Neuronal Survival and Cell Death Signaling Pathways

Richard S. Morrison; Yoshito Kinoshita; Mark D. Johnson; Saadi Ghatan; Joseph

Neuronal Survival and Cell Death Signaling Pathways

Morrison RS, Kinoshita Y, Johnson MD, et al.

Abstract

Neuronal viability is maintained through a complex interacting network of signaling pathways that can be perturbed in response to a multitude of cellular stresses. A shift in the balance of signaling pathways after stress or in response to pathology can have drastic consequences for the function or the fate of a neuron. There is significant evidence that acutely injured and degenerating neurons may die by an active mechanism of cell death. This process involves the activation of discrete signaling pathways that ultimately compromise mitochondrial structure, energy metabolism and nuclear integrity. In this review we examine recent evidence pertaining to the presence and activation of anti- and pro-cell death regulatory pathways in nervous system injury and degeneration.

Introduction

Neuronal viability is maintained through a complex interacting network of signaling pathways that can be perturbed in response to a multitude of cellular stresses. A shift in one or more of these signaling pathways can alter the fate of a neuron resulting in cell death or continued survival. The nature of the stresses affecting neurons, the duration of the stresses, the developmental stage of the neuron and a variety of other factors influence the signaling pathways that are ultimately affected. These diverse parameters may also regulate the temporal response as well as the final disposition of the affected neurons.

Apoptosis is a mechanism of cell death that plays a fundamental role during the development of many tissues including the central nervous system. Apoptosis has traditionally been distinguished in developing tissues on the basis of specific morphological and biochemical criteria, including perinuclear chromatin condensation, cell shrinkage and endonuclease-mediated internucleosomal DNA fragmentation into a “ladder” pattern. More recently, the term “apoptosis” has been used to describe the programmed biochemical pathways of cell death that accompany development, tissue injury and degeneration. In the context of this review, apoptosis will refer to the biochemical pathways of cell death. There is accumulating evidence that acutely injured and degenerating neurons may die by a process of apoptosis, contributing to the loss of neurons observed under these conditions. The possibility that neurons may succumb under some circumstances by an active mechanism of cell death has raised interest in the regulatory pathways governing these processes.

In this review we examine recent evidence pertaining to the presence, activation and contribution of these regulatory pathways to nervous system development, injury and degeneration. The review begins with a description of cell death signals initiating through plasma membrane receptors followed by a description of relevant pro- and anti-apoptotic signal transduction pathways activated by various cellular stresses and trophic factor receptors. The review then considers signal transduction cascades activated in the nucleus and finally the critical mediators of viability that are related to mitochondrial function and disassembly of essential cellular components.

Death Receptor-Mediated Neuronal Apoptosis

Members of the tumor necrosis factor receptor (TNFR) family are involved in a number of physiological processes, including neuronal cell death during development and after injury. This family of receptors includes Fas/CD95/Apo1, TNFR1, TNFR2, DR3/TRAMP/Wsl-1, TRAIL-R1/DR4, TRAIL-R2/DR5/Killer/TRICK2, TRAILR3/DcR1/TRID, TRAIL-R4/DcR2/TRUNDD, DcR3, osteoprotegerin, DR6, p75^NTR and others.¹ The prototypic members of the TNF receptor family are transmembrane proteins containing an extracellular cysteine-rich domain and an intracellular cytoplasmic protein-binding sequence called the death domain (DD). Activation of Fas/CD95, TNFR1, TRAIL-R1, TRAIL-R2 and DR6 receptors by their respective ligands leads to the recruitment of an intracellular protein complex known as the DISC (death-inducing signaling complex), followed by downstream caspase activation.

Fas/CD95/Apo1

Fas receptors and Fas ligand (FasL) are expressed on both astrocytes and neurons in normal rat and human brain.² Exogenous application of FasL can cause neuronal apoptosis in vivo and in vitro, and this death can be blocked by a selective caspase-8 inhibitor.³ Embryonic motoneurons co-express Fas and FasL at the time of programmed cell death, and their death may be reduced by blocking antibodies to the Fas receptor or by the use of a specific caspase-8 inhibitor.⁴ Other studies have revealed a role for Fas in neuronal death after injury. In spinal cord ischemia, studies suggest the rapid formation of a complex containing the Fas receptor and pro-caspase-8, followed by activation of caspases -8 and -3 and neuronal apoptosis.⁵ FasL is also up-regulated in cortical neurons after cerebral ischemia.^6,7 In lpr mice expressing a dysfunctional Fas receptor, infarct volume after ischemia was reduced when compared to wild-type control mice.⁷ Nervous system infections may also be a stimulus for activation of the Fas/FasL pathway. Both Fas and FasL are up-regulated in the brains of patients with HIV.⁸ In vitro studies indicate that neuronal death in HIV dementia is not due to direct viral infection of neurons but may be due to the release from activated microglia of other cytokines such as TNF and/or FasL.⁹

Most of the studies elucidating the mechanisms underlying Fas/FasL receptor signaling have been performed in non-neuronal cells (for review, see Refs. 10,11). The binding of FasL to its receptor leads to trimerization of the receptor and results in the recruitment of an intracellular adapter protein, FADD (Fas-associated death domain). FADD binds to the DD of the Fas/CD95/Apo1 receptor via homophilic DD-DD interactions. FADD also contains a separate death effector domain (DED) at its N-terminal, which interacts directly with a homologous region in the prodomain of pro-caspase-8. The association of the trimerized Fas/CD95/Apo1 receptor, FADD and pro-caspase-8 forms the DISC. Pro-caspase-8 subsequently undergoes autocatalytic cleavage to yield its active form. Caspase-8 may then cleave and activate caspases -3, -6 and -7 directly, thereby leading to cell death.¹² Alternatively, caspase-8 may cleave Bid to form truncated Bid (t-Bid; see Ref. 13). Some investigators have argued that there are at least two Fas/CD95/Apo1L signaling pathways that occur after DISC formation.¹⁴ One pathway involves mitochondrial amplification of caspase activation, while the other results in mitochondrial dysfunction that occurs only after activation of caspases -8 and -3. Whereas the former apoptotic pathway is inhibitable by bcl-2 overexpression, the latter is not. The reasons for these cell type-specific differences in the order of caspase activation and mitochondrial dysfunction after Fas activation are not completely known. Nevertheless, it is apparent that caspase-8 is the primary apical caspase involved in signaling by Fas/CD95/Apo1L and other members of the TNFR superfamily. Caspase-10 also contains several DEDs and can be recruited to the DISC after activation of TNF and TRAIL (TNF-related apoptosis-inducing ligand) receptors.¹⁵ Thus, caspase-10 may also serve as an apical caspase in some cells, as has been demonstrated in lymphocytes.¹⁶ However, the fact that caspase-8 gene deletion completely abrogates TNFR1 and Fas receptor-induced apoptosis indicates that caspase-8 is primarily responsible for death receptor signaling by the TNFR superfamily.¹²

TNF/TNFRs

Recent studies suggest that TNF may play an important role in the control of neuronal survival. For example, TNFa induced apoptosis in cultured primary rat cortical neurons and in differentiated PC12 cells.¹⁷ Function-blocking antibodies to TNFa or TNFR1 partially protected embryonic mouse sympathetic and sensory neurons from apoptosis induced by NGF withdrawal.¹⁸ Moreover, fewer sensory and sympathetic neurons died during the phase of naturally occurring cell death in TNFa-deficient mice than in wild-type mice, and sympathetic neurons derived from such mice survived better than their wild-type counterparts. Other investigators have found that TNFR1^-/- and TNFR2^-/- mice have an increased number of apoptotic cells in the injured mouse spinal cord.¹⁹ The proposed explanation for this result was that activation of TNFRs in the wild-type animals resulted in NF-κB activation and increased expression of c-IAP2, thereby inhibiting caspase-mediated apoptosis.

Like the Fas/CD95/Apo1L receptor, TNF receptors (TNFRs) also signal cell death via the formation of an intracellular protein complex.¹⁵ After binding its ligand, TNFR1 trimerizes and recruits the intracellular DD-containing protein, TRADD (TNFR-associated death domain). TRADD, in turn, recruits FADD, pro-caspase-8 and the serine-threonine kinase RIP (receptor-interacting protein). TNFR1 also couples to a number of other intracellular signaling pathways. The serine-threonine kinase interacting protein (RIP) also interacts with the NF-κB pathway.²⁰ Activation of NF-κB is thought to oppose TNF-induced apoptosis in many cell types. TNFR1 can also associate with another factor, TRAF2 (TNF receptor-associated factor 2), which appears to couple TNFR1 to both NF-κB and c-Jun N-terminal kinase (JNK) activation.²¹ Like TNFR1, DR6 has also been shown to activate NF-κB and JNK (Pan et al., 1998). Additional studies indicate that TNFRs may even couple to the nuclear transcription factor AP-1.²²

TNFR2 lacks a cytoplasmic death domain. Thus, it was initially unclear how activation of the receptor leads to cell death. However, a recent study by Grell et al.²³ has indicated that activation of TNFR2 leads to increased synthesis of TNF, which then acts in an autocrine fashion on TNFR1 receptors to initiate cell death.

TRAIL/TRAIL-Rs

TRAIL/Apo-2L is a type II transmembrane protein which, based on homology to TNF, was predicted (and subsequently shown) to be most effective in activating TRAIL receptors as a multimer.²⁴ The tissue distribution of TRAIL receptors and their ligand varies widely, with both being commonly expressed in the same tissues.^24,25 There are currently five known TRAIL receptors, including TRAIL-R1/DR4, TRAILR2/DR5/Killer, TRAIL-R3/DcR1/LIT, TRAIL-R4/DcR2/TRUNDD, DcR3 and osteoprotegerin, a soluble receptor that also binds osteoclast differentiation factor.^15,24,26 TRAIL-R1 and TRAIL-R2 are 58% identical and are type I transmembrane proteins that contain extracellular cysteine-rich domains and intracellular cytoplasmic domains containing a DD similar to that of the TNFRs.²⁴ TRAILR3/DcR1 and TRAIL-R4/DcR2 have a somewhat different structure than that seen in TRAIL-R1 or TRAIL-R2. TRAIL-R3/DcR1 contains only a partial cytoplasmic DD, while TRAIL-R4/DcR2 lacks any transmembrane component and is instead attached to the cell surface by a glycosylphosphatidylinositol linker.^1,24,27

Interestingly, TRAIL preferentially induces apoptosis in tumorigenic cells and not in most normal tissues, including the brain.^28,29 However, this finding has recently been challenged by reports of TRAIL-induced apoptosis in normal human hepatocytes and in epileptic human brain.³⁰ Messenger RNA for TRAIL and its receptors have been localized in many tissues, including normal human brain tissue.³¹ Exposure of epileptic human brain slices to TRAIL induces apoptosis,³² and TRAIL induces cell death in primary mouse cortical neurons and neuroblastoma cells in vitro.^6,33

The mechanisms underlying TRAIL/Apo-2L receptor signaling are not as well understood as those for CD95/Fas/Apo1 and TNFRs. As mentioned previously, TRAIL receptor activation leads to activation of caspase-8,^1,34 and caspase inhibition blocks TRAIL-induced apoptosis.³⁵ Studies have demonstrated inhibition of TRAIL signaling in FADD-deficient cells, as well as direct binding of FADD and TRADD to TRAIL-R1 and TRAIL-R2.^34,36 Thus, it seems likely that TRAIL receptor activation results in DISC formation, caspase-8 activation and cell death in a manner similar to that observed with TNFRs and the Fas receptor. There may be other signaling pathways coupled to TRAIL receptors as well, although their functional role remains to be determined. For example, TRAIL-R4 has been shown to activate NF-κB, despite having an incomplete DD.²⁷

The factors determining sensitivity to TRAIL are only now being elucidated. TRAIL itself is up-regulated after cerebral ischemia,⁶ and both Trail-R2/DR5 and TRAIL-R3/DcR1 can be induced in a variety of tissues in a p53-dependent or p53-independent manner.³⁷^-³⁹ These findings suggest that sensitivity to TRAIL-mediated apoptosis may be regulated in the nervous system, at least in part by injury-induced regulation of the expression of TRAIL and its receptors.

Both TRAIL-R3 and TRAIL-R4 have been called decoy receptors because their ligation fails to induce apoptosis. One hypothesis is that these non-functional “decoy” receptors bind the available TRAIL, thereby protecting TRAIL-sensitive cells from the ligand.^40,41 However, studies examining expression of mRNA and protein for TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in a variety of cell types have not found a significant correlation with TRAIL sensitivity.^29,42,43 Thus, the function of TRAIL-R3 and TRAIL-R4 has not been conclusively demonstrated.

p75^NTR

Recent studies have provided evidence that the low affinity neurotrophin receptor, p75^NTR, has significant homology to other members of the TNFR superfamily, and that it may play a role as a death receptor under some circumstances. In addition to its well-established role as a component of neurotrophin receptors, p75^NTR may induce the death of selected neuronal populations when expressed in the absence of Trkreceptors. For example, embryonic or neonatal motor neurons derived from wild-type, but not p75^NTR-deficient mice undergo apoptosis after NGF exposure.^44,45 These cells also show increased survival after axotomy when compared to wild-type motor neurons, suggesting that p75^NTR may promote neuronal cell death after injury.⁴⁶ p75^NTR knockout mice also show decreased cell death of a subset of spinal cord interneurons, retinal neurons, and sympathetic neurons.^47,48

The mechanism by which p75^NTR signals cell death is poorly understood. It has a different death domain structure than that of other TNFRs, and there is conflicting evidence as to whether it signals apoptosis when occupied or unoccupied.¹¹ Studies have implicated NF-κB activation, ceramide production and caspase activation in the p75^NTR signaling pathway (for review, see Ref. 49). In immortalized striatal neurons containing an inducible p75^NTR, expression of the receptor was found to activate caspases -9, -6 and -3 in an NGF-independent manner.⁵⁰ Cell death was inhibited by Bcl-X_L, by a dominant-negative form of caspase-9 and by a viral FLIP, E8. The protection by viral FLIP suggests that DEDs are involved in the signaling cascade. However, FADD, TRADD and caspase-8 were not involved. Other proteins identified that interact with p75^NTR and promote apoptosis include NRIF (neurotrophin receptor interacting factor, Ref. 51), NADE (p75NTR-associated cell death executor, Ref. 52), and NRAGE (neurotrophin receptor-interacting MAGE homolog, Ref. 53), while RIP2⁵⁴ was shown to suppress apoptosis.

Signal Transduction Pathways

Signaling Kinases Pro- and Anti-Apoptotic Effectors in the Nervous System

The mitogen-activated protein (MAP) kinases and phosphatidylinositol-3 kinase (PI3K) are serine/threonine protein kinases that play critical roles in neuronal growth, differentiation and survival (Figs. 1 and 2). In general, activation of the extracellular signal-regulated kinase members of the MAP kinase family (ERK or p42/p44 MAP kinase) and the PI3KAkt signaling pathway promote cell survival, while members of the MAP kinase family known as the stress-activated protein kinases (SAPK's), c-Jun N-terminal kinases (JNK's) and the p38 MAP kinase (p38 MAPK), promote cell death. In this section, we will review the molecular pathways and contributions of these kinases based upon evidence from animal studies, as well as neuronal cell lines and various cultured neuronal populations.

Figure 1

Cell death signaling pathways in neurons. Schematic representation of proposed cell death pathways initiated by excitotoxic and genotoxic damage in the nervous system. In this model, excitotoxicity and DNA damage are common activators of neuronal damage, (more...)

Figure 2

Survival signaling pathways in neurons. Schematic representation of pathways that promote neuronal survival. In this model, a major factor contributing to the survival of CNS neurons is the stimulation of the PI3K-Akt kinase pathway following trophic (more...)

PI3K-Akt Signaling

Observations over the past decade have identified the PI3K-Akt pathway's importance in mediating survival in PC12 cells⁵⁵ and cultured neurons from the peripheral⁵⁶ and central nervous systems.⁵⁷ A recent study by Kuruvilla et al.⁵⁸ demonstrated the importance of PI3K activity under conditions that may more accurately simulate in vivo function. By applying NGF exclusively onto the distal axon compartment, the authors were able to demonstrate that PI3K activation in the axons and its retrograde signaling play a critical role in survival of both the axons and cell body.⁵⁸

Neurotrophic factors such as NGF, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor I (IGFI), activate the PI3-Akt signaling cascade through corresponding receptor tyrosine kinases such as the high affinity neurotrophin receptors (Trk's) (reviewed in Ref. 59). After receptor dimerization, PI3K is recruited to the plasma membrane where its catalytic subunit generates lipid second messengers, phosphoinositide phosphates (PIP₂, PIP₃), at the inner surface of the plasma membrane. Phosphoinositide-dependent protein kinase-1 (PDK1) then acts in concert with PIP₂ and PIP₃ to phosphorylate and activate Akt (a.k.a. protein kinase B, PKB), a protein kinase first identified in the AKT virus (reviewed in Ref. 60). Alternately, Trk receptors may stimulate PI3K via the Ras G-protein, insulin receptor substrate (IRS) signaling, and Gab-1, an adaptor protein, which binds to Trk and directly stimulates PI3K.^61,62

Dominant-negative and constitutively active forms of Ras,⁶² PI3K,^63,64 and Akt⁶⁵ have been used to study signaling through the PI3K-Akt pathway. These and other studies have demonstrated downstream signaling effects that regulate cellular survival, proliferation, and metabolism. For example, Akt phosphorylates and inactivates FKHRL1, a member of the family of Forkhead transcriptional regulators. Inactivated FKHRL1 is unable to induce the expression of death genes in cerebellar granule neurons.⁶⁶ In primary hippocampal neurons subjected to hypoxia or nitric oxide, p53 activation and p53-mediated Bax upregulation are also blocked by Akt signaling.⁶⁷ Akt activates the cAMP-responsive element binding protein (CREB) and nuclear factor-κB (NF-κB), additional transcriptional regulators that may promote neuronal survival.^68,69In addition, Akt can directly inhibit the apoptotic machinery by phosphorylation at sites both upstream (BAD⁷⁰) and downstream (Caspase-9⁷¹) of mitochondrial cytochrome c release. Finally, there is evidence to support the role of Akt in promoting neuronal survival through metabolic effects, by regulating glucose metabolism in neurons.⁷²

ERK/MAP Kinase Pathways

The ERK cascade represents one of the evolutionarily conserved MAP kinase pathways, first found to be important in the regulation of neuronal survival in response to neurotrophin withdrawal.⁷³ A wide body of evidence has also been collected to demonstrate the role of ERK in neuronal plasticity and memory formation.^74,75 For the purposes of this discussion, we will focus on the data supporting ERK's role in promoting survival although ERK's can be activated by toxic stimuli as well and have been implicated in cell death.⁷⁶^-⁷⁸

At least six ERK isoforms have been identified.⁷⁹ In general, the common pathway leading to ERK activation by trophic factor signaling involves engagement of the membrane receptor at the cell surface, activation of small (p21^{ras, rap}) GTPases, which in turn activate the protein kinase Raf. Raf, in turn, phosphorylates and activates MAP kinase kinase 1/2 (MKK 1/2), which phosphorylates and activates ERK (reviewed in Ref. 80). The ERK pathway can also be stimulated and modulated by intracellular Ca²⁺ levels, protein kinase A, diacylglycerol, and cAMP (reviewed in Refs. 74,75,81).

Constitutively activated or dominant-negative mutants directed at Ras, Raf, and MEK 1/2 (a.k.a. MKK1/2) have been used to determine the effects of the ERK pathway in the setting of neurotrophin withdrawal. Current evidence indicates a direct role for the ERK pathway in protecting neurons after injury. Activation of the ERK pathway is essential for BDNF-mediated protection of cortical neurons from DNA damage,⁸⁴ glutamate-induced excitotoxicity and ischemic preconditioning.⁸⁵ The role of ERK in suppressing neuronal apoptosis, however, has been controversial. The importance of this pathway as a neuroprotective mechanism appears related to the nature of the cell type and exposure. For example, a MKK1 protein kinase inhibitor protects against damage resulting from focal cerebral ischemia.⁸² These conflicting findings may stem from the use of different experimental systems and the possibility that the regulation of ERK is complex and might also be controlled by MKK1-independent mechanisms.⁸³ While PI3K-Akt is thought to be more important in protecting cells from trophic factor withdrawal, ERK is thought to protect against injury-induced apoptosis.⁸⁴

The mechanisms by which ERK mediates its protective effect are poorly understood. Downstream signaling may be directed through the serine/threonine kinase p90/rsk2 and CREB phosphorylation,⁶⁸ but a direct link between CREB and ERK has not yet been established in neurons. Alternately, ERK can phosphorylate Bad resulting in its sequestration by 14-3-3 and prevents Bad-mediated induction of apoptosis.⁶⁸

JNKs

As their name implies, stress-activated protein kinases, such as the c-Jun N-terminal kinase (JNK) are activated by numerous noxious stimuli including trophic factor withdrawal⁷³, excitotoxicity,^86,87 seizures,⁸⁸ irradiation,⁸⁶ hypoxia,⁸⁹ exposure to β-amyloid (Abeta),^90,91 arsenite toxicity,⁹² and axotomy⁹³ (reviewed in Ref. 94). At least ten isoforms of JNK are expressed in the human adult brain, encoded by three separate genes through alternative splicing mechanisms.⁹⁵

The relevance of JNK signaling to neuronal cell death has been demonstrated using JNK-deficient mice, JNK inhibitors and dominant-negative forms of upstream activating kinases. Transfecting cortical neurons with various dominant-negative forms of components comprising the JNK signaling pathway significantly decreased the number of neurons undergoing apoptosis in response to the β-amyloid peptide.⁹⁰ Similarly, treatment with an inhibitor of JNK activation, CEP-1347, effectively blocked increases in cellular JNK activity and protected PC12 cells, sympathetic neurons,⁹¹ and cortical neurons⁹⁶ from β-amyloid-induced death. These results are consistent with the demonstration that JNK is activated and redistributed in the brains of patients with Alzheimer's disease.⁹⁷ Moreover, CEP-1347 also inhibited MPTP-mediated MKK4 and JNK signaling and attenuated MPTP-induced dopaminergic cell loss,⁹⁸ suggesting that the JNK pathway may be activated in the degenerative process in Parkinson's disease. Cultured rat sympathetic neurons and neuronally differentiated PC12 cells were protected from nerve growth factor withdrawal, exposure to ultraviolet irradiation, and oxidative stress following treatment with the JNK inhibitor CEP-1347⁹⁸. Interestingly, CEP-1347 failed to protect undifferentiated PC12 cells induced to die by serum withdrawal or Jurkat T cells from Fas activation, even though each injury stimulus activated JNK and was inhibited by CEP-1347. Blocking JNK activation also protects neurons from cell death induced by the withdrawal of survival signals.^73,99,100 These results are consistent with the data obtained applying NGF withdrawal to JNK3-deficient sympathetic neurons.¹⁰¹ Moreover, the deficiency in JNK3 activity⁸⁷ and the expression of a mutant form of jun that is resistant to phosphorylation by JNK¹⁰² confers resistance to kainic acid-induced seizures and subsequent neuronal damage. Collectively, these results illustrate the importance of JNK signaling to the regulation of neuronal injury responses.

Upstream regulation of JNK activation is poorly understood. As in the ERK system, the immediate upstream activators of JNK are MKK's. While the ERK pathway is stimulated by MKK's 1/2, JNK appears to be activated by MKK4¹⁰³ or MKK7,¹⁰⁴ depending upon the cell type, developmental stage,¹⁰⁵ and stressful stimulus.¹⁰⁶ Other kinases such as MLK3¹⁰⁷, MEKK1,¹⁰⁸ and ASK1^109,110 lie further upstream of the MKKs and probably activate both MKK's and JNK in a similar cell type- and stimulus-dependent manner.¹⁰⁶ ASK1 also activates p38 MAPK and is up-regulated in PC12 cells following NGF withdrawal.¹¹¹ Overexpression of a constitutively active mutant of ASK1 activates JNK and induces apoptosis in differentiated PC12 cells and sympathetic neurons.¹¹¹ JNK activity is further modified by JNK interacting proteins (JIP's), which are scaffolding proteins thought to sequester all of the necessary kinases for JNK activation.¹¹²

The downstream effectors of the JNK pathway are numerous and include both cytoplasmic and nuclear targets. In the nucleus, JNK's phosphorylate and activate the transcription factor c-jun,¹¹³ a component of the AP-1 transcription factor, which regulates genes involved in apoptosis. For example, in PC12 cells, JNK activation is followed by induction of Fas ligand (FasL) expression¹⁰⁰ (see Fas/CD95/Apo1 in the previous section). Cytoplasmic targets include p53¹¹⁴ (see Nuclear Signaling Pathways in the following section), which is stabilized and activated by JNK,¹¹⁵ and a cell death domain protein MADD, which co-translocates with JNK to the nucleus after hypoxic stress.¹¹⁶

p38 MAP Kinases

p38 MAPK's, like JNK's, are stress-activated kinases that are highly conserved in evolution, exist in multiple isoforms, and are activated in response to numerous cellular stresses (reviewed in Refs. 94,106). Like other members of the MAP kinase family, upstream regulation involves phosphorylation by MKK's, specifically MKK3, MKK4, and MKK6.¹¹⁷^-¹¹⁹ The downstream targets of the p38 MAPK pathway are similar to the targets of JNKs and involve transcription factors, kinases, and pathways influencing pro-inflammatory cytokines (reviewed in Ref. 106). However, little is known about the downstream targets of p38 MAPK signaling in the brain.

Inhibition of p38 MAPK has been shown to attenuate neuronal cell death in response to a variety of different cellular stresses. The p38 MAPK inhibitor SB203580 significantly reduced apoptosis in potassium-deprived cerebellar granule cells^120,121 and prevented the activation of downstream effectors such as caspase-3.¹²⁰ p38 MAPK inhibition also conferred protection against neuronal cell death induced by C2-ceramide,¹²² oxidative damage,¹²³ and trophic factor deprivation.^73,124 Focal ischemia induces p38 MAPK activity and treatment with a second-generation p38 MAPK inhibitor, SB 239063, significantly reduced infarct size and behavioral deficits.¹²⁵ Inhibiting p38 MAPK activity in the focal ischemia model was also associated with decreased expression of IL-1beta and TNFa, cytokines known to contribute to stroke-induced brain injury. Glutamate-mediated cell death, an important contributor to stroke-induced changes in viability, is also attenuated in cerebellar granule cells by p38 MAPK inhibition.¹²⁶

Mechanistic aspects of the contribution of p38 MAPK activation to cell death were further revealed in a study from our laboratory.¹²⁷ We showed that nitric oxide (NO)-induced cell death of human neuroblastoma cells and murine cortical neurons in culture was dependent upon p38 MAPK activity and Bax. We further demonstrated that inhibition of p38 MAPK activity blocked Bax translocation from the cytosol to the mitochondria and thereby conferred protection from cell death. Cheng et al. recently reported a similar result.¹²⁸ Thus, p38 MAPK represents a potential target for interrupting Bax translocation and preserving neuronal viability following neuronal injury involving NO toxicity. Collectively, the stress-activated kinases, p38 MAPK and JNK, play important roles in the neuronal response to injury and newly developed inhibitors for these enzymes hold promise for therapeutic intervention.

Glycogen Synthase Kinase-3β

Another emerging kinase that has been implicated in neuronal cell death is glycogen synthase kinase-3β (GSK-3β). GSK-3β regulates a diverse array of proteins including many transcription factors (for review, see Ref. 129) and is inhibited by the PI3K pathway.^130,131 Its function in the nervous system has been linked to neuropathology, such as Alzheimer's disease, based on its ability to promote tau hyperphosphorylation,^132,133 although the significance of this finding is still controversial.¹³⁴ Several cellular stresses including serum deprivation stimulate GSK-3β activity in cortical neurons prior to the induction of apoptosis.⁷² Expression of an inhibitory GSK-3β binding protein or a dominant interfering form of GSK-3β reduced neuronal apoptosis, suggesting that GSK-3β contributes to trophic factor deprivation-induced apoptosis. Moreover, overexpressing GSK-3β in neurons was sufficient to trigger neuronal cell death. Elucidating the intriguing role played by this kinase in neuronal cell survival/death is certainly warranted and will require the identification of specific substrates and their functions in the context of a specific survival or death-promoting stimulus.

Nuclear Signaling Pathways

Cyclins, Cyclin-Dependent Kinases, Rb and E2F1

What is seemingly a contradiction in our understanding of cell death in post-mitotic neurons is the demonstration that neuronal damage results in the elaboration of events that are normally associated with proliferating cells. Neurons express a range of proteins after injury that normally function to control cell cycle progression (for review, see Ref. 135). Alterations in the levels of cyclin-dependent kinases (CDK's) and their respective cyclin partners have been observed in neurons in response to trophic factor deprivation,¹³⁶ ischemia and seizures,¹³⁷ stroke,¹³⁸ exposure to the β-amyloid peptide,^139,140 and DNA damage.¹⁴¹ In addition, abnormal expression of these cell cycle regulators has also been implicated in neurodegenerative diseases such as Alzheimer's disease¹⁴²^-¹⁴⁸ and amyotrophic lateral sclerosis (ALS).¹⁴⁹ Pharmacological and molecular manipulation of CDK activity has been used to evaluate the relationship between cell cycle regulatory proteins and neuronal cell death.

The use of CDK inhibitors or dominant-negative forms of CDK 4/6 protects cultured postmitotic sympathetic neurons from death evoked by NGF withdrawal.¹⁵⁰ Pharmacological inhibitors of cyclin-dependent kinases, such as flavopiridol, olomoucine, and roscovitine, prevent cerebellar granule neuron apoptosis induced by non-depolarizing KCl.¹⁵¹ Moreover, camptothecin-induced cell death of PC12 cells, sympathetic neurons, and cerebral cortical neurons is also suppressed by the CDK-inhibitors flavopiridol and olomoucine.^152,153 However, these cell cycle inhibitors did not prevent cell death induced by a strong oxidative stress (SOD1 depletion),¹⁵⁴ suggesting that cell cycle proteins are not involved in all forms of neuronal cell death.

The mechanism by which the CDK's facilitate neuronal cell death is under active investigation. Increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease¹⁴⁸ and ALS¹⁴⁹ through its ability to hyperphosphorylate tau, although the finding that cdk5 is a major protein tau kinase has been disputed.¹⁵⁵ However, Cdk5 may alter neuronal viability through its effects on other signaling cascades, consistent with the finding that it directly phosphorylates β-catenin and regulates the latter's binding to presenilin-1.¹⁵⁶

Cdk4/6 activation results in the phosphorylation of the Rb tumor suppressor protein in neurons following DNA damage.¹⁴¹ One consequence of Rb phosphorylation is the activation of E2F family members. Overexpression of E2F1 is sufficient to induce neuronal cell death,¹⁵⁷ ¹⁵⁹ while E2F1 deficiency protects neurons from multiple cellular stresses including DNA damage,¹⁶⁰ ischemia,^161,162 staurosporine,¹⁵⁷ reduced potassium¹⁵⁸ and β-amyloid toxicity.¹⁶³ In addition, overexpression of dominant-negative versions of DP-1, a binding partner for E2F family members, also protects neurons from cell death induced by DNA damage.^139,160 Interestingly, overexpression of E2F activity in dissociated sensory neurons from adult rats stimulated their entry into S-phase, although the authors found no evidence for subsequent mitotic events in the E2F-overexpressing cells.¹⁵⁹ These results suggest that other molecular signals are responsible for sister chromatid separation and continued cycling in neurons. The absence of these additional mitotic factors in damaged or degenerating neurons might provide an explanation for why the inappropriate activation of cell cycle regulatory genes promotes neuronal cell death.

The CDK-dependent inactivation of Rb is likely to have many other effects beyond the activation E2F family members. Rb has been shown to repress NF-κB transcriptional activity¹⁶⁴ as well as JNK activity,¹⁶⁵ both important neuronal cell death mediators. The relevance of Rb phosphorylation to the process of neuronal cell death is illustrated by the finding that overexpressing a mutant form of Rb lacking critical phosphorylation sites protects neurons from cell death induced by DNA damage.^160,166 Moreover, deregulation of the Rb cell cycle pathway during development promotes ectopic cell cycle entry and elevated apoptosis which is, in part, p53-dependent.^167,168 The latter finding is consistent with a role for the Rb-regulated E2F1 protein as a specific inducer of apoptosis and p53 accumulation.¹⁶⁹

p53 Tumor Suppressor Gene

The p53 Gene

The p53 tumor suppressor gene encodes a nuclear phosphoprotein that functions as a key regulator of DNA repair, cell cycle progression and apoptosis. The p53 protein is up-regulated in response to a diverse array of cellular stresses, including DNA damage, hypoxia, oxidative stress, ribonucleotide depletion and oncogene activation.^170,171 p53 protein levels are primarily regulated by changes in protein degradation in response to injury. In response to cellular stress, p53 induces its biological response largely through the transactivation of specific target genes. These downstream effectors have been characterized with respect to p53-mediated growth arrest,¹⁷² but the pathways associated with p53-mediated apoptosis have not been completely elucidated.¹⁷³ In addition to its transactivating activity, p53 may also promote apoptosis by repressing the expression of select genes.^174,175 Moreover, p53-mediated apoptosis may also occur through transcription-independent pathways requiring direct protein-protein interactions.^176,177

p53 Expression and Neuronal Injury

Alterations in p53 mRNA and protein expression have been associated with neuronal damage in a variety of model systems (for review, see Ref. 178). Neuronal damage resulting from stimulation by excitatory amino acids (excitotoxicity) has been strongly associated with p53 accumulation. The systemic injection of kainic acid, an excitotoxin that produces seizures, induced p53 expression in neurons exhibiting morphological evidence of damage.^179,180 Activation of glutamate receptors by intrastriatal infusion of either N-methyl-D-aspartate (NMDA), the NMDA receptor agonist quinolinic acid (QA) or kainic acid produced a significant elevation in p53 levels in striatal neurons.¹⁸¹^-¹⁸³ Elevated expression of the p53 gene has also been observed in models of experimental traumatic brain injury.¹⁸⁴^-¹⁸⁶ As early as 6 h post-injury, p53 mRNA is induced predominantly in neurons that are vulnerable to traumatic brain injury. Transient or permanent occlusion of the middle cerebral artery causes ischemia-induced cell death in striatal and cerebral cortical neurons, which is associated with a significant increase in the expression of p53 mRNA¹⁸⁷ and protein.¹⁸⁸

Elevated p53 immunoreactivity has also been detected in brain tissue derived from animal models of human neurodegenerative disease or from patients that have been diagnosed with a neurodegenerative disorder. Patients with Alzheimer's disease^189,190 show increased p53 immunoreactivity in morphologically damaged neurons, consistent with the presence of increased p53 immunoreactivity in neurons from mice overexpressing the β-amyloid peptide (Ab 142).¹⁹¹ Mutation of the E6AP ubiquitin ligase in a mouse model of Angelman syndrome results in increased cytoplasmic abundance of the p53 protein in hippocampal pyramidal neurons and cerebellar Purkinje neurons.¹⁹² Animals expressing the Angelman mutation display motor dysfunction, inducible seizures and a deficiency in contextual learning. Thus, increased levels of the p53 protein in Angelman syndrome resulting from abnormalities in the ubiquitination process may contribute to neuronal dysfunction.

The brains of patients with Down's syndrome, a genetic disorder manifesting a similar pathology to Alzheimer's disease, have also been shown to express elevated levels of apoptosis effectors, including the p53 protein.^190,193,194Motor neuron degeneration observed in amyotrophic lateral sclerosis has been associated with increased levels of p53 in motor neurons of the spinal cord and motor cortex.¹⁹⁵ Recent evidence also suggests that p53 could be involved in the pathogenesis of Huntington's disease. The Huntington's disease protein, huntingtin, was found to interact with p53 and the CREB-binding protein,¹⁹⁶ and to repress transcription of several p53-regulated promoters. These results raise the possibility that the mutant huntingtin protein may cause neuronal dysfunction and cell death by interacting with transcription factors and altering gene transcription.

The results obtained with in vitro models of neuronal injury are consistent with the data described above for the in vivo models. Excitotoxicity, which figured so prominently in the whole animal studies, is a potent inducer of p53 protein in cultured cerebellar granule neurons.^197,198 Another potent stimulus for elevating p53 expression in cultured neurons is DNA damage induced by cytosine arabinoside,^199,200 ionizing radiation,¹⁶⁶ camptothecin,¹⁵³ and the absence of essential DNA repair proteins.²⁰¹ Hypoxia in culture, which models the ischemia produced by middle cerebral artery occlusion, increases p53 protein expression in rat embryonic cortical neurons.²⁰²

p53 Expression and Neuronal Cell Death

The physiological relevance of p53 expression to neuronal cell death has been evaluated in numerous models of injury and disease. p53-deficient mice or neurons derived from these mice have been used most often, but inhibitors of p53 expression or p53 function have also been used to evaluate the role of p53 in the context of neuronal injury. The absence of p53 has been shown to protect neurons from a wide variety of toxic insults including focal ischemia,²⁰³ ionizing radiation,²⁰⁴^-²⁰⁸ kainate-induced seizures,²⁰⁹ MPTP-induced neurotoxicity,²¹⁰ methamphetamine-induced neurotoxicity,²¹¹ adrenalectomy,²¹² traumatic brain injury,¹⁸⁶ DNA-damaging agents,^{199,200,206,213}^-²¹⁵ glutamate,^197,198 hypoxia,^202,216 and NGF withdrawal.^217,218 A role for p53 has also been demonstrated for apoptosis associated with developmental neuronal death in certain subpopulations of neurons²¹⁷ as well as cell death occurring during abnormal development.^167,219 In addition to the use of p53-deficient mice, the application of antisense oligonucleotides^{197,200,202,220,221} or a synthetic inhibitor of p53²²² have been successfully used to protect neurons against cell death induced by a variety of cellular stresses.

However, the absence of p53 does not protect neurons against all forms of cellular stress. Cerebellar neurons lacking p53 die when transferred to a low potassium medium,²¹³ and postnatal cortical and hippocampal neurons also die after staurosporine exposure in a p53-independent manner.²⁰⁴ Cerebellar granule neuron death induced by methylazoxymethanol is not alleviated in p53-null mice. Moreover, two separate reports failed to demonstrate that p53 is involved in the death of neurons in a mutant SOD1 transgenic mouse model of familial ALS.^223,224 Despite evidence that p53 plays an important role in mediating cell death after acute neuronal injury, additional studies will be required to fully evaluate the role of p53 in late-onset neurodegenerative diseases.

p53-Mediated Cell Death Signaling Pathways

The mechanism by which p53 specifies the neuronal response to injury is poorly understood. However, currently available data suggest that the Bcl2 family member, Bax, is involved in p53-mediated neuronal death. Bax-deficient neurons are protected from cell death induced by DNA-damaging agents^204,215,225 and adenovirus-mediated p53 overexpression.^215,226 Moreover, various forms of neuronal injury are associated with Bax translocation from the cytosol to the mitochondria.^{127,153,227,228} The redistribution of Bax to the mitochondria has been associated with a reduction in mitochondrial membrane potential, mitochondrial release of cytochrome c, and activation of caspases,²²⁹^-²³³ suggesting that caspases may also be a component of a p53-induced cell death pathway. Recent studies indeed demonstrated that p53 is required for caspase activation in response to genotoxic stress.^{215,225,226,234} These findings suggest that some forms of neuronal injury invoke a common pathway involving signal transduction through p53, Bax, mitochondrial dysfunction, cytochrome c release, and caspase activation.

Clearly, additional studies are required to elucidate the downstream effectors mediating neuronal cell death in response to p53 activation. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene has been identified as a p53-inducible gene in cultured cerebellar granule neurons subjected to DNA damage.²⁰⁰ Apaf1, a key element of the apoptosome, which regulates initiation of the caspase-9 cascade, is induced in neurons in response to p53 activation or DNA damage.²³⁵ Other genes, such as DR5, Fas, Fas ligand,¹⁷³ PERP,²³⁶ Noxa and PUMA²³⁷ have been shown to be induced by apoptotic stimuli as a result of p53 activation in a variety of non-neuronal cell types, but the involvement of these genes in p53-dependent neuronal apoptosis is not known.

It is also critical that the pathways responsible for activating and suppressing p53 activity be identified. In this regard it is interesting to note that the important survival-promoting protein, Akt, can protect neurons from cell death by inhibiting p53-dependent transcriptional activity.⁶⁷ These results demonstrate the interconnection that exists between pathways that govern cell death and viability and serve to remind us that the response and the outcome of neurons to stress are exceedingly complex.

DNA Damage-Activated Enzymes

Excessive DNA damage may result from exposure to free radicals generated by oxidative stress and thereby may be a common initiating stimulus for neuronal apoptosis. Oxidative stress is associated with a variety of neuropathological conditions including acute excitotoxicity (stroke, traumatic brain injury) and progressive neurodegeneration (Alzheimer's, Parkinson's, Huntington's, ALS).^238,239 Several enzymes are activated in response to DNA damage (strand breaks) and are associated with the control of cell cycle checkpoints, DNA repair programs and the regulation of apoptosis. These include the gene product for ataxia telangiectasia (ataxia telangiectasia mutated, ATM), DNA-dependent protein kinase (DNA-PK), and poly(ADP-ribose) polymerase-1 (PARP1). Interestingly, all three of these enzymes are substrates for caspase-3,^240,244 implicating them in the regulation of apoptosis.

ATM

ATM (ataxia telangiectasia mutated), a protein kinase belonging to the PI3 kinase family, is activated by DNA double-strand breaks secondary to ionizing radiation (IR) and radiomimetics. Multiple phosphorylation substrates have been identified including p53, MDM2, and BRCA1, proteins involved in DNA repair and cell cycle checkpoint control (for a review, see Ref. 245). The generation of an ATM-deficient mouse verified the relationship of ATM function to DNA damage-induced responses. For example, ATM^−/− mice display extreme sensitivity to IR due to its toxic effects on the gastrointestinal tract, and fibroblasts derived from the mice show compromised G1/S checkpoint function.²⁴⁶ In contrast to these results from non-neuronal and proliferative cell types, developing CNS neurons in ATM^−/− mice turn out to be radioresistant compared to those in wild-type mice which show massive cell death, indicating that IR-induced neuronal apoptosis is ATM-dependent.²⁰⁸ IR-induced, ATM-mediated neuronal cell death also depends on p53, Bax and caspase-3.²²⁵ ATM is known to phosphorylate p53 at Ser-15 directly^247,248 and Ser-20 through the activation of CHK2.²⁴⁹ ATM also phosphorylates MDM2 at Ser-395.²⁵⁰ These phosphorylation events are thought to collectively contribute to p53 stabilization and transcriptional activation.^251,252 However, it is still not known precisely how these ATM-dependent modifications of p53 result in the induction of apoptotic mediators.

Interestingly, IR-induced death of neural progenitor cells is not dependent on ATM, while p53 is required for apoptosis in both neural progenitor and post-mitotic neuronal populations.^225,253 Thus, the linkage between ATM and p53-dependent cell death is not observed in undifferentiated, multipotent precursors but appears to develop in newly formed post-mitotic neurons; it still remains to be established if ATM is also required for IR-induced apoptosis in fully differentiated neurons in adults. Indeed, adult neurons, irrespective of the ATM genotype, don't seem to be sensitive to IR.²⁴⁶ This implies that a critical function of ATM in neuronal apoptotic signaling may be the elimination of neurons born with a substantial amount of DNA damage accumulated during development.²⁵³ Consistent with this idea is the observation that ATM deficiency in Ataxia Telangiectasia (AT) patients causes early-onset progressive neurodegeneration, which is apparently due to compromised genomic integrity based on the phenotypic similarities of A-T to pathological conditions caused by genetic defects in DNA repair and cell cycle checkpoints.²⁵⁴ ATR, an ATM-related protein primarily involved in the response to UV-induced DNA damage,²⁵⁵ has not been evaluated in the process of neuronal apoptosis and remains to be examined.

DNA-Dependent Protein Kinase

DNA-dependent protein kinase (DNA-PK) is a member of the PI3 kinase family and is activated in response to DNA strand breaks,²⁵⁶ and is essential for DNA repair.²⁵⁷ DNA-PK phosphorylates p53,²⁵⁸ MDM2,²⁵⁹ and other factors implicated in the regulation of cell death including c-abl²⁶⁰ and IkB.²⁶¹ However, several recent reports suggest that DNA-PK is not required for neuronal apoptosis but may instead be important for neuronal survival after genotoxic insult. Neurons derived from scid mice, which express a truncated form of DNA-PK with impaired kinase activity, show higher rates of spontaneous cell death and increased vulnerability to apoptotic insults in culture as well as in vivo.²⁶²^-²⁶⁴ Although an inhibitor of DNA-PK reportedly reduces the manifestation of apoptotic nuclear changes in staurosporine-treated neuroblastoma cells,²⁶⁵ this finding may be due to the tumorigenic and proliferative properties of neuroblastoma cells. These results suggest that the compromise in DNA repair activity associated with the scid mutation facilitates the accumulation of unrepaired DNA strand breaks, which eventually culminates in neuronal apoptosis.

Poly(ADP-Ribose) Polymerase

Poly(ADP-ribose) polymerase-1 (PARP-1) is the principal member of an expanding family of PARP enzymes,²⁶⁶ which are activated in response to single-strand DNA breaks. In response to DNA damage, PARP-1 modifies a variety of nuclear proteins, including itself, by attaching poly(ADP-ribose) chains. The activated enzyme plays an important role in base excision repair and transcriptional regulation, accounting for its contribution to genomic stability.²⁶⁷^-²⁶⁹ Apart from its involvement in DNA repair, PARP1 has been proposed to have a major impact on cell viability as excessive activation of the enzyme depletes cellular NAD and ATP, causing necrotic cell death²⁷⁰ (for a review, see Ref. 271). In agreement with this idea, PARP-1 inhibitors attenuate neuronal cell death caused by excitotoxicity in culture.^272,273 More compelling results were obtained from studies involving PARP-1 knockout mice, in which PARP-1 deficiency resulted in substantially reduced areas of infarct in an animal model of stroke.^274,275 In addition, the absence of PARP-1 also dramatically reduces the size of lesions induced by intrastriatal NMDA injection²⁷⁶ and the extent of MPTP-induced loss of dopaminergic neurons.²⁷⁷ The protection conferred by genetic or pharmacological suppression of PARP-1 activity in models of stroke,²⁷⁸ NMDA excitotoxicity²⁷⁶ and traumatic brain injury²⁷⁹ appears to be long-lasting based on anatomical and behavioral analyses.

The cleavage of PARP-1 by caspase-3 during the process of apoptosis is thought to serve as a switch between necrotic vs. apoptotic cell death, ensuring that cells maintain sufficient ATP levels to die by apoptosis. Consistent with this idea, PARP-1 inhibitors attenuate necrotic, but not apoptotic, neuronal death in an in vitro model of cerebral ischemia.²⁸⁰ What is not clear in the stroke model, however, is whether subsequent to PARP1 inhibition, neurons in the core region of an infarct that would otherwise die by necrosis are rescued completely or die by apoptosis. Even though ATP depletion is prevented by PARP-1 inhibition, neurons in the core region still suffer from extensive oxidative damage, which should eventually activate apoptotic pathways. Apparently, however, even the apoptotic process is efficiently blocked since the protection afforded by PARP-1 inhibition is sustained for at least 2-3 weeks,^276,278,279 beyond the typical time course of apoptosis. Thus, these results suggest that PARP-1 inhibition may also work to suppress apoptotic neuronal cell death. This would be consistent with the finding that PARP-1 inhibition can partially suppress p53-dependent neuronal cell death²³⁴ as well as p53 induction.²⁸¹

A number of important issues remain to be examined with respect to the role of PARP in neuronal injury. For example, it is not clear whether NAD/ATP depletion can account for the entire cascade of PARP-mediated changes in neuronal viability. In addition, it is not clear if other members of the PARP family are involved in the neuronal response toxic insults. Nevertheless, data obtained thus far, suggests that PARP-1 plays an important role in several forms of neuronal apoptosis.

Bcl-2 Family Members and Mitochondrial Integrity

Properties of the Bcl-2 Family

Proteins belonging to the Bcl-2 family are key regulators of neuronal cell death and survival, and individual family members can serve to inhibit or promote apoptosis.²⁸² The prototypical anti-apoptotic member, Bcl-2, was first discovered because of its involvement in 95% of follicular B-cell lymphomas as a result of a chromosomal translocation t(14;18). High levels of bcl-2 did not act to increase proliferation but rather increased cell survival, thus defining a new class of oncogenes.²⁸³^-²⁸⁵ Proteins in this family share a number of Bcl-2 homology domains (BH domains). In general, the anti-apoptotic members, Bcl-2 and Bcl-X_L, contain BH1-4, whereas the pro-apoptotic members, Bax and Bak, contain BH1-3. A third subset of family members including Bad, Bik, Blk, Hrk, Bim, Bid²⁸⁶ and Noxa²⁸⁷ contain only the BH3 domain. Many of the family members contain a hydrophobic C-terminal tail, which may serve to anchor these proteins into intracellular membranes.

Bcl-2 proteins regulate the apoptotic cascade mainly at the level of the mitochondria. Upon activation by an apoptotic signal, pro-apoptotic Bcl-2 proteins accumulate at the mitochondrial membrane.²⁸⁸^-²⁹¹ This is associated with alterations in mitochondrial membrane potential and the release of several pro-apoptotic factors including cytochrome c, which, in conjunction with Apaf-1, facilitates the activation of the caspase cascade (Fig. 1).^292,293

The impact of pro-apoptotic Bcl-2 proteins on mitochondria can be inhibited by Bcl-2 and/or Bcl-X_L.^294,295 Although the mechanism underlying the regulation of mitochondrial membrane permeability is not completely understood, current hypotheses suggest that Bcl-2 family members regulate the outer mitochondrial membrane by interaction with the voltage-dependent anion channel (VDAC),²⁹⁶ and other components of the mitochondrial permeability transition pore (PTP) complex.²⁹⁷ Furthermore, there is evidence indicating that some Bcl-2 family members can form hetero- and homodimers with inherent ion channel-forming abilities.²⁹⁸^-³⁰⁰

The ability of Bcl-2 family members to form dimers is essential to their function and regulation. Enforced homodimerization of Bax stimulates Bax translocation, caspase activation and cell death.³⁰¹ On the other hand, heterodimerization of anti-apoptotic members such as Bcl-2 or Bcl-X_L with pro-apoptotic members such as Bax can inhibit or activate apoptosis depending on the relative levels of each protein (Rheostat model³⁰²). Mutational analyses have shown that the BH domains are important for the dimerization of Bcl-2 family members. The BH1 and BH2 domains in Bcl-2 and Bcl-X_L are essential for their dimerization to Bax.³⁰³ NMR and X-ray crystallographic analyses of Bcl-X_L show that the α-helical BH1-3 domains are closely juxtaposed to form a hydrophobic binding pocket.^304,305 Mutation of the BH3 domain in Bax, Bad and Bid have shown that this domain is essential for both the binding ability of these proteins and their pro-apoptotic function.^306,307 These findings support the rheostat model for Bcl-2 proteins in apoptotic regulation. This hypothesis proposes that anti-apoptotic and pro-apoptotic Bcl-2 proteins exist in a delicate balance at the mitochondrial membrane. A shift in one direction or the other may determine the fate of the cell.

However, the rheostat model is perhaps too simple, as more recent evidence has shown the regulation of these proteins to be much more complex. Pro-apoptotic proteins such as Bad and Bid contain the critical amphipathic BH3 domain but lack a C-terminal hydrophobic tail present in many of the other membrane-anchored family members. These proteins can move between the cytosol and membranes in a regulated fashion. For example, the pro-apoptotic activity of Bad is regulated by phosphorylation. When Bad is phosphorylated on two specific serine residues, it is sequestered by binding to 14-3-3 and can no longer bind Bcl-2 or Bcl-X_L.³⁰⁸ Phosphorylation also appears to play an important role in regulating other Bcl-2 family proteins. For example, Bcl-2 can be phosphorylated at serine 70 to activate Bcl-2's anti-apoptotic activity or it can be hyperphosphorylated on multiple sites to inhibit Bcl-2 activity.^309,310 Furthermore, we have shown that p38 MAP kinase activity is involved in regulating Bax function.¹²⁷ These changes in phosphorylation state may very well affect the protein-protein interactions of these Bcl-2 family members.

The BH3 domain-only proteins function by modulating the activity of other Bcl-2 family members. Bcl-2 proteins such as Bad, Bim, Bid, Bik, Blk and Hrk can bind to the hydrophobic groove of the anti-apoptotic family members as well as pro-apoptotic family members such as Bax and Bak.^311,312 For example, Bid is a pro-apoptotic Bcl-2 homolog that, once cleaved by caspase-8, is targeted to mitochondria by cardiolipin.³¹³ Truncated Bid binds and activates pro-apoptotic members, Bax and Bak.³¹⁴^-³¹⁶ Other members, such as Bim, Bad and Noxa bind and inhibit the anti-apoptotic members, Bcl-2 and Bcl-X_L.^287,317,318 These interactions suggest that the BH3 domain-only proteins play an important role regulating the function of other Bcl-2 family members.

Bcl-2 Family Members in the Nervous System

Members of the Bcl-2 family are expressed in the nervous system during development and in the adult. Bcl-2 is widely expressed during development, whereas in the adult, its expression is low in the CNS but remains high in the PNS. Conversely, Bcl-X_L remains highly expressed in the nervous system throughout both development and adult life.³¹⁹ Bax is down-regulated in the adult CNS but highly expressed in the nervous system during the period of natural cell death when the number of neurons is reduced by 20-80%, presumably to match the number of innervating neurons with the size of the target tissue.³²⁰

Studies with transgenic mice have demonstrated the importance of Bcl-2 family members in regulating neuronal cell death. Animals overexpressing Bcl-2 contained 30–40% more neurons than wild-type animals following the period of developmental neuronal death. Furthermore, neurons overexpressing Bcl-2 are protected from apoptosis after facial and sciatic nerve axotomy as well as optic nerve transection. Although the exact mechanism is not known, it may be related to the effects of Bcl-2 on intracellular Ca²⁺ homeostasis.³²¹ High levels of Bcl-2 expression have also proven protective in other models of neuronal cell death, for example, following ischemia and excitotoxic injury.³²²

Conversely, the absence of pro-apoptotic family members such as Bax tends to be protective. For example, Bax deficiency protects neurons from ionizing radiation, changes in extracellular ionic conditions, excitotoxicity and ischemia.^204,215,323 One of the important initiators of cell death following stroke is thought to be DNA damage. DNA damage can independently activate cyclin-dependent kinases and p53 pathways, and both mediate Bax activation in neurons.^153,215 Recently, a novel splice variant of Bax, Bax k, has been shown to promote death following ischemia, and its mRNA was distributed in selectively vulnerable hippocampal CA1 neurons that are destined to die following global ischemia.³²⁴

Recent evidence has shown Bim to be important in regulating neuronal cell death. Upon withdrawal of nerve growth factor (NGF), neurons activate c-Jun and induce BIM_EL expression, and subsequently die in a Bax-dependent manner.³²⁵ Furthermore, overexpression of BIM_EL induces cytochrome c release and apoptosis even in the presence of NGF. Finally, neurons treated with Bim antisense oligonucleotides and neurons from Bim^−/− mice die more slowly following NGF withdrawal.^326,327 These studies suggest that Bim and perhaps other BH3 domain-only proteins play redundant roles upstream of Bax in the apoptotic pathway following growth factor withdrawal, and that these proteins may be regulated by kinases such as JNK.³²⁸

On the other hand, there are some Bcl-2 family members that act quite different in neuronal versus non-neuronal cells. Bid is an example of a Bcl-2 family member that seems to have cell type-specific functions. One report suggests that, unlike in other cell types, Bid does not play an essential role in either naturally occurring or AraC-induced cell death in neurons.³²⁹ Conversely, another report suggests that cleavage of Bid may amplify caspase-8-induced neuronal death in a seizure model.³³⁰ Interestingly, another BH3 domain-only protein, N-Bak, plays opposite roles in neuronal versus non-neuronal cells. N-Bak is a neuron-specific, BH3-only isoform of Bak that is anti-apoptotic in neurons, but pro-apoptotic in non-neuronal cells.³³¹ This was the first example of a neuron-specific Bcl-2 family member.

Bcl-2 family members have also been implicated in various neurodegenerative diseases such as Parkinson's Disease (PD), Alzheimer's Disease (AD) and motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Overexpression of Bcl-2 and ablating Bax expression protected dopaminergic neurons from MPTP toxicity, a model of PD.^332,333 Presenilin-1 and -2, genes involved in AD, have also been shown to interact with Bcl-X_L.³³⁴ Mutations in the superoxide dismutase-1 (SOD-1) gene are linked to familial ALS, and spinal cord motor neuron death in mutant SOD-1 mice is associated with a decrease in Bcl-2 expression and an increase in Bax expression.³³⁵^-³³⁷ Furthermore, in the spinal cords of these mice, Bax translocates from the cytosol to the mitochondria during the progression of the disease.³³⁸ Overexpression of Bcl-2 delays caspase expression, increases motor neuron survival, and improves muscle function in mutant SOD-1 mice.^335,339 However, the disease eventually progresses, and duration of the disease is not altered.³⁴⁰ Survival of motor neuron protein (SMN), which has been implicated in SMA, has also been shown to interact with Bcl-2.³⁴¹ In fact, Bcl-2 acts synergistically with SMN to inhibit Bax-induced or Fas-mediated toxicity.³⁴² These studies suggest a possible role for Bcl-2 family members in a variety of neuropathological processes. Moreover, manipulating the expression and/or activity of Bcl-2 family members may prove instrumental in maintaining neuronal survival after injury and in response to disease.

Mitochondrial Pro-Apoptotic Factors

Mitochondria play a pivotal role in the amplification of an apoptotic signal since cytochrome c is normally sequestered in the mitochondrial intra-membrane space. In many cell types, apoptosis is associated with a loss in the normal electrochemical gradient employed by mitochondria to generate ATP (the proton motive force).³⁴³ Loss of mitochondrial membrane potential leads to opening of a protein complex known as the permeability transition pore.³⁴⁴ When this channel is opened, cytochrome c is released into the cytoplasm, leading to the formation of the apoptosome and activation of caspase-9.³⁴³ In neurons, mitochondrial cytochrome c release can be independent of permeability transition.³⁴⁵ Cytochrome c release in this situation may be mediated by the action of pro-apoptotic bcl-2 family members³⁰⁰ (discussed elsewhere).

In addition to cytochrome c, several other factors are released from mitochondria in response to apoptotic stimuli. One pro-apoptotic protein released by mitochondria is the apoptosis-inducing factor (AIF). AIF is released from mitochondria after permeability transition and translocates to the nucleus where it promotes chromatin condensation and DNA fragmentation in a caspase-independent manner.³⁴⁶ A second factor normally sequestered in mitochondria is known by the dual name of Smac/DIABLO. When released into the cytoplasm, this small protein up-regulates the proteolytic activity of caspases by interacting with members of the IAP family. A third pro-apoptotic factor normally resident in mitochondria, endonuclease G, was recently identified as an important apoptosis initiator.³⁴⁷ At this point, little is known about the role of the mitochondrial proteins Smac/DIABLO, AIF or endonuclease G in excitotoxicity or death receptor-mediated neuronal apoptosis. Caspase-2-deficient neurons demonstrate increased levels of smac, which is thought to allow developmental neuronal death to proceed in the absence of caspase-2 by releasing caspase-9 from IAP inhibition.³⁴⁸ A mutation in the C. elegans homologue for endonuclease G also prevents normal progression to neuronal apoptosis during development.³⁴⁹

Proteolytic Enzymes

Caspases

Pioneering studies aimed at identifying genes required for programmed neuronal death during the course of C. elegans development led to the discovery that one of these genes shared strong molecular homology with interlukin-1 converting enzyme (ICE).³⁵⁰ ICE turned out to be a member of a family of proteases later designated caspase enzymes.³⁵¹ Caspase stands for cysteine-dependent, aspartate-specific protease. To date, fourteen family members have been identified. Some family members (caspases -4 and -5) are thus far unique to humans, and others, caspases -12 and -13, have not yet been identified in the human genome. All caspases are synthesized as inactive proenzymes and activated by proteolytic cleavage. The pro-enzyme is cleaved into an N-terminal peptide, a large subunit containing the catalytic portion of the enzyme and a small subunit at the C-terminal end of the peptide. The active enzyme consists of a tetrameric complex of two large subunits and two small subunits.³⁵² Caspase enzymes have been divided into several distinct groups based on protein structure and putative function. Caspases -1, -4, -5, -11 and -13 are classified as cytokine activators, caspases -2, -8, -9, -10 and 12 are believed to act as initiators of the apoptotic cascade, and caspases -3, -6 and -7 are designated executioners of apoptosis. Caspase-14 appears to have a unique role in supporting the terminal differentiation of keratinocytes in the epidermis and cornea without promoting the typical features of apoptosis.^353,354 Not all studies have supported these designations, and the biologic function associated with some of the less well-studied members of this enzyme family have not been evaluated in neurons or neural tissues. In addition, some debate remains about the possibility that caspases known as cytokine activators, primarily caspase-1, may also participate as apoptotic initiators in neurons³⁵⁵ or oligodendrocytes.^356,357

The initiator caspases can be further subdivided into two distinct groups. One group is activated following ligand binding to members of the tumor necrosis factor α (TNF-α) receptor (TNF-R) family including FAS, TNF-R and TRAIL receptors. These caspases (-8 and -10) contain death effector domains (DED) in the N-terminal region of the pro-enzyme. The DED confers homomeric binding ability to adaptor proteins in the death-inducing signaling complex (DISC) at the cytoplasmic region of TNF-R family members known as death receptors. Ligand binding of death receptor family members promotes receptor clustering. The predominant theory of caspase activation is that death receptor clustering brings molecules of pro-caspase-8 or pro-caspase-10 in such close proximity that they can be activated by autocleavage.³⁵²

A second group of caspases (-2 and -9) contain a domain known as the caspase recruitment domain (CARD). The CARD domain appears to confer homomeric binding capabilities between caspases and their specific regulatory complexes. Caspase-2 is recruited to the DISC through interactions between its CARD and the CARD on an adapter protein called RAIDD. RAIDD contains both a CARD domain and a DED domain allowing it to interact with caspase-2 on one end and a DED containing serine/threonine kinase, RIP, that is associated with TNF-R1.³⁵⁸ Thus, despite lacking a DED domain, caspase-2 may still be activated by proximity-induced auto-processing. Pro-caspase-9 binds apoptotic protease activating factor-1 (Apaf-1) by CARD domain interaction, and Apaf-1 oligomerization promotes proximity-induced caspase-9 activation.^359,360 Apaf-1 is a required peptide co-factor for caspase-9 activation, but caspase-9 activation also requires two additional factors, cytochrome c and dATP.^361,362 The complex of these factors, designated the apoptosome, is under intense investigation and additional regulatory proteins continue to be identified.³⁶³ In addition, there is some evidence that human caspase activity is regulated by phosphorylation⁷¹ although the putative phosphorylation sites are not conserved in several other species.³⁶⁴

Once initiator caspases are activated, they can proteolytically process executioner caspases (caspases -3, -6 and -7) into their active form. This group of caspases contains truncated N-terminal sequences without known regulatory function. Once activated, their substrates include nuclear and cytoskeletal components that must be proteolyzed for a cell to develop the phenotypic characteristics of apoptosis. The activation of executioner caspases has been demonstrated in variety of neuronal death paradigms. A large number of studies have demonstrated that caspase-3 is activated in the CNS in response to a variety of injurious stimuli including ischemia, excitotoxic insult, trauma and neurodegenerative diseases.^365,371 Caspase-3 is also required for normal developmental cell death in the CNS.³⁷² Caspase-6 may play a role in the pathogenesis of Alzheimer's and Huntington's disease.^373,374 Caspase-7 is activated during motoneuron degeneration in a mouse model of amyotrophic lateral sclerosis.³³⁸ However, the inhibition or ablation of executioner caspases may not improve neuronal survival, even though the morphologic changes associated with apoptosis do not develop.^375,376

Endogenous Caspase Inhibitors

Apoptosis appears to be a tightly regulated cellular process. In addition to specific patterns of caspase activation, several endogenous inhibitors of caspase enzyme activity have also been described. One class of caspase inhibitor acts by forming proteolytically inactive heterodimeric complexes with active caspases. Examples of this strategy include splice variants of caspase-2³⁷⁷ and caspase-14.³⁵⁴ Very little is known about the presence of caspase-inhibitory splice variants in the central or peripheral nervous system. The FLICE (caspase-8) inhibitory protein (FLIP) shares a high degree of sequence homology with caspase-8 and inhibits caspase-8 activity by promoting the formation of a caspase-8/FLIP heterodimers devoid of proteolytic activity.³⁷⁸ Motoneurons expressing high levels of FLIP are resistant to FasL-induced apoptosis,⁴ and FLIP is expressed in neuroblasts in rat cortex during the early postnatal period.³⁷⁹ The localization of FLIP in developing neural tissue suggests that FLIP may play a regulatory role in cell survival during nervous system development. However, FLIP knockout mice have an embryonic lethal phenotype similar to that observed in caspase-8-deficient mice,³⁸⁰ suggesting that endogenous caspase inhibition is also required for normal caspase function, perhaps by preventing proteolytic inactivation of other important apoptosis regulators.

The peptide family of inhibitor of apoptosis (IAP) genes³⁸¹ employs a second strategy for endogenous caspase inhibition. This family of proteins consists of at least seven mammalian members including X-chromosome-linked inhibitor of apoptosis (XIAP). All members of this family contain at least one BIR (baculovirus inhibitor of apoptosis repeat) domain that binds and inhibits the proteolytic activity of caspases -3 and -7. Some of the IAP proteins also inhibit caspase-9 activity (for review, see Ref. 382). Inhibition by IAP family members can be overcome by several different mechanisms. The C-terminal region of some IAPs contains a domain referred to as the RING (Really Interesting New Gene) domain, which contains ubiquitin E3 ligase activity that targets them for rapid proteasomal degradation.³⁸³ Two other proteins that bind IAPs and prevent caspase inhibition have also been identified. The XIAP-associated factor 1 (XAF1) binds XIAP in the nucleus, potentially promoting release of caspase inhibition once the enzymes have been able to translocate into the nuclear compartment.³⁸⁴ The second mitochondria-derived activator of caspase (Smac) or DIABLO protein is normally sequestered in mitochondria but will bind and inhibit the anti-apoptotic activities of IAPs when it is released into the cytoplasm.^385,386

Calpains

In addition to caspases, there are additional classes of proteases associated with neuronal cell death. The calpains are calcium-activated cysteine proteases implicated in neuronal cell death following acute neurological insults. Administration of calpain inhibitors during the initial 24-h period following injury can attenuate injury-induced derangements of neuronal structure and function following traumatic brain injury and excitotoxicity.³⁸⁷^-³⁹³ At least one substrate, fodrin (spectrin), appears to be shared between the calpains and caspases, but it is not presently known if the caspases and calpains activate one another. In addition, a novel serine protease has been identified which is expressed at ten-fold higher concentrations in the CNS than in peripheral tissues of the rat and human.³⁹⁴ The mRNA for this protease was found to be significantly elevated by excitotoxic injury. It seems clear that proteases can have profound effects on neuronal viability following injury although additional studies will be required to determine if they represent a rate-limiting step in the apoptotic process.

Abbreviations

AIF, apoptosis-inducing factor; ALS, amyotrophic lateral sclerosis; Apaf-1, apoptotic protease activating factor-1; ATM, ataxia telangiectasia mutated; BH domain, Bcl-2 homology domain; CARD, caspase recruitment domain; CDK, cyclin-dependent kinase; DD, death domain; DISC, death-inducing signaling complex; DNA-PK, DNA-dependent protein kinase; ERK, extracellular signal-regulated kinase; FADD, Fas-associated death domain; FasL, Fas ligand; FLIP, FLICE (caspase-8) inhibitory protein; IAP, inhibitor of apoptosis; IR, ionizing radiation; JNK, c-Jun N-terminal kinase; MKK, MAP kinase kinase; NMDA, N-methyl-D-aspartate; PARP, poly(ADP-ribose) polymerase; PI3K, phosphatidylinositol-3 kinase; RIP, receptor-interacting protein; TNFR, tumor necrosis factor receptor; TRADD, TNFR-associated death domain; TRAF2, TNF receptor-associated factor 2

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Publication Details

Author Information and Affiliations

Authors

Richard S. Morrison, Yoshito Kinoshita, Mark D. Johnson, Saadi Ghatan, and Joseph.

Copyright

Publisher

Landes Bioscience, Austin (TX)

NLM Citation

Morrison RS, Kinoshita Y, Johnson MD, et al. Neuronal Survival and Cell Death Signaling Pathways. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.