The Role of Glutamate in Neuronal Ischemic Injury: the Role of Spark in ﬁre

Home , Gavestinel, Lubeluzole, Traxoprodil

Neurol Sci (2012) 33:223–237 DOI 10.1007/s10072-011-0828-5

REVIEW ARTICLE

The role of glutamate in neuronal ischemic injury: the role of spark in ﬁre

Botros B. Kostandy

Received: 7 June 2010 / Accepted: 20 October 2011 / Published online: 2 November 2011 Ó Springer-Verlag 2011

Abstract Although being a physiologically important cellular and synaptic functions, cell death and survival, excitatory neurotransmitter, glutamate plays a pivotal role motor functions, learning and memory [1]. Its level in in various neurological disorders including ischemic neu- mammalian brain is 1,000 times more than other important rological diseases. Its level is increased during cerebral neurotransmitters such as dopamine or serotonin. Although ischemia with excessive neurological stimulation causing glutamate has important physiological functions in the the glutamate-induced neuronal toxicity, excitotoxicity, CNS it is suggested to take part in the pathophysiology of and this is considered the triggering spark in the ischemic many disease processes such as epilepsy, neurodegenera- neuronal damage. The glutamatergic stimulation will lead tive disorders and cerebrovascular stroke [2]. Playing an to rise in the intracellular sodium and calcium, and the important role in ischemic neuronal damage, this review elevated intracellular calcium will lead to mitochondrial focuses on the mechanisms of injurious effect of glutamate dysfunction, activation of proteases, accumulation of during ischemia and possible therapeutic approaches. reactive oxygen species and release of nitric oxide. Inter- ruption of the cascades of glutamate-induced cell death during ischemia may provide a way to prevent, or at least Synthesis, release and breakdown of glutamate reduce, the ischemic damage. Various therapeutic options are suggested interrupting the glutamatergic pathways, e.g., Glutamate is not synthesized in neurons nor acquired from inhibiting the glutamate synthesis or release, increasing its circulation [3], but rather synthesized in astrocytes through clearance, blocking of its receptors or preventing the rise in the intermediate metabolite glutamine. The process of intracellular calcium. Development of these strategies may glutamate synthesis and breakdown can be summarized in provide future treatment options in the management of Fig. 1. Activated cells release glutamate through hemi- ischemic stroke. channels of gap junctions [4]. The released glutamate produced by the depolarization of synaptosomes consists of Keywords Glutamate Á Excitotoxicity Á two components. A physiologically relevant Ca2?-depen- Cerebral ischemia Á Neuroprotectants dent component is produced by the exocytosis of synaptic vesicles containing glutamate. N-type Ca2? channels, a type of voltage-dependent Ca2? channels (VDCCs), are Introduction principally responsible for release of synaptic glutamate in the most of the CNS [5]. The other component, Ca2?- Glutamate is one of the major excitatory neurotransmitters independent one, can be attributed to the reversal of the in the central nervous system (CNS). It controls various glutamate transporter [6]. The released glutamate is effectively removed by ﬁve different Na?-dependent high- afﬁnity glutamate transporters [7]: EAAT 1 (GLAST), & B. B. Kostandy ( ) EAAT2 (GLT-1), EAAT3 (EAAC1) and EAAT4 and Department of Pharmacology, Faculty of Medicine, University of Assiut, Assiut 71526, Egypt EAAT5. EAAT2 has the predominant role in clearing e-mail: [email protected] glutamate throughout the neuroaxis [8]. 123 224 Neurol Sci (2012) 33:223–237

Astrocyte Glutametergic NMDARs have been shown to be organized into multi- neuron protein signaling complexes within a specialized structure located beneath the postsynaptic membrane aligned with Glutamine Glutamine Glutamine active zones of presynaptic terminals with the CNS, known as the postsynaptic density (PSD) [15]. PSDs are involved in

Glutamine Glutaminase several functions including: cell-to-cell adhesion, regulation synthase of receptor clustering and modulation of receptor function. Glutamate Glutamate Glutamate Of the proteins involved in the PSD, PSD-95 plays a prom- inent organizational role by coupling the NR2 subunits of aminotransferase NMDARs to intracellular proteins and signaling enzymes such as neuronal nitric oxide synthase (nNOS) [16, 17]. α-ketoglutaric AMPARs are composed of four subunits GluR1–4 or A–D subunits encoded by four genes [18]. The perme- Fig. 1 Schematic representation of the process of glutamate ability to Ca2? depends on the presence or absence of a synthesis and glutamate-glutamine cycle between neurons and astrocytes (arrow release, dashed arrow enzymatic conversion, dot- GluR2 subunit. Its presence renders an AMPAR channel ted arrow uptake) impermeable to Ca2?. Activation of AMPARs after ischemia allows Na? influx which leads to the depolarization of the neuron and subsequent influx of Ca2? Glutamate receptors through Ca2? channels. Drugs interfering with the function of NMDAR and AMPAR are summarized in Table 1. Glutamate acts on four different postsynaptic receptors: Molecular cloning of Kainate receptors (KARs) has NMDA (N-methyl D-aspartate), AMPA (a-amino-3- identified five subtypes: GluK1-5, which co-assemble in hydroxy-5-methyl-4-isoxazole propionic acid), kainate and various combinations to form functional receptors. KAR metabotropic receptors. The three former receptors are subunits that were formally known as GluR5–7 (or linked to membrane ion channels, whereas metabotropic GLUK5–7) are now named GluK1–3 and those previously receptors are coupled with a G-protein. called KA1 and KA2 (or GLUK1 and GLUK2) are now NMDA receptors (NMDARs) are composed of seven named GluK4 and GluK5 [19]. Functional studies suggest NMDAR subunits have been identified so far: one NR1, that KARs mainly have a modulatory role in synaptic four NR2 (A–D), and two NR3 (A and B). Their functional transmission (excitatory and inhibitory) rather than being characteristics are determined by the receptors specific the major postsynaptic target for synaptically released combination of NR1 and NR2 subunits. Most native glutamate as is the case for NMDARs and AMPARs [20]. NMDARs appear to function as heterotetrameric assem- KARs regulate glutamate release. They can function as blies composed of two glycine-binding NR1 and two glu- facilitatory autoreceptors or inhibitory autoreceptors during tamate-binding NR2 subunits [9]. NR2A containing repetitive synaptic activation and can respond to ambient receptors are characterized by faster decay times than levels of L-glutamate to provide a tonic regulation of 2? NR2B-containing ones, as evidenced by different Ca L-glutamate release. KARs contribute to excitatory synap- influx [10]. Moreover, in mature cultured neurons, NR2A- tic transmission at certain synapses. containing NMDARs promote trafficking of the AMPA Metabotropic receptors (mGluRs) are grouped to three receptors (AMPARs) subunit GluR1 while NR2B-con- families, group I (mGluR1 and mGluR5), II (mGluR2 and taining NMDARs inhibit such trafficking [11]. NR3 sub- mGluR3) and III (mGluR4–mGluR8). Group I receptors units reduce the amplitude and dramatically decrease the are linked to phospholipase C and localized in the post- Ca2? permeability of NMDAR-associated channels and it synaptic density area at excitatory synaptic sites. Group I is suggested that endogenous NR3A protects neurons [12]. mGluRs may also have effects on NMDA signaling [21]. There are also modulatory sites on the NMDAR, including They potentiate NMDA-mediated neurotoxicity, increase those for glycine, Mg2? and polyamines. arachidonic acid release and inflammation [22]. Groups II The subcellular localization of NMDARs also affects and III, primarily localized presynaptically, are negatively the nature of NMDAR signaling. Evidence suggests that linked to adenylate cyclase [23]. They are blocked by synaptic NMDAR activity is extremely important for various agents, including for example alpha-methyl- neuronal survival while the extrasynaptic NMDARs are cyclopropyl glycine (MCCG) [24] and a-cyclopropyl- coupled to cell-death pathways [13]. Extrasynaptic 4-phosphonophenylglycine (CPPG) [25], respectively. NMDARs oppose synaptic NMDARs by triggering cAMP- Activation of these receptors results in feedback inhibition responsive element-binding protein (CREB) shutoff and of glutamate release, through the inhibition of voltage- cell-death pathways [14]. gated Ca2? entry into the cell. 123 Neurol Sci (2012) 33:223–237 225

Table 1 NMDAR and AMBAR blockers NMDAR blockers AMPAR blockers Competitive Noncompetitive Glycine site Polyamine site Competitive Noncompetitive blockers blockers blockers blockers antagonists antagonists

AP-7 Cerestat ACEA 1021 Traxoprodil NBQX GYKI 52466 CGP 37849 Dextromethorphan Felbamate Liprodil PNQX SYM2206 CGP 39551 Dextrorphan Gavestinel CGS 19755 Dizocilpine HA966 CPP Ketamine L-687,414 Midafotel Memantine, (CPPene) Phencyclidine Remacemide ACEA 1021 5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione, AP-7 2-amino-7-phosphonoheptanoic acid, CGP 37849 (E)-(±)-2-amino-4- methyl-5-phosphono-3-pentenoic acid, CGP 39551 (E)-(±)-2-amino-4-methyl-5-phosphono-3-pentenoic acid ethyl ester, CPP 3-(2-carbo- xypiperazin-4-yl)propyl-1-phosphonic acid, CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid), GYKI 52466 1-(4-aminophenyl)- 4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine, HA966 3-amino-1-hydroxypyrrolid-2-one, L-687,414 3R(?)cis-4-methyl-pyrrollid-2-one, NBQX 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione, PNQX 9-methyl-amino-6-nitro-hexahydrobenzo(F)quinoxalinedione, SYM2206 4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine

Elevation of glutamate level during ischemia substrate is enhanced by depolarization-stimulated Ca2? entry, which results in the facilitation of glutamate release Normally, neurons are exposed to small, transient impulses [36]. The rise in extracellular glutaminase, released from of glutamate due to efficient uptake removal mechanisms damaged neurons will increase the hydrolysis of glutamine, [26], but neurons are subjected to excessive glutamatergic with the formation of extracellular glutamate [37]. stimulation during ischemia due to the elevation of extra- Ischemia with subsequent poverty of ATP leads to col- cellular glutamate with overstimulation of its receptors, lapse of Na?/K? electrochemical gradient. As a result, the and this phenomenon has been recognized since the 1980s glutamate transporters operate in a reverse direction, and [27]. Ischemia-induced elevations in glutamate occur in all this process is suggested by some authors to be the main brain areas which have been investigated and in response to mechanism responsible for glutamate accumulation [38] all experimental approaches employed to provide low and the excess extracellular glutamate is the end result. The levels of cerebral blood flow [28]. Both focal and global expression of the glutamate transporters EAAT1 and cerebral ischemia cause rise in extracellular glutamate EAAT2 in astrocytes is rapidly altered following hypoxic- levels [29]. Glutamate levels return to normal shortly after ischemic injury [39], and the reduced expression of these reperfusion has been initiated [30]. transporters leading to the accumulation of their substrate The release of glutamate occur when blood supply to extracellularly [40]. neurons decrease below certain level (threshold relation- Although the glutamate physiological antidote, gamma ship). The neurotransmitter is released at a blood flow amino butyric acid (GABA), increase extracellularly in below 20 ml/100gm/min [31]. It is worth mentioning that response to ischemia, this increase is not as large as that of neuronal necrosis occur at a blood flow of 17 ml/100gm/ glutamate and does not persist as long [41]. Since gluta- min maintained for 180 min or more [32]. mate uptake via EAAC1 has been posited to provide a The increased formation and release of glutamate from source for GABA synthesis, decreases in EAAC1 may lead glutamine in intact glutamatergic neurons has been sug- to decreased GABA-ergic activity. The resultant increased gested to account for the increased extracellular glutamate extracellular glutamate and relative poverty in GABA may [33]. Reduction in adenosine triphosphate (ATP), resulting lead to overexcitation and neuronal death [39]. from anoxia, causes failure in the energy-mediated processes in the cell, e.g., Na? pumps [34], in addition to swelling-induced opening of anion channels [35], lead to Glutamate-induced cell injury accumulation of ions inside the neurons with facilitation of cellular membrane depolarization. This is followed by The concept that glutamate is a potent neurotoxin has long exocytosis and expulsion of the glutamate loaded in been recognized for more than half centaury [42]. It is intracellular vesicles. Protein kinase C (PKC)-dependent responsible for toxic neuronal death of postsynaptic neu- phosphorylation of myristoylated alanine-rich C kinase rons. Neuronal death in cell culture occurs at doses above 123 226 Neurol Sci (2012) 33:223–237

10 lM[43]. Prolonged and excessive stimulation of influx, resulting in cytotoxic edema [48]. NMDARs play glutamatergic receptors occur when extracellular glutamate a key role in mediating at least some aspects of gluta- approaches 100 lM[44]. Even ambient glutamate con- mate neurotoxicity [49]. Many studies have referred centrations may be neurotoxic in energy-deprived cells to NMDAR-triggered, Ca2?-mediated excitotoxicity as [45]. This action has been denominated excitotoxicity and ‘‘rapidly triggered’’, emphasizing the speed at which it can occurs as a consequence of a prolonged or a strong acti- occur. In cortical cell cultures, 3–5 min of sustained vation of glutamate postsynaptic receptors [46]. The NMDAR activation is sufficient to destroy most neurons. pathophysiology of several acute or chronic neurological In contrast, AMPAR-triggered, Ca2?-mediated excitotox- disorders has been linked to excitotoxicity [1, 47]. icity typically occurs more slowly, requiring hours of The build-up of glutamate results in overstimulation of sustained receptor activation to induce lethal injury in the AMPARs, KARs and NMDARs on neurons, with conse- same cell cultures [50]. The whole story of ischemia- quent influx of Na? and Ca2? ions through the channels triggered glutamate release and excitotoxicity can be gated by these receptors. Water passively follows the ion summarized in Fig. 2.

Ischemia

Energy failure Ion pump failure Reduction of transporter s’ function and number

Reversal of Na+ Accumulation of intracellular Ca2+ exchanger ions i.e. Na+ Activation of Na+/ H+ Reduced glutamate clearance exchanger Membrane depolarization

Excessive release of glutamate

Glutamate accumulation

Excessive glutamatergic receptor activation

Metabotropic receptors I

Phospholipase C ATP NMDAR Increase in AMPAR/ KAR activation depletion and activation IP3 activation energy failure

Rise in intracellular Ca2+ Activation. of Membrane. Rise in + Activation of VDCC depolarization intracellular Na cPLA2 and cyclooxygenase Rise in Activation of mitochondrial mtNOS Activation nNOS Ca2+

Excess entry of water Mitochondrial Formation dysfunction of peroxy- Release of NO Generation of nitrite superoxide and toxic prostanoids Oxidative stress Release of Calapain Formation of Cytotoxic oedema cytochrome c activation nitrotyrosine adducts and DNA damage

Degradation of Activation of cytoskeletal and caspase-3 extracellular matrix Release of AIF proteins Neuronal cell death

DNA fragmentation

Fig. 2 Summary of role of glutamate during neuronal ischemia (mtNOS mitochondrial NOS, cPLA2 cytosolic phospholipase 2, IP3 inositol triphosphate, AIF apoptosis-inducing factor) 123 Neurol Sci (2012) 33:223–237 227

The role of elevated intracellular Ca21 bisphosphate. TRPM7 also contributes to increase in intracellular sodium content during ischemia [67]. The rise in intracellular Ca2? seems to play a major role in the pathological events following excitotoxicity [51]. NMDAR activation is the major source of Ca2? intracel- Mitochondrial dysfunction lularly in normal and ischemic cells [52]. The rise in intracellular Ca2? occurs as a consequence of direct entry Mitochondrial dysfunction is a consequent of rise in through NMDARs and VDCCs, which are activated due to intracellular Ca2? [68]. The rise in intracellular Ca2? above membrane depolarization secondary to NMDAR activation certain threshold (*0.5 lM) will increase its uptake into [53]. Of the VDCCs, the L-type mediates long-lasting Ca2? mitochondria [69]. The mitochondrial Ca2? content currents in response to depolarization in excitable cells increases from 1 to 3 nmol/mg protein to 6–9 nmol/mg [54]. These inward Ca2? currents are considered to be an protein after 24 h of reperfusion [70]. This rise causes important mediator of excitotoxicity and neuronal damage devastating effects, including opening of the mitochondrial [55]. A recent research, surprisingly, showed that certain permeability transition pore, complete loss of respiration, types of L-type VDCC in retinal neurons are subjected to and release of both NADH and cytochrome c [71]. rapid glutamate-induced internalization, serving as a pro- Impairment of mitochondrial function and collapse of tective negative feedback mechanism [56]. mGluRs can mitochondrial membrane potential lead to failing mainte- also raise intracellular Ca2? via the inositol triphosphate- nance of anti-oxidant pathways and generation of ROS (see mediated release of Ca2? from the endoplasmic reticulum below), and inhibition of Ca2? rise intracellularly will [57]. inhibit this cascade [72]. Loss of mitochondrial NADH, Other mechanisms may be responsible for the rise in which is necessary for the numerous dehydrogenases intracellular Ca2? following ischemia. The increased present within the mitochondrial matrix, occurs as a result activity of Na?/H? exchanger triggered by cellular ische- of the opening of the permeability transition pore (PTP). mia, with subsequent accumulation of Na? intracellularly, The PTP is activated by abnormally high concentrations of will secondarily activate Na?/Ca2? exchanger (NCX), that Ca2?, oxidative stress [73] and low ATP, conditions that will work in a reverse manner, leading to increased Na? are present during and immediately following hypoxic expulsion in exchange with Ca2? [58]. This reversal mode ischemia [74]. Contribution of PTP opening to ischemic of NCX seems to be responsible for the early rise in and traumatic brain injury is supported by the neuropro- intracellular Ca2?. It should be stated that Na?/H? tection observed with PTP inhibitors, e.g., cyclosporins exchanger activation seems to be subsequent to glutamate [75], which bind to cyclophilin D, the one well-established stimulation (rather than being directly activated by ische- protein associated with pore opening. Cyclophilin D mia), as toxic glutamate doses causes cellular acidiﬁcation knockout mice are resistant to ischemic brain injury [76]. and H? overload, with increased Na?/H? exchange [59]. Respiratory inhibition stimulates lactate dehydrogenase This is supported by the recent evidences of the neuro- (LDH), resulting in lactic acidosis, which further inhibits protective effect of Na?/H? exchanger blockers against oxidative phosphorylation and promotes oxidative stress glutamate-induced neuronal damage [60]. Ca2? may be [77]. Mitochondrial dysfunction will result in release of also released from intracellular stores due to stimulation of cytochrome c through Bax or Bak megapores present in the ryanodine receptors [61]. outer membrane. The cytochrome c, once released, inter- The initial rise in Ca2? precedes a second wave, and so acts with deoxyadenosine triphosphate and binds to Apaf-1 the rise in the intracellular Ca2? occurs in two phases [62]. to activate caspase-9. The activated caspase-9 then acti- Cell death, however, does not depend on the initial Ca2? vates caspase-3 which leads to (assisted by the release of rise, but rather invariably follows the delayed massive apoptosis-inducing factor triggered by cytochrome c) accumulation of Ca2?, called delayed Ca2? deregulation apoptotic cell death [78] (see Fig. 2). Stabilization of (DCD), occurring a few hours after the toxic challenge, mitochondrial function is associated with neuroprotection causing a no-return transition into the death process [63]. [79]. Activation of transient receptor potential channels of the melastatin family (TRPM), especially TRPM2 and TRPM7, is implicated in the DCD. TRPM2 can be acti- The role of caspases and calpains vated by H2O2 [64], reactive oxygen species (ROS) [65] and intracellular Ca2? [66]. Blockade of TRPM7 channels The role of caspase-3 has been greatly supported in animal in vitro can prevent Ca2? inﬂux and decrease cell death studies. Caspase-3 knockout mice had smaller infarct following oxygen glucose deprivation. TRPM7 can be volumes than wild-type mice [80], and administration activated by ROS as well as phosphatidylinositol of caspase enzyme inhibitors would decrease infarction 123 228 Neurol Sci (2012) 33:223–237

size [81], while mice overexpressing human caspase-3 Rise in intracellular Ca2+ exhibited increased apoptosis and larger lesion volumes following cerebral ischemia [82]. In addition, caspase-3 activation, especially if combined with mitochondrial Calpain activation dysfunction, will lead to DNA fragmentation and neuronal apoptosis [83] (see Fig. 2). Pre-incubation with caspase-3- Upregulation of Bax Degrading Calpastatin (calpain specific inhibitor z-DEVD or pan-caspase inhibitor z-VAD activity endogenous inhibitor) prior to glutamate exposure resulted in a 50% reduction in cell death [84]. Caspase-3 activation In addition to mitochondrial dysfunction the elevated 2? Ca can activate degradative proteolytic enzymes, Fig. 4 Synergistic activity of calpain and caspase including the family of cytosolic proteases, calpains [85]. Calpain I (l-calpain) and II (m-calpain) are found while caspase-3 specific cleavage generates a 120 kDa throughout the brain and are activated by Ca2? at neutral SBDP [101]. pH. Inhibitors of NMDA-mediated Ca2? influx was associated with decreased calpains activity [86]. While calpains do play a role in normal cell function [87], over activation Production of ROS of these proteases has been implicated in both necrosis [88] and apoptosis [89]. They participate in excitotoxic cell Glutamate overactivity is associated with production of death by cleaving the NCX [90] and mGluRI [91]. The various ROS. ROS cause damage and oxidization of lipids, calpain-induced degradation of NCX, in addition to inter- DNA, and proteins. Accumulated Ca2? induces the acti- nalization of plasma membrane Ca2? ATPase is suggested vation of the neutral protease, calpain, which results in the to participate in DCD by causing critical rise in Ca2? [92] conversion of xanthine dehydrogenase into xanthine oxi- (see Fig. 3), although recent studies raised some doubt dase. Xanthine oxidase catalyzes the oxidation of xanthine about such assumption [93]. and hypoxanthine into uric acid, producing superoxide as a Calpains also truncate a number of anti-apoptotic pro- by-product [102]. Excess Ca2? in the mitochondria inter- teins, such as Bcl-2, Bcl-XL and Bid, which could account rupts the electron transport chain, therefore free electrons for the role of calpains in apoptosis. Activated calpains are accumulated in the mitochondria, which react with hydrolyze peptide bonds in cytoskeletal and structural oxygen that is supplied after reperfusion, and causes the proteins, membrane proteins and other regulatory and production of superoxide. The superoxide is further pro- signaling proteins [94]. Calpains have been observed to cessed to produce the hydroxyl radical by a Fenton reaction activate calcineurin through direct cleavage [95] and by or peroxynitrite by reacting with nitric oxide. The ROS degrading its endogenous inhibitor Cain/Cabin1 [96]. They also inhibit the electron transport chain in the mitochon- also upregulate proapoptotic Bax activity [97], which dria, and amplify a generation of mitochondrial free radi- eventually leads to downstream activation of caspase-3 cals [103]. [98]. While caspase-3 may cleave calpastatin, the endog- Zn2? is stored in the presynaptic vesicles of glutama- enous inhibitor of calpains [99] and so the two proteolytic tergic neurons, released with glutamate in an activity- enzymes may act synergistically [100] (see Fig. 4). dependent manner, and translocated into adjacent neurons Calpains and caspase-3 cleave the cytoskeletal protein [104]. The Zn2? translocation was observed in degenerat- a-spectrin at specific sites, and calpain-specific cleavage ing neurons after transient forebrain ischemia [105]. Zn2? generates a 145 kDa spectrin breakdown product (SBDP) ions enter into target cells through VGCC, NMDARs that are permeable to Ca2?,Na?/Ca2? exchanger, or Zn2? 2? Rise in intracellular Ca2+ transporter [106]. The entry and accumulation of Zn into neurons result in the transient generation of ROS, by activation of cyclooxygenases (COX) and PKC that Internalization of plasma Degradation of mediate latent neuronal death [107]. membrane Ca2+ ATPase NCX

Activation of arachidonic acid pathway Activation of Calpains

2? 2? Accumulated Ca in neurons results in the translocation Fig. 3 Relationship between Ca and calpains. Being activated by 2? high intracellular Ca2? level, calpains, in turn, will contribute to the of cPLA2 into the plasma membrane. And the Ca catastrophic rise in Ca2? overload through glutamate receptors inducing the 123 Neurol Sci (2012) 33:223–237 229

activation of cPLA2 will lead to production of neurotoxic NMDAR activation metabolites, such as prostaglandins, leukotrienes, reactive oxygen species, and platelet activating factors through the metabolism of arachidonic acid and lysophospholipids. The Rise in intracellular 2+ pharmacological inhibitors of cPLA2 reduce excitotoxic Ca and hypoxic-ischemic neuronal death [108]. Furthermore, the activation of NMDAR will cause an increased Activation of NOS expression and activation of COX-2 [109]. Increased COX-2 expression in ischemic brain was found to be due to excess glutamate [110]. Absence of COX-2 or its inhibition Increase in NO attenuates NMDA-mediated excitotoxicity and hypoxic- ischemic injury [111]. Increase in Neuronal peroxynitrite Death The role of NO Fig. 5 Closed circuit is present between peroxynitrite and intracellular Ca2? leading finally to cell death The role of NO, generated by NMDARs activation, in neuronal damage produced by glutamate has been established in the early 1990s. PSD-95 binds to the amino ter- minal of nNOS, a Ca2?-activated form of NOS, through its Inhibition of soluble guanylate cyclase and cGMP produc- PDZ domain. Therefore, PSD-95 may concentrate nNOS tion prevents glutamate and NO neurotoxicity [122]. near the NMDARs at postsynaptic sites in neurons [112]. Primary brain cultures treated with NOS inhibitors or cultures from mice with targeted disruption of nNOS are The role of AMPAR and KAR resistant to NMDA neurotoxicity [113]. NOS activity is enhanced by the elevation of intracel- Although most of the excitotoxic glutamate-induced dam- lular Ca2? [114], by a Ca2?/calmodulin-dependent mech- age is due to NMDAR activation, the AMPA/kainate anism [115]. Mitochondrial NOS is also activated by rise in pathway is not innocent. Activation of this pathway will intramitochondrial Ca2? [116]. Activation of mGluR1 was lead to the rise in the intracellular Na content, with sec- 2? found to be associated with increased NO level [117]. ondary rise in Ca concentration due to reverse operation ? 2? NO exacerbates brain damage by reducing neuronal of the Na /Ca exchanger [123]. These events will join energy production by inhibiting glycolytic and mitochon- the downstream cascade as evoked by NMDA receptor drial enzymes, by increasing DNA damage, and through activation. Glutamate acts on AMPARs to produce mem- the conversion of superoxide to peroxynitrite, being a brane depolarization and sufficient membrane depolariza- 2? highly reactive molecule, increasing the levels of toxic free tion removes the Mg block of NMDA channels, allowing 2? radicals [94]. Increased peroxynitrite production has been the influx of extracellular Ca into the cell [124]. shown to increase cytosolic Ca2?, contributing to cell death There are evidences suggesting the formation of ROS [118] (see Fig. 5). NO together with the generation of ROS after AMPAR activation [125]. The sole rise in the intra- ? (secondary to increased Ca2? and mitochondrial dysfunc- cellular Na after AMPAR stimulation may cause neuronal tion) such as hydroxyl radicals, superoxide anions can damage due to cellular swelling [126]. cause lipid peroxidation and membrane damage [119]. Stimulation of KRs by kainic acid (KA), a cyclic analog Despite the mentioned harmful effects of NO, low NO of glutamic acid, caused excitotoxic neurodegeneration was found neuroprotective. Low concentration of NO accompanied by the expression of various genes, including played a protective role in glutamate neurotoxicity by immediate-early genes (IEG) and stress protein genes closing the NMDA receptor-gated ion channels, suggesting [127]. the dual effect of NO [120]. KA-induced neurodegeneration was not prevented either Another mediator in NO and glutamate neurotoxicity is by MK-801 or CNQX, blockers of NMDAR and AMPA/ cGMP. The rise in intracellular (not extracellular) cGMP, KA receptors, respectively. It indicated that CNQX-resis- being stimulated by NO, plays a role in the mechanisms of tant KARs might be involved in the observed neurode- glutamate, NMDA and kainate neurotoxicity in cultured generation. Accordingly, more specific inhibitors of KARs cerebral cortical neurons [121]. There is a good correlation might be effective in preventing KA-induced neuronal between inhibition of cGMP formation and neuroprotection. [128].

123 230 Neurol Sci (2012) 33:223–237

Glutamate and preconditioning glutamate is induced by NMDAR-gated Ca2? influx [124]. Ischemia induces rapid upregulation of CREB-targeted It is known that transient, sub-lethal or non-catastrophic genes [135]. CREB phosphorylation and subsequent gene brain ischemia can induce a protected state against sub- expression may play an important role in the acquisition of sequent episodes of ischemia (ischemic tolerance); this is ischemic tolerance [136]. Cpg15 is an immediate-early called ischemic preconditioning [129]. Preconditioning is gene induced by Ca2? influx through NMDAR and L-type associated with changes in the glutamatergic system, VDCC and is regulated by multiple transcription factors mainly increase in glutamate release through the non-Ca2?- including CREB [137]. CPG15 expression is upregulated dependent mechanisms [130] associated with increased after transient global ischemia protects cortical neurons expression and activity of glutamate transporters [131] (see from apoptosis by preventing activation of caspase path- Fig. 6). Although the malfunction of glutamate transporters ways [138]. appears to be important in glutamate accumulation during ischemic injury, the increased expression of glutamate expression has been shown to be important in acquisition Drugs inhibiting glutamate-induced cell injury of ischemic tolerance by removing glutamate from the extracellular space and maintaining the glutamate below Despite the extensive and continued research the mecha- neurotoxic level in the brain. The EAAT2 inhibitor, nisms of excitotoxicity, there are currently no pharmaco- dihydrokainate, prevents preconditioning in a dose-depen- logical interventions capable of providing significant dent manner [132]. The role of ciliary neurotrophic factor neuroprotection in the clinical setting of brain ischemia or in neuronal protection may be due to enhancement of injury. As shown in this review, glutamate induces neu- glutamate transporters’ activity [133]. ronal cell damage through multiple and complex pathways, The involvement of NMDAR in ischemic tolerance has which makes the interruption of these pathways rather been suggested, but not fully explained. The different roles difficult. Therapeutic targeting of a single pathway may not of NMDAR subtypes might be largely dependent on the provide satisfactory neuroprotection, due to the presence of activation of their downstream signaling pathways. NR2B alternative neurotoxic pathways by which glutamate can subunit of NMDAR may be particularly important in bypass the inhibited one. This suggests that combination neuronal ischemic preconditioning [134]. Phosphorylation therapies may have better outcomes than monotherapy of CREB in neurons after ischemia or exposure to [139]. Due to the unsatisfactory results reported by glutamate receptor blockers as neuroprotectants against ischemia, efforts have been made to attach glutamate during Transient periods of earlier stage (prereceptor level) by inhibition of synthesis ischemia or release. Although less specific, interruption of signaling pathways following glutamate receptor interaction (postreceptor level) were also suggested, e.g., by antioxidants Release of Glutamate (see Fig. 7). Neuroprotective agents against glutamate- induced excitotoxicity are summarized in Table 2. As early as 1980s, glutamate receptor antagonists had been found to attenuate the ischemic damage observed NMDAR activation Upregulation of in animal models. Many problems still face its clinical glutamate transporters Inhibitors of Glutamate synthesis Pre receptor binding Enhancement of Glutamate clearance Phosphorylation of level CBEB Inhibitors of Glutamate release Enhancement reuptake of glutamate during further periods of ischemia Receptor binding Glutamate receptor blockers level Cpg15

Inhibition of signaling pathways Post receptor binding Protection against level apoptosis Antioxidants

Fig. 7 Mechanism and stages of action of neuroprotectants against Fig. 6 Possible role of glutamate in preconditioning glutamate-induced neurotoxicity 123 Neurol Sci (2012) 33:223–237 231

Table 2 Examples of neuroprotective agents against glutamate-induced neurotoxicity Neuroprotective agents Reference

Inhibitors of glutamate formation 6-Diazo-5-oxo-L-norleucine (DON) Decreased glutamate release from activated microglia [140] Methionine sulfoximine Inhibited the synthesis of glutamate in vivo in mice [141] Phenylsuccinate Inhibited cytosolic glutamate formation from glutamine [142] Inhibitors of glutamate release Carbenoxolone Gap junction blocker which decrease glutamate microglial release [140] Dextromethorphan Inhibited glutamate release by suppression of presynaptic VDCC [143] Enhancement of glutamate clearance Ceftriaxone Increased the glial GLT expression enhancing glutamate uptake [144] Maslinic acid Promoted glutamate clearance by NF-jB-mediated EAAT 2 upregulation [145] Rosiglitazone Increased the GLT-1 expression by acting as PPAR agonist [146] NMDAR blockers ACEA 1021 Reduced the infarction size [147] Bis(7)tacrine Showed neuroprotective effect against retinal ischemic damage [148] Cerestat Had a good preclinical profile, but low clinical efficacy [149] Dextromethorphan It reduced infarction size but with a variety of dose-related reversible adverse events [150] Dizocilpine Reduced the infarction volume, but most effective given early after ischemia with [151] neuropsychiatric problems Gavestinel Generally better tolerated without neuropsychiatric adverse effects but it had little effects [152] of on infarct volume in patients with ischemic stroke Ketamine and thiopental Showed neuroprotective effects against NMDA-induced neurotoxicity [153] Magnesium Beside NMDAR blocking, it inhibited excitatory neurotransmitter release, blocked Ca2? [154] channels, dilated the cerebral vasculature Memantine Improved neurochemical and neurobehavioral functions after ischemia [155] Dizocilpine Reduced the infarction size [156] Remacemide Reduced the infarction size [157] Traxoprodil Modest neuroprotective activity in humans with stroke [158] AMPAR blockers NBQX Had neuroprotective capacities but was associated with nephrotoxicity [159] PNQX Showed neuroprotective effects against oxygen–glucose deprivation [160] KAR blockers LY37770 Selective GluK1 antagonist which is neuroprotective under various states [161] Modulation of mGluRs ACPD Activated the mGluR protecting against NMDA receptor activation [162] CHPG Acted as mGluR5 agonist reducing the infarction volume [163] LY379268 Group II mGluR agonist, stimulating astrocytes to produce neuroprotective factors and [164] reducing glutamate release PPG A novel group III mGluR agonist which is neuroprotective against hypoxic injury [165] YM-202074 mGluR1 antagonist which decreased infarction size [166] Inhibitors of Na?/H? exchanger KR-33028 The novel Na?/H? exchanger inhibitor inhibit intracellular Ca2? overload [60] Inhibition of postreceptor signaling mechanisms Lubeluzole It inhibited glutamate-induced activation of NOS reducing infarction volume in [167] experimental animals PSD-95 inhibitors, e.g., N-alkylated Reduced infarction size even after ischemic insult and improve neurobehavioral changes [168] tetrapeptides Inhibitors of Ca2? influx 2-Cyclopropylimino-3-methyl-1,3- Decreased Ca2? influx, mitochondrial dysfunction, overproduction of NO and oxidative [169] thiazoline hydrochloride stress in cultured glial cells Lacidipine Reduced infarction size before or after ischemic injury [170]

123 232 Neurol Sci (2012) 33:223–237

Table 2 continued Neuroprotective agents Reference

Nimodipine Reduced Ca2? inﬂux, neuroprotective effect in kainate, glutamate, and NMDA-induced [171] neurotoxicity NGP1-01 Inhibited major neuronal calcium channels; L- and N-types. It also inhibited NMDA- [172] induced Ca2? inﬂux by 52% ACPD (1-aminocyclopentyl-1,3-dicarboylic acid), CHPG 2-chloro-5-hydroxyphenylglycine, KR-33028 4-cyano (benzobthiophene-2- carbonyl)guanidine, NGP1-01 8-benzylamino-8,11-oxapentacycloundecane, LY379268 2-oxa-4-aminobicyclohexane-4,6-dicarboxylate, PPG 4-phosphonophenylglycine, YM-202074 N-cyclohexyl-6-{[(2-methoxyethyl)(methyl)amino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2- carboxamide

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