Coupling of the NMDA receptor to neuroprotective and neurodestructive events

C i. Activation of the PI3K-Akt pathway

The PI3K (phosphoinositide-3-kinase) - Akt kinase cascade is an important signalling pathway which contributes to the pro-survival effects of NMDAR activity [29, 30]. It is activated by NMDAR in many neuronal types, but not in all [27]. Briefly, in response to extracellular stimuli, PI3K, which can be activated in a Ca²⁺-dependent manner by Ca²⁺/calmodulin [31], catalyses the phosphorylation of the lipid PIP2 (phosphatidylinositol 4,5-biphosphate) to PIP3 (phosphatidylinositol 3,4,5-triphosphate) in the membrane. The kinase PDK1 (phosphoinositide-dependent protein kinase) [32] as well as its substrate Akt/PKB (Protein Kinase B) are recruited to the membrane via their interactions with PIP3 through their pleckstrin homology (PH) domains. PDK1, along with PDK2 (which is possibly rictor-mTOR- [33]) then phosphorylates and activates Akt. Akt promotes cell survival and growth via phosphorylation and activation or inactivation of its many targets: Akt phosphorylates and inactivates GSK3β (Glycogen synthase kinase-3 beta; [34]), an event that also contributes to NMDAR signalling to neuroprotection [35]. Akt mediates further anti-apoptotic events: it inactivates the pro-apoptotic Bcl-2 family member BAD (Bcl2/Bcl-X_L-antagonist causing cell death) by phosphorylation, thus stopping its interaction with the pro-survival Bcl-2 family members Bcl-2 and Bcl-X_L [36]. The JNK/p38 activator ASK1 can also be inhibited by Akt phosphorylation [37] and p53 activity is suppressed by Akt, leading to reduction of Bax expression and of neuronal death [38].

C ii. Suppression of pro-death gene expression

As well as affecting post-translational events that can promote neuroprotection, Akt can also modify gene expression with neuroprotective effect by promoting the phosphorylation and subsequent nuclear export of the FOXO (forkhead box O) subfamily of forkhead transcription factors. FOXOs control the expression of pro-death genes such as Fas ligand [39] and Bim [40], and these are down-regulated by Akt activation. Akt-dependent FOXO export has also been observed in the context of NMDAR signalling [35, 41] which triggers the transcriptional suppression of Thioredoxin-interacting protein (Txnip), a thioredoxin-inhibiting FOXO1 target gene whose product renders neurons vulnerable to oxidative stress [41]. Interestingly, we recently found that FOXO1 itself is a FOXO target gene, meaning that FOXO export triggered by synaptic activity also suppresses FOXO1 gene expression, likely to lead to long-lasting down-regulation of FOXO target gene expression [42].

Importantly, the transcription of the pro-death transcription factor p53 gene has recently been shown to be suppressed by synaptic NMDAR activity [43]. P53 is a transcription factor which regulates a number of pro-apoptotic genes such as Bax and Puma. Knock-down of P53 expression was found to be neuroprotective, preventing mitochondrial depolarisation in response to excitotoxic trauma. In a similar vein, we have observed that key components of the intrinsic apoptosis pathway are transcriptionally repressed by physiologically relevant synaptic NMDAR activity in vitro and in vivo but via a p53-independent mechanism (GEH, unpublished results). Thus, activity-dependent suppression of pro-death genes may represent an important route to neuroprotection, acting in concert with the induction of pro-survival genes, which is discussed below.

C iii. Nuclear Ca²⁺ and activation of CREB (cAMP response element-binding protein)

An important mediator of activity-dependent gene expression is the transcription factor CREB, which binds to the cAMP response element (CRE) [44]. CRE-dependent gene expression is strongly induced by synaptic NMDAR activity [45]. Ca²⁺ influx through the synaptic NMDAR activates the Ras/ERK pathway in the cytoplasm and the nuclear Ca²⁺/calmodulin dependent protein kinases, principally CaMKIV. CaMKIV mediates fast CREB phosphorylation at Ser133, whereas the ERK1/2 pathway promotes CREB phosphorylation in a slower, more long lasting manner [46, 47]. Ser133 phosphorylation of CREB is necessary to recruit CBP (CREB-binding protein), a transcriptional cofactor, to CREB. The transactivation potential of CBP is itself positively regulated by NMDAR activity [48] by a mechanism involving its phosphorylation on Ser301 by CaMKIV [49]. Gene expression mediated by CREB can also be mediated by another family of CREB co-activators, the TORCs (Transducers of Regulated CREB activity, [50, 51] and references therein). TORC2 translocates to the nucleus as a result of the synergistic action of Ca²⁺ and cAMP signalling, through calcineurin-mediated TORC2 dephosphorylation and cAMP-mediated inhibition of the TORC2 kinase SIK2, respectively [50]. In hippocampal neurons TORC1, which is expressed more abundantly than TORC2, senses the coincidence of Ca²⁺ and cAMP signalling and converges this dual signalling to target gene expression by translocating to the nucleus and potently coactivating CREB [51]. The potential of CREB family-regulated gene products to promote neuronal survival was first demonstrated in the context of neurotrophin signalling [52, 53] and exogenous overexpression [54]. In addition, studies of mice where CREB and/or CREB family members have been deleted also point to a pro-survival role for CREB in vivo both pre- and post-natally [55, 56].

CREB-dependent gene expression is causally linked to the long-lasting phase of activity-dependent neuroprotection against apoptotic insults [29]. This long-lasting phase is dependent on nuclear Ca²⁺/calmodulin signalling [29], consistent with the known requirement for nuclear Ca²⁺ in CREB activation via CaMKIV [28, 57-59]. Activity-dependent CRE activation in developing CNS neurons also protects against excitotoxic trauma [60] which would otherwise result in rapid cell death resembling necrosis. Expression of the inhibitory CREB family member, ICER, increases in neurons in response to apoptotic and excitotoxic insults and can promote neuronal death [61]. Thus, CREB activation by NMDAR signalling may be important in antagonizing the CREB-inhibiting effects of ICER accumulation.

The importance of NMDAR signalling to CREB in promoting survival in vivo is not as well established. Activation of CREB in response to transient ischemic episodes is proposed to contribute to the establishment of ischemic tolerance or preconditioning and may well be mediated by NMDAR activation [62]. Interestingly, resistance to hypoxic/ischemic death in different neuronal populations correlates positively with an ability to sustain active (phosphorylated) CREB [63]: in neurons destined to die CREB is only transiently phosphorylated [63].

The identity of the CRE-regulated gene(s) responsible for long lasting protection against apoptosis is beginning to be understood. Genome-wide profiling studies have identified a large number of CREB target genes, although only a subset of these are induced by CREB-activating stimuli [64-66]. Furthermore, the identity of the genes within the subset is cell-type specific and may be stimulus-specific, so is hard to predict in any particular scenario. A recent study identified two genes that contribute to synaptic NMDAR-dependent neuroprotection [67]: Btg2, a potentially anti-apoptotic CREB target gene, and Bcl6, a transcriptional repressor implicated in suppression of p53, also potentially a CREB target. Both Btg2 and Bcl6 genes confer neuroprotection in the face of apoptotic stimuli like trophic deprivation and staurosporine treatment, and siRNA-mediated knockdown demonstrated that their induction was also necessary for synaptic NMDAR-mediated neuroprotection. While the molecular mechanism of how Btg2 protects is not completely clear, its expression has recently been shown to render neurons resistant to excitotoxicity-induced mitochondrial permeability transitions [43] indicating that it may enhance mitochondrial function, suppress permeability transition, or enhance respiratory capacity. Another CRE-regulated candidate is the pro-survival neurotrophin BDNF (brain-derived neurotrophic factor), which is upregulated by NMDAR activity [45] and is known to promote neuronal survival [27, 68, 69]. NMDAR blockade in vivo reduces BDNF mRNA expression and in vitro supplementation of neurons with BDNF can rescue them from NMDAR antagonist-induced neuronal death [70]. Still more protective CREB target genes are likely to emerge; it is possible that different genes are responsible for neuroprotection against different types of insult (e.g. apoptotic-like, excitotoxic/necrotic-like, oxidative stress).

C iv. Synaptic NMDAR activity boosts intrinsic antioxidant defences

Correct redox regulation is essential in all cells, especially in post-mitotic cells such as neurons where harmful oxidative damage can accumulate. Oxidative damage and stress occurs when there is an imbalance between production of reactive oxygen species (ROS) and the cell’s capacity to neutralize them through its intrinsic antioxidant defenses. Neurons are particularly susceptible to oxidative damage due to high levels of ROS production (through respiration and metabolism) and relatively low levels of certain antioxidant enzymes, particularly catalase [71, 72]. Oxidative damage is implicated in the pathogenesis of several neurodegenerative diseases as well as acute cerebrovascular disorders[71, 72]. Appropriate redox balance depends on the activity of antioxidant systems. Key among these are the thiol reducing systems based round thioredoxin and glutathione respectively, which are important reducers of many oxidative stressors such as peroxides [72, 73]. The principle source of peroxide is from spontaneous and SOD-catalysed dismutation of superoxide generated in active mitochondria. However, other sources of peroxides exist, including products of metabolic pathways involving oxidases/oxygenases (e.g. monoamine oxidase).

We recently showed that the vulnerability of neurons to oxidative death triggered by exposure to hydrogen peroxide was regulated by synaptic activity acting via NMDA receptor (NMDAR) signalling ([41], see also commentary [41]). Neurons that were experiencing (or had recently experienced) higher levels of synaptic NMDAR activity were far more likely to withstand the oxidative insult than electrically quiet neurons. Neurons experiencing complete NMDAR blockade were highly vulnerable to peroxide-induced apoptosis in vitro, and NMDAR blockade in vivo promoted neuronal apoptosis associated with markers of oxidative damage [41]. It is not completely clear as to why synaptic activity, acting via Ca²⁺ signalling, should act to boost antioxidant defences. One possibility is to protect against increased ROS generation associated with high energy demand. Synaptic activity and activity-dependent plasticity place high energy demands on a neuron which must be largely met through oxidative phosphorylation- a process that generates ROS. Thus, active neurons would ordinarily need to have stronger antioxidant defenses than inactive ones in order to maintain the correct redox balance.

Investigations into the mechanism behind this revealed that synaptic activity exerted a number of changes to the thioredoxin-peroxiredoxin system which contributed to the activity-dependent protection. Synaptic activity enhanced thioredoxin activity and facilitated the reduction of hyperoxidized peroxiredoxins, an important class of antioxidant enzymes. These effects were mediated by a coordinated program of gene expression changes ([41], Fig. 2). Synaptic activity triggered the transcriptional suppression of the thioredoxin inhibitor Txnip, which, as mentioned above, is a FOXO target gene. Furthermore, enhanced reduction of hyperoxidized peroxiredoxins was associated with transcriptional induction of two genes, sulfiredoxin and sestrin 2, whose products are reported to mediate this reaction [74-76]. The induction of sulfiredoxin was mediated primarily through two AP-1 sites, and partly via Nrf2 [77], while sestrin2 was activated via two C/EBPβ sites [41]. We did not address which of sestrin 2 or sulfiredoxin was responsible for reducing peroxiredoxin hyperoxidation. However, we recently showed that specific induction of sulfiredoxin is sufficient to prevent peroxiredoxin hyperoxidation in neurons [77], so this is likely to be the most important, particularly as recent doubt has been cast on the ability of sestrin 2 to catalyze this reaction [78]. In any case, induction of one (or both) of these genes cooperated with the suppression of Txnip in boosting antioxidant defenses. The gene expression changes described above did not account for all the antioxidant effects of synaptic NMDAR activity. Work is ongoing in the laboratory to investigate another major antioxidant system which is subject to activity-dependent enhancement-that centred on glutathione (GH, unpublished observations).

D i. Mitochondrial dysfunction and reactive oxygen/nitrogen species generation

Mitochondrial dysfunction caused by excessive Ca²⁺ uptake by the mitochondria through the potential-driven uniporter is a key event in severe excitotoxicity [83, 84]. The mitochondrial membrane gets depolarised due to this uptake, which inhibits ATP production, and can even cause depletion of cytosolic ATP due to reversal of the mitochondrial ATPase. This loss of ATP further limits the ability of the neuron to regulate intracellular Ca²⁺ levels. It had been generally accepted that mitochondrial Ca²⁺ uptake can also promote ROS production, triggering further mitochondrial damage and delayed Ca²⁺ deregulation (DCD). However, recent studies have made the case that enhanced ROS production following a strong excitotoxic insult occurs after DCD, and that most superoxide production is in any case non-mitochondrial in origin [84, 85]. This does not rule out a role for oxidative stress, since prior oxidative stress greatly potentiates NMDAR-mediated toxicity, possibly by reducing mitochondrial ATP supply [86]. Recent evidence for a role for ROS generation upstream of NMDAR-mediated cell death also focused on a non-mitochondrial source: NADPH oxidase (NOX). It was reported that activation of NADPH oxidase is a major source of superoxide production by NMDA receptor overactivation, and that inhibiting this process was neuroprotective [87].

NMDAR activity-regulated overactivation of the Ca²⁺-dependent nNOS (neuronal nitric oxide synthase) also has toxic downstream responses, including mitochondrial dysfunction, p38 mitogen-activated protein kinase signalling and TRPM (transient receptor potential melastatin) channel activation [88]. nNOS is bound by its N-terminal PDZ domain to the post-synaptic density protein PSD-95, which brings it in the proximity of the synaptic NMDAR that is also bound to PSD-95 via the extreme C-terminal amino acids of its NR2 subunit (its so-called PDZ ligand). nNOS activation leads to Nitric Oxide (NO) production, which in excess can be toxic both on its own and when combined with other reactive oxygen species (ROS) such as superoxide to form ONOO⁻ (peroxynitrite). Both NO and ONOO⁻ can damage cellular components, inhibit mitochondrial respiratory chain enzymes and promote mitochondrial depolarisation [10]. ONOO⁻ also damages DNA, resulting in single strand breaks and overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1). Excessive PARP-1 activation can promote neuronal death by depleting cellular NAD(+) levels and also by triggering the mitochondrial release of apoptosis activating factor, (AIF) [89, 90]. Another important target of NMDAR signalling to NO production in excitotoxicity is the cation channel TRPM7 [91]. NMDAR-dependent Ca²⁺ influx triggers both NO production via nNOS activation and superoxide production via mechanisms described above, which combine to form ONOO⁻, an activator of TRPM7. Since TRPM7 itself passes Ca²⁺, this results in a positive feedback loop.

D ii. Calpain activation

As well as promoting mitochondrial dysfunction and ROS generation, excessive Ca²⁺ influx also causes efflux to be impaired [92, 93]. In neurons, Ca²⁺ exit from the cell is achieved through the plasma membrane Ca²⁺ ATPase pump (PMCA) and Na⁺/Ca²⁺ exchangers (NCXs). Calpains, Ca²⁺-dependent proteases, are activated by the excessive NMDAR-mediated Ca²⁺ influx and cleave a major isoform of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX3) impairing its function in cerebellar granule neurons (CGNs) [92]. The PMCA, which would utilise the energy from ATP hydrolysis to transport Ca²⁺ across the plasma membrane, is inactivated by excitotoxic insults via mechanisms attributed to both caspases [94] and calpains [93]. Other important calpain targets have recently been revealed: the metabotropic glutamate receptor mGluR1α [95] and striatal-enriched protein tyrosine phosphatase (STEP) [96]. NMDAR-dependent, calpain-mediated truncation of the mGluR1α C-terminal tail does not disrupt coupling to Gq protein activation, but results in other altered signalling properties of the receptor, including a disruption of PI3K-Akt signalling, as well as relocating the receptor from dendrites to axons [95]. The functional result of this truncation is that while wild-type mGluR1α is neuroprotective against NMDAR excitotoxicity, the truncated form has the opposite effect. NMDAR-mediated calpain cleavage of STEP led to the sustained activation of the stress-activated pro-death kinase, p38 (see below). Furthermore, inhibition of STEP cleavage is neuroprotective and suppresses p38 activation [96].

D iii. Stress-activated protein kinases

Stress-activated protein kinases (SAPKs) are another class of signalling molecules that are implicated in NMDAR-dependent cell death [97-101]. In CGNs, NMDAR-dependent excitotoxic death relies upon p38 activation, leading to caspase-independent cell death [97, 99]. In cortical neurons another SAPK family, the c-Jun N-terminal kinases (JNKs) contribute to NMDAR-dependent cell death in vitro and in vivo as well as p38 [100, 101]. New components of the excitotoxic cell death pathway signalling to p38 are emerging; Rho, a member of the Rho-family of GTPases, was recently found to contribute to glutamate-induced p38α-dependent excitotoxic neuronal death [102]. As mentioned above, calpain cleavage of STEP has also been implicated in p38 activation [96]. In addition, studies in CGNs and cortical neurons have reported that NMDAR-dependent p38 activation is dependent on activation of nNOS, which is associated with the NMDA receptor signaling complex through its association with PSD-95. PSD-95 is itself linked to NMDARs through interaction with the PDZ ligand of the NR2 subunit. Disruption of NR2 PDZ ligand:PSD-95 interactions that can uncouple the NMDAR from NO production also inhibit p38 activation [101, 103]. Interestingly, disruption of interactions between the NR2 PDZ ligand and downstream proteins such as PSD-95 has a remarkably specific effect on the p38 pathway. NMDAR signalling to pro-survival pathways resulting in activation of Akt or of CREB are not interfered with [101, 104]. However, the neuroprotection afforded by such as strategy is imperfect: activation of JNK does not require interactions with PSD-95. Indeed, NMDAR-induced death via JNK can be reconstituted in non-neuronal cells simply by expressing functional NMDARs [101]. The mitochondrially targeted antioxidant MitoQ [105] inhibited NMDAR-activated JNK signalling in cortical neurons, potentially placing ROS generation upstream of JNK activation [101].

D iv. So many pathways to excitotoxic cell death?

The myriad potential routes to NMDAR-mediated excitotoxicity begs the question as to which is the most important or dominant mechanism. While cell-type specific differences may offer a partial explanation, another important consideration is the intensity of the excitotoxic insult [106]. Extremely high and sustained levels of NMDAR-induced Ca²⁺ influx will likely cause rapid mitochondrial dysfunction and bioenergetic failure leading to DCD. On the other hand, incomplete mitochondrial depolarisation or depolarisation of only a subpopulation of mitochondria may lead to the release of apoptotic factors such as cytochrome c or AIF, and the activation of apoptotic-like biochemical cascades. Activation of SAPK-dependent neuronal death may also be associated with more modest excitotoxic insults near the threshold of toxicity, and take neurons down a more delayed route to death. Consistent with this, peptide inhibitors of JNK and also of NR2 PDZ ligand interactions are neuroprotective in excitotoxic models in vitro and in vivo when administered after the insult [7, 100]. Furthermore, the protective effects of these peptides can be overcome by increasing the severity of the toxic insult [101], as can neuroprotection by pharmacological inhibition of p38 [98]. Another consideration is that studies on apparently different mechanisms of excitotoxicity may in some cases involve different parts of the same pathway, as illustrated in Fig. 3. For example, NMDAR-dependent generation of oxidative/nitrosidative stress can lead on to mitochondrial dysfunction, as well as SAPK and PARP-1 activation. Similarly, calpain activation acts as a signalling hub for many pro-death events, including p38 activation, disruption of Akt activation, and Ca²⁺ dyshomeostasis.

F i. Stimulus intensity

The magnitude of activation, be it intensity or duration, is very important in determining the nature of the response to an episode of NMDAR activity. The classical bell-shaped curve model of the neuronal response to NMDA or glutamate contends that intermediate, modest/physiological NMDAR activity levels can promote neuroprotection whereas too low or too high pathological NMDAR activity promotes cell death. This implies that the effectors of the pro-survival Ca²⁺ signalling have a higher affinity (or considerably lower requirements for Ca²⁺) than the Ca²⁺ effectors of death. Therefore the Ca²⁺ concentration threshold for activating pro-survival signalling by PI3K, ERK1/2 and CaMKIV must be lower than that necessary to trigger toxic levels of calpain activation, mitochondrial Ca²⁺ uptake or NO production. The key Ca²⁺-dependent components of the survival pathways are mainly activated not by Ca²⁺ directly, but by Ca²⁺/Calmodulin. Calmodulin is a ubiquitous Ca²⁺ binding protein which changes conformation when it binds to Ca²⁺. Ca²⁺/Calmodulin then activates a number of downstream signals, including CaMKIV, PI3K and upstream activators of ERK1/2. As a physiological sensor of elevated Ca²⁺ levels, calmodulin is designed to be activated by relatively modest increases in Ca²⁺. In contrast, central mediators of NMDAR-dependent cell death, calpains, are not Ca²⁺/Calmodulin dependent but are activated by Ca²⁺ directly, and require higher levels to be fully induced [107]. The same can be said of the mitochondrial uniporter, which senses Ca²⁺ directly and is fully activated only by high concentrations [108]. Consistent with this, when increasing doses of NMDA are applied to neurons, only the higher, toxic doses evoke sustained loss of mitochondrial membrane potential and increases in mitochondrial Ca²⁺ [35]. Another Ca²⁺-dependent promoter of neuronal death, nNOS, is Ca²⁺/Calmodulin-dependent and indeed plays important roles in non-pathological signalling processes such as synaptic plasticity. However, nNOS is also regulated by phosphorylation, and a recent study showed that, in contrast to non toxic stimuli, high levels of glutamate fail to trigger an inhibitory phosphorylation event that ensures that nNOS activation is transient [109]. Thus, nNOS becomes excessively active and contributes to excitotoxic cell death [109].

F ii. NMDAR location

Aside from stimulus intensity, the location of the NMDAR may also profoundly affect the signals that emanate from the NMDAR. Developing neurons have sizeable pools of NMDARs at extrasynaptic, as well as synaptic locations, which signal very differently. Ca²⁺ influx dependent on intense synaptic NMDAR activation is well tolerated by cells whereas equivalent activation of extrasynaptic NMDARs, either on their own or in the presence of synaptic NMDAR activation, is less well tolerated, triggering mitochondrial membrane potential and cell death [45]. The findings from this study were based on hippocampal neurons and have since been recapitulated independently in cortical neurons [110].

F iii. Does subunit composition matter?

While extrasynaptic NMDARs are preferentially enriched with NR2B-containing NMDARs, there is little evidence that differences in subunit composition are dramatic enough to explain the effects observed. However, a recent study has contended that NR2B-containing NMDARs tend to promote neuronal death, irrespective of location (synaptic or extrasynaptic) while NR2A-containing NMDARs promote survival [127]. The cytoplasmic c-terminal tails of NR2A and NR2B are large, show considerable sequence divergence and have been reported to differentially associate with signalling molecules [128], offering scope for subunit-specific routes to survival/death. The investigation of subunit-specific differences in NMDAR signalling is hampered by the lack of a NR2A-specific antagonist that is sufficiently selective to discriminate in the physiological scenario of trans-synaptic stimulation [129, 130]. The antagonist NVP-AAM077 has some selectivity for NR2A-containing NMDARs but studies disagree as to the degree of selectivity, possibly because of some laboratories studying rodent NMDARs and others human NMDARs expressed in oocytes [130, 131]. However, it is likely that the difference in the equilibrium constants (NR2A vs. NR2B) is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors being trans-synaptically activated [130]. Moreover, NR2A-NMDARs have been shown to be capable of mediating excitotoxicity [132], and NR2B-NMDARs have been shown to be capable of mediating both pro-survival and pro-death signalling, depending on the stimulation paradigm [133]. As such, the issue of whether the NR2 subunit influences whether an NMDAR signals to survival or death (controlling for the amount of Ca²⁺ passed) remains unresolved.