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The p53 tumor suppressor gene is the most frequently mutated gene in human tumors. As a tumor suppressor, p53 plays a key role in DNA damage repair, cell cycle regulation, and cellular apoptosis. The mechanisms that underly the ability of p53 to subserve these functions in cycling, nonneuronal cells have been intensively studied and are the subject of several recent reviews.1,2,3 We will not cover these topics in detail here, but will instead focus upon the role of p53 in neurons, a cell type that is ‘forever’ postmitotic, and that must survive and maintain its genome over as long as a century in humans. Moreover, we will discuss emerging evidence that the recently-described p53 family member, p73, also plays a major role in regulating development and survival in the nervous system.

The p53 family and developmental apoptosis in the nervous system

During nervous system development both progenitor cells and postmitotic neurons are overproduced, and the nervous system then chooses, through a process of elimination, those cells that have differentiated and made appropriate connections. It is now clear that the nervous system selects cells during two major periods of apoptosis. The first takes place in the ventricular and subventricular zones of the developing nervous systems, where neural stem and progenitor cells differentiate to produce the neurons and glial cells that will migrate and populate the brain and spinal cord. It is likely that this period of apoptosis serves two functions; to eliminate those progenitors that do not differentiate appropriately, and to ensure that the appropriate cell number is generated in rapidly-growing tissues such as the cerebral cortex. The existence of this period of apoptotic death has only recently been appreciated, and the mechanisms that control the life versus death of any given cell are still only poorly understood.

The second period of apoptotic death in the nervous system occurs once newly-born neurons have migrated to their final destinations, have extended their axons, and have attempted to establish appropriate connections. This period of naturally-occurring neuronal death eliminates approximately half of the neurons in any given population.4 In the peripheral nervous system, where this process has been extensively studied, neurons compete for limiting amounts of target-derived neurotrophins such as nerve growth factor (NGF), and their ultimate survival is dependent upon the interplay between receptor-mediated prosurvival and proapoptotic signals.5 Over the past several years, evidence has emerged implicating the p53 family in both of these periods of developmental apoptosis.

p53 is essential for the elimination of neural progenitor cells that fail to differentiate appropriately

Mice that carry a null mutation in the p53 gene are born and survive until early adulthood, when they succumb to a variety of tumors,6 an observation that initially led to the conclusion that p53 was not involved in development. However, closer examination revealed that a significant portion of p53−/− embryos developed craniofacial abnormalities and died as a consequence of an overproduction of neural tissue and failed neural tube closure (exencephaly),7,8 indicating that p53 was important in regulating neural development.

Additional evidence supporting a role for p53 in progenitor cell development came from studies of mice lacking the retinoblastoma tumor suppressor protein (pRb).9,10,11 The Rb−/− mice die during embryogenesis, and have a striking nervous system phenotype consisting of ectopic mitoses and massive neural apoptosis. Further studies indicated (i) that this phenotype was due to the inability of newly-born neurons to undergo terminal mitosis in the absence of pRb,12,13,14 and (ii) that coincident deletion of p53 rescued the apoptotic phenotype in the Rb−/− CNS.15,16 Thus, inappropriate terminal differentiation in the absence of pRb led to activation of a p53 default apoptotic pathway. The existence of this p53 apoptotic pathway in progenitors suggests that a major role for p53 is to eliminate neural progenitors that fail to differentiate appropriately. A deficit in this pathway in the p53−/− mice would provide at least a partial explanation for their exencephaly.

Insights into other potential players in this progenitor cell apoptotic pathway derive from recent studies of mice carrying null alleles in genes that are part of a death receptor-independent, intrinsic apoptotic pathway.17 This pathway, which can be activated by p5318 independently of its transcriptional function,24 involves release of cytochrome c from the mitochondria, oligomerization and activation of Apaf-1 and caspase 9, and subsequent activation of caspase 3 and other effector caspases.17 Remarkably, animals mutant in each of Apaf1,19,20 caspase 9,21 and caspase 3,22,23 all display a dramatic overgrowth of neural tissue during embryogenesis, as a consequence of decreased progenitor cell apoptosis. Moreover, the Apaf1−/− mice display abnormalities in craniofacial structures,19,20 a phenotype also observed in the p53−/− embryos.8 These data therefore indicate that the intrinsic apoptotic pathway is critical during neural progenitor cell development and suggest that p53 is at least partially responsible for its activation.

An essential role for p53 and p73 during naturally-occurring sympathetic neuron death

A potential role for p53 in the apoptosis of postmitotic neurons was originally suggested by two sets of studies. First, a large number of studies documented increases in p53 following neural injury (reviewed below; Table 1). Second, overexpression of p53 was sufficient to induce the apoptosis of postmitotic sympathetic,25 hippocampal,26 and cortical27 neurons. Since these original reports, a number of studies have been published demonstrating that p53 is necessary for neuronal apoptosis, either following neural injury (reviewed below; Table 1), or during naturally-occurring neuronal death. With regard to the latter, the best-characterized example involves sympathetic neurons of the peripheral nervous system, which we will focus upon here.

Table 1 p53 is upregulated in response to many types of neural damage, and is necessary for the subsequent neuronal apoptosis
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During development, peripheral sympathetic neurons become postmitotic, extend their axons to appropriate target tissues, and then about half of these neurons undergo apoptosis during the first 3 postnatal weeks. The survival of any given neuron during this period is determined by its ability to compete for limiting amounts of target-derived nerve growth factor (NGF).5 NGF binds to the neuronal TrkA tyrosine kinase receptor, leading to the activation of a number of survival pathways, the most important of which is the Ras-PI3-kinase-Akt pathway.28,29 This pathway supports sympathetic neuron survival by overriding a receptor-mediated apoptotic signaling cascade that originates from a second neurotrophin receptor, the p75 neurotrophin receptor (p75NTR).28,29 Genetic support for this model derives from the findings that (i) all sympathetic neurons die in the TrkA−/−30 and NGF−/−31 mice, (ii) naturally-occurring sympathetic neuron death is greatly delayed in p75NTR−/− mice,32 and (iii) the coincident deletion of p75NTR rescues the sympathetic neuron death in the TrkA−/− mice (M Majdan and F Miller, unpublished observations). Thus, sympathetic neurons are ‘destined to die’ as a consequence of an ongoing, p75NTR-mediated apoptotic signal, and survive only if they sequester sufficient NGF to robustly activate TrkA.

A number of recent studies indicate that p53 and the related p73 play a key role in regulating the survival of sympathetic neurons during this developmental period. First, overexpression of p53 is sufficient to cause the death of sympathetic neurons in the presence of NGF.25 Second, Vogel and Parada33 demonstrated that embryonic p53−/− sympathetic neurons showed enhanced survival in culture in the absence of NGF, their obligate survival factor. Third, Aloyz et al 34 demonstrated that p53 levels increased when sympathetic neurons underwent apoptosis in response to either NGF withdrawal or activation of p75NTR, and that apoptosis could be inhibited if this increase in p53 levels was prevented. Moreover, developmental sympathetic neuron death was delayed (but not prevented) in the p53−/− mice.34 Thus, p53 is important in an apoptotic signaling cascade that is activated following NGF withdrawal or p75 neurotrophin receptor activation.

What is this apoptotic signaling cascade? Evidence indicates that NGF withdrawal activates at least two apoptotic signaling pathways, both of which may converge onto p53 (Figure 1). One of these pathways, which is also activated by p75NTR, involves JNK-p53-Bax.34,35 MEKK and JNK function upstream of p53 in p75NTR-mediated apoptosis,34 while cdc42/Rac1,36 Ask1,37 MKK, JNK, c-jun,38,39,40 and p5334 have been shown to act in a signaling pathway regulating NGF withdrawal-induced apoptosis. TrkA can silence the JNK-p53 arm of this pathway via Ras activation.29,41 A second pathway shown to be important for NGF withdrawal involves the activation of the cell cycle regulatory molecules CDK4/6, which activate pRb by phosphorylation, and subsequently cause sympathetic neuron apoptosis.42,43 Since pRb dysregulation (i) is known to cause p53 activation via p19ARF in nonneuronal cells,44 and (ii) leads to p53-dependent apoptosis in the embryonic nervous system (reviewed above), then it follows that this cell cycle pathway might also converge onto p53. If this were the case, then p53 would play a pivotal role in integrating neuronal apoptotic stimuli, perhaps thereby ensuring that apoptosis ensues only when these stimuli reach a certain critical threshold (Figure 1).

Figure 1
figure 1

The role of p53 and p73 in developmental neuron death. Naturally-occurring sympathetic neuron death is regulated by the balance of signals deriving from the NGF/TrkA prosurvival receptor and the proapoptotic, p75 neurotrophin receptor.5,28,29 Withdrawal of the survival ligand, NGF, or activation of the p75 neurotrophin receptor trigger two apoptotic signaling cascades, the JNK pathway and cell cycle deregulation, both of which are essential for neuronal apoptosis. P53 plays an essential proapoptotic role in this process, while a naturally-occurring truncated p73 isoform, ΔNp73, plays an essential anti-apoptotic role, potentially by antagonizing p53. A more extensive discussion of these pathways is found in the text

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Surprisingly, the p53 family member p7345,46,47 also plays an essential role in this system, but whereas p53 is proapoptotic, p73 is anti-apoptotic. A recent study by Pozniak et al 48 indicates that the predominant isoform of p73 in the developing brain and sympathetic ganglia is truncated at the amino-terminus (ΔNp73), and lacks the transactivation domain.49 Levels of ΔNp73β are high in sympathetic neurons when they are maintained in NGF, but decrease dramatically when NGF is withdrawn; if this decrease is prevented by ectopic expression of ΔNp73, neurons are rescued from apoptosis. Moreover, in p73−/− mice,49 developmental sympathetic neuron death is enhanced, indicating an essential anti-apoptotic role for p73 in these neurons. How does ΔNp73 inhibit sympathetic neuron apoptosis? ΔNp73 can directly bind to p53, at least in vitro, and can rescue p53-mediated death of sympathetic neurons.48 Thus, one mechanism whereby ΔNp73 might inhibit apoptosis is by binding to p53 and inhibiting its proapoptotic actions (Figure 1).

Does p73 play a similar anti-apoptotic role in other populations of developing or mature neurons? Although this question has not yet been answered, the phenotype of the p73−/− mice indicates that p73 is essential for normal neural development.49 These mice display hippocampal dysgenesis, absence of certain neuronal subtypes in both the central and peripheral nervous systems, and many die showing greatly enlarged ventricles and decreased cortical tissue. Although there are several potential explanations for these phenotypes, they could be explained by the absence of an anti-apoptotic activity in selected populations of CNS neurons and/or progenitors. Moreover, the truncated form of p73β that is predominantly observed in the developing brain48 is generated from the same gene as the full-length, proapoptotic form of p73 by alternative promoter usage,49 providing a mechanism for rapidly altering the ratios of the proapoptotic versus anti-apoptotic isoforms of p73 in the nervous system. In this regard, one potential explanation for the partial penetrance of the neural phenotype observed in the p53−/− embryos is that p73 may be able to compensate for the absence of p53 in the nervous system, at least with regard to developmental apoptosis. p73 may also play a role in differentiation; a recent publication132 indicates that full-length p73 causes differentiation of neuroblastoma cells, a finding that may well have implications for neural development.

p53 and neuronal injury: p53 mediates neuronal apoptosis in response to DNA damage

The apoptotic mechanisms underlying p53-mediated apoptosis in injured neurons are perhaps best-understood in the case of DNA damage. These studies have not only shed light on intracellular mechanisms that become increasingly important in long-lived cells, but have also provided insights into the neuronal apoptosis observed in acute injury and some forms of neurodegeneration. In this regard, we will discuss first the mechanistic insights gained studying DNA damage in neurons, and then the implications that these have for the traumatized nervous system.

A large body of work indicates that almost any DNA-damaging agent can cause the apoptosis of postmitotic neurons, including sympathetic, cortical, hippocampal and cerebellar neurons, and that in all cases this apoptosis is dependent upon p53 (Table 1). Examples include ionizing radiation,80,81,82 cytosine arabinoside (araC),83,84,85,86 DNA topoisomerase II inhibitors such as etoposide,81 cisplatin,87 and the topoisomerase I inhibitor camptothecin.88 Recent studies indicate that this activity is essential for the nervous system; absence of the DNA repair protein XRCC4 led to a massive neural apoptosis, presumably as a consequence of the accumulated DNA damage, and this apoptosis was rescued by coincident deletion of p53.89

How does DNA damage cause the activation and stabilization of p53? This question has perhaps been best answered for camptothecin-induced death of cortical neurons (Figure 2), but similar findings have been reported in other systems. Camptothecin leads to a rapid phosphorylation of pRb and p107,90 and increased levels of p53. This increase is likely to be at least partially mediated via a CDK4/6-pRb-E2F-p53 pathway, since camptothecin-induced apoptosis can be inhibited by dominant-inhibitory CDK4 or 691,92 and dominant-inhibitory DP1,90 a binding partner for E2Fs.93,94 Moreover, both ionizing radiation-induced death of hippocampal neurons26 and camptothecin-induced death of cortical neurons90 can be partially rescued by expression of a pRb mutant lacking phosphorylation sites, including the CDK4/6 site. Once p53 levels are increased, this in turn results in increased Bax levels and caspase activation; these downstream events are eliminated in p53−/− neurons.95 The activation of Bax is essential for apoptosis in response to both ionizing radiation96 and camptothecin.95 However, caspase inhibitors either have no or limited effects, suggesting the possibility of caspase-independent apoptosis downstream of Bax.97,98 That these alterations are essential for p53-mediated apoptosis is further supported by a recent study showing that the apoptosis observed following ectopic overexpression of p53 was abolished in Bax−/− cerebellar granule neurons, and reduced in caspase 3−/− neurons.99

Figure 2
figure 2

p53 is essential for neuronal apoptosis in response to DNA damage, excitotoxicity, and oxidative damage. Both oxidative damage and excitotoxicity cause DNA damage, and both cause neuronal apoptosis via a p53-dependent mechanism. DNA damage activates at least three signaling pathways, a cell cycle pathway, the JNK pathway, and ATM/CHK2, all of which are involved in the subsequent neuronal apoptosis. These pathways may be distinct from each other (as shown), or they may intersect upstream of p53. A more extensive discussion of these pathways is found in the text

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A second potential mechanism for stabilization of p53 in response to DNA damage involves the product of the ataxia telangiectasia mutated (ATM) gene.100 AT is characterized by a spectrum of disorders, including progressive neurodegeneration that is most pronounced in the cerebellum. ATM is required for p53 stabilization in response to DNA damage,101 and can phosphorylate p53.102,103 The importance of this interaction in neurons derives from recent reports by McKinnon and colleagues101,104 who show that ionizing radiation is unable to induce neural apoptosis in ATM−/− mice, a phenotype similar to that seen in p53−/− mice.101 Moreover, the increase in p53 that is observed following ionizing radiation in wild-type mice is absent in ATM−/− mice, leading to the conclusion that ATM is upstream of p53. Interestingly, the Bax−/− mice display a similar resistance to ionizing radiation,104 supporting the existence of an ATM-p53-Bax pathway (Figure 2). The recently-identified checkpoint kinase CHK2 is another potential player in this pathway; like ATM, CHK2 is required for p53 stabilization and apoptosis following ionizing radiation,105 supporting the notion that interactions between all three of these proteins are necessary for an appropriate DNA damage response.106 The point at which the CDK4/6-pRb-E2F1-p53 pathway intersects with the ATM/CHK2-p53 pathway in regulating neuronal apoptosis has not been defined (Figure 2).

Interestingly, the DNA damage-induced apoptosis of neurons can be rescued by growth factors. TGFβ rescues ionizing radiation-induced apoptosis of hippocampal neurons,26 and NGF and BDNF rescue araC and camptothecin-induced apoptosis of sympathetic107 and cortical neurons,108 respectively. In these latter two situations, the rescue is mediated as a consequence of Trk receptor-mediated activation of MEK.

An essential role for p53 in neuronal apoptosis due to excitotoxicity and ischemia

The first indication that p53 might be important for neuronal apoptosis following ischemia or excitotoxicity came from studies showing that p53 levels increased in response to these insults (Table 1). A similar elevation of p53 was observed in response to dopamine-neurotoxicity,79 traumatic brain injury,73 and adrenalectomy,77 the latter of which causes selective apoptosis of hippocampal granule cells (summarized in Table 1). In many of these injury paradigms, p53 is essential for apoptosis (Table 1). Of particular interest are studies demonstrating (i) that kainate was able to induce death of p53+/+ but not p53−/− cortical and hippocampal neurons, even though intracellular calcium was elevated to the same degree in both populations of neurons,50 and (ii) that kainate-induced seizures led to significant apoptosis in p53+/+ but not p53−/− mice.52 These studies definitively established that p53 was an essential downstream component of NMDA receptor-mediated excitotoxicity. Similar studies have confirmed that p53 is also essential for neuronal apoptosis following ischemia,65 adrenalectomy,78 and hypoxia.72

What is the molecular mechanism linking excitotoxin exposure to p53 activation? A number of lines of evidence indicate that it likely involves DNA damage. First, excitotoxicity may be associated with the accumulation of single-strand DNA breaks.109 Second, the alterations downstream of excitotoxicity are similar to those downstream of DNA damage in the same neurons. For example, in cortical neurons, excitotoxicity-induced apoptosis is blocked in Bax−/− neurons,95 as it is in p53−/− neurons,50 and there is little or no inhibition of this apoptosis with caspase inhibitors,97,98 findings very similar to those observed for camptothecin.97,98 One potential difference between these two pathways is upstream of p53; kainate-induced apoptosis is inhibited in JNK3−/− mice,110 but camptothecin-induced neuronal apoptosis is thought to be triggered by a CDK4/6-pRb-E2F-p53 pathway (Figure 2). However, a recent report indicates that DNA damage-induced apoptosis is inhibited in JNK1−/−, JNK2−/− cells,111 suggesting either that there are two pathways to p53 following DNA damage, as there are following growth factor withdrawal (Figure 1), or that these two pathways intersect upstream of p53.

These findings suggest that the formation of DNA strand breaks is a key molecular event linking excitotoxic injury and the induction of apoptosis. How does this occur? Accumulating evidence has invoked a role for oxidative damage in the response to neuronal damage and, potentially in the degeneration of neurons in neurodegenerative diseases (discussed below). In this regard, glutamate receptor activation leads to the generation of reactive oxygen species.112,113 Reactive oxygen species are known to induce DNA strand breaks, and exposure of neurons to reactive oxygen species leads to neuronal apoptosis.114,115,116,117 Enhancement of p53 expression has been observed in numerous cell types following exposure to reactive oxygen species.118 Moreover, p53 is essential for neuronal apoptosis after exposure to stimuli that increase reactive oxygen species, such as ionizing radiation.80,81 Taken together, these data suggest that any form of neuronal injury that produces an excess of free radicals, such as excitotoxic insults, could generate DNA strand breaks, which in turn could provide a signal for stabilizing and activating p53.

Is p53 involved in neurodegeneration?

Together, the aforementioned studies make a strong case that p53 is likely to be involved in the acute neuronal apoptosis that is observed following stroke or traumatic brain injury. However, the evidence implicating p53 in chronic neurodegeneration is largely indirect, and can be summarized as follows. First, p53 is increased in appropriate regions in the brains of individuals suffering from a number of neurodegenerative disorders.119,120,121 For example, p53 is increased in the temporal and frontal lobes of brains from Alzheimer's disease (AD) patients, in the spinal cord of ALS patients, and in the striatum of Parkinsons disease patients.121 Although there is as yet some controversy as to whether this increase is localized to neurons and/or glial cells, it is clear that p53 is elevated. Second, there is accumulating evidence that some neurodegenerative conditions involve oxidative or excitotoxic mechanisms,122,123,124125 both of which would be predicted to cause DNA damage, and to potentially lead to p53-dependent apoptosis. For example, nitric oxide has recently been invoked in the motor neuron apoptosis observed in both familial and sporadic ALS.126 Finally, it is clear that any situation that leads to an increase in the DNA mutation load in neurons is likely to lead to neuronal death. In this regard, cancer chemotheraphy with agents such as cisplatin87 might trigger both short-term and long-term p53-dependent neuronal apoptosis. Alternatively, mutations that interfere with the repair system itself would be predicted to increase the mutation load, and potentially lead to a chronic and progressive neurodegeneration. The best example of this is the neurodegenerative disorder AT;100 it is likely that the inability to repair DNA using the ATM/p53 system ultimately causes neurodegeneration in a p53-independent fashion. In support of this idea, a recent study indicates that there is chronic and progressive neurodegeneration in the p53−/− mice.127 Although this finding may seem counterintuitive, it is likely that the inability to properly scan and repair DNA in the absence of p53 would ultimately lead to a nonfunctional neuron that would die by a p53-independent mechanism.

Of the few experimental studies exploring the role of p53 in neurodegeneration, most are focused upon AD. In one study, Xu et al 128 demonstrated that transfection of wild-type amyloid precursor protein (APP) into neuroblastoma cells was sufficient to rescue them from apoptosis induced by UV irradiation or by p53 itself. However, a mutant form of APP found in familial Alzheimer's had no effect, leading to the hypothesis that APP protects neurons from apoptosis by controlling p53 and that mutations in APP could enhance neuronal vulnerability to p53-mediated apoptosis. In a second study, LaFerla et al 129 examined a transgenic mouse expressing beta-amyloid protein (Abeta) in neurons, and found a correlation between Abeta accumulation in neurons, activation of p53 and DNA fragmentation. Both of these studies would predict that the elevated p53 found in AD cortex might be important in the neurodegeneration. Arguing against this conclusion are two studies reporting that Abeta-mediated apoptosis did not require p53.130,131

Together, these studies highlight the difficulties of ascertaining the involvement of a given signaling pathway in human neurodegeneration. Nonetheless, the accumulating evidence that oxidative stress and excitotoxicity lead to p53-dependent apoptosis, and that perturbations in DNA repair can also lead to longterm neuronal degeneration, provide a strong argument for pursuing the potential involvement of p53 in a set of debilitating degenerative diseases for which we currently have no treatment.