Excitatory Amino Acid Neurotoxicity

Historical Perspective

The excitatory potency of the acidic amino acids glutamate and aspartate in various regions of the central nervous system (CNS) has been recognized since the 1960s.^1,2 Nevertheless, the earlier findings that these amino acids are (1) constituents of intermediary metabolism and are (2) located in the brain ubiquitously in high concentrations rendered them unlikely candidates as neurotransmitters. These findings fueled a sustained debate about their physiological role as neurotransmitters in the 1970s. Today, L-glutamate is accepted as the predominant fast excitatory neurotransmitter in the vertebrate brain.

In parallel to studies on the physiological role of these amino acids, it has been observed since the 1950s that glutamate can exert toxic effects on the nervous system under certain conditions. Following the systemic application of glutamate to mice, toxic effects on retinal neurons were described.³ Further studies in the 1970s corroborated these toxic effects and extended this view by showing neuronal cell death following oral intake of glutamate or aspartate in brain regions devoid of the blood-brain barrier in mice and nonhuman primates (Fig. 1).^4–9

Figure 1

A, Tissue section through arcuate nucleus (arc) of hypothalamus from a 10-day-old mouse (control; ×150). No signs of pathology are present. B, Section trough arcuate nucleus (arc) from a 10-day-old mouse treated orally with 1 g/kg sodium glutamate (more...)

Thus, L-glutamate is the primary excitatory transmitter in the mammalian CNS but is cytotoxic under certain conditions. This relation between the physiological function as excitatory amino acid (EAA) and the pathological effect is reflected by the term “excitotoxicity” introduced in the 1970s by Olney et al.¹⁰

With the introduction of structural transmitter analogues, local injections of the glutamate agonist kainate were shown in the late 1970s and 1980s to induce cell death with a similar pattern of damage in different brain regions, thus confirming the neurotoxic effect.^11–14 Today, it is well recognized, that exogenous or endogenous agonists of EAA receptors can induce cell death in CNS neurons.

Our knowledge about the role of glutamate as an excitatory neurotransmitter and its cytotoxic effects increased in parallel during the 1980s, and notably, the development of substances that antagonized the excitatory function¹⁵ also stimulated studies on mechanisms underlying the toxic effects.^16–18 The discovery of different EAA receptor subtypes in conjunction with the introduction of selective receptor antagonists revealed that the glutamate-induced cell death was induced by excessive ionotropic glutamate receptor activation.^19–26

The view that activation of different ionotropic EAA receptors can induce excitotoxic cell death was supported by subsequent studies on the ionic mechanisms underlying excitotoxicity. It was demonstrated that excessive calcium loading plays a pivotal role in neuronal cell death following the intense stimulation of ionotropic glutamate receptors,^27,28 and since then the implication of ion homeostasis dysregulation and dysfunction in calcium signaling have been studied extensively (Fig. 2 ).^29–33

Figure 2

Cortical neurons in cell culture before (top row) and 1 day after (bottom row) a 5-min incubation in 500 mM glutamate. A, Na ions replaced with equimolar choline. B, 1 mM tetrodotoxin added. C, Ca²⁺ ions omitted. Considerable cell death occurred even (more...)

Regarding the mode of cell death, glutamate-induced neuronal cell death has been judged originally as necrotic from the morphological appearance.^6,34–36 However, the observation of a delayed neuronal cell death in the penumbra of ischemic lesions³⁷ and after EAA exposure^38,39 has stimulated studies in the 1990s focusing on the mode of cell death. Today, there is compelling evidence that failure in extracellular glutamate homeostasis can result in different modes of cell death with morphological and biochemical features of either apoptosis or necrosis depending on the severity of the insult, with more fulminant insults causing rapid energy failure because of lack of ionic homeostasis and thus necrosis.^40–44

Clinical Relevance of Excitatory Amino Acid Neurotoxicity

There is evidence that excitotoxicity is involved in acute brain damage under pathophysiological conditions following status epilepticus, mechanical trauma or ischemia (Fig. 3).⁴⁵ Moreover, glutamate cytotoxicity seems to be partly involved in many neurodegenerative diseases.

Figure 3

Glutamate induces acute CNS injury. This historical schematic summarizes that seizures, hypoxia, hypoglycemia and trauma share common mechanisms of acute injury. The excitatory activity of the transmitter glutamate is linked to its toxic effects (excitotoxicity). (more...)

Epilepsy

Histopathological studies on the brains of patients suffering from chronic epilepsy have revealed that certain brain regions show structural alterations with severe loss of neurons and reactive gliosis.^46,47 Brain pathology in epilepsy is described best for human temporal lobe epilepsy, resulting in sclerosis of the hippocampus that extends into the amygdala and the parahippocampal gyrus and is termed “mesial temporal sclerosis”, “hippocampal sclerosis” or “Ammon's horn sclerosis”. Pronounced brain damage has been observed following sustained epileptiform activity with seizures lasting more than 30 min, called “status epilepticus” (SE).^48–50 Importantly, neuronal cell loss after SE is not distributed equally across all hippocampal subfields and the extent of damage is in the order CA1 > CA4 > CA3 > CA2, thus indicating different susceptibility to SE-induced cell death.^12,50

In animal models of epilepsy using chemoconvulsant-induced or electrical stimulation-induced SE, similar patterns of brain damage were observed.^51–54 Experiments aimed at the observation of ultrastructural changes have demonstrated that certain features of cell damage, such as swelling of dendrites and soma, are independent of the mechanism used to induce SE.^51–56

Evidence for the implication of EAA-mediated excitotoxicity in SE-induced neuronal cell death arises from several experiments. First, the morphological appearance of cell damage following SE is similar to damage following systemic or local application of the glutamate receptor agonists L-glutamate, NMDA or kainate.^{6,36,51,53,55,56} Second, the administration of ionotropic glutamate receptor antagonists that inhibit excitotoxic cell death in cultured neurons can prevent cell death induced by epileptiform activity.^57–60

Apart from the above-mentioned evidence for excitotoxicity in epilepsy, there has been a considerable debate regarding the mode of cell death. There is now a large body of evidence suggesting the implication of both, necrotic and apoptotic cell death following pathologically relevant EAA receptor activation. Recently, several reports have added evidence for the implication of apoptotic pathways in epilepsy-associated neuronal cell death.^61–66 In conclusion, the mode of cell death in SE-induced brain pathology may be the result of intensity and duration of glutamate receptor activation, with a shift from apoptotic death to necrotic death with increasing intensity and duration of receptor activation.^40,43

Traumatic Brain Injury

Following head trauma, mechanical brain injury can be accompanied by secondary changes as ‘metabolic’ glutamate leaks uncontrollably from the neuronal cytoplasm⁶⁷ causing subsequent excitotoxic damage of surrounding neurons. However, compared to research on mechanisms underlying the cell damage in epilepsy, studies on traumatic injury have been sparse and there is less evidence for the implicated modes of cell death. Some studies have suggested the sudden release of excitatory amino acids from the cytoplasm into the extracellular space and subsequent bioenergetic failure as well as ultrastructural damage that can be diminished by NMDA antagonists, all indicative of excitotoxicity,^67–69 whereas others have demonstrated activation of caspase enzymes, internucleosomal DNA fragmentation and induction of immediate early genes, indicative of apoptosis.^69–71 Most recently, in neonatal models of traumatic brain injury (TBI) (mortality and morbidity from head trauma is highest in children), the resulting excitotoxicity has been shown to elicit both apoptosis and necrosis. Necrosis occurs localized to the site of impact, and within 4 hr of the insult, whereas a secondary apoptotic damage occurs between 6–24 hr and is found in the areas surrounding the primary necrosis.⁷² In this model, the secondary damage was more severe than the primary damage, suggesting a preponderance of apoptosis over necrosis.

Hypoxia/Ischemia

Under conditions of local or global ischemia, neurons are deprived of glucose and oxygen, resulting in bioenergetic failure and subsequent decrease of ion gradients across the plasma membrane.⁷³ The resulting plasma membrane depolarization leads to increased synaptic release of glutamate, and the diminished Na gradient is followed by attenuated Na-dependent glutamate uptake or even reversed glutamate transport in terms of transporter-mediated release of glutamate from neurons and astrocytes.^74–76 Moreover, osmotic cell swelling following the influx of Na, Cl and H₂O can result in plasma membrane rupture and further release of cytoplasmic glutamate into the extracellular space. In summary, hypoxia/ischemia results in a secondary net increase in the extracellular glutamate concentration.^77–79 As in the other above-mentioned acute CNS insults, this increase in extracellular glutamate results in excitotoxic damage.^80–83 The implication of excitotoxicity in hypoxia/ischemia is corroborated by the observation that NMDA-type glutamate receptor antagonists can reduce neuronal death in animal models of cerebral ischemia.^84–87 However, the actual set of circumstances in humans appears to differ from the defined animal models.⁸⁸ In addition to the well-accepted concept of excitotoxicity-associated acute necrotic cell death, there are several lines of evidence that neurons can undergo apoptosis following ischemia.^41,89 Among various biochemical markers of apoptosis, DNA fragmentation^90,91 and activation of caspase-3 have been observed^63,92 in models for cerebral ischemia. Moreover, application of caspase-inhibitors results in reduced infarct size^42,93,94 and decreases cell death in cultured neurons following ischemia.⁹⁵

Neurodegenerative Diseases

In addition to acute neurological disorders, many chronic neurodegenerative diseases may exhibit a component of glutamate-dependent neuronal damage, including apoptosis or injury to dendrites and axons (Table 1). This arises when the primary disease causes neuronal injury which in turn may cause the leak or release of excessive glutamate. Additionally, elevated inflammatory responses in many of these diseases can also contribute to excessive glutamate release or decreased glutamate clearance from the synaptic cleft.^96,97

Table 1

Neurodegenerative diseases thought to be mediated at least in part through stimulation of glutamate receptors.

Intoxication with Exogenous Excitatory Amino Acids

Several structural analogues of endogenous EAAs have been introduced to the neurosciences during the 1980s, which exhibit similar or even higher excitatory and neurotoxic potency compared to the endogenous EAAs. In 1987, an outbreak of domoic acid poisoning following ingestion of mussels occurred in Canada. Patients suffered from acute headache, seizures, sensory dysfunctions and showed motor signs. In four fatal cases, neuropathological studies revealed lesions predominantly in the hippocampus and amygdala, resembling the lesion pattern after application of the exogenous excitotoxin kainate.⁹⁸ It was reconstructed that the mussels had accumulated domoic acid, synthesized by the phytoplankton Nitzchia pungens.⁹⁸ Domoic acid is a structural analogue of kainic acid, which is also synthesized by seaweed, but compared to kainate, domoic acid has a higher excitatory potency.^99,100 In parallel to its high excitatory potency, domoic acid exerts potent excitotoxic effects on CNS neurons (Table 2). Experimental administration of domoic acid to rodents and monkeys has resulted in brain damage with ultrastructural features resembling L-glutamate excitotoxicity.^101–103

Table 2

Exogenous excitotoxins.

Ingestion of the chick pea Lathyrus sativus, which contains another EAA structural analogue results in acute spastic motor signs following the consumption. The clinical features of this motor disorder called “lathyrism” were known even by the ancient Greeks but the toxic component, the amino acid β-N-oxalyl-L-alanine (BOAA) was identified only some decades ago (Table 2).¹⁰⁴ Experimental administration of BOAA is known to induce the features of lathyrism in nonhuman primates¹⁰⁵ and application of BOAA to cell cultures results in cell death with structural features of excitotoxicity¹⁰⁶ that can be attenuated by non-NMDA receptor antagonists.^107,108 Importantly, the high excitotoxic potency of this glutamate receptor ligand is in accordance with its high excitatory potency as agonist at AMPA receptors.¹⁰⁹

Implication of Distinct Glutamate Receptor Classes in Excitotoxicity

L-Glutamate, the most abundant excitatory transmitter in the brain, binds to different classes of receptors comprising different types of ionotropic receptors as well as metabotropic receptors. Ionotropic glutamate receptors are ligand-gated ion channels, which are named after agonists that bind preferentially to these receptor subtypes. They include α-amino-3-hydroxy-5-methyl-isoxalole-4-propionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors.

AMPA receptors are widely distributed in the CNS and are expressed on many different types of neurons. They control a cation channel that is permeable to Na and K ions with a single channel conductance <20 pS.¹¹⁰ Activation of AMPA receptors results in the fast onset of an excitatory postsynaptic current (EPSC) with rapid desensitization.¹¹¹ This current shapes the fast component of glutamatergic EPSCs in CNS neurons.¹¹² Importantly, certain AMPA receptor subtypes can exhibit substantial Ca²⁺ permeability and thereby contribute to the Ca²⁺-dependent form of excitotoxic cell damage.^113–116 Ca²⁺-permeable channels are formed from the receptor subunits GluR1 or GluR3, whereas coassembly with GluR2 results in only poor Ca²⁺ permeability.^117–119

Another type of ionotropic glutamate receptor termed kainate receptor is less well understood because of lack of sufficiently selective agonists and antagonists. As with AMPA receptors, kainate receptors control a cation channel that desensitizes rapidly. Since the agonist kainate activates kainate receptors but also AMPA receptors, their physiological and pathophysiological role remains elusive. Recently, results based on gene-targeted rodents lacking certain kainate receptor subtypes have extended our knowledge of localization and function of kainate receptors.^120,121 From these studies one can conclude that kainate receptors are not only localized to postsynaptic sites but also appear to be localized presynaptically, suggesting a role for modulation of synaptic strength (for review see refs. 122,123).

The third type of ionotropic glutamate receptor, the NMDA receptor differs fundamentally from AMPA and kainate receptors in several ways. First, the pore is significantly permeable to Ca²⁺ ions,^124,125 but also to K and Na ions.¹²⁶ In contrast to AMPA receptors, NMDA receptors exhibit a high single channel conductance (50 pS) and desensitize much slower (for review see refs. 127,128). NMDA receptors are widely distributed in different types of CNS neurons and shape the late component of glutamatergic EPSCs. Second, the opening of the ligand-gated cation channel does not only depend on binding of agonist but is voltage-dependent, since the channel is blocked by Mg²⁺ions at resting membrane potentials and a depolarization of the plasma membrane is required to relieve the Mg²⁺-dependent block. Third, activation of NMDA receptors requires binding of a coagonist to the so-called glycine-binding site of the NMDA receptor. Very recently, the amino acid D-serine has been suggested to be an endogenous ligand for the glycine-binding site.¹²⁹ This is supported by the findings that (1) D-serine has a high potency to potentiate NMDAR-mediated neurotransmission, (2) D-serine is colocalized with NMDARs in the forebrain and (3) enzymatic degradation of the amino acid attenuates NMDAR-mediated neurotransmission.

As discussed at the start of this chapter, experimental application of the endogenous nonselective agonist glutamate has been shown to induce excitotoxic cell damage.⁴⁸ As with L-glutamate, administration of the agonists kainate^11,12,14,20 or NMDA¹⁹ induced brain damage, thus confirming the implication of different glutamate receptor classes.

The implication of different ionotropic glutamate receptor types in induction of brain damage has been further studied by the use of selective receptor antagonists. Experimental administration of competitive or uncompetitive NMDA antagonists^{21,22,85,86,130} or application of kainate antagonists attenuates cell death in cell cultures as well as brain damage.²⁰ Unfortunately, the clinical use of these receptor antagonists has been hampered by the inhibition of physiological NMDAR-mediated neurotransmission, resulting in various adverse effects.¹³¹ However, the uncompetitive NMDA antagonist memantine (1-amino-3,5-dimethyladamantane hydrochloride), which has already been used for years for treatment of Parkinson's disease, is sufficiently tolerated. Memantine is an open-channel blocker, but has faster kinetics than MK-801. This results in substantial inhibitory drug action under conditions of prolonged exposure to glutamate but much less inhibition under millisecond exposure to glutamate.¹³² This raises the hypothesis, that memantine should be suited to treat disease conditions associated with elevated glutamate concentrations for prolonged periods. Indeed, several groups have demonstrated its neuroprotective effectiveness combined with only few adverse effects.^133–135

Whereas the role of ionotropic glutamate receptors in excitotoxicity has been studied extensively, the significance of metabotropic glutamate receptors (mGluRs) in excitotoxicity is less well understood. Importantly, there is no indication, that activation or inhibition of mGluRs by itself exerts excitotoxicity.¹³⁶ Instead, in view of the literature it appears more likely, that specific metabotropic receptor subtypes can be involved in modulation of ionotropic receptor-mediated excitotoxicity. This may be due to the coupling of different mGluR subtypes to different signal transduction pathways and effectors. Group I mGluRs comprise the subtypes mGluR₁ and mGluR₅ which in heterologous expression systems couple to phospholipid hydrolysis through phospholipase C. Among other effects, this results in second messenger-mediated activation of protein kinase C and Ca²⁺ release from IP_3-sensitive Ca²⁺ stores.^137–139 Application of recently developed selective receptor ligands suggests, that activation of group I, and particularly mGluR1 receptors amplifies NMDA-mediated excitotoxicity.^{136,140–144}

Group II receptors comprise the subtypes mGluR₂ and mGluR₃ that are negatively coupled to the adenylate cyclase pathway. Recent pharmacological studies using selective agonists indicate that group II receptor activation results in protection against NMDA-mediated excitotoxicity.^{141,145–147} Likewise, group III receptors (mGluR₄ and mGluR_6–8) are negatively coupled to adenylyl cyclase, and activation of mGluR4 or mGluR8 group III receptors results in attenuation of NMDA-mediated neurotoxicity.^147–149

Ionic Dependence of Excitotoxic Cell Damage

Activation of different ionotropic receptor types is linked to excitotoxic cell damage through the underlying ion currents. Depending on the predominance of either Na or Ca²⁺ influx, two different components of excitotoxicity have been suggested. Na ion influx mediated by activation of NMDA-type and non-NMDA-type glutamate receptors is followed by secondary influx of Cl and H₂O and results in swelling of neurons.^{28,34,150,151} This acute form of cell damage depends on the transmembrane Na and Cl gradients²⁸ and can be prevented by extracellular substitution of Na and Cl with impermeant ions.^152,153 In contrast to the acute, primarily Nadependent osmotic damage, a more delayed mode of cell death has been attributed to Ca²⁺ influx.^28,153 This delayed Ca²⁺⁻-dependent cell death can be largely attenuated by inhibition of NMDA receptors,^19,29,84,154 removal of extracellular Ca²⁺ ions^23,155,156 or buffering of cytoplasmic Ca²⁺ by membrane-permeable chelators.^157,158 Therefore, the Ca²⁺-dependent component was traditionally believed to be induced exclusively by NMDA receptor activation.

The finding that the late component can be mimicked by calcium ionophores in presence of Ca²⁺ ions has corroborated the Ca²⁺ dependence of the delayed cell death.²⁸ However, the discovery of Ca²⁺-permeable AMPA/kainate receptors, has lead to the view that neurons expressing these Ca²⁺-permeable nonNMDA receptors can also undergo Ca²⁺-dependent delayed cell death.^32,159 In summary, different routes of Ca²⁺ influx such as through NMDA receptors, Ca²⁺-permeable AMPA/kainate receptors and voltage-dependent Ca²⁺ channels, which are all involved in physiological signaling, are implicated in excitotoxic cell death.

The suggested key role of Ca²⁺ in excitotoxicity has been subsequently confirmed by the finding, that the extent of excitotoxic cell death correlates with the total amount of Ca²⁺ uptake and is independent of the route of entry. In some cases Zn²⁺ can substitute for Ca²⁺ as the cation inducing excitotoxic damage.^29,159,160

The Ca²⁺ overload observed following sustained stimulation of NMDA receptors results from the inability of cellular Ca²⁺ homeostasis, such as extrusion of Ca²⁺ across the plasma membrane by Na/Ca²⁺ antiporter^161–163 and Ca²⁺ ATPase¹⁶⁴ or Ca²⁺ sequestration by the endoplasmic reticulum and mitochondria^165,166 to remove the large influx of Ca²⁺.

Interestingly, during sustained exposure to glutamate, the intracellular calcium concentration [Ca²⁺]_c rises rapidly to a peak value and thereafter slowly recovers to an elevated plateau level.¹⁶⁷ However, the [Ca²⁺]_c can also manifest a second, delayed increase,^30,168 immediately preceding cell death (Fig. 4 ). This delayed [Ca²⁺]_c increase, termed “delayed calcium deregulation” (DCD) is irreversible and reflects irreversible loss of cellular Ca²⁺ homeostasis. Although the interval between the initial Ca²⁺ spike and the DCD varies within a cell population exposed to NMDA receptor agonists, once DCD occurs within a neuron, it invariably precedes cell death. The temporal relation between neuronal Ca²⁺ levels and delayed cell death suggests, that Ca²⁺-dependent effector mechanisms are involved, which do not need sustained high Ca²⁺ levels but are triggered by transient changes in [Ca²⁺]_c.

Figure 4

Time course of glutamate-induced Ca²⁺ overload. A, intracellular free Ca²⁺ concentration [Ca²⁺]_c of a hippocampal neuron. [Ca²⁺]_c was measured using the calcium-sensitive fluorochrome indo1. Glutamate (100 mM) was applied for 5 min by superfusion (see (more...)

Since many enzymes are activated by transient or sustained [Ca²⁺] elevation various effector mechanisms may be implicated in Ca²⁺-mediated excitotoxic cell death. Consequently, a variety of Ca²⁺-dependent hydrolytic enzymes, including lipases and proteases, have been suggested to be involved in excitotoxic neuronal damage. Activation of the Ca²⁺-dependent phospholipase A2 has been observed following NMDA receptor activation¹⁶⁹ and the subsequent catabolism of released arachidonic acid by lipoxygenases and cyclooxygenases (COX), is also associated with concomitant production of reactive oxygen species (ROS).^169–171 In addition, activation of phospholiase A2 and subsequent release of arachidonic acid may inhibit transporter-mediated glutamate uptake from the extracellular space.^33,172,173

Among several Ca²⁺-activated proteases, the activity of the Ca²⁺-dependent cysteine protease calpain is increased following glutamate receptormediated Ca²⁺ loading. ^174–177 Calpain activation results in proteolysis of structural proteins and degradation of the neuronal cytoskeleton.¹⁷⁷ Furthermore, calpain may direct the mode of cell death to necrosis by preventing the cytochrome c-mediated activation of caspases (see following).¹⁷⁴

Ca²⁺-mediated activation of nitric oxide synthase may be another pathway involved in excitotoxic cell death since neurons constitutively express the isoenzyme called “neuronal nitric oxide synthase” (nNOS). nNOS is activated by glutamate receptor-mediated [Ca²⁺]_c increases^178,179 and there is compelling evidence that nNOS activation is linked to excitotoxic damage³¹ since inhibition of NO formation results in protection of neurons from glutamate receptor-mediated cell death.^179–182 Studies on nNOS-deficient neuronal cultures confirmed the role of NO in glutamate receptor-mediated neurotoxicity since in nNOS-deficient cortical cultures the toxic effects elicited by administration of NMDA are markedly attenuated.¹⁸³ The role of NO in excitotoxicity is mediated at least partly through the reaction of NO with superoxide anions (O₂ ) to form peroxynitrite (ONOO).¹⁸⁴

Other classes of enzymes are also thought to be involved in the Ca²⁺-mediated cell death but in less direct ways. Calcineurin is a Ca²⁺/calmodulin-activated phosphatase which can dephosphorylate nNOS, thereby increasing its activity¹⁸⁵ and potentially increasing excitotoxic damage. Calcineurin has been convincingly demonstrated to be involved in neuronal cell death since pharmacological experiments using the calcineurin inhibitors cyclosporin A and FK-506 have revealed attenuation of excitotoxic cell death.^186,187 Accordingly, inhibition of calmodulin can also be shown to decrease excitotoxic cell death.^182,188

Mitochondrial Dysfunction

Studies on the time course of glutamate receptor-induced [Ca²⁺]_c increase have demonstrated that if the neuronal cell does not succumb to the insult, [Ca²⁺]_c slowly recovers after the termination of agonist application.^30,167 This recovery is due to several mechanisms that ensure the cellular Ca²⁺ homeostasis under physiological conditions (Fig. 5) such as extrusion across the plasma membrane by the Na/Ca²⁺ exchanger¹⁸⁹ and the Ca²⁺-ATPase, or sequestration of Ca²⁺ into the endoplasmic reticulum^189,190 and mitochondria.^191,192

Figure 5

Calcium transport in neurons. Influx of Ca²⁺ ions through receptor-operated channels (ROC) and voltage-dependent calcium channels (VDCC) activates extrusion mechanisms and calcium sequestration into mitochondria, endoplasmic reticulum (ER) and nucleus. (more...)

In theory, the driving force for Ca²⁺ ions to enter the mitochondrial matrix in energized mitochondria is formed by the strong electrochemical gradient for Ca²⁺ across the inner mitochondrial membrane, mainly due to the mitochondrial membrane potential Δψ_m(Δψ_m Å-180 mV). Therefore, normal energized mitochondria can take up substantial amounts of calcium.¹⁹³ Net uptake of calcium occurs in isolated mitochondria whenever [Ca²⁺]_c rises above a set point in the high nanomolar range (>0.5 μM), indicating the dynamic equilibrium of mitochondrial uptake and extrusion mechanisms.¹⁹⁴ [Ca²⁺]_c can be shown to increase above this set point¹⁹⁵ during excessive cytoplasmic Ca²⁺ loading following ionotropic glutamate receptor activation and results in net mitochondrial Ca²⁺ uptake.^191,192

It has been proposed that mitochondrial Ca²⁺ uptake can shape the time course of cytoplasmic [Ca²⁺]_c.¹⁹⁶ During excessive Ca²⁺ loading into neurons, mitochondrial Ca²⁺ uptake appears to blunt the [Ca²⁺]_c increase since mitochondrial depolarization by protonophores prior to Ca²⁺ loading results in increased [Ca²⁺]_c.^{163,196–199} After termination of the Ca²⁺ influx, [Ca²⁺]_c slowly decreases and Ca²⁺ redistribution from mitochondria into the cytoplasm begins when [Ca²⁺]_c decreases below the set point.^192,196

Ca²⁺ uptake into mitochondria results in a decrease in Δψ_m that can be monitored in isolated energized mitochondria^200–202 as well as in situ.^203–207 This depolarization is due to the balancing of the charge transfer carried by Ca²⁺ ions by re-entry of protons into the mitochondrial matrix. Under normal conditions, Ca²⁺⁻induced depolarization is transient and serves to activate mitochondrial dehydrogenases^208–214 and the mitochondrial ATP synthase,²¹⁵ which in turn results in increased electron transport through the respiratory chain and consequently increased outward flux of protons.

However, studies on in situ mitochondrial membrane potential using cationic fluorochromes have demonstrated convincingly that the time course of Δψ_m depolarization can vary considerably, and depends on the time of Ca²⁺ loading. Short pulses of NMDA result in at least partial recovery of Δψ_m²⁰³ whereas prolonged NMDA exposure or higher agonist concentrations result in sustained depolarization with negligible recovery.^40,203,206 This sustained depolarization indicates that excessive Ca²⁺ uptake triggers severe mitochondrial bioenergetic dysfunction. Sustained depolarization has been ascribed to various underlying mechanisms, and ROS seem to play a key role in several of the suggested hypotheses.

In neurons as in other tissues, mitochondria are a major source of ROS;^216,217 nonetheless, mitochondria are themselves susceptible to oxidative damage. Excessive Ca²⁺ uptake into isolated mitochondria is known to result in increased formation of ROS (Fig. 6),^218,219 which in turn inhibit pyruvate dehydrogenase²²⁰ and tricarboxylic acid cycle enzymes^221,222 as well as complex I of the respiratory chain.^223–225 In intact neurons, ROS formation has been monitored with redox-sensitive fluorochromes, which are oxidized by ROS to form fluorescent molecules.^226–228 These studies indicate that ionotropic glutamate receptor-induced Ca²⁺ loading enhances the production of O₂^{227,229–231}

Figure 6

Electron paramagnetic resonance spectra from intact, coupled mitochondria isolated from rat cerebral cortex. A, no signals indicative of free radicals were detected in the absence of Na and Ca²⁺. B, following exposure to 14 mM Na and 2.5 mM Ca²⁺ signals (more...)

The main site of ROS formation within mitochondria is within the respiratory chain. Complex I^232,233 as well as complex III^234–236 are thought to participate in one electron reduction of molecular oxygen, resulting in the generation of O₂ which in turn can lead to other ROS. Nevertheless, the exact biophysical link between mitochondrial Ca²⁺ uptake and increased mitochondrial ROS production remains unclear.

The Role of Reactive Oxygen Species in Excitotoxicity

Evidence for the implication of ROS in excitotoxic cell damage arises from experiments showing enhanced production of O₂^{227,230,231,237} following ionotropic glutamate receptor overstimulation and from studies using radical scavengers or inhibitors of the formation of certain ROS.²²⁷ These studies unequivocally demonstrate that removal of ROS results in attenuation of glutamate receptor-induced cell death.^238,239

Several Ca²⁺-dependent processes that increase the endogenous production of ROS have been described, which are all assumed to be implicated in excitotoxic cell death following NMDA receptor activation. As stated above, Ca²⁺ loading into mitochondria appears to be one important mechanism of ROS production under conditions associated with excitotoxicity.^{227,229–231,237,240} Apart from this mechanism, activation of Ca²⁺-dependent phospholipase A2 has been observed following NMDA receptor activation¹⁶⁹ and catabolism of released arachidonic acid by lipoxygenases and cyclooxygenases has been implicated in excitotoxicity through the concomitant production of ROS.^169–171

Some ROS exhibit a high reactivity with correspondingly short halflives and can undergo many different reactions. Generally speaking, ROS can exert multiple damaging reactions to proteins, lipids, carbohydrates and nucleic acids, thereby disrupting cellular functions. The increased production of ROS is therefore a potential threat to cellular homeostasis and neuronal survival if production is not balanced by the capacity of endogenous antioxidant mechanisms. Some basic characteristics of activated oxygen species are listed in Table 3.

Table 3

Characteristics of reactive oxygen species.

From a multitude of ROS-mediated disturbances following NMDA receptor activation, only some examples will be discussed. Since mitochondria appear to be the major source of Ca²⁺-induced increase in ROS formation and ROS are highly reactive, mitochondria are prone to damage by ROS. As discussed above, several mitochondrial enzymes, such as NADH:CoQ oxidoreductase, succinate dehydrogenase, ATP synthase, pyruvate dehydrogenase and the citric acid cycle enzyme aconitase, are inhibited by ROS including O₂ , H₂O₂ or -OH.^{220,221,223,241,242} The delayed deregulation of cellular Ca²⁺ homeostasis (DCD) that has been observed after NMDA exposure could be caused by ROS-dependent mechanisms. This hypothesis is supported by the finding that experimentally induced production of O₂ by menadione results in enhancement of DCD,²³⁸ whereas dismutation of O₂ by the manganoporphyrin Mn-TBAP attenuates DCD.²³⁸ Importantly, the redox-sensitive mechanism underlying DCD is still unresolved. Although oxidative stress-induced dysfunction of the plasma membrane Ca²⁺⁻ATPase has been demonstrated²⁴³ and this dysfunction has been proposed as a mechanism underlying the NMDA receptor-induced DCD,^238,244 the conclusion that mitochondrially generated ROS are involved in plasma membrane protein dysfunction should be judged cautiously because of the small reaction range.

Ca²⁺⁻-induced increase in ROS can result in mitochondrial dysfunction including dissipation of Δψ_m, bioenergetic failure and disturbance of cellular Ca²⁺ homeostasis. Apart from the inhibition of enzyme activity, the opening of a large nonselective pore in the inner mitochondrial membrane termed “mitochondrial permeability transition” (mPT) has been suggested as a key mechanism in neuronal excitotoxicity.^203,206 The basic concept of a pore-mediated permeabilization of the inner mitochondrial membrane rather than membrane damage had been suggested as early as the 1970s. The early studies by Haworth and Hunter²⁴⁵ showed that various divalent cations could induce or prevent mitochondrial swelling and that the assumed pore is selective for the permeation of solutes below 1500 kDa. The idea of a large nonspecific channel was confirmed almost one decade later by electrophysiological experiments. Single-channel patch-clamp recordings from the inner mitochondrial membrane revealed a large conductance channel (1.3 nS),²⁴⁶ which opened in response to the addition of Ca²⁺ to the mitochondrial preparation. Notably, the activation and inhibition characteristics of this “mitochondrial megachannel” are similar to the proposed mPT. Application of Ca²⁺, ROS, inorganic phosphate and the adenine nucleotide translocator ligand atractylate induce permeability transition and opening of the megachannel, whereas Mg²⁺, antioxidants, ADP, cyclosporin A and the adenine nucleotide translocator ligand bongkrekic acid (BA) inhibit permeability transition and channel opening.^{219,247–256} This strikingly similar behavior has resulted in the conclusion that the mPT and the mitochondrial megachannel are virtually identical.²⁵⁷

Dissipation of Δψ_m, bioenergetic failure and disturbance of mitochondrial Ca²⁺ homeostasis following activation of NMDA receptors all could be explained by opening of the mPT.^203,206 However, conclusions regarding the involvement of mPT in neuronal excitotoxicity until recently were based on pharmacological experiments using the mPT inhibitor cyclosporin A and should be treated cautiously since cyclosporin A also inhibits calcineurin and the multidrug resistance channel.^258,259 Recently, however, our group succeeded in demonstrating that BA, a more specific inhibitor of mPT, could prevent NMDA-induced neuronal apoptosis, suggesting the involvement of mPT in this form of excitotoxic cell death.²⁶⁰

In summary, several ROS-dependent mechanisms have been suggested, mainly based on data from isolated mitochondria. In any event, the complex environment of in situ mitochondria, containing, for example, mPT enhancers as well as inhibitors, prevents a definitive interpretation of the physiological and pathophysiological relevance of the proposed mechanisms at this time.

Role of Nitric Oxide and other Reactive Nitrogen Species in Excitotoxicity

Nitric oxide (NO) is a well-recognized messenger molecule in the CNS that affects various cellular functions, e.g., neuronal transmitter release, synaptic plasticity and gene expression (see refs. 261–263). NO is produced by several types of cells expressing three distinct isoforms of the enzyme nitric oxide synthase (NOS, NADPHdiaphorase) that converts L-arginine into NO and citrulline: (1) neuronal NOS (nNOS), (2) inducible or immunologic NOS (iNOS) in microglia and astrocytes and (3) endothelial NOS (eNOS) predominantly in endothelial cells of brain blood vessels. Unlike classical neurotransmitters, NO is freely diffusible and therefore cannot be stored in synaptic vesicles. Once synthesized, NO diffuses across cell membranes and thus can reach (1) various compartments within the NO-producing cells and (2) different types of surrounding cells (Fig. 7). In contrast to conventional transmitters, the activity of the messenger molecule is not terminated by reuptake or enzymatic degradation but by chemical reactions with target molecules and its spontaneous formation of nitrite. The absence of control over its activity by release and reuptake mechanisms is functionally compensated by a tight regulation of its synthesis. However, the regulatory mechanisms involved in physiological signaling may also implicated under pathophysiological conditions. During the last decade, pharmacological experiments using NO donors, NOS inhibitors or exogenous substrates (reduced hemoglobin) for competitive reactions with NO suggested that NO is linked to neuronal cell death.^31,43,182 Even more important, cell death under conditions assumed to induce excitotoxicity could be largely attenuated by inhibition of NO formation or blockade of NO effects.^31,182,264 Subsequent studies on nNOS-deficient neuronal cultures confirmed the role of NO in glutamate receptor-mediated neurotoxicity since toxic effects elicited by administration of NMDA are markedly attenuated in nNOS-deficient cortical cultures compared with wild-type neurons.¹⁸³ Since nNOS activity is known to increase with increasing calcium concentrations, the NMDAR-mediated calcium uptake provides the link between conditions associated with excitotoxicity and NO-mediated cell damage.¹⁷⁸

Figure 7

Schematic presentation of a nitric oxide (NO) producing neuron. NO formed from arginine by the enzyme nitric oxide synthase freely diffuses across membranes and thus can affect various organelles within the same neuron as well as other surrounding neurons (more...)

Although NO is a free radical, it is not as reactive as most ROS²⁶⁵ and calculations based on its reactivity had estimated a reaction range of about 100 mm.^261,263The well-accepted implication of NO in excitotoxicity has been suggested to depend on increased NO formation and the concomitant production of O₂. Under this condition, O₂ reacts with NO extremely fast to form peroxynitrite (ONOO).^266–268 ONOO can undergo a multitude of different chemical reactions with various substrates, e.g., hydroxylation or nitration of tyrosine residues, lipid peroxidation or apparent decomposition into NO₂ and -OH and subsequently, this toxic molecule has been implicated in NO-induced cell damage.^{43,182,184,269}

So far, several pathways of ONOO toxicity have been suggested but with respect to its high reactivity, it appears unlikely that these are the only mechanisms involved in NO-mediated cell death.

Intense exposure to NO/O₂ with resultant ONOO formation has been demonstrated convincingly to result in neuronal necrosis because of energy failure, while mild insults lead to apoptosis.⁴³ This bioenergetic failure may be due to (1) increased ATP consumption under the condition of poly-(ADP ribose) polymerase (PARP) activation,²⁷⁰ which occurs following ONOO-mediated DNA damage and (2) decreased ATP synthesis through NO-mediated inhibition of the respiratory chain machinery.^271–273

Very recently, p38 mitogen-activated protein (MAP) kinase has been suggested as another pathway implicated in NO-induced cell death. Application of a p38 MAP kinase inhibitor significantly attenuated NO-induced caspase activation, Bax translocation, and neuronal cell death.²⁷⁴

Excitotoxicity, Calcium Loading and Apoptosis

A great advance in excitotoxicity came with the understanding that neurons can die by apoptosis. In response to excitotoxicity the neuron activates apoptosis, i.e., dismantling of itself including DNA, cytoskeleton, and production of ATP. While in acute neurological situations such as stroke the cells in the ischemic core die rapidly by necrosis, at least in animal models, the cells in the penumbra also go on to die but show markers of apoptosis, such as oligonucleosomal DNA damage.^275,276 Further evidence of apoptosis in excitotoxicity came with the examination of the activation of caspases in animal models of stroke.²⁷⁷ Inhibition of caspases in general by intracerebroventricular injections of N-benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone (z-VAD.FMK), or selective inhibition of caspase-3 with N-benzyloxycarbonyl-Asp-Glu-Val-Aspfluoromethylketone (z-DEVD.FMK) reduced the ischemic infarct volume by as much as 60%.²⁷⁷ These caspase inhibitors remain effective even when applied hours after the insult, making them effective later than NMDA receptor blockade with MK801.⁹⁴ While the morphology of dying ischemic cells does not precisely match that of “classical” apoptosis,²⁷⁸ and in vivo excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, studies with caspase inhibitors clearly indicate a role for apoptosis in ischemia.

Apoptosis is also a feature of neurons dying in neurodegenerative disorders. DNA fragmentation has been detected in human post mortem samples of Parkinson's disease,^279,280 Alzheimer's disease,^281,282 Huntington's disease,^282,283 and amyotrophic lateral sclerosis.²⁸⁴ However, the presence of DNA fragmentation is not a guarantee that the cells are dying by apoptosis,²⁸⁵ and DNA fragmentation can be influenced by antemortem hypoxia.²⁸⁶ More recently other recognized apoptotic molecules such as caspases,^287,288 and BclX_L²⁸⁹ have been shown in Alzheimer's disease. It is now largely accepted that neuron loss in chronic neurodegeneration is mediated by apoptosis.

Key Signaling Players in Neuronal Apoptosis

Caspases are central to the induction of neuronal cell death.^94,277,290 Their actions on key intracellular substrates, including other protease zymogens, make caspases the primary executioners of the cell death program. The 13 known caspases can be separated into two functional groups; those that initiate apoptosis by receiving the initiating signals, and those that effect the dismantling of the cell. The caspases that receive or integrate apoptotic signals (caspases-8, -10, -2, -9) cleave and activate the downstream effector caspases (caspases-3, -7, -6). Perhaps the last reversible step in the death of neurons is the activation of the caspase-3,²⁹¹ although caspase-independent mechanisms almost certainly exist.^174,292 As discussed above, peptide inhibitors of caspase-3 block the death of neurons in many different situations (for review see ref. 293). The central importance of caspase-3 in neurons was clearly shown in caspase-3-deficient mice which have a doubling of brain size, correlated with decreased apoptosis and premature death.²⁹⁴

Mitochondria appear to provide a link between the initiator caspases and the downstream effector caspases (Fig. 8). In nonneuronal cells, mitochondria have been shown to accelerate activation of effector caspases by releasing proapoptotic molecules, such as cytochrome c,^295,296 the apoptosis-inducing factor,²⁹⁷ and SMAC/diabolo.²⁹⁸ Currently in neuronal systems, only the pathway utilized by cytochrome c has been fully elucidated. Cytochrome c triggers activation of caspase-9 which in turn cleaves caspases-3, -6, -7.²⁹⁹ Release of cytochrome c from mitochondria has received much attention as a commitment to apoptosis.^{295,296,300,301} Nevertheless, the release of cytochrome c from mitochondria has been shown to be not sufficient for neuronal apoptosis,^260,302 and microinjection of cytochrome c into sympathetic neurons does not lead to death in the absence of additional stimuli.³⁰³ Therefore, cytochrome c requires other factors to initiate the caspase cascade. These factors, Apaf-1, dATP and procaspase-9 along with cytochrome c are known collectively as the apoptosome.³⁰⁴ The processed caspase-9 is then able to cleave and activate caspase-3.

Figure 8

Model incorporating several features of apoptotic injury in neurons. After a relatively mild insult by glutamate at the NMDA receptor (initiating excessive Ca²⁺ influx and generating NO and O₂to form ONOO), the mitochondrial membrane potential transiently (more...)

Considerable uncertainty surrounds the mechanism of release of proapoptotic factors from the mitochondria. Possibilities include (i) transport by poreforming Bcl-2 family proteins such as Bax,³⁰⁵ (ii) opening of the permeability transition pore (PTP) in the inner mitochondrial membrane leading to rupture of the outer mitochondrial membrane,³⁰⁶ or (iii) transport coupled to the voltage-dependent anion channel in the outer mitochondrial membrane.^301,307 This remains an important question to answer, since cytochrome c release from the mitochondria will eventually bring about mitochondrial dysfunction and an energetic deficit. Studies with nonneuronal cells have shown that mitochondrial membrane potential is maintained by ATP hydrolysis following the exit of cytochrome c.³⁰⁸ The release of cytochrome c from mitochondria can be stimulated by some caspases and by the proapoptotic members of the Bcl-2 family Bid and Bax.^300,305,306 On the contrary, survival factors can act to prevent release of cytochrome c, e.g., channel activity of Bax is inhibited by anti-apoptotic Bcl-2 family members such as Bcl-2 or Bcl-xL, and ordinarily Apaf-1 is sequestered at the membrane by Bcl-xL, and in the absence of cytochrome c cannot interact with procaspase-9.³⁰⁹ While the role of signaling molecules in neuronal apoptosis has been studied extensively in cell culture models, the in vivo data is sparse for many of the apoptotic pathways. However, several of the key signaling molecules in apoptosis have been detected following ischemia, e.g., the release of cytochrome c,^310,311 Fas/TNFR,³¹² Bax,³¹³ phospho-JNK,³¹⁴ and caspase-9.³¹⁵

The initiators of apoptosis in neurons are diverse including the withdrawal of trophic factors, and signaling through the transmembrane “death” receptors such as Fas/TNFR family as well as intrinsic signaling pathways that sense overload of ROS, Ca²⁺, and H (for review see ref. 290). In vitro, neuronal cell death can be induced by a variety of treatments: axotomy, excitotoxins, trophic deprivation, etc. These diverse stimuli have demonstrated that neurons indeed contain numerous apoptotic signaling pathways. Excitotoxicity has been achieved in cultures of primary neurons using either a direct insult such as activation of glutamate receptors, or with a secondary insult such as with Aβ (amyloid peptide), hypoxia, etc. These various paradigms which ultimately overstimulate glutamate receptors all result in the activation of caspases. Upstream of the caspases however, excitotoxic insults can simultaneously activate more than one pathway.

As discussed in earlier sections, overstimulation of NMDA receptors results in an influx of calcium. Calcium at high (>500 nM) concentrations inappropriately activates many enzymes, is accumulated within the nucleus, endoplasmic reticulum and mitochondria, and as a general disturbance to the cell's ionic homeostasis causes a large energy drain as the cell tries to extrude the excess calcium (reviewed in ref. 316). All of these occurrences can and do trigger the neuron to apoptosis. From an extensive and rapidly unfolding literature, the following points demonstrate some of the pathways that are concurrently activated by excitotoxic entry of calcium.

Isolated brain mitochondria when exposed to high calcium release cytochrome c.³¹⁷ Accordingly, prolonged activation of NMDA receptors in cultured neurons and subsequent calcium loading results in release of cytochrome c from mitochondria followed by the activation of caspase-3.^260,318 Thus in excitotoxicity, cytochrome c release from mitochondria, in the presence of the other members of the apoptosome, is a direct signal to apoptosis. Apart from the mitochondrion as a key player in the apoptosis of neurons, the endoplasmic reticulum (ER) seems to participate in apoptotic signaling. The ER regulates cellular responses to stress and intracellular calcium levels. Treatment of cultured neurons with glutamate or thapsigargin induces the expression of caspase-12 on the ER outer membrane.^319,320 This results in cleavage and activation of cytosolic caspase-3.³¹⁹

As discussed in an earlier section, glutamate receptor activation also activates the Ca²⁺-dependent cysteine protease calpain.^174–177 Calpain inhibitors appear to be at least as effective as caspase inhibitors in preventing neuronal death and DNA fragmentation,²⁹³ and have been demonstrated to reduce neuronal cell death caused by hypoxia.³²¹

Other concomitantly activated pathways include the p38 MAP kinase and c-jun N-terminal kinase or stress-activated kinase (JNK/SAPK) which promote cell death.³²² SAPK/JNK is activated by tyrosine kinase and elevated intracellular calcium, and in primary striatal cultures, treatment with glutamate causes both increased phosphorylation of its substrate cjun and increased activity of JNK.³²³ Glutamate has been shown to activate p38 MAP kinase in rat cerebellar granule neurons,³²⁴ and inhibition of p38 MAP kinase has been demonstrated to protect in vitro retinal ganglion neurons from NMDA-induced apoptosis.³²⁵

DNA damage is a common trigger for apoptosis in many different tissues, and in the mature nervous system, p53 is essential for neuronal death in response to DNA damage, ischemia and excitotoxicity.³²⁶ Lately, signaling pathways that are responsible for triggering p53-dependent neuronal apoptosis are starting to be elucidated and involve cell cycle deregulation and also activation of the JNK pathway.

Conclusions

A large body of evidence has accumulated showing that excessive activation of EAA receptors by the primary excitatory amino acid L-glutamate under pathophysiological conditions or intoxication with exogenous agonists results in excitotoxic cell death. Studies on the ion currents occurring under conditions of excitotoxicity have revealed that imbalanced Na and Cl gradients as well as Ca²⁺ overloading are implicated in cell death. Calcium ions (and in some cases zinc ions) seem to play a pivotal role in EAA neurotoxicity, since this ubiquitous intracellular messenger can activate a multitude of enzymatic and nonenzymatic signaling and execution pathways implicated in neuronal cell death. There is compelling evidence that the mode of cell death that neurons undergo in excitotoxicity varies considerably along an apoptosis-necrosis continuum, depending on the severity of the insult. To date, numerous mechanisms involved in necrosis as well as various signaling and execution pathways that are implicated in apoptosis have been shown convincingly. However, while the role of signaling molecules in neuronal apoptosis has been studied extensively in in vitro models, the in vivo data is sparse for many of the pathways.

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