain of the proenzyme form of caspase-8 (Boldin et al., 1996). Following its interaction with DED, caspase-8 becomes

activated through self-cleavage (Chinnaiyan et al., 1996; Hsu et al., 1996a, 1996b; Muzio et al., 1998). The active form of caspase-8 then proteolytically activates downstream caspases ultimately leading to the activation of the e€ector protease, caspase-3 (Li et al., 1997a, 1997b; Muzio et al., 1998). Activation of caspase-8 may also occur through receptor binding of TNFR1 (also known as p55 or CD120a; Fig. 4; Ashkenazi and Dixit, 1998). TNFmediated caspase activation occurs subsequent to association of the TNFR1 death domains and subsequent binding of an adapter termed TNFRassociated death domain (TRADD) (Hsu et al., 1996a). TRADD functions as a docking protein for a number of signaling molecules including FADD, TNFR-associated factor-2 (TRAF-2), and receptor interacting protein (RIP) (Rothe et al., 1995; Hsu et al., 1996a). FADD mediates activation of caspase-8 as described above. In contrast, TRAF-2 and RIP stimu-

Fig. 4. Mechanisms of fas and TNF-a receptor caspase activation. (A) Binding of fas to its receptor leads to caspase-8 activation via procaspase8 interaction with the fas-associated death domain (FADD). Activated caspase-8 cleaves caspase-3 to its activated form leading to subsequent apoptotic cell death. (B) Binding of TNF-a to its receptor also leads to caspase-8 activation via procaspase-8 interaction with the TNF-receptor and fas-associated death domains (TRADD and FADD). Alternatively, TNF-receptor binding may lead to activation of the nuclear transcription factor KB (NF-?B) and jun kinases forming pathways that inhibit apoptosis.

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late anti-apoptotic pathways involving activation of NF-kappa B and Jun kinase/AP1 (Rothe et al., 1995; Hsu et al., 1996a, 1996b). The DR3 receptor exhibits close sequence homology with the TNFR1 receptor (Pan et al., 1997a). Upon binding of Apo3 ligand, the DR3 receptor can activate caspase-8 or inhibit apoptotis via mechanisms similar to that of TNFR1 (Chicheportiche et al., 1997; Marsters et al., 1998). It is unlikely that DR3 is important in neuronal apoptotic cell death as it is predominantly expressed in the spleen, thymus, and the peripheral blood (Tartaglia and Goeddel, 1992; Pan et al., 1997a). Caspase activation leading to apoptotic cell death may also occur via binding of Apo2 ligand (TRAIL) to DR4 or DR5 TNF family receptors (Marsters et al., 1996; Mariani et al., 1997; Martinez-Lorenzo et al., 1998). The mechanism by which binding of these receptors induces caspase activation is unclear. There is con¯icting evidence as to whether these receptors interact with adapter molecules, such as FADD, TRADD, TRAF2, or RIP (Pan et al., 1997a, 1997b; Walczak et al., 1997; Schneider et al., 1997). However, there is evidence that DR4 may induce apoptosis via a FADD-independent pathway (Yeh et al., 1998). 7.2.2. Cytotoxic caspase activation Caspase-3 activation also occurs in a manner that is independent of caspase-8 activation (Fig. 3; see above). Following mitochondrial release of cytochrome c in response to cytotoxic stimuli, cytochrome c forms a trimeric complex with apoptosis protease activating factor-1 (Apaf-1), and the proenzyme form of caspase9 (Li et al., 1997a, 1997b). The resulting complex cleaves and activates caspase-9 that then is available to activate caspase-3 (Li et al., 1997a, 1997b). 7.3. Role of cytokines and caspases in hypoxia-induced apoptosis There is a vast body of literature documenting an increase in cytokines following hypoxic-ischemic injury (Stoll et al., 1998). TNF-a, fas, TRAIL, and TNFR1 are increased in brain and spinal cord following an hypoxic-ischemic insult and correlate with the occurrence of apoptosis (Matsuyama et al., 1994, 1995; Botchinka et al., 1997; Sairanen et al., 1997; Chao et al., 1998; Sakurai et al., 1998; Zhai et al., 1997; Herr et al., 1999; Martin-Villalba et al., 1999). However, some controversy exists as to whether cytokine release, particularly TNF-a, enhances neuronal injury or promotes neuronal survival. The majority of in vivo studies demonstrate the detrimental e€ects of TNF-a. Administration of TNF-a enlarges infarct size while TNFR1 receptor blockers and TNF antibodies reduce infarct size (Barone et al., 1997; Meistrell et al., 1997; Nawa-

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shiro et al., 1997; Lavine et al., 1998; Yang et al., 1998; Herr et al., 1999). Apoptosis induced by these death-inducing ligands has been attributed to ceramide and nitric oxide generation and caspase activation (Nomura, 1998; Herr et al., 1999). In contrast, data exist supporting a protective e€ect of TNF-a. TNF-a, for example, has been shown to prevent neuronal apoptosis in vivo and in vitro (Tamatani et al., 1999). Furthermore, mice lacking TNFR1 (p55) have increased damage in response to hypoxic-ischemic injury compared to wild-type controls (Gary et al., 1998). TNF-a may exert its protective e€ects via NFkappaB mediated activation of bcl-2 and bcl-xl expression (Tamatani et al., 1999). There is no clear answer as to why TNF-a possesses these two opposing e€ects. One possible explanation may be that persistent, expression of NF-kappaB induced by TNF-a promotes neuronal apoptosis while transient elevations of NF-kappa B have been associated with neuronal survival (Clemens et al., 1997a, 1997b). Another possibility is that TNF-a may be acting on di€erent receptors, although there is no clear evidence currently to support this hypothesis. It is becoming clearer that caspases are likely the critical e€ector molecules in hypoxia-induced apoptosis. Activation of caspase-3 has been shown in apoptotic cells following hypoxic insults (Chen et al., 1998b; Namura et al., 1998; Pulera et al., 1998; Schulz et al., 1998; Tamatani et al., 1998). Inhibitors of caspase-3 have shown some protective e€ect against hypoxic neuronal injury (Cheng et al., 1998; Nath et al., 1998; Tamatani et al., 1998). Current evidence suggests that both cytokine receptor activation and mitochondrial permeability transition contribute to the activation of caspase-3 during cerebral hypoxia-ischemia. Caspases contribute to the genesis of apoptosis (Table 3) by: (a) activation of caspases and other death promoting agents, (b) destruction of cytoskeletal elements, and (c) inactivation of anti-apoptotic proteins. For example, caspase-3 is converted from its inactive, or procaspase, form by caspase-8 following Fas receptor activation (Muzio et al., 1997; Takahashi et al., 1997). Similarly, following mitochondrial cytochrome c release and caspase-9 activation, the activated form of caspase-9 cleaves procaspase-3, producing activated caspase-3 (Li et al., 1998). The activated form of caspase-3 targets multiple proteins for proteolysis. Actin is a cytoskeletal protein shown to be cleaved by caspase-3, a mechanism that may be important to the structural changes observed in apoptotic cells (Villa et al., 1997; Thornberry and Lazebnik, 1998). Caspase-3 has been also shown to cleave the anti-apoptotic protein, bcl-2, releasing a fragment that promotes apoptosis (Cheng et al., 1997; Xue and Horvitz, 1997; Adams and Cory, 1998). Determination of the exact pathway(s) leading to caspase activation in

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neurons exposed to hypoxia is still under investigation. However, it is likely that both cytokine- mediated and cytotoxic-mediated mechanisms will be involved.

8. Future directions Hypoxia-induced neuronal apoptosis involves an ever-growing broad range of biochemical and genetic mechanisms. Despite the e€orts by numerous investigators, most of our knowledge in this area remains descriptive. Clearly, these studies are important but many important questions regarding the functions of genes, proteins, and other mechanisms in determining cell survival or death following hypoxia-ischemia remain unanswered. Based on current studies, there are a number of areas of great interest regarding the mechanisms inducing apoptosis in response to hypoxia. 1. Does hypoxia directly induce apoptosis in neurons? Current evidence seems to suggest that hypoxia, in and of itself, does not induce apoptosis. The possibilities of energy depletion, or altered ionic homeostasis, or ``oxygen-sensing'' molecules as the initiating events should be pursued further. 2. What is the role of altered ionic homeostasis, particularly potassium and sodium, in neuronal apoptosis and through what channel or transporter does the ¯ux take place? There is strong evidence supporting the relationship between changes in ionic ¯ux and apoptosis. Increases in intracellular calcium have been linked with gene activation. As it has been recently observed, it is possible to consider that changes in intracellular sodium and potassium may play similar roles. 3. What is the role of altered intracellular pH in apoptosis? Current evidence links decreased intracellular pH with caspase and endonuclease activation, a process that can be abrogated by intracellular alkalization. With growing evidence that other cations may alter gene expression, the possibility that changes in intracellular pH may do so as well does not seem improbable. 4. What is the role of immediate-early genes and related transcription factors in hypoxia-induced apoptosis? The varied expressions of these genes may impact on neuronal survival. Which of these genes promote and which inhibit apoptosis remains unclear. In addition, the downstream genes activated by the IEGs require further investigation. 5. What are the mechanisms controlling the di€erences in gene expression in regions of the brain more susceptible to injury from hypoxia versus regions resistant to hypoxic injury? 6. What is the role of altered gene expression in

regions of the brain outside those regions supplied by an occluded blood vessel? 7. What are the conditions that determine whether cytokines contribute to further neuronal death or neuronal survival following hypoxic-ischemic injury? 8. What is the functional importance of apoptotic neuronal death following hypoxia-ischemia? It is generally accepted that apoptosis contributes to an increase in infarct size. The question that remains is whether inhibition of neuronal apoptosis following hypoxia-ischemia will contribute to improved neurologic outcome. 9. What are some of the speci®c markers of apoptosis? Can we develop methods to delineate apoptosis in a better fashion than we have at present, keeping in mind the new knowledge that is forthcoming? 10. What are some of the pharmacological interventions that are potentially helpful in interrupting apoptosis? What are some of the key steps that could be used in gene targeting or experimental or therapeutic designs to alleviate cell death?

References Adams, J.M., Cory, S., 1998. The bcl-2 protein family: arbiters of cell survival. Science 281, 1322±1326. An, W.G., Kanekal, M., Simon, M.C., Maltepe, E., Blgosklonny, M.V., Neckers, L.M., 1998. Stabilization of wild type p53 by hypoxia-inducible 1. Nature 392, 405±408. Angel, P., Imagawa, M., Chui, R., Stein, B., Imbra, R.J., Rhamsdorf, H.J., Jonal, C., Herrlich, P., Karin, M., 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49, 729± 739. 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, 961±973. Ankarcrona, M., Dypbukt, J.M., Orrenius, S., Nicotera, P., 1996. Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Lett 394, 321±324. Antonawich, F.J., Krajewski, S., Reed, J.C., Davis, J.N., 1998. Bclx(l) Bax interaction after transient global ischemia. J. Cereb. Blood Flow Metab 18, 882±886. Antonawich, F.J., Federo€, H.J., Davis, J.N., 1999. BCL-2 transduction, using a herpes simplex virus amplicon, protects hippocampal neurons from transient global ischemia. Exp. Neurol 156, 130± 137. Antonsson, B., Conti, F., Ciavatta, A., Montessuit, S., Lewis, S., Martinou, I., Bernasconi, L., Bernard, A., Mermod, J.J., Mazzei, G., Maundrell, K., Gambale, F., Sadoul, R., Martinou, J.C., 1997. Inhibition of Bax channel-forming activity by Bcl-2. Science 277, 370±372. Apte, S.S., Mattei, M.G., Olsen, B.R., 1995. Mapping of the human BAX gene to chromosome 19q13.3-q13.4 and isolation of a novel alternatively spliced transcript, BAX delta. Genomics 26, 592± 594. Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling ang modulation. Science 281, 1305±1308.

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 Avantaggiati, M.L., Ogryzko, V., Gardner, K., Giordano, A., Levine, A.S., Kelly, K., 1997. Recruitment of p300/ CPB in p53dependent signal pathways. Cell 89, 1175±1184. Avdonin, V., Kasuya, J., Ciorba, M.A., Kaplan, B., Hoshi, T., Iverson, L., 1998. Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels. Proc. Natl. Acad. Sci. U.S.A 95, 11703±11708. Baker, S.J., Kerppola, T.K., Luk, D., Vandenberg, M.T., Marshak, D.R., Curran, T., Abate, C., 1992. Jun is phosphorylated by several protein kinases at the same sites thae are modi®ed in serumstimulated ®broblasts. Mol. Cell Biol 12, 4694±4705. Bakhshi, A., Jemesen, J.P., Goldman, P., Wright, J.J., McBride, O.W., 1985. Cloning the chromosomal breakpoint of t(14:18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 41, 899±906. Bamji, S.X., Majdan, M., Pozniak, C.D., Belliveau, D.J., Aloyz, R., Kohn, J., Causing, C.G., Miller, F.D., 1998. The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J. Cell Biol 140, 911±923. Ban, J., Eckhart, L., Weninger, W., Mildner, M., Tschachler, E., 1998. Identi®cation of a human cDNA encoding a novel Bcl-x isoform. Biochem. Biophys. Res. Commun 248, 147±152. Banasiak, K.J., Haddad, G.G., 1998. Hypoxia-induced apoptosis: e€ect of hypoxic severity and role of p53 in neuronal cell death. Brain Res 797, 295±304. Banasiak, K.J., Cronin, T., Haddad, G.G., 1999. bcl-2 prolongs neuronal survival during hypoxia-induced apoptosis. Mol. Brain Res 72, 214±225. Barde, Y.A., 1989. Trophic factors and neuronal survival. Neuron 2, 1525±1534. Bardelli, A., Longati, P., Albero, D., Goruppi, S., Schneider, C., Ponzetto, C., Comoglio, P.M., 1996. HGF receptor associates with the anti-apoptotic protein BAG-1 and prevents cell death. Embo J 15, 6205±6212. Barone, F.C., Arvin, B., White, R.F., Miller, A., Webb, C.L., Willette, R.N., Lysko, P.G., Feuerstein, G.Z., 1997. Tumor necrosis factor alpha. A mediator of focal ischemic brain injury. Stroke 28, 1233±1244. Barry, M.A., Eastman, A., 1992. Endonuclease activation during apoptosis: the role of cytosolic Ca2+ and pH. Biochem. Biphys. Res. Commun 186, 783±789. Basanez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S., Wood, K.A., Hsu, Y., Zimmerberg, J., Youle, R.J., 1999. Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA 96, 5492±5497. Basu, S., Bayoumy, S., Zhang, Y., Lozano, J., Kolesnick, R., 1998. BAD enables ceramide to signal apoptosis via Ras and Raf-1. J. Biol. Chem 273, 30419±30426. Baudier, J., Delphin, C., Grunwald, D., Khochbin, S., Lawrence, J.J., 1992. Characterization of the tumor suppressor gene p53 as protein kinase c substrate and an S100b-binding protein. Proc. Natl. Acad. Sci. USA 89, 11627±11631. Behrens, M.M., Strasser, U., Lobner, D., Dugan, L.L., 1999. Neurotrophin-mediated potentiation of neuronal injury. Micr. Res. Tech 45, 276±284. Belliveau, D.J., Krivko, I., Kohn, J., Lachance, C., Pozniak, C., Rusakov, D., Kaplan, D., Miller, F.D., 1997. NGF and neurotrophin-3 both activate TrkA on sympathetic neurons but di€erentially regulate survival and neuritogenesis. J. Cell Biol 136, 375± 388. Bernardi, P., Broekemeier, K.M., Pfei€er, D.R., 1994. Recent progress on regulation of the mitochondrial permeability transition pore: a cyclosporin-sensitive pore in the inner mitochondrial membrane. J. Bioenerget. Biomembr 26, 509±517. Bischo€, J.R., Friedman, P.N., Marshak, D.R., Prives, C., Beach,

237

D., 1990. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc1. Proc. Natl. Acad. Sci. USA 87, 4766±4770. Boise, L.H., Gonzalez-Garcia, M., Postema, C.E., Ding, L., Lindsten, T., Turka, L.A., Mao, X., Nunez, G., Thompson, C.B., 1993. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74, 597±608. Boise, L.H., Thompson, C.B., 1997. Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation. Proc. Natl. Acad. Sci. USA 94, 3759±3764. Bokesch, P.M., Marchand, J.E., Connelly, C.S., Wura, W.H., Kream, R.M., 1994. Dextramethorphan inhibits ischemia-induced c-fos expression and delayed neuronal death in hippocampal neurons. Anesthesiology 81, 470±477. Boldin, M.P., Goncharov, T.M., Goltsev, Y.V., Wallach, D., 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803±815. Boniece, I.R., Wagner, J.A., 1993. Growth factors protect PC12 cells against ischemia by a mechanism that is independent of PKA, PKC, and protein synthesis. J. Neurosci 13, 4220±4228. Bortner, C.D., Hughes, F.M., Cidlowski, J.A., 1997. A primary role for K+ and Na+ e‚ux in the activation of apoptosis. J. Biol. Chem 272, 32426±32442. Bossenmeyer-Pourie, C., Daval, J.L., 1998. Prevention from hypoxiainduced apoptosis by pre-conditioning: a mechanistic approach in cultured neurons from fetal rat forebrain. Mol. Brain Res 58, 237±239. Bossenmeyer-Pourie, C., Chihab, R., Schroeder, H., Daval, J.L., 1999. Transient hypoxia may lead to neuronal proliferation in the developing mammalian brain: from apoptosis to cell cycle completion. Neuroscience 91, 221±231. Bossy-Wetzel, E., Newmeyer, D.D., Green, D.R., 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-speci®c caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 17, 37±49. Botchinka, G.I., Meistrtell, M.E., Botchinka, I.L., Tracey, K.J., 1997. Expression of TNF and TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia. Mol. Med 3, 765±781. Boyd, J.M., Gallo, G.J., Elangovan, B., Houghton, A.B., Malstrom, S., Avery, B.J., Ebb, R.G., Subramanian, T., Chittenden, T., Lutz, R.J., et al., 1995. Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. Oncogene 11, 1921±1928. Brady, H.J., Salomons, G.S., Bobeldijk, R.C., Berns, A.J., 1996. T cells from bax alpha transgenic mice show accelerated apoptosis in response to stimuli but do not show restored DNA-damaged induced cell death in the absence of p53. Embo J 15, 1221±1230. Buckbinder, L., Talbott, R., Valesco-Miguel, S., Takenaka, I., Faha, B., Seizinger, B.R., Kley, N., 1995. Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377, 646±649. Calmels, S., Hainaut, P., Ohshima, H., 1998. Nitric oxide induces conformational and functional modi®cations of wild type p53 tumor suppressor protein. Cancer Res 15, 3365±3369. Collaco-Moraes, Y., Aspey, B.S., deBellaroche, J.S., Harrison, M.J., 1994. Focal ischemia causes an extensive induction of immediate early genes that are sensitive to MK-801. Stroke 25, 1855±1860. Carmeliet, P., Dor, Y., Herbert, J.M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C.J., Ratcli€e, P., Moons, L., Jain, R.K., Collen, D., Keshet, E., 1998. Role of HIF-1alpha in hypoxiamediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394, 485±490. Carmody, R.M., McGowan, A.J., Cotter, T.G., 1999. Reactive oxygen species as mediators of photoreceptor apoptosis in vitro. Exp. Cell Res 248, 520±530. Carrier, F., Smith, M.L., Bae, I., Kilpatrick, K.E., Lansing, T.J.,

238

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249

Chen, C.Y., 1994. Characterization of gadd45, a p53-regulated protein. J. Biol. Chem 269, 32672±32677. Carrier, F., Georgel, P.T., Pourquier, P., Blake, M., Kontny, H.U., Antinore, M.J., Gariboldi, M., Myers, T.G., Weinstein, J.N., Pmmier, Y., Fornace, A.J., 1999. Gadd45, a p53-responsive stress protein, modi®es DNA accessibility on damaged chromatin. Mol. Cell. Biol 19, 1673±1685. Casaccia-Bonne®l, P., Gu, C., Khursigara, G., Chao, M.V., 1999. p75 neurotrophin receptor as a modulator of survival and death decisions. Micr. Res. Tech 45, 217±224. Chakrabarti, S., Hoque, A.N., Karmazyn, M., 1997. A rapid ischemia-induced apoptosis in isolated rat hearts and its attenuation by the sodium-hydrogen exchange inhibitor HOE 642 (cariporide). J. Mol. Cell Cardiol 29, 3169±3174. Chao, D.T., Linette, G.P., Boise, L.H., White, L.S., Thompson, C.B., Korsmeyer, S.J., 1995. Bcl-XL and Bcl-2 repress a common pathway of cell death. J. Exp. Med 182, 821±828. Chao, D.T., Korsmeyer, S.J., 1998. Bcl-2 family: regulators of cell death. Annu. Rev. Immunol 16, 395±419. Chao, G., Zhen, Q., Lorris Betz, A., Xiao-Hong, L., Guo-Yan, Y., 1998. Cellular localization of tumor necrosis alpha following focal cerebral ischemia in mice. Brain Res 801, 1±8. Chen, I.T., Smith, M.L., O'Connor, P.M., Fornace, A.J., 1995a. Direct intersction of gadd45 with PCNA and evidence for competitive interaction of gadd45 and p21Waf1/Cip1 with PCNA. Oncogen 11, 1931±1937. Chen, J., Graham, S.H., Chan, P.H., Lan, J., Zhou, R.L., Simon, R.P., 1995b. bcl-2 is expressed in neurons that survive focal ischemia in the rat. Neuroreport 6, 394±398. Chen, J., Zhu, R.L., Nakayama, M., Kawaguchi, K., Jin, K., Stetler, R.A., Simon, R.P., Graham, S.H., 1996a. Expression of the apoptosis-e€ector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons following global ischemia. J. Neurochem 67, 64±71. Chen, P., Nordstrom, W., Gish, B., Abrams, J.M., 1996b. Grim, a novel cell death gene in Drosophila. Genes Dev 10, 1773±1782. Chen, J., Graham, S.H., Nakayama, M., Zhu, R.L., Jin, K., Stetler, R.A., Simon, R.P., 1997. Apoptosis repressor genes Bcl-2 and Bcl-x-long are expressed in the rat brain following global ischemia. J. Cereb. Blood Flow Metab 17, 2±10. Chen, J., Uchimura, K., Stetler, R.A., Zhu, R.L., Nakayama, M., Jin, K., Graham, S.H., Simon, R.P., 1998a. Transient global ischemia triggers expression of the DNA damage-inducible gene gadd45 in the rat brain. J. Cereb. Blood Flow Metab 18, 846± 857. Chen, J., Nagayama, T., Jin, K., Stetler, R.A., Zhu, R.L., Graham, S.H., Simon, R.P., 1998b. Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci 18, 4914±4928. Cheng, B., Mattson, M.P., 1994. NT-3 and BDNF protect CNS neurons against metabolic/excitotoxic insults. Brain Res 640, 56± 67. Cheng, B., Mattson, M.P., 1991. NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemic damage by stabilizing calcium homeostasis. Neuron 7, 1031±1041. Cheng, E.H., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B., Bedi, A., Ueno, K., Hardwick, J.M., 1997. Conversion of Bcl-2 to a Bax-like death e€ector by caspases. Science 278, 1966±1968. Cheng, Y., Deshmukh, M., D'Costa, A., Demaro, J.A., Gidday, J.M., Shah, A., Sun, Y., Jacquin, M.F., Johnson, E.M., Holtzman, D.M., 1998. Caspase inhibitor a€ords neuroprotection with delayed administration in a rat model of neonatal hypoxicischemic brain injury. J. Clin. Invest 101, 1992±1999. Cheung, N.S., Carroll, F.Y., Larm, J.A., Beart, P.M., Giardina, S.F., 1998. Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells. J. Neurosci. Res 52, 69±82.

Chicheportiche, Y., Bourdon, P.R., Xu, H., Hsu, Y.M., Scott, H., Hession, C., Garcia, I., Browning, J.L., 1997. TWEAK, a new secreted ligand in the tumor necrosis factor family tha weakly induces apoptosis. J.Biol. Chem 272, 32401±32410. Chihab, R., Bossenmeyer, C., Oillet, J., Daval, J.L., 1998. Lack of correlation between the e€ects of transient exposure to glutamate and those of hypoxia/reoxygenation in immature neurons in vitro. J. Neurochem 71, 1177±1186. Chin, L.S., Park, C.C., Zitnay, K.M., Sinha, M., DiPatri Jr, A.J., Perillan, P., Simard, J.M., 1997. J. Neurosci. Res 48, 122±127. Chinnaiyan, A.M., O'Rourke, K., Tewari, M., Dixit, V.M., 1995. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81, 505± 512. Chinnaiyan, A.M., Tepper, C.G., Seldin, M.F., O'Rourke, K., Kischkel, F.C., Hellbardt, S., Krammer, P.H., Peter, M.E., Dixit, V.M., 1996. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J. Biol. Chem 271, 4961±4965. Chittenden, T., Harrington, E.A., O'Connor, R., Flemington, C., Lutz, R.J., Evan, G.I., Guild, B.C., 1995a. Induction of apoptosis by the Bcl-2 homologue Bak. Nature 374, 733±736. Chittenden, T., Flemington, C., Houghton, A.B., Ebb, R.G., Gallo, G.J., Elangovan, B., Chinnadurai, G., Lutz, R.J., 1995b. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 14, 5589±5596. Chiu, R., Boyle, W.J., Meek, J., Smeal, T., Hunter, T., Karin, M., 1988. The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1-responsive genes. Cell 54, 541±552. Chiu, R., Angel, P., Karin, M., 1989. Jun B di€ers in its biological properties from, and is a nefgative regulator of c-jun. Cell 59, 979±986. Choi, D.W., 1987. Ionic dependence of glutamate neurotoxicity. J. Neurosci 7, 369±379. Choi, D.W., 1996. Ischemia-induced neuronal apoptosis. Curr. Opin. Neurobiol 5, 667±672. Chow, E., Haddad, G.G., 1998. Di€erential e€ects of anoxia and glutamate on cultured neocortical neurons. Exp. Neurol 150, 52± 59. Chun, L.L., Patterson, P.H., 1977. Role of nerve growth factor in the development of rat sympathetic neurons in vitro. J. Cell Biol 75, 705±711. Cleary, M.L., Sklar, J., 1985. Nucleotide sequence of a t(14:18) chromosomal breakpoint in follicular lymphoma and a demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18. Proc. Natl. Acad. Sci. USA 82, 7439±7443. Clem, R.J., Cheng, E.H., Karp, C.L., Kirsch, D.G., Ueno, K., Takahashi, A., Kastan, M.B., Grin, D.E., Earnshaw, W.C., Veliuona, M.A., Hardwick, J.M., 1998. Modulation of cell death by Bcl-XL through caspase interaction. Proc. Natl. Acad. Sci. USA 95, 554±559. Clemens, J.A., Stephenson, D.T., Smalstig, E.B., Dixon, E.P., Little, S.P., 1997a. Global ischemia activates nuclear factor-kappa B in forebrain neurons of rats. Stroke 28, 1073±1080. Clemens, J.A., Stephenson, D.T., Dixon, E.P., Smalstig, E.B., Mincy, R.E., Rash, K.S., Little, S.P., 1997b. Global cerebral ischemia activates nuclear factor-kappa B prior to evidence of DNA fragmentation. Mol. Brain Res 48, 187±196. Colom, L.V., Diaz, M.E., Beers, D.R., Neely, A., Xie, W.J., Appel, S.H., 1998. Role of potassium channels in amyloid-induced cell death. J. Neurochem 70, 1925±1934. Cosulich, S.C., Worrall, V., Hedge, P.J., Green, S., Clarke, P.R., 1997. Regulation of apoptosis by BH3 domains in a cell-free system. Curr. Biol 7, 913±920. Crook, T., Parker, G.A., Rozycka, M., Crossland, S., Allday, M.J., 1998. A transforming p53 mutant, which binds DNA, transacti-

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 vates and induces apoptosis reveals a nuclear:cytoplasmic shuttling defect. Oncogene 16, 1429±1441. Cryns, V., Yuan, J., 1998. Proteases to die for. Genes Dev 12, 1551± 1570. D'Sa-Eipper, C., Subramanian, T., Chinnadurai, G., 1996. b¯-1, a bcl-2 homologue, suppresses p53-induced apoptosis and exhibits potent cooperative transforming activity. Cancer Res 56, 3879± 3882. D'Sa-Eipper, C., Chinnadurai, G., 1998. Functional dissection of B¯-1, a Bcl-2 homolog: anti-apoptosis, oncogene-cooperation and cell proliferation activities. Oncogene 16, 3105±3114. Davis, P.K., Johnson, V.W., 1999. Energy metabolism and protein phosphorylation during apoptosis: a phosphorylation study of tau and high-molecular-weight tau in di€erentiated PC 12 cells. Biochem. J 340, 51±58. Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Ko¯er, R., Kroemer, G., 1997. Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res 57, 62±67. DeCoster, M.A., Schabelman, E., Tombran-Tink, J., Bazan, N.G., 1999. Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons. J. Neurosci. Res 56, 604±610. Delphin, C., Cahen, P., Lawrence, J.J., Baudier, J., 1994. Characterization of baculovirus recombinant wild type p53. Dimerization of p53 is required for high-anity DNA binding and cysteine oxidation inhibits p53 DNA binding. Eur. J. Biochem 223, 683±692. Derijard, B.B., Hibi, M., Wu, I.H., Barrett, T., Su, B., Deng, T., Karin, M., Davis, R.J., 1994. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-jun activation domain. Cell 76, 1025±1037. Didier, M., Bursztajn, S., Adamec, E., Passani, L., Nixon, R.A., Coyle, J.T., Wei, J.Y., Berman, S.A., 1996. DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures. J. Neurosci 16, 2238±2250. Distelhorst, C.W., McCormick, T.S., 1996. Bcl-2 acts subsequent to and independent of Ca2+ ¯uxes to inhibit apoptosis in thapsigargin- and glucocorticoid-treated mouse lymphoma cells. Cell Calcium 19, 473±483. Dixon, E.P., Stephenson, D.T., Clemens, J.A., Little, S.P., 1997. BclXshort is elevated following severe global ischemia in rat brains. Brain Res 776, 222±229. Dragunow, M., Young, D., Hughes, P., MacGibbon, G., Lawlor, P., Singleton, K., Sirimanne, E., Gluckman, P., 1993. Is c-jun involved in nerve cell death following status epilepticus and hypoxic-ischemic brain injury? Mol. Brain Res 18, 347±352. Dragunow, M., Beilharz, E., Sirimanne, P., Lawlor, P., Williams, C., Bravo, R., Gluckman, P., 1994. Immediate-early gene protein expression in nueorns undergoing delayed death, but not necrosis, following hypoxic-ischemic injury to the young rat brain. Mol. Brain Res 25, 19±33. 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 glutamatemediated apoptosis of cultured cerebellar granule neurons. Proc. Natl. Acad. Sci. U.S.A 94, 11657±11662. Dubinsky, J.M., Kristal, B.S., Elizondo-Fournier, M., 1995. An obligate role for oxygen in the early stages of glutamate-induced delayed neuronal death. J. Neurosci 15, 7071±7078. El-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B., 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817±825. Elangovan, B., Chinnadurai, G., 1997. Functional dissection of the pro-apoptotic protein Bik. Heterodimerization with anti-apoptosis

239

proteins is insucient for induction of cell death. J. Biol. Chem 272, 24494±24498. Eskes, R., Antonsson, B., Osen-Sand, A., Montessuit, S., Richter, C., Sadoul, R., Mazzei, G., Nichols, A., Martinou, J.C., 1998. Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions. J. Cell Biol 143, 217±224. Fabisiak, J.P., Kagan, V.E., Ritov, V.B., Johnson, D.E., Lazo, J.S., 1997. Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis. Am. J. Physiol 272, 675±684. Ferrer, I., Pozas, E., Lopez, E., Ballabriga, J., 1997. Bcl-2, Bax and Bcl-x expression following hypoxia-ischemia in the infant rat brain. Acta Neuropathol 94, 583±589. Ferrer, I., Lopez, E., Blanco, R., Rivera, R., Ballabriga, J., Pozas, E., Marti, E., 1998. Bcl-2, Bax, and Bcl-x expression in the CA1 area of the hippocampus following transient forebrain ischemia in the adult gerbil. Exp. Brain Res 121, 167±173. Figiel, I., Kaczmarek, L., 1997. Cellular and molecular correlates of glutamate-evoked neuronal programmed cell death in the in vitro cultures of rat hippocampal dentate gyrus. Neurochem 31, 229± 240. Finucane, D.M., Bossy-Wetzel, E., Waterhouse, N.J., Cotter, T.G., Green, D.R., 1999. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by BclxL. J. Biol. Chem 274, 2225±2233. Friedman, J.E., Haddad, G.G., 1993. Major di€erences in Cai2‡ between neonatal and adult rat CA1 neurons in response to anoxia. J. Neurosci 13, 63±72. Friedman, J.E., Haddad, G.G., 1994a. Anoxia induces an increase in intracellular sodium in rat central neurons. Brain Res 663, 329± 334. Friedman, J.E., Haddad, G.G., 1994b. Removal of extracellular sodium prevents anoxia-induced injury in rat CA1 hippocampal neurons. Brain Res 641, 57±64. Furlong, I.J., Ascaso, R., Rivas, A.L., Collins, M.K.L., 1997. Intracellular acidi®cation induces apoptosis by stimulating ICElike protease activity. J. Cell Science 110, 653±661. Furlong, I.J., Lopez-Mediavilla, C., Ascaso, R., Lopez-Rivas, A., Collins, M.K., 1998. Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidi®cation. Cell Death Di€er 5, 214±221. Gary, D.S., Bruce-Keller, A.J., Kindy, M.S., Mattson, M.P., 1998. Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J. Cerb. Blood Flow Metab 18, 1283±1287. Gass, P., Spranger, M., Herdegen, T., Kck, P., Bravo, R., Hacke, W., Kiessling, M., 1992. Induction of fos and jun proteins after focal ischemia in rat: di€rential e€ect of the N-methyl-D-asparte receptor antagonist MK801. Acta Neuropathol 84, 545±553. Gavrieli, Y., Sherman, Y., Ben-Sasson, S.A., 1992. Identi®cation of programmed cell death in situ via speci®c labeling of nuclear DNA fragmentation. J. Cell Biol 119, 493±501. Giaccia, A.J., Kastan, M.B., 1998. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev 12, 2973±2983. Gibson, L., Holmgreen, S.P., Huang, D.C., Bernard, O., Copeland, N.G., Jenkins, N.A., Sutherland, G.R., Baker, E., Adams, J.M., Cory, S., 1996. bcl-w, a novel member of the bcl-2 family, promotes cell survival. Oncogene 13, 665±675. Gil-Gomez, G., Berns, A., Brady, H.J., 1998. A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis. Embo J 17, 7209±7218. Gillardon, F., Lenz, C., Waschke, K.F., Krajewski, S., Reed, J.C., Zimmermann, M., Kuschinsky, W., 1996. Altered expression of Bcl-2, Bcl-X, Bax, and c-Fos colocalizes with DNA fragmentation

240

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249

and ischemic cell damage following middle cerebral artery occlusion in rats. Mol. Brain Res 49, 254±260. Goga, A., Liu, X., Hambuch, T.M., Senechal, K., Major, E., Berk, A.J., Witte, O.N., Sawyers, C.L., 1995. p53-dependent growth suppression by c-abl nuclear tyrosine kinase. Oncogene 11, 791± 799. Goldman, S.C., Chen, C.Y., Lansing, T.J., Gilmer, T.M., Kastan, M.B., 1996. The p53 signal transduction pathway is intact in human neuroblastoma despite cytoplamsic localization. Am. J. Pathol 148, 1381±1385. Gonzalez-Garcia, M., Garcia, I., Ding, L., O'Shea, S., Boise, L.H., Thompson, C.B., Nunez, G., 1995. bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death. Proc. Natl. Acad. Sci. USA 92, 4304±4308. Goping, I.S., Gross, A., Lavoie, J.N., Nguyen, M., Jemmerson, R., Roth, K., Korsmeyer, S.J., Shore, G.C., 1998. Regulated targeting of BAX to mitochondria. J. Cell Biol 143, 207±215. Gottron, F.J., Ying, H.S., Choi, D.W., 1997. Caspase inhibition selectively reduces the apoptotic component of oxygen±glucose deprivation-induced cortical neuronal cell death. Mol. Cell Neurosci 9, 159±169. Graeber, T.G., Peterson, J.F., Tsai, M., Fornace, A.J., Giaccia, A.J., 1994. Hypoxia induces the accumulation of p53 protein, but activation of G1-phase checkpoint by low oxygen conditions is independent of p53 status. Mol. Cell Biol 14, 6264±6277. Graybiel, A.M., Moratalla, R., Robertson, H.A., 1990. Amphetamine and cocaine induce drug-speci®c activation of the c-fos gene in striasome matrix compartments and limbic subdivisions of the striatum. Proc. Natl. Acad. Sci 87, 6912±6916. Greenberg, M.E., Greene, L.A., Zi€, E.B., 1985. Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells. J. Biol. Chem 260, 14101±14110. Greenberg, M.E., Zi€, E.B., Greene, L.A., 1986. Stimualtion of the neuronal acetylcholine receptors induces rapid gene transcription. Science 234, 80±83. Griths, G.J., Dubrez, L., Morgan, C.P., Jones, N.A., Whitehouse, J., Corfe, B.M., Dive, C., Hickman, J.A., 1999. Cell damageinduced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J. Cell Biol 144, 903±913. Grillot, D.A., Gonzalez-Garcia, M., Ekhterae, D., Duan, L., Inohara, N., Ohta, S., Seldin, M.F., Nunez, G., 1997. Genomic organization, promoter region analysis, and chromosome localization of the mouse bcl-x gene. J. Immunol 158, 4750±4757. Gross, A., Jockel, J., Wei, M.C., Korsmeyer, S.J., 1998. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. Embo J 17, 3878±3885. Gross, A., Yin, X.M., Wang, K., Wei, M.C., Jockel, J., Milliman, C., Erdjument-Bromage, H., Tempst, P., Korsmeyer, S.J., 1999. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem 274, 1156± 1163. Gu, W., Roeder, R.G., 1997. Activation of p53 sequence speci®c binding by acetylation of the p53 C-terminal domain. Cell 90, 595±606. Gubits, R.M., Burke, R.E., Casey-MacIntosh, E., Bandele, A., Munell, F., 1993. Immediate-early genes induction after neonatal hypoxia-ischemia. Mol. Brain Res 18, 228±238. Guegan, C., Ceballos-Picot, I., Chevalier, E., Nicole, A., Onteniente, B., Sola, B., 1999. Reduction of ischemic damage in NGF-transgenic mice: correlation with enhancement of antioxidant enzyme activities. Neurobiol. Dis 6, 180±189. Gwag, B.J., Lobner, D., Koh, J.Y., Wie, M.B., Choi, D.W., 1995. Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen±glucose deprivation in vitro. Neurosci 68, 615±619. Haddad, G.G., Jiang, C., 1993. Mechanisms of anoxia-induced de-

polarization in brainstem neurons: in-vitro current and voltage clamp studies in the adult rat. Brain Res 625, 261±268. Hagg, T., Varon, S., 1993. Ciliary neurotrophic factor prevents degeneration of adult rat substantia nigra dopaminergic neurons in vivo. Proc. Nat. Acad. Sci. USA 90, 6315±6319. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M., Mak, T.W., 1998. Di€erential requirement for caspase-9 in apoptotiv pathways in vivo. Cell 94, 339±352. Hainaut, P., Milner, J., 1993a. A structural role for metal ions in the ``wild-type'' conformation of the tumor suppressor protein p53. Cancer Res 53, 1739±1742. Hainaut, P., Milner, J., 1993b. Redox modulation of p53 conformation and sequence speci®c DNA binding in vitro. Cancer Res 53, 4469±4473. Hall, P.A., Kearsey, J.M., Coates, P.J., Norman, D.G., Warbrick, E., Coz, L.S., 1995. Characterization of the interaction between PCNA and gadd45. Oncogene 10, 2427±2433. Hall, S.R., Campbell, L.E., Meek, D.W., 1996. Phosphorylation of p53 at the casein kinase II site selectively regulates p53-dependent transcriptional repression but not transactivation. Nucleic Acids Res 24, 1119±1126. Hansen, R., Oren, M., 1997. p53: from inductive signal to cellular e€ect. Curr. Opin. Genet. Cell Dev 7, 46±51. Haupt, Y., Maya, R., Kazaz, A., Oren, M., 1997. Mdm2 promotes the rapid degradation of p53. Nature 387, 296±299. Hara, A., Iwai, T., Niwa, M., Uematsu, T., Yoshimi, N., Tanaka, T., Mori, H., 1996. Immunohistochemical detection of Bax and Bcl-2 proteins in gerbil hippocampus following transient forebrain ischemia. Brain Res 711, 249±253. He, H., Lam, M., McCormick, T.S., Distelhorst, C.W., 1997. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J. Cell Biol 138, 1219±1228. Heacock, C.S., Sutherland, R.M., 1986. Induction characteristics of oxygen regulated proteins. Int. J. Radiat. Oncol. Biol. Phys 12, 1287±1290. Hegde, R., Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T., Alnemri, E.S., 1998. Blk, a BH3-containing mouse protein that interacts with Bcl-2 and Bcl-xL, is a potent death agonist. J. Biol. Chem 273, 7783±7786. Heiden, M.G., Chandel, N.S., Schumacker, P.T., Thompson, C.B., 1999. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Mol. Cell 3, 159±167. Hecker, D., Page, G., Lohrum, M., Weiland, S., Scheidtmann, K.H., 1996. Complex regulation of the DNA-binding activity of p53 by phosphorylation: di€erential e€ects of individual phosphorylation sites on the interaction with di€erent binding motifs. Oncogene 12, 953±961. Herdegen, T., Leah, J.D., 1998. Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by jun, fos, and krox, and CREB/ATF proteins. Brain Res. Rev 28, 370±490. Herr, I., Martin-Villalba, A., Kurz, E., Roncaioli, P., Schenkel, J., Cifone, M.G., Debatin, K.M., 1999. FK506 prevents strokeinduced generation of ceramide and apoptosis signaling. Brain Res 826, 210±219. Hibi, M., Lin, A., Smeal, T., Minden, A., Karin, M., 1993. Identi®cation of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-jun activation domain. Genes Dev 7, 2135±2148. Hida, H., Takeda, M., Soliven, B., 1998. Ceramide inhibits inwardly rectifying K+ currents via a Ras-and Raf-1-dependent pathway in cultured oligodendrocytes. J. Neurosci 18, 8712±8719. Hill, I.E., MacManus, J.P., Rasquinha, I., Tuor, U.I., 1995. DNA

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat. Brain Res 676, 398±403. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D., Korsmeyer, S.J., 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334±336. Hockenbery, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241±251. Hohfeld, J., Jentsch, S., 1997. GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1. Embo J 16, 6209± 6216. Holinger, E.P., Chittenden, T., Lutz, R.J., 1999. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. J. Biol. Chem 274, 13298±13304. Hollander, M.C., Alamo, I., Jackman, J., Wang, M.G., McBride, O.W., Fornace, A.J., 1993. Analysis of the GADD45 gene and its response to DNA damage. J. Biol. Chem 268, 24385±24393. Hollstein, M., Rice, K., Greenblatt, M.S., Soussi, T., Fuchs, R., Sorli, T., Hovig, E., Smith-Sorensen, B., Montessano, R., Harris, C.C., 1994. Databsae of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res 22, 3551±3555. Honda, R., Tanaka, H., Yasuda, H., 1997. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 420, 25± 27. Honkaniemi, J., States, B.A., Weinstein, P.R., Espinoza, J., Sharp, F.R., 1997. Expression of zinc ®nger immediate early genes in rat brain after permanent middle cerebral artery occlusion. J. Cereb. Blood Flow Metab 17, 636±646. Hou, S.T., Tu, Y., Buchan, A.M., Huang, Z., Preston, E., Rasquinha, I., Robertson, G.S., MacManus, J.P., 1997. Increases in DNA lesions and the DNA damage indicator gadd45 following transient cerebral ischemia. Biochem. Cell Biol 75, 383±392. Hsu, H., Shu, H.B., Pan, M.G., Goeddel, D.V., 1996a. TRADDTRAF2 and TRADD-FADD interactions de®ne two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299±308. Hsu, H., Huang, J., Shu, H.B., Baichwal, V., Goeddel, D.V., 1996b. TNF- dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387±396. Hsu, S.Y., Kaipia, A., McGee, E., Lomeli, M., Hsueh, A.J., 1997a. Bok is a pro- apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc. Natl. Acad. Sci. USA 94, 12401±12406. Hsu, Y.T., Wolter, K.G., Youle, R.J., 1997b. Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc. Natl. Acad. Sci. USA 94, 3668±3672. Hsu, S.Y., Lin, P., Hsueh, A.J., 1998. BOD (Bcl-2-related ovarian death gene) is an ovarian BH3 domain-containing proapoptotic Bcl-2 protein capable of dimerization with diverse antiapoptotic Bcl-2 members. Mol. Endocrinol 12, 1432±1440. Hu, Y., Benedict, M.A., Wu, D., Inohara, N., Nunez, G., 1998. BclXL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase9 activation. Proc. Natl. Acad. Sci. USA 95, 4386±4391. Hughes, P., Dragunow, M., 1995. Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmac. Rev 47, 133±178. Hughes, P.E., Alexi, T., Walton, M., Williams, C.E., Dragunow, M., Clark, R.G., Gluckman, P.D., 1998. Activity and injury-dependent expression of inducible transcription factors, growth factors, and apoptosis-related genes within the central nervous system. Prog. Neurobiol 57, 421±450. Hupp, T.R., Lane, D.P., 1994. Regulation of the crytic sequencespeci®c DNA binding function of p53 by protein kinases. Cold Spring Harb. Symp. Quant. Biol 59, 195±206. Ibanez, C.F., Ebendal, T., Barbany, G., Murray-Rust, J., Blundell, T.L., Persson, H., 1992. Disruption of the low anity receptor-

241

binding site in NGF allows neuronal survival and di€erentiation by binding to the trk gene product. Cell 69, 329±341. Ikeda, J., Terakawa, S., Murota, S., Morita, I., Hirakawa, K., 1996. Nuclear disintegration as a leading step of glutamate excitotoxicity in brain neurons. J. Neurosci. Res 43, 613±622. Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., Dikranian, K., Tenkova, T.I., Stefovska, V., Turski, L., Olney, J.W., 1999. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283, 70±74. Inohara, N., Ding, L., Chen, S., Nunez, G., 1997. harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L). Embo J 16, 1686±1694. Inohara, N., Gourley, T.S., Carrio, R., Muniz, M., Merino, J., Garcia, I., Koseki, T., Hu, Y., Chen, S., Nunez, G., 1998a. Diva, a Bcl-2 homologue that binds directly to Apaf-1 and induces BH3-independent cell death. J. Biol. Chem 273, 32479±32486. Inohara, N., Ekhterae, D., Garcia, I., Carrio, R., Merino, J., Merry, A., Chen, S., Nunez, G., 1998b. Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL. J. Biol. Chem 273, 8705±8710. Isenmann, S., Stoll, G., Schroeter, M., Krajewski, S., Reed, J.C., Bahr, M., 1998. Di€erential regulation of Bax, Bcl-2, and Bcl-X proteins in focal cortical ischemia in the rat. Brain Pathol 8, 49± 62. Ishaque, A., Al-Rubeai, M., 1998. Use of intracellular pH and annexin-V ¯ow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J. Immunol. Methods 221, 43±57. Jayamaran, L., Murthy, K.G., Zhu, C., Curran, T., Xanthoudakis, S., Prives, C., 1997. Identi®cation of redox/repair protein ref-1 as a potent activator of p53. Genes Dev 11, 558±570. Jin, K., Chen, J., Kawaguchi, K., Zhu, R.L., Stetler, R.A., Simon, R.P., Graham, S.H., 1996. Focal ischemia induces expression of the DNA damage-inducible gene GADD45 in the rat brain. Neuroreport 7, 1707±1802. Johnson, D., Lanahan, A., Buck, C.R., Sehgal, A., Morgan, C., Mercer, E., Bothwell, M., Chao, M., 1986. Expression and structure of the human NGF receptor. Cell 47, 545±554. Joo, C.K., Choi, J.S., Ko, H.W., Park, K.Y., Sohn, S., Chun, M.H., Oh, Y.J., Gwag, B.J., 1999. Necrosis and apoptosis after retinal ischemia: involvement of NMDA-mediated excitotoxicity and p53. Invest. Ophthalmol. Vis. Sci 40, 713±720. Juin, P., Pelletier, M., Oliver, L., Tremblais, K., Gregoire, M., Me¯ah, K., Vallette, F.M., 1998. Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system. J. Biol. Chem 273, 17559±17564. Jurgensmeier, J.M., Krajewski, S., Armstrong, R.C., Wilson, G.M., Oltersdorf, T., Fritz, L.C., Reed, J.C., Ottilie, S., 1997. Bax- and Bak-induced cell death in the ®ssion yeast Schizosaccharomyces pombe. Mol. Biol. Cell 8, 325±339. Jurgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., Reed, J.C., 1998. Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. USA 95, 4997±5002. Kamijo, T., Weber, J.D., Zambetti, G., Zindy, F., Rossel, M.F., Sherr, C.J., 1998. Functional and physical interactions of the ARF tumor suppressor with p53 and mdm2. Proc. Natl Acad. Sci. USA 95, 8292±8297. Kane, D.J., Sara®an, T.A., Anton, R., Hahn, H., Gralla, E.B., Valentine, J.S., Ord, T., Bredesen, D.E., 1993. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262, 1274±1277. Kasof, G.M., Goyal, L., White, E., 1999. Btf, a novel death-promoting transcriptional repressor that interacts with Bcl-2-related proteins. Mol. Cell Biol 19, 4390±4404. Kastan, M.B., Onyekwere, O., Sidransky, D., Vogelstein, B., Craig,

242

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249

R.W., 1991. Participation of p53 in the cellular response to DNA damage. Cancer Res 51, 6304±6311. Kastan, M.B., Zhan, Q., El-Deiry, W., Carrier, F., Jacks, T., Walsh, W.V., Plunkett, B.S., Vogelstein, B., Fornace, A.J., 1992. A mammalian cell cycle checkpoint pathway utilizing p53 and gadd45 is defective in ataxia-telangiectasia. Cell 71, 587±597. Kearsey, J.M., Coates, P.J., Prescott, A.R., Warbrick, E., Hall, P.A., 1995. Gadd45 is a nuclear cell cycle regulated protein which interacts withp21/cip1. Oncogene 11, 1675±1683. Kelekar, A., Chang, B.S., Harlan, J.E., Fesik, S.W., Thompson, C.B., 1997. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol. Cell Biol 17, 7040±7046. Kharbanda, S., Pandey, P., Scho®eld, L., Israels, S., Roncinske, R., Yoshida, K., Bharti, A., Yuan, Z.M., Saxena, S., Weichselbaum, R., Nalin, C., Kufe, D., 1997. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis. Proc. Natl. Acad. Sci. USA 94, 6939±6942. Kiefer, M.C., Brauer, M.J., Powers, V.C., Wu, J.J., Umansky, S.R., Tomei, L.D., Barr, P.J., 1995. Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature 374, 736±739. Kim, C.N., Wang, X., Huang, Y., Ibrado, A.M., Liu, L., Fang, G., Bhalla, K., 1997. Overexpression of Bcl-X(L) inhibits Ara-Cinduced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis. Cancer Res 57, 3115±3120. Kinouchi, H., Sharp, F.R., Chan, P.H., Koistinaho, A., Sagar, S.M., Yoshimoto, T., 1994. Induction of c-fos, junB, c-jun, and hsp70 mRNA in cortec, thalamus, basal ganglia, and hippocampus following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab 14, 808±817. Kitagawa, K., Matsumoto, M., Tsujimoto, Y., Ohtsuki, T., Kuwabara, K., Matsushita, K., Yang, G., Tanabe, H., Martinou, J.C., Hori, M., Yanagihara, T., 1998. Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice. Stroke 29, 2616±2621. Kitanaka, C., Namiki, T., Noguchi, K., Mochizuki, T., Kagaya, S., Chi, S., Hayashi, A., Asai, A., Tsujimoto, Y., Kuchino, Y., 1997. Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: di€erential e€ects of Bcl-2 and Bcl-xL on Baxinduced caspase activation and apoptosis. Oncogene 15, 1763± 1772. Kiyota, Y., Takami, K., Iwane, M., Shino, A., Miyamoto, M., Tsukuda, R., Nagaoka, A., 1991. Increased in basic ®broblastt growth factor-like immunoreactivity in rat brain after forebrain ischemia. Brain Res 545, 322±328. 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, 1132±1136. Knippschild, U., Milne, D.M., Campbell, L.E., DeMaggio, A.J., Christenson, E., Hoekstra, M.F., Meek, D.W., 1997. p53 is phosphorylated in vitro and in vivo by the delta and epsilon forms of casein kinase I and enhances the level of casein kinase delta in response to topisomerase-directed drugs. Oncogene 15, 1727±1736. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J., Williams, L.T., 1997. Caspase-3-generated fragment of gelsolin: e€ector of morphological change in apoptosis. Science 278, 294±298. Kovary, K., Bravo, R., 1991. Expression of di€erent jun and fos proteins during the G0±G1 transition in mouse ®broblasts: in vitro and in vivo associations. Mol. Cell Biol 11, 2451±2459. Krajewski, S., Tanaka, S., Takayama, S., Schibler, M.J., Fenton, W., Reed, J.C., 1993. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 53, 4701±4714. Krajewski, S., Mai, J.K., Krajewska, M., Sikorska, M.,

Mossakowski, M.J., Reed, J.C., 1995. Upregulation of bax protein levels in neurons following cerebral ischemia. J. Neurosci 15, 6364±6376. Krajewski, S., Krajewski, M., Ellerby, L.M., Welsh, K., Xie, Z., Deveraux, Q.L., Salvesen, G.S., Bredesen, D.E., Rosenthal, R.E., Fiskum, G., Reed, J.C., 1999. Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc. Natl. Acad. Sci. U.S.A 96, 5752±5757. Kruman, I., Guo, Q., Mattson, M.P., 1998. Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells. J. Neurosci. Res 51, 293± 308. Kubbutat, M.H., Jones, S.N., Vousden, K.H., 1997. Regulation of p53 stability by Mdm2. Nature 387, 299±303. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., Su, M.S., Rakic, P., Flavell, R.A., 1998. Reduced apoptosis and caspase-mediated activation in mice lacking caspase-9. Cell 94, 325±337. Kuo, T.H., Kim, H.R., Zhu, L., Yu, Y., Lin, H.M., Tsang, W., 1998. Modulation of endoplasmic reticulum calcium pump by Bcl-2. Oncogene 17, 1903±1910. Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M., Avruch, J., Woddgett, J.R., 1994. The stress-activated protein kinase subfamily of c-jun kinases. Nature 369, 156± 160. Lam, M., Dubyak, G., Chen, L., Nunez, G., Miesfeld, R.L., Distelhorst, C.W., 1994. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ ¯uxes. Proc. Natl. Acad. Sci. USA 91, 6569±6573. Lam, M., Bhat, M.B., Nunez, G., Ma, J., Distelhorst, C.W., 1998. Regulation of Bcl-xl channel activity by calcium. J. Biol. Chem 273, 17307±17310. 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, 617±622. Lavine, S.D., Hofman, F.M., Zlokovic, B.V., 1998. Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury. J. Cereb. Blood Flow Metab 18, 52±58. 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, 486±496. Lawrence, M.S., McLaughlin, J.R., Sun, G.H., Ho, D.Y., McIntosh, L., Kunis, D.M., Sapolsky, R.M., Steinberg, G.K., 1997. Herpes simplex viral vectors expressing Bcl-2 are neuroprotective when delivered after a stroke. J. Cereb. Blood Flow Metab 17, 740± 744. Lees-Miller, S.P., Chen, Y.R., Anderson, C.W., 1990. Human cells contain a DNA- activated protein kinase that phosphorylates simian virus 40T antigen, mouse p53, and the Ku autoantigen. Mol. Cell Biol 10, 6472±6481. 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, 750±764. Levi-Montalcini, R., Booker, B., 1960. Destruction of the sympathetic ganglia in mammals by an anti-serum to the never growth factor. Proc. Nat. Acad. Sci. USA 46, 384±391. Levi-Montalcini, R., 1987. The nerve growth factor 35 years later. Science 237, 154-62. Levi-Montalcini, R., Angeletti, P.U., 1966. Immunosympathectomy. Pharmacol. Rev 18, 619±628. Levine, A.J., 1997. p53, the cellular gatekeeper for growth and division. Cell 88, 328±331. Li, F., Srinivasan, A., Wang, Y., Armstrong, R.C., Tomaselli, K.J., Fritz, L.C., 1997a. Cell-speci®c induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release. J. Biol. Chem 272, 30299±30305.

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 Li, P., Nijhawan, D., Budihardjo, D., Srinivasula, S.M., Ahmad, M., Almeri, E.S., Wang, X., 1997b. Cytochrome c and ATP-dependent formation of Apaf1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479±489. Li, H., Zhu, H., Xu, C.J., Yuan, J., 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491±501. Li, R., Bernd, P., 1999. Neurotrophin-3 increases neurite outgrowth and apoptosis in explants of the chicken neural plate. Dev. Neurosci 21, 12±21. Li, Y., Chopp, M., Jiang, N., Zhang, Z.G., Zaloga, C., 1995. Induction of DNA fragmentation after 10 to 120 min of focal cerebral ischemia in rats. Stroke 26, 1252±1257. Lill, N.L., Grossman, S.R., Ginsberg, D., DeCaprio, J., Livingston, D.M., 1997. Binding and modulation of p53 by p300/CPB coactivators. Nature 387, 823±827. Lin, A., Frost, J., Deng, T., Smeal, T., Al-Alawi, N., Kikkawa, U., Hunter, T., Brenner, D., Karin, M., 1992. Casein kinase II is a negative regulator of c-jun DNA binding and AP-1 activity. Cell 70, 777±789. Lin, T.N., Chen, J.J., Wang, S.J., Cheng, J.T., Chi, S.I., Shyu, A.B., Sun, G.Y., Hsu, C.Y., 1996. Expression of NGFI-B mRNA in a rat focal ischemia-reperfusion model. Mol. Brain Res 43, 149± 156. Lindsay, R.M., 1988. Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. J. Neurosci 8, 2394±2405. Linette, G.P., Li, Y., Roth, K., Korsmeyer, S.J., 1996. Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation. Proc. Natl. Acad. Sci. USA 93, 9545±9552. Linnik, M.D., Zobrist, R.H., Hat®eld, M.D., 1993. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 24, 2002±2008. Linnik, M.D., Zahos, P., Geschwind, M.D., Federo€, H.J., 1995. Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia. Stroke 26, 1670± 1674. Long, X., Boluyt, M.O., Hipolito, M.L., Lundberg, M.S., Zheng, J.S., O'Neill, L., Cirielli, C., Lakatta, E.G., Crow, M.T., 1997. p53 and the hypoxia-induced apoptosis of cardiac myocytes. J. Clin. Invest 99, 2635±2643. Ludwig, R.L., Bates, S., Vousden, K.H., 1996. Di€erential activation of target cellular promotersby p53 mutants with impaired apoptotic function. Mol. Cell Biol 16, 4952±4960. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., Wang, X., 1998. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481±490. Ma, E., Haddad, G.G., 1997. Anoxia regulates gene expression in the central nervous system of Drosophila melanogaster. Mol. Brain Res 46, 325±328. MacManus, J.P., Rasquinha, I., Black, M.A., Laferriere, N.B., Monette, R., Walker, T., Morley, P., 1997. Glutamate-treated rat cortical neuronal cultures die in a way di€erent from the classical apoptosis induced by staurosporine. Exp. Cell Res 233, 310±320. Maheswaran, S., Englert, C., Bennet, P., Heinrich, G., Haber, D.A., 1995. The wt1 gene product stabilizes p53 and inhibits p53mediated apoptosis. Genes Dev 9, 2143±2156. Maki, G.G., Howley, P.M., 1997. Ubiquitination of p53 and p21 is di€erentially a€ected by ionizing radiation and UV radiation. Mol. Cell Biol 17, 355±363. Maltzman, W., Czyzyk, L., 1984. UV radiation stimulates levels of p53 tumor antigen in nontransformed mouse cells. Mol. Cell Biol 4, 1689±1694. Manev, H., Kharlamov, E., Uz, T., Mason, R.P., Cagnoli, C.M.,

243

1997. Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells. Exp. Neurol 146, 171±178. Manon, S., Chaudhuri, B., Guerin, M., 1997. Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these e€ects by coexpression of Bcl-xL. FEBS Lett 415, 29±32. Mariani, S.M., Matiba, B., Armandola, E.A., Krammer, P.H., 1997. Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells. J. Cell Biol 137, 221±229. Marin, M.C., Fernandez, A., Bick, R.J., Brisbay, S., Buja, L.M., Snuggs, M., McConkey, D.J., von Eschenbach, A.C., Keating, M.J., McDonnell, T.J., 1996. Apoptosis suppression by bcl-2 is correlated with the regulation of nuclear and cytosolic Ca2+. Oncogene 12, 2259±2266. Marsters, S.A., Sheridan, J.P., Donahue, C.J., Pitti, R.M., Gray, C.L., Goddard, A.D., Bauer, K.D., Ashkenazi, A., 1996. Apo-3, a new member of the tumor necrosis factor receptor family, contains a death domain and activates apoptosis and NF-kappa B. Curr. Biol 6, 1669±1676. Marsters, S.A., Sheridan, J.P., Pitti, R.M., Brush, J., Goddard, A., Ashkenazi, A., 1998. Identi®cation of a ligand for the deathdomain-containing receptor Apo3. Curr. Biol 8, 525±528. Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry, O.H., Carter, J.G., Johnson, E.M., 1988. Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by never growth factor deprivation. J. Cell Biol 106, 829±844. Martin-Villalba, A., Herr, I., Jeremias, I., Hahne, M., Brandt, R., Vogel, J., Schenkel, J., Herdegen, T., Debatin, K.M., 1999. CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons. J. Neurosci 19, 3809±3817. Martinez-Lorenzo, M.J., Alava, M.A., Gamen, S., Kim, K.J., Chuntharapai, A., Pineiro, A., Naval, J., Anel, A., 1998. Involvment of Apo2 ligand/TRAIL in actication-induced death of Jurkat and human peripheral blood T cells. Eur. J. Immunol 28, 2714±2725. Martinou, J.C., Dubois-Dauphin, M., Staple, J.K., Rodriguez, I., Frankowski, H., Missotten, M., Albertini, P., Talabot, D., Catsicas, S., Pietra, C., et al., 1994. Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 13, 1017±1030. Martinou, I., Missotten, M., Fernandez, P.A., Sadoul, R., Martinou, J.C., 1998. Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons. Neuroreport 9, 15±19. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., Prevost, M.C., Xie, Z., Matsuyama, S., Reed, J.C., Kroemer, G., 1998. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 2027±2031. Mason, R.P., Leeds, P.R., Jacob, R.F., Hough, C.J., Zhang, K.G., Mason, P.E., Chuang, D.M., 1999. Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells. J. Neurochem 72, 1448±1456. Matsushita, K., Matsuyama, T., Kitagawa, K., Matsumoto, M., Yanagihara, T., Sugita, M., 1998. Alterations of Bcl-2 family proteins precede cytoskeletal proteolysis in the penumbra, but not in infarct centres following focal cerebral ischemia in mice. Neuroscience 83, 439±448. Matsuyama, S., Xu, Q., Velours, J., Reed, J.C., 1998. The Mitochondrial F0F1- ATPase proton pump is required for function of the proapoptotic protein Bax in yeast and mammalian cells. Mol. Cell 1, 327±336. Matsuyama, T., Hata, R., Tagay, M., Yamamoto, Y., Nakajima, T., Furuyama, J., Wanaka, A., Sugita, M., 1994. Fas antigen mRNA induction in postischemic murine brain. Brain Res 657, 342±346. Matsuyama, T., Hata, R., Yamamoto, Y., Tagaya, M., Akita, H.,

244

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249

Uno, H., Wanaka, A., Furuyama, J., Sugita, M., 1995. Localization of Fas antigen mRNA induced in postischemic murine forebrain by in situ hybridization. Mol. Brain Res 54, 166± 172. Mattson, M.P., Sche€, S.W., 1994. Endogenous neuroprotection factors and traumatic brain injury: mechanisms of action and implications for therapy. J. Neurotrauma 11, 3±33. McCarthy, N.J., Whyte, M.K., Gilbert, C.S., Evan, G.I., 1997. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol 136, 215±227. McCormick, W., Penman, S., 1969. regulation of protein synthesis in HeLa cells: translation at elevated temperatures. J. Mol. Biol 39, 315±333. McFarlane, M., Ahamad, M., Srinivasula, S.M., Fernandez-Alnemri, T., Cohen, G.M., Alnemri, E.S., 1997. Identi®cation and cloning of two novel receptors for the cytotoxic ligand TRAIL. J.Biol. Chem 272, 25417±25420. Meers, P., Mealy, T., 1993. Calcium-dependent annexin V binding to phospholipids: stoichiometry, speci®city, and the role of negative charge. Biochemistry 32, 11711±11721. Meistrell, M.E., Botchinka, G.I., Wang, H., Di Santo, E., Cockroft, K.M., Bloom, O., Vishnubhakat, J.M., Ghezzi, P., Tracey, K.J., 1997. Tumor necrosis factor is a brain damaging cytokine in cerebral ischemia. Shock 8, 341±348. Merad-Boudia, M., Nicole, A., Santiard-Baron, D., Saille, C., Ceballos-Picot, I., 1998. Mitochondrial impairment as an early event in the process of apoptosis induced by glutathione depletion in neuronal cells: relevance to Parkinson's disease. Biochem. Pharmacol 56, 645±655. Metz, R., Bannister, A.J., Sutherland, J.A., Hagemeier, C., O'Rourke, E.C., Cook, A., Bravo, R., Kouazrides, T., 1994. Cfos-induced activation of a TATA box-containing promoter involvesbdirect contact with TATA-box-binding protein. Mol. Cell Biol 14, 6021±6029. Meyer, K.M., Fiskum, G., Liu, Y., Simmnes, S.J., Bredesesn, D.E., Murphy, A.N., 1995. Bcl-2 protects neural cells from cyanide/ aglycemia-induced lipid, oxidation, mitochondrial injury, and loss of viability. J. Neurochem 65, 2432±2440. Midgley, C.A., Lane, D.P., 1997. p53 protein stability in tumor cells is not determined by mutation but is dependent on mdm2 binding. Oncogene 15, 1179±1189. Milne, D.M., Palmer, R.H., Campbell, D.G., Meek, D.W., 1992. Phoisphorylation of the p53 tumor-suppressor protein at three Nterminal sites by a novel casein kinase-I-like enzyme. Oncogene 7, 1361±1369. Minden, A., Lin, A., Smeal, T., Derijard, B., Cobb, M., Davis, R., Karin, M., 1994. C-jun-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases. Mol. Cell Biol 14, 6683±6688. Mitchell, J.J., Paiva, M., Walker, D.W., Heaton, M.B., 1999. BDNF and NGF a€ord in vitro neuroprotection against ethanol combined with acute ischemia and chronic hypoglycemia. Dev. Neurosci 21, 68±75. Moll, U.M., Riou, G., Levine, A.J., 1992. Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc. Natl. Acad. Sci. USA 89, 7262±7266. Moll, U.M., LaQuaglia, M., Bernard, J., Riou, G., 1995. Wild type p53 undergoes cytoplasmic sequestration in undi€erentiated neuroblastomas but not in di€erentiated tumors. Proc. Natl. Acad. Sci. USA 92, 4407±4411. Momand, J., Zambetti, G.P., Olson, D.C., Geoge, D.L., Levine, A.J., 1992. The mdm-2 oncogene product forms a complex with p53 protein and inhibits p53-mediated transactivation. Cell 69, 12337±12345. Monaghan, P., Robertson, D., Amos, T.A., Dyer, M.J., Mason,

D.Y., Greaves, M.F., 1992. Ultrastructural localization of bcl-2 protein. J. Histochem. Cytochem 40, 1819±1825. Montal, M., 1998. Mitochondria, glutamate neurotoxicity and the death cascade. Biochim. Biophys. Acta 1366, 113±126. Morana, S.J., Wolf, C.M., Li, J., Reynolds, J.E., Brown, M.K., Eastman, A., 1996. The involvement of protein phosphatases in the activation of ICE/CED-3 protease, intracellular acidi®cation, DNA digestion, and apoptosis. J. Biol. Chem 271, 18263±18271. Morgan, J.I., Curran, T., 1986. Role of ion ¯ux in the control of cfos expression. Nature 322, 552±555. Morgan, J.I., Curran, T., 1991. Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu. Rev. Neurosci 14, 421±451. Moss, S.F., Agarwal, B., Arber, N., Guan, R.J., Krajewska, M., Krajewski, S., Reed, J.C., Holt, P.R., 1996. Increased intestinal Bak expression results in apoptosis. Biochem. Biphys. Res. Commun 223, 199±203. Muchmore, S.W., Sattler, M., Liang, H., Meadows, R.P., Harlan, J.E., Yoon, H.S., Nettesheim, D., Chang, B.S., Thompson, C.B., Wong, S.L., Ng, S.L., Fesik, S.W., 1996. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381, 335±341. Murphy, A.N., Bredesen, D.E., Cortopassi, G., Wang, E., Fiskum, G., 1996. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl. Acad. Sci. USA 93, 9893±9898. Muzio, M., Salvesen, G.S., Dixit, V.M., 1997. FLICE-induced apoptosis in a cell-free system, Cleavage of caspase zymogens. J. Biol. Chem 272, 2952±2956. Muzio, M., Stockwell, B.R., Stennicke, H.R., Salvesen, G.S., Dixit, V.M., 1998. An induced proximity model for caspase-8 activation. J. Biol. Chem 273, 2926±2930. Myashita, T., Reed, J.C., 1995. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293± 299. Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355±365. Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan, J., Moskowitz, M.A., 1998. Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci 18, 3659±3668. Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R.J., Matsuda, H., Tsujimoto, Y., 1998. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. USA 95, 14681±14686. Nath, R., Probert Jr, A., McGinnis, K.M., Wang, K.K., 1998. Evidence for activation of caspase-3-like protease in excitotoxinand hypoxia/hypoglycemia-injured neurons. J. Neurochem 71, 186±195. Nawashiro, H., Martin, D., Hallenbeck, J.M., 1997. Neuroprotective e€ects of TNF binding protein in Focal cerebral ischemia. Brain Res 778, 265±271. Neame, S.J., Rubin, L.L., Philpott, K.L., 1998. Blocking cytochrome c activity within intact neurons inhibits apoptosis. J. Cell Biol 142, 1583±1593. Nicholson, D.W., Thorn berry, N.A., 1997. Caspases: killer proteases. Trends Biochem Sci 22, 299±306. Nicotera, P., Zhivotovsky, B., Orrenius, S., 1994. Nuclear calcium transport and the role of calcium in apoptosis. Cell Calcium 16, 279±288. Nicotera, P., Rossi, A.D., 1994. Nuclear Ca2+: physiological regulation and role in apoptosis. Mol. Cell. Biochem 135, 89±98. Nicotera, P., Leist, M., Manzo, L., 1999. Neuronal cell death: a demise with di€erent shapes. Trends Pharmacol. Sci 20, 46±51. Nikolakaki, E., Co€er, P.J., Hemelsoet, R., Woodgett, J.R., De®ze, L.H.K., 1993. Glycogen synthase kinase 3 phosphorylates jun

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 family members in vitro and nebatively regulates their transactivating potential in intact cells. Oncogene 8, 833±840. Nitta, A., Ohmiya, M., Sometani, A., Itoh, M., Nomoto, H., Furukawa, Y., Furukawa, S., 1999. Brain-derived neurotrophic factor prevents neuronal cell death induced by corticosterone. J. Neurosci. Res 57, 227±235. Niwa, M., Hara, A., Iwai, T., Sassa, T., Mori, H., Uematsu, T., 1997. Expression of Bax and Bcl-2 protein in the gerbil hippocampus following transient forebrain ischemia and its modi®cation by phencyclidine. Neurol Res 19, 629±633. Ng, F.W., Nguyen, M., Kwan, T., Branton, P.E., Nicholson, D.W., Cromlish, J.A., Shore, G., 1997. p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. J. Cell Biol 139, 327±338. Ng, F.W., Shore, G.C., 1998. Bcl-XL cooperatively associates with the Bap31 complex in the endoplasmic reticulum, dependent on procaspase-8 and Ced-4 adaptor. J. Biol. Chem 273, 3140±3143. Nonaka, S., Hough, C.J., Chuang, D.M., 1998. Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptor-mediated calcium in¯ux. Proc. Natl. Acad. Sci. U.S.A 95, 2642±2647. Nomura, Y., 1998. A transient brain ischemia- and bacterial endotoxin-induced glial iNOS expression and NO-induced neuronal apoptosis. Toxixol. Lett 102, 65±69. O'Connor, L., Strasser, A., O'Reilly, L.A., Hausmann, G., Adams, J.M., Cory, S., Huang, D.C., 1998. Bim: a novel member of the Bcl-2 family that promotes apoptosis. Embo J 17, 384±395. O'Reilly, L.A., Huang, D.C., Strasser, A., 1996. The cell death inhibitor Bcl-2 and its homologues in¯uence control of cell cycle entry. Embo J 15, 6979±6990. Ohgoh, M., Kimura, M., Ogura, H., Katayama, K., Nishizawa, Y., 1998. Apoptotic cell death of cultured cerebral cortical neurons induced by withdrawal of astroglial trophic support. Exp. Neurol 149, 51±63. Oliner, J.D., Pitenepol, J.A., Thiagalingam, S., Gyuris, J., Kinzler, K.W., Vogelstein, B., 1993. Oncoprotein mdm2 conceals the activation domain of tumor suppressor p53. Nature 362, 857±860. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609±619. Orlov, S.N., Thorin-Trescases, N., Kotelevtsev, S.V., Tremblay, J., Hamet, P., 1999. Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3. J. Biol. Chem 274, 16545±16552. Orth, K.I., Chinnayan, A.M., Garg, M., Froehilch, C.J., Dixit, V.M., 1996. The ced-3/ICE-like protease mch2 is activated during apoptosis and cleaves the death substrate lamin. J. Biol. Chem 271, 16443±16446. Ottilie, S., Diaz, J.L., Horne, W., Chang, J., Wang, Y., Wilson, G., Chang, S., Weeks, S., Fritz, L.C., Oltersdorf, T., 1997. Dimerization properties of human BAD. Identi®cation of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCLXL proteins. J. Biol. Chem 272, 30866±30872. Otter, I., Conus, S., Ravn, U., Rager, M., Olivier, R., Monney, L., Fabbro, D., Borner, C., 1998. The binding properties and biological activities of Bcl-2 and Bax in cells exposed to apoptotic stimuli. J. Biol. Chem 273, 6110±6120. Pan, G., O'Rourke, K., Chinnaiyan, A.M., Gentz, R., Ebner, R., Ni, J., Dixit, V.M., 1997a. The receptor for the cytotoxic ligand TRAIL. Science 276, 111±113. Pan, G., Ni, J., Wei, Y.F., Yu, G., Gentz, R., Dixit, V.M., 1997b. An antagonist decoy receptor and a death domain-containing receptor for trail. Science 277, 815±817. Pan, G., O'Rourke, K., Dixit, V.M., 1998. Caspase-9, Bcl-XL, and Apaf-1 form a ternary complex. J. Biol. Chem 273, 5841±5845. Park, C.O., Xiao, X.H., Allen, D.G., 1999. Changes in intracellular

245

Na+ and pH in rat heart during ischemia: role of Na+/H+ exchanger. Am. J. Physiol 276, H1581±H1590. Perlman, H., Sata, M., Le Roux, A., Sedlak, T.W., Branellec, D., Walsh, K., 1998. Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity. Embo J 17, 3576±3586. Perlman, H., Zhang, X., Chen, M.W., Walsh, K., Buttyan, R., 1999. An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis. Cell Death Di€er 6, 48±54. Perez-Sala, D., Collado-Escobar, D., Mollinedo, F., 1995. Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cell through the inactivation of pH-dependent endonuclease. J. Biol. Chem 270, 6235±6242. Petterson, E.O., Juul, N.O., Ronning, O.W., 1986. Regulation of protein metabolism of human cells during and after acute hypoxia. Cancer Res 46, 4346±4351. Piantadosi, C.A., Zhang, J., Levin, E.D., Folz, R.J., Schmechel, D.E., 1997. Apoptosis and delayed neuronal damage after carbon monoxide poisoning in the rat. Exp. Neurol 147, 103±114. Pomerantz, J., Schreiber-Agus, N., Liegeois, N.J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Lee, H.W., Cordon-Cardo, C., DePinho, R.A., 1998. The ink4a tumor suppressor gene product, p19arf interacts with mdm2 and neutralizes mdm2's inhibition of p53. Cell 92, 713±723. Portera-Cailliau, C., Price, D.L., Martin, L.J., 1997. Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum. J. Comp. Neurol 378, 70±87. Priault, M., Camougrand, N., Chaudhuri, B., Manon, S., 1999. Role of the C- terminal domain of Bax and Bcl-XL in their localization and function in yeast cells. FEBS Lett 443, 225±228. Price, B.D., Calderwood, S.K., 1992. Gadd45 and gadd153 messenger RNA levels are increased during hypoxia and after exposure of cells to agents which elevate the levels of the glucose-regulated proteins. Cancer Res 52, 3814±3817. Price, B.D., Calderwood, S.K., 1993. Increased speci®c DNA-binding activity after DNA damage is attenuated by phorbol esters. Oncogene 8, 3055±3062. Price, B.D., Hughes-Davies, L., Park, S.J., 1995. Cdk2 kinase phosphorylates serine 315 of human p53 in vitro. Oncogene 11, 73±80. Pulera, M.R., Adams, L.M., Liu, H., Santos, D.G., Nishimura, R.N., Yang, F., Cole, G.M., Wasterlain, C.G., 1998. Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia. Stroke 29, 2622±2630. Puthalakath, H., Huang, D.C., O'Reilly, L.A., King, S.M., Strasser, A., 1999. The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Mol. Cell 3, 287±296. Radeke, M.J., Misko, T.P., Hsu, C., Herzenberg, L.A., Shooter, E.M., 1987. Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 325, 593±597. Reynolds, J.E., Li, J., Craig, R.W., Eastman, A., 1996. BCL-2 and MCL-1 expression in Chinese hamster ovary cells inhibits intracellular acidi®cation and apoptosis induced by staurosporine. Exp. Cell Res 225, 430±436. Rheaume, E., Cohen, L.Y., Uhlmann, F., Lazure, C., Alam, A., Hurwitz, J., Sekaly, R.P., Denis, F., 1997. The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis. Embo J 16, 6346±6354. Rong, P., Bennie, A.M., Epa, W.R., Barrett, G.L., 1999. Nerve growth factor determines survival and death of PC12 cells by regulation of the bcl-x, bax, and caspase-3 genes. J. Neurochem 72, 2294±2300. Rosenbaum, D.M., Rosenbaum, P.S., Gupta, H., Singh, M., Aggarwal, A., Hall, D.H., Roth, S., Kessler, J.A., 1998. The role of the p53 protein in the selective vulnerability of the inner retina to transient ischemia. Invest. Ophthalmol. Vis. Sci 39, 2132±2139.

246

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249

Roth, J., Dobbelstein, M., Freedman, D.A., Shenk, T., Levine, A.J., 1998. Nucleo- cytoplasmic shuttling of the mdm2 on coproteinregulates the levels of p53 protein via pathways used by the human immunode®ciency virus rev protein. Embo J 17, 554±564. Rothe, M., Pan, M.G., Henzel, W.J., Ayres, T.M., Goeddel, D.V., 1995. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243±1252. Rotonda, J., Nicholson, D.W., Fazil, K.M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E.P., Rasper, D.M., Ruel, R., Vaillancourt, J.P., Thornberry, N.A., Becker, J.W., 1995. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol 3, 619±625. Rouayrenc, J.F., Boise, L.H., Thompson, C.B., Privat, A., Patey, G., 1995. Presence of the long and the short forms of Bcl-X in several human and murine tissues. C. R. Acad. Sci. III 318, 537±540. Roux, P.P., Colicos, M.A., Barker, P.A., Kennedy, T.E., 1999. p75 neurotrophin receptor expression is induced in apoptotic neurons after seizure. J Neurosci 19, 6887±6896. Rudel, T., Bokoch, G.M., 1997. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571±1574. Ruit, K.G., Osborne, P.A., Schmidt, R.E., Johnson, E.M., Snider, W.D., 1990. Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse. J. Neurosci 10, 2412±2419. Ryan, K.M., Vousden, K.H., 1998. Characterization of structural p53 mutants which show selective defects in apoptosis but cell cycle arrest. Mol. Cell Biol 18, 3692±3698. Ryseck, P., Bravo, R., 1991. c-Jun, jun B, and jun D di€er in their binding anities to AP-1 and CRE consensus sequences: e€ect of fos proteins. Oncogene 6, 533±542. Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J.M., Venkatachalam, M.A., 1998. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17, 3401±3415. Santella, L., Carafoli, E., 1997. Calcium signaling in the cell nucleus. FASEB J 13, 1091±1109. Sairanen, T.R., Lindsberg, P.J., Brenner, M., Siren, A.L., 1997. Global forebrain ischemia results in di€erential cellular expression of interleukin 1 beta (IL-beta) and its receptor at mRNA and protein level. J. Cereb. Blood Flow Metab 17, 1107±1120. Sakurai, M., Hayashi, T., Abe, K., Sadahiro, M., Tabayashi, K., 1998. Delayed selective motor neuron death and fas antigen induction after spinal cord ischemia in rabbits. Brain Res 797, 23± 28. Sassone-Corsi, P., Sisson, J.C., Verna, I.M., 1988. Transcriptional autoregulation of the proto-oncogene c-fos. Nature 334, 314±319. Sato, T., Hanada, M., Bodrug, S., Irie, S., Iwama, N., Boise, L.H., Thompson, C.B., Golemis, E., Fong, L., Wang, H.G., 1994. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc. Natl. Acad. Sci USA 91, 9238±9242. Satoh, T., Sakai, N., Enokido, Y., Uchiyama, Y., Hatanaka, H., 1996. Free radical-independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide-triggered apoptosis. J. Biochem. (Tokyo) 120, 540±546. Sattler, M., Liang, H., Nettesheim, D., Meadows, R.P., Harlan, J.E., Eberstadt, M., Yoon, H.S., Shuker, S.B., Chang, B.S., Minn, A.J., Thompson, C.B., Fesik, S.W., 1997. Structure of Bcl-xLBak peptide complex: recognition between regulators of apoptosis. Science 275, 983±986. Schendel, S.L., Xie, Z., Montal, M.O., Matsuyama, S., Montal, M., Reed, J.C., 1997. Channel formation by antiapoptotic protein Bcl-2. Proc. Natl. Acad. Sci. USA 94, 5113±5118. Schlesinger, P.H., Gross, A., Yin, X.M., Yamamoto, K., Saito, M., Waksman, G., Korsmeyer, S.J., 1997. Comparison of the ion

channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc. Natl. Acad. Sci. U.S.A 94, 11357±11362. Schutte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., Minna, J., 1989. Jun-B inhibits and c-fos stimulates the transforming and transactivating activities of c-jun. Cell 59, 987±997. Scolnick, D.M., Chehab, N.H., Stavridi, E.S., Lien, M.C., Caruso, L., Moran, E., Berger, S.L., Halazonetis, T.D., 1997. CREB binding protein and p300/CPB-associated factor are transcriptional activators of the p53 tumor suppressor protein. Cancer Res 57, 3693±3696. Scotto, C., Deloulme, J.C., Rousseau, D., Chambaz, E., Baudier, J., 1998. Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis. Mol. Cell. Biol 18, 4272±4281. Scheid, M.P., Duronio, V., 1998. Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation. Proc. Natl. Acad. Sci. USA 95, 7439±7444. Schneider, P., Bodmer, J.L., Thome, M., Hofmann, K., Holler, N., Tschopp, J., 1997. Characterization of two receptors for TRAIL. FEBS Lett 416, 329±334. Schulz, J.B., Bremen, D., Reed, J.C., Lommatzsch, J., Takayama, S., Wullner, U., Loschmann, P.A., Klockgether, T., Weller, M., 1997. Cooperative interception of neuronal apoptosis by BCL-2 and BAG-1 expression: prevention of caspase activation and reduced production of reactive oxygen species. J. Neurochem 69, 2075±2086. Schulz, J.B., Weller, M., Matthews, R.T., Heneka, M.T., Groscurth, P., Martinou, J.C., Lommatzsch, J., von Coelln, R., Wullner, U., Loschmann, P.A., Beal, M.F., Dichgans, J., Klockgether, T., 1998. Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia. Cell Death Di€er 5, 847±857. Screaton, G.R., Mongkolsapaya, J., Xu, X.N., Cowper, A.E., McMichael, A.J., Bell, J.I., 1997. TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL. Curr. Biol 7, 693±696. Sedlak, T.W., Oltvai, Z.N., Yang, E., Wang, K., Boise, L.H., Thompson, C.B., Korsmeyer, S.J., 1995. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc. Natl. Acad. Sci. USA 92, 7834±7838. Sendtner, M., Arakawa, Y., Stockli, K.A., Kreutzberg, G.W., Thoenen, H., 1991. E€ect of ciliary neurotrophic factor (CNTF) on motoneuron survival. J. Cell Sci 15 (Suppl), 103±109. Sheng, M., Dougan, S.T., Mc Fadden, G., Greenberg, M.E., 1988. Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences. Mol. Cell Biol 8, 2787±2796. Sheng, M., Greenberg, M.E., 1990. The regulation and function of cfos and other immediate early genes in the nervous system. Neuron 4, 477±485. Sheridan, J.P., Marsters, S.A., Pitti, R.M., Gurney, A., Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C.L., Baker, K., Wood, W.I., Goddard, A.D., Godowski, P., Ashkenazi, A., 1997. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277, 818±821. Shi, B., Triebe, D., Kajiji, S., Iwata, K.K., Bruskin, A., Mahajna, J., 1999. Identi®cation and characterization of baxepsilon, a novel bax variant missing the BH2 and the transmembrane domains. Biochem. Biophys. Res. Commun 254, 779±785. Shieh, S.Y., Ikeda, M., Taya, Y., Prives, C., 1997. DNA damageinduced phosphorylation of p53 alleviates inhibition by mdm2. Cell 91, 325±334. Shimazaki, K., Ishida, A., Kawai, N., 1994. Increase in bcl-2 oncoprotein and the tolerance to ischemia-induced neuronal death in the gerbil hippocampus. Neurosci. Res 20, 95±99. Shimizu, S., Eguchi, Y., Kamiike, W., Funahashi, Y., Mignon, A., Lacronique, V., Matsuda, H., Tsujimoto, Y., 1998. Bcl-2 prevents

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 apoptotic mitochondrial dysfunction by regulating proton ¯ux. Proc. Natl. Acad. Sci. U.S.A 95, 1455±1459. Smith Jr, M.L., Chen, I.T., Zhan, Q., Bae, I., Chen, C.Y., Gilmer, T.M., Kastan, M.B., O'Connor, P.M., Fornace, A.J., 1993. Interaction of the p53-regulated protein gadd45 with proliferating cell nuclear antigen. Science 266, 1376±1380. Snider, W.D., 1994. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 77, 627±638. Simonen, M., Keller, H., Heim, J., 1997. The BH3 domain of Bax is sucient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis. Eur. J. Biochem 249, 85±91. Sobko, A., Peretz, A., Attali, B., 1998. Constitutive activation of delayed-recti®er potassium channels by a Src family tyrosine kinase in Schwann cells. EMBO J 17, 4723±4734. Sondell, M., Lundborg, G., Kanje, M., 1999. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system. J. Neurosci 19, 5731±5740. Song, Q., Kuang, Y., Dixit, V.M., Vincenz, C., 1999. Boo, a novel negative regulator of cell death, interacts with Apaf-1. Embo J 18, 167±178. Sonnenberg, J.L., Macgregor-Leon, P.F., Hempstead, J., Morgan, J.I., Curran, T., 1989. Glutamate receptor antagonists increase the expression of fos, fra, and AP-1 DNA binding activity in the mammalian brain. J. Neurosci. Res 24, 72±80. St. Clair, E.G., Anderson, S.J., Oltvai, Z.N., 1997. Bcl-2 counters apoptosis by Bax heterodimerization-dependent and -independent mechanisms in the T-cell lineage. J. Biol Chem 272, 29347±29355. Stoler, D.L., Anderson, G.R., Russo, C.A., Spina, A.M., Beerman, T.A., 1992. Anoxia-inducible endonuclease activity as a potential basis of the genomic instability of cancer cells. Cancer Res 52, 4372±4378. Stoll, G., Jander, S., Schroeter, M., 1998. In¯ammation and glial responses in ischemic brain lesions. Prog. Neurobiol 56, 149±171. Stuart, J.K., Myszka, D.G., Joss, L., Mitchell, R.S., McDonald, S.M., Xie, Z., Takayama, S., Reed, J.C., Ely, K.R., 1998. Characterization of interactions between the anti-apoptotic protein BAG-1 and Hsc70 molecular chaperones. J. Biol. Chem 273, 22506±22514. Sza¯arski, J., Burtrum, D., Silverstein, F.S., 1995. Cerebral hypoxiaischemia stimulates cytokine gene expression in perinatal rats. Stroke 26, 1093±1100. Tabuchi, A., Oh, E., Taoka, A., Sakurai, H., Tsuchiya, T., Tsuda, M., 1996. Rapid attenuation of AP-1 transcriptional factors associated with nitric oxide (NO)- mediated neuronal cell death. J. Biol. Chem 271, 31061±31067. Takahashi, A., Alnemri, E.S., Lazebnik, Y.A., Fernandez-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufman, S.H., Earnshaw, W.C., 1996. Cleavage of lamin by mch2a but not CPP32: multiple interleukin-1b enzyme-related proteases with distinct substrate activities are active in apoptosis. Proc. Natl. Acad. Sci. USA 93, 8395±8400. Takahashi, A., Hirata, H., Yonehara, S., Imai, Y., Lee, K.K., Moyer, R.W., Turner, P.C., Mesner, P.W., Okazaki, T., Savai, H., Kishi, S., Yamamoto, K., Okuma, M., Sasada, M., 1997. Anity labeling displays the stepwise activation of ICE-related proteases by fas, staurosporine, and CrmA-sensitive caspase-8. Oncogene 14, 2741±2752. Takahashi, S.I., Kuro-o, M., Hiroi, Y., Yamazaki, T., Noguchi, T., Miyagishi, A., Nakahara, K.I., Aikawa, M., Manabe, I., Yazaki, Y., Nagai, R., 1995. Activation of Na+±H+ antiporter (NHE-1) gene expression during growth, hypertrophy and proliferation of the rabbit cardiovascular system. J. Mol. Cell Cardiol 27, 729± 742.

247

Takei, N., Endo, Y., 1994. Ca2+ inophore-induced apoptosis on cultured embryonic rat cortical neurons. Brain Res 651, 65±70. Tamatani, M., Ogawa, S., Tohyama, M., 1998. Roles of Bcl-2 and caspases in hypoxia-induced neuronal cell death: a possible neuroprotective mechanism of peptide growth factors. Mol. Brain Res 58, 27±39. Tamatani, M., Che, Y.H., Matsuzaki, H., Ogawa, S., Okado, H., Miyake, S., Mizuno, T., Tohyama, M., 1999. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFkappaB activation in primary hippocampal neurons. J. Biol. Chem 274, 8531±8538. Tan, S., Sagara, Y., Liu, Y., Maher, P., Schubert, D., 1998. The regulation of reactive oxygen species production during programmed cell death. J. Cell Biol 141, 1423±1432. Tan, Y.J., Beerheide, W., Ting, A.E., 1999. Biophysical characterization of the oligomeric state of Bax and its complex formation with Bcl-XL. Biochem. Biophys. Res. Commun 255, 334±339. Tartaglia, L.A., Goeddel, D.V., 1992. Two TNF receptors. Immunol. Today 13, 151±153. Tao, W., Kurschner, C., Morgan, J.I., 1997. Modulation of cell death in yeast by the Bcl-2 family of proteins. J. Biol. Chem 272, 15542±15547. Tenneti, L., D'Emilia, D.M., Troy, C.M., Lipton, S.A., 1998. Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons. J. Neurochem 71, 946±959. Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T., Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, S.M., Weidner, J.R., Aunines, J., et al., 1992. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, 768±774. Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T., Nicholson, D.W., 1997. A combinatorial approach de®nes speci®cities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem 272, 17907±17911. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 281, 1312±1316. Thress, K., Henzel, W., Shillinglaw, W., Kornbluth, S., 1998. Scythe: a novel reaper-binding apoptotic regulator. EMBO J 17, 6135± 6143. Thut, C.J., Goodrich, J.A., Tijan, R., 1997. Represion of p53mediated transcription by mdm2: a dual mechanism. Genes Dev 11, 1974±1986. Tomasevic, G., Kamme, F., Stubberod, P., Wieloch, M., Wieloch, T., 1999. The tumor suppressor p53 and its response gene p21Waf1/Cip1 are not markers of neuronal death following transient global cerebral ischemia. Neuroscience 90, 781±792. Trump, B.F., Berezesky, I.K., Cowley, R.A., 1982. The cellular and subcellular characteristics of acute and chronic injury with emphasis on the role of calcium. In: Cowley, R.A., Trump, B.F. (Eds.), Pathophysiology of shock, anoxia, and ischemia. Williams and Wilkins, Baltimore, MD, pp. 6±46. Tsujimoto, Y., Croce, C.M., 1986. analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. USA 83, 5214±5218. Uberti, D., Belloni, M., Grilli, M., Spano, P., Memo, M., 1998. Induction of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurones triggered by excitatory amino acids. Eur. J. Neurosci 10, 246±254. van Lookeren-Campagne, M., Gill, R., 1998a. Increased expression of cyclin G1 and p21Waf1/Cip1 in neurons following transient forebrain ischemia: comparison with early DNA damage. J. Neurosci. Res 53, 279±296. van Lookeren-Campagne, M., Gill, R., 1998b. Cell cycle-related gene

248

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249

expression in adult rat brain: selective induction of cyclin G1 and p21Waf1/Cip1 in neurons following focal cerebral ischemia. Neuroscience 84, 1097±1112. Vander Heiden, M.G., Chandel, N.S., Williamson, E.K., Schumacker, P.T., Thompson, C.B., 1997. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91, 627±637. Verhaegh, G.W., Richard, M.J., Hainaut, P., 1997. Regulation of p53 by metal ions and by antioxidants: dithiocarbamate downregulates p53 DNA-binding activity by increasing the intracellular level of copper. Mol. Cell Biol 17, 5699±5706. Vermes, I., Haanen, C., Ste€ens-Nakken, H., Reutelingsperger, C., 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using ¯uorescein labelled Annexin V. J. Immunol. Methods 184, 39±51. Villa, P., Kaufmann, S.H., Earnshaw, W.C., 1997. Caspases and caspase inhibitors. Trends Biochem. Sci 22, 388±393. Vucic, D., Kaiser, W.J., Miller, L.K., 1998. Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol. Cell Biol 18, 3300± 3309. Walczak, H., Degli-Esposti, A., Johnson, R.S., Smolak, P.J., Waugh, J.Y., Boiani, N., Timour, M.S., Gerhart, M.J., Schooley, K.A., Smith, C.A., Goodwin, R.G., Rauch, C.T., 1997. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. Embo J 16, 5386±5397. Walker, N.I., Harmon, B.V., Gobe, G.C., Kerr, J.F.R., 1988. Patterns of cell death. Methods Achiev. Exp. Pathol 13, 18±54. Walker, N.P., Talanian, R.V., Brady, K.D., Dang, L.C., Bump, N.J., Ferenz, C.R., Franklin, S., Ghayur, T., Hackett, M.C., Hamill, L.D., et al., 1994a. Cell 78, 343±352. Walker, P.R., Weaver, V.M., Lach, B., LeBlanc, J., Sikorska, M., 1994b. Endonuclease activities associated with high molecular weight and internucleosomal DNA fragmentation in apoptosis. Exp. Cell Res 213, 100±106. Walton, M., Connor, B., Lawlor, P., Young, D., Sirimanne, E., Gluckman, P., Cole, G., Dragunow, M., 1999. Neuronal death and survival in two models of hypoxic- ischemic brain damage. Brain Res. Rev 29, 137±168. Wang, H.G., Rapp, U.R., Reed, J.C., 1996a. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 87, 629±638. Wang, H.G., Takayama, S., Rapp, U.R., Reed, J.C., 1996b. Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc. Natl. Acad. Sci. USA 93, 7063±7068. Wang, K., Yin, X.M., Chao, D.T., Milliman, C.L., Korsmeyer, S.J., 1996c. BID: a novel BH3 domain-only death agonist. Genes Dev 10, 2859±2869. Wang, H.G., Reed, J.C., 1998. Bc1-2, Raf-1 and mitochondrial regulation of apoptosis. Biofactors 8, 13±16. Wang, H.G., Pathan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T.F., Reed, J.C., 1999a. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339±343. Wang, L., Zu, D., Dai, W., Lu, L., 1999b. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J. Biol. Chem 274, 3678±3685. Wang, X.W., Yeh, H., Schae€er, L., Roy, R., Moncollin, V., Egly, J.M., Wang, J.M., Wang, Z., Friedberg, E.C., Evans, M.K., Ta€e, B.G., et al., 1995. p53 modulation of TFIIH associated nucleotide excision repair activity. Nature Genet 10, 189±195. Wang, X.W., Zhan, Q., Coursen, J.D., Khan, M.A., Kontny, H.U., Yu, L., Hollander, M.C., O'Connor, P.M., Fornace, A.J., Harris, C.C., 1999. Gadd45 induction of a G2/M xcell cycle checkpoint. Proc. Natl. Acad. Sci. USA 96, 3706±3711. Wei, H., Wei, W., Bredesen, D.E., Perry, D.C., 1998. Bcl-2 protects against apoptosis in neuronal cell line caused by thapsigargin-

induced depletion of intracellular calcium stores. J. Neurochem 70, 2305±2314. Weinmann, P., Gaehtgens, P., Walzog, B., 1999. Bcl-Xl- and Baxalpha-mediated regulation of apoptosis of human neutrophils via caspase-3. Blood 93, 3106±3115. Wen, L.P., Fahrni, J.A., Troie, S., Guan, J.L., Orth, K., Rosen, G.D., 1997. Cleavage of focal adhesion kinase by caspases during apoptosis. J. Biol. Chem 272, 26056±26061. Whit®eld, P.C., Williams, R., Pickard, J.D., 1999. Delayed induction of junB precedes CA1 neuronal death after global ischemia in the gerbil. Brain Res 18, 450±458. Wiedau-Pazos, M., Trudell, J.R., Altenbach, C., Kane, D.J., Hubbell, W.L., Bredesen, D.E., 1996. Expression of bcl-2 inhibits cellular radical generation. Free Radic. Res 24, 205±212. Wiley, S.R., Schooley, K., Smolak, P.J., Din, W.S., Huang, C.P., Nicholl, J.K., Sutherland, G.R., Smith, T.D., Rauch, C., Smith, C.A., et al., 1995. Identi®cation and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673±682. Wilson, K.P., Black, J.A., Thomson, J.A., Kim, E.E., Grith, J.P., Navia, M.A., Murcko, M.A., Chambers, S.P., Aldape, R.A., Raybuck, S.A., et al., 1994. Nature 370, 270±275. Wing, J.P., Zhou, L., Schwartz, L.M., Nambu, J.R., 1998. Distinct cell killing properties of the Drosophila reaper, head involution defective, and grim genes. Cell Death Di€er 5, 930±939. Wood, A.M., Bristow, D.R., 1998. N-methyl-d-aspartate receptor desensitisation is neuroprotective by inhibiting glutamate-induced apoptotic-like death. J. Neurochem 70, 677±687. Wu, R.C., Schonthal, A.H., 1997. Activation of p53-p21waf1 pathway in response to disruption of cell-matrix interactions. J. Biol. Chem 272, 29091±29098. Wyllie, A.H., 1980. Gluocorticoid-induced thymic apoptosis is associated with endogenous nuclease activation. Nature 284, 555± 556. Wyllie, A.H., 1981. Cell death: a new classi®cation separating apoptosis from necrosis. In: Bowen, I.D., Lockshin, R.A. (Eds.), Cell Death in Biology and Pathology. Chapman and Hall, London, pp. 9±34. Xiang, H., Kinoshita, Y., Knudson, C.M., Korsmeyer, S.J., Schwartzkroin, P.A., Morrison, R.S., 1998. J. Neurosci 18, 1363± 1373. Xu, Q., Reed, J.C., 1998. Bax inhibitor-1, a mammalian apoptosis suppressor identi®ed by functional screening in yeast. Mol. Cell 1, 337±346. Xue, D., Horvitz, H.R., 1997. Caenoehabditis elegans CED-9 protein is a bifunctional cell death inhibitor. Nature 390, 305±308. Yang, D.D., Juan, C.-Y., Whitmarsh, A.J., Rincon, M., Zheng, T.S., Davis, R.J., Rakic, P., Flavell, R.A., 1997a. Nature 389, 865±870. Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P., Wang, X., 1997b. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129±1132. Yang, G.Y., Gong, C., Qin, Z., Ye, W., Mao, Y., Bertz, A.L., 1998. Inhibition of TNF alpha attenuates infarct volume and ICAM-1 expression in ischemic mouse brain. Neuroreport 9, 2131±2134. Yeh, W.C., Pompa, J.L., McCurrach, M.E., Shu, H.B., Elia, A.J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W.S., Lowe, S.W., Goeddel, D.V., Mak, T.W., 1998. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 279, 1954±1958. Yin, X.M., Oltvai, Z.N., Korsmeyer, S.J., 1994. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369, 321±323. Yu, S.P., Yeh, C.H., Sensi, S.L., Gwag, B.J., Canzoniero, L.M.T., Farhangrazi, Z.S., Ying, H.S., Tian, M., Dugan, L.L., Choi, D.W., 1997. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science 278, 114±117.

K.J. Banasiak et al. / Progress in Neurobiology 62 (2000) 215±249 Yu, S.P., Farhangrazi, Z.S., Ying, H.S., Yeh, C.H., Choi, D.W., 1998. Enhancement of outward potassium current may participate in beta-amyloid peptide-induced cortical neuronal death. Neurobiol. Dis 5, 81±88. Yu, S.P., Yeh, C.H., Strasser, U., Tian, M., Choi, D.W., 1999. NMDA receptor-mediated K+ e‚ux and neuronal apoptosis. Science 284, 336±339. Yuan, J., Shaham, S., Ledoux, S., Ellis, H.M., Horvitz, H.R., 1993. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta converting enzyme. Cell 75, 641± 652. Zamzami, N., Brenner, C., Marzo, I., Susin, S.A., Kroemer, G., 1998. Subcellular and submitochondrial mode of action of Bcl-2like oncoproteins. Oncogene 16, 2265±2282. Zhang, H., Heim, J., Meyhack, B., 1998a. Redistribution of Bax from cytosol to membranes is induced by apoptotic stimuli and is an early step in the apoptotic pathway. Biochem. Biophys. Res. Commun 251, 454±459. Zha, J., Harada, H., Yang, E., Jockel, J., Korsmeyer, S.J., 1996. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 87, 619±628. Zha, H., Reed, J.C., 1997. Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells. J. Biol. Chem 272, 31482±31488. Zha, J., Harada, H., Osipov, K., Jockel, J., Waksman, G., Korsmeyer, S.J., 1997. BH3 domain of BAD is required for heterodimerization with BCL-XL and pro- apoptotic activity. J. Biol. Chem 272, 24101±24104. Zhai, Q.H., Futrell, N., Chen, F.J., 1997. Gene expression of IL-10 in relation to TNF- alpha, IL-1 beta and IL-2 in the rat brain following middle cerebral artery occlusion. J. Neurol. Sci 152, 119± 124. Zhan, Q., Antinore, N.J., Wang, X.W., Carrier, F., Smith, M.L., Harris, C.C., Fornace, A.J., 1999. Anssociation with cdc2 and inhibition of cdc2/cyclinB1 kinase activity by the p53-regulated protein gadd45. Oncogene 18, 2892±2900.

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Zhang, Y., Xiong, Y., Yarbrough, W.G., 1998b. ARF promotes mdm2 degradation and stabilizes p53: ARF-ink4a locus deletion impairs both the RB and p53 tumor suppression pathways. Cell 92, 725±734. Zhong, L.T., Sara®an, T., Kane, D.J., Charles, A.C., Mah, S.P., Edwards, R.H., Bredesen, D.E., 1993. bcl-2 inhibits death of central neural cells induced by multiple agents. Proc. Natl. Acad. Sci. USA 90, 4533±4537. Zhou, M., Demo, S.D., McClure, T.N., Crea, R., Bitler, C.M., 1998. A novel splice variant of the cell death-promoting protein BAX. J.Biol. Chem 273, 11930±11936. Zindy, F., Eische, C.M., Randle, D., Kamijo, T., Cleveland, J.L., Sherr, C.J., Roussel, M.F., 1998. Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev 12, 2424±2433. Zong, W.X., Edelstein, L.C., Chen, C., Bash, J., Gelinas, C., 1999. The prosurvival Bcl-2 homolog B¯-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Dev 13, 382±387. Zorati, M., Szabo, I., 1994. Electrophysiology of the inner mitochondrial membrane. J. Bioenerget. Biomembr 26, 543±553. Zundel, W., Giaccia, A., 1998. Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress. Genes Dev 12, 1941±1946.

Further reading Marti, H.H., Risau, W., 1998. Systemic hypoxia changes the organspeci®c distribution of vascular endothelial growth factor and its receptors. Proc. Natl. Acad. Sci. USA 95, 15809±15814. Mukhopadhyay, D., Tslokas, L., Zhou, X.M., Foster, D., Brugge, J.S., Sukhatme, V.P., 1995. Hypoxic induction of human vascular endothelial growth factor expression through c-src activation. Nature 375, 577±581. Nagata, S., Golstein, P., 1995. The Fas death factor. Science 267, 1449±1456.