Beyond NMDA and AMPA glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke

Review

Beyond NMDA and AMPA glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke Elaine Besancon1, Shuzhen Guo1,2, Josephine Lok1,3, Michael Tymianski4 and Eng H. Lo1,2 1

Neuroprotection Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA Departments of Radiology and Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA 3 Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA 4 Toronto Western Research Institute and Department of Neurosurgery, University of Toronto, Ontario M5T 2S8, Canada 2

The glutamate receptor was one of the most intensely investigated targets for neuroprotection. However, numerous clinical trials of glutamate receptor antagonists for the treatment of stroke were unsuccessful. These failures have led to pessimism in the field. But recent advances could provide hope for the future. This minireview looks beyond the traditional mechanism of glutamate-receptor-driven excitotoxicity to identify other mechanisms of ionic imbalance. These advances include findings implicating sodium–calcium exchangers, hemichannels, volume-regulated anion channels, acid-sensing channels, transient receptor potential channels, nonselective cation channels and signaling cascades that mediate crosstalk between redundant pathways of cell death. Further in vivo validation of these pathways could ultimately lead us to new therapeutic targets for stroke, trauma and neurodegeneration. Introduction The glutamate receptor is one of the most intensely investigated targets for neuroprotection. The standard excitotoxic model comprises a series of pathways that link energetic stress to neuronal cell death. Although excitotoxicity has been proposed to underlie a wide range of central nervous system (CNS) disorders ranging from acute brain injury to chronic neurodegeneration, the largest amount of data is to be found in investigations of stroke. Loss of cerebral blood flow rapidly triggers energy deficits and neuronal depolarization that release large amounts of glutamate into extracellular space. Overactivation of the NMDA (N-methyl-D-aspartic acid) and AMPA propionic (DL-a-amino-3-hydroxy-5-methyl-4-isoxazole acid) receptors then lead to a generalized ionic imbalance within neurons, especially of calcium. The calcium overload is then thought to induce a wide range of cell death executioners including ATPases, proteases, lipases and DNAses (Figure 1). But whereas blockade of glutamate receptors showed dramatic neuroprotective effects in the lab, clinical trials aimed at reducing ischemic brain injury by targeting NMDA and AMPA glutamate receptors were disappointing [1,2]. Treatment of ischemic stroke remains Corresponding authors: Tymianski, M. ([email protected]); Lo, E.H. ([email protected]).

268

limited to the use of tissue-type plasminogen activator to dissolve clots in approximately 3%–4% of all patients. More than two decades of neuroprotection research has not yielded any clinical therapies. The translation of promising mechanistic data into true clinical therapies for stroke and other CNS disorders is extremely challenging, and there are many complex reasons that might partially explain some of our failures to date. A rigorous dissection of this issue lies outside the scope of the present analysis, and the reader is referred to other in-depth papers that attempt to address this difficult question [2–7]. The purpose of the present paper is to survey several newly emerging mechanisms of ionic imbalance that might mediate neuronal death beyond the proximal glutamate receptor per se. Some limitations of the NMDA–AMPA model It is increasingly clear that the standard model of excitotoxicity based solely on NMDA and AMPA receptors will not suffice. During acute phases after stroke, overactivation of these glutamate signals might indeed be neurotoxic to select subsets of neurons. However, some aspects of these glutamergic signals also could be beneficial as the injured brain attempts to recruit endogenous recovery mechanisms [8]. For example, it is now known that activation of a given neuronal NMDA receptor can result in either cell survival or cell death depending on whether the receptor is synaptic or extrasynaptic [9,10]. The activation of synaptic NMDA receptors promotes cell survival by activation of the CaM kinase and Ras–ERK1/2 (extracellular signal-regulated kinase) pathways and subsequent expression of BDNF (brain-derived neurotrophic factor) [9]. In contrast, the activation of extrasynaptic NMDA receptors inactivates the CREB pathway and downregulates BDNF [9]. Beyond the synapse different mechanisms also might operate in other parts of the neuron. In the axon, the release of glutamate and subsequent AMPA activation is initiated by a large Na+ influx and the reverse of Na+-dependent glutamate transporters [11–13]. Under some experimental conditions, imbalances in sodium might even be more important than calcium in axonal compartments. In neuronal dendrites, overactivation of NMDA receptors are damaging. However, activation of

0165-6147/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2008.02.003 Available online 1 April 2008

Review

Trends in Pharmacological Sciences

Vol.29 No.5

cell bodies are mediated by AMPA/kainate receptors [25]. Additionally, glia express many different subtypes of mGlu receptors that exhibit a variety of modulatory functions [23]. Because of all these complexities in glial glutamate regulation and signaling, it might perhaps be difficult to design a single NMDA or AMPA antagonist that would protect all neurons and glia after stroke. In the final analysis the standard NMDA–AMPA model of excitotoxicity is perhaps oversimplistic and does not take into account the complex interactions with other parallel routes of ionic entry and imbalance within the injured cell. In this minireview, we will survey six emerging mechanisms of ionic imbalance that deserve further attention as we continue to search for neuroprotectants against stroke and brain injury: sodium-calcium exchangers (NCX), hemichannels, volume-regulated anion channels (VRACs), acidsensing ion channels (ASICs), transient receptor potential channels (TRPs) and nonselective cation channels (Figure 2). Figure 1. Standard NMDA and AMPA glutamate receptor model of neuronal cell death. This so-called standard model emphasizes the proximal role of the NMDA and AMPA receptor subtypes that become activated by elevated levels of extracellular glutamate. Dysregulated currents into these two channels lead to a generalized calcium imbalance within the neuron. Subsequently, these calcium abnormalities induce an upregulation of a wide range of death executioners, including ATPases that serve to further deplete energy stores, lipases that damage lipid membranes of organelles and the cell surface itself, proteases that dismantle the cytoarchitecture of the neuron, and DNAses that damage the nucleus.

kainate-type receptors, which are closely related to the AMPA receptors, might actually promote growth and remodeling [14,15]. Ultimately, of course, glutamergic signaling is not only mediated by NMDA and AMPA currents. Many other glutamate receptors and transporters exist in brain. Careful targeting of other neuronal glutamate receptors and transporters, including five metabotropic glutamate (mGlu) receptor subtypes and the EAAT2 transporter (excitatory amino acid transporter 2), also could prove fruitful, based on similar variations in their capacity to trigger death or survival [16–18]. And beyond the glutamate-associated channels, many other voltage-gated calcium channels also can carry large currents in damaged neurons (for a detailed review, see Ref. [19]). Altogether, it is important to realize that NMDA–AMPA pathways only comprise a subset of the multiple routes of ionic imbalance that are induced in brain injury and neurodegeneration. Another potential limitation of the standard NMDA– AMPA model is the focus on neurons alone. Astrocytes and oligodendrocytes play crucial roles in glutamate regulation, and their roles also must be considered. For example, glutamate uptake by astrocytes via GLAST and GLT-1 (rodent form of the EAAT2 transporter) transporters normally keep extracellular glutamate below toxic levels. If these mechanisms become impaired by ischemia, neuronal excitotoxicity can be amplified [20,21]. As both astrocytes and oligodendrocytes express NMDA and AMPA/kainate receptors, they also are vulnerable to high levels of glutamate [20–24]. But as seen in neurons, the processes of oligodendrocytes and astrocytes might respond differently compared to the cell body. It has been proposed that NMDA receptors comprising NR2C and NR3A subunits mediate injury in the glial processes, whereas damage to the glial

Sodium-calcium exchangers NCX is a transmembrane protein that is widely expressed in the heart and brain. Using the energy of the Na+ gradient generated by the Na+/K+ ATPase, NCX catalyzes the extrusion of one intracellular Ca2+ and the influx of three extracellular Na+ in each reaction cycle. NCX can function in the forward and reverse direction, and its activity is regulated by many factors including Na+, Ca2+, intracellular pH, and ATP. Studies suggest that NCX plays a role in glial and neuronal damage induced by hypoxiareoxygenation, glucose deprivation and excitotoxicity, although controversy remains whether net NCX activity is beneficial or detrimental. For example, NCX blockade worsens infarction volumes in rodent models of focal cerebral ischemia [26]. But there are also conflicting reports in which NCX inhibitors reduced infarct volume in animal stroke models [27]. Some of these variations in outcome could be due to the differential NCX responses in ischemic brain. In areas of mild injury where Na+/K+ ATPase is preserved, NCX operates in a forward mode, so inhibition of NCX reduces calcium extrusion and ends up enhancing calciummediated cell injury. In contrast, in areas of more severe ischemia where ATP levels are low and Na+/K+ ATPase activity is reduced, intracellular sodium loading causes NCX to operate in the reverse mode as a calcium-influx pathway. Under these conditions NCX inhibition could be protective. Recent studies are now beginning to define the spatial and temporal profiles of expression in different NCX subtypes after focal cerebral ischemia [28]. Whether targeting these differential responses leads to net benefit or brain injury remain to be fully elucidated. Hemichannels Gap junctions are ubiquitously found in neurons, astrocytes, oligodendrocytes and microglia [29]. A gap junction is made by the joining of two hemichannels. Each hemichannel typically consists of a hexamer made of members of the transmembrane protein family known as the connexins [29]. Ischemic conditions result in an increased opening of hemichannels (unopposed half-gap junctions) 269

Review

Trends in Pharmacological Sciences Vol.29 No.5

Figure 2. Non-glutamate receptor pathways of neuronal ionic imbalance. The traditional model of excitotoxicity emphasized events at the NMDA and AMPA receptor channels. Emerging data now suggest that these proximal loci might not be optimal targets for neuroprotection. Emerging novel routes of ionic imbalance include NCXs, hemichannels, VRACs, ASICs, TRPs and other nonselective cation channels. Furthermore, intracellular signals from perturbed mitochondria and endoplasmic reticulum (ER) compartments also might contribute to overall neuronal dysfunction. Significant interactions and feedback exists between these multiple mechanisms so that targeting a single step in the cascade might not be sufficient.

to the extracellular environment but might simultaneously result in decreased cell–cell communication via gap junctions [30]. Hemichannel function can be influenced by many events known to be triggered after stroke, including elevation in reactive oxygen species (ROS), alterations in phosyphorylation pathways and changes in redox state that can modulate the nitrosylation state of connexins [29,31]. Although the research done on hemichannels thus far indicates that they are certainly involved in the response to ischemia in the brain, the specific role they play is still controversial. Two modes of action have been proposed: the ‘Good Samaritan effect’ or the ‘bystander effect’ [32]. The ‘Good Samaritan effect’ postulates that after cerebral ischemia, astrocytes are able to remove cytotoxic substances from extracellular space via hemichannels, thereby rescuing neighboring cells that otherwise would have died. In support of this view, knockout mice lacking connexin 43 (Cx43) expression in astrocytes have increased infarct volume and apoptosis after experimental stroke [33]. Consistent with a neuroprotective role for connexins, mice lacking Cx32 have markedly increased neuronal death in the hippocampus after transient global brain ischemia [34]. Cx43-positive astrocytes have been observed recently in human stroke brain samples, further supporting the notion that these endogenous responses could play important roles in neuronal injury [35]. In contrast, the ‘bystander effect’ postulates that under some conditions gap junctions exacerbate ischemic damage by allowing the dumping of internal cell contents and the spread of cytotoxic substances to cells that otherwise would have been 270

unaffected [32,36]. In theory, abnormal gap-junction opening can allow the influx of calcium and sodium, as well as the efflux of metabolites and potentially excitotoxic glutamate between adjacent cells [29]. Thus, application of gapjunction blockers decreased cell death after hypoxic insults in culture [37,38]. In vivo knockdown of Cx43 as well as simultaneous knockdown of Cx32 and Cx36 with antisense oligonucleotides led to decreased neurotoxicity in rodent models of cerebral ischemia [37]. In models of transient global brain ischemia, pyramidal neurons in the CA1 sector of the hippocampus are particularly vulnerable. It has been shown that astrocytes in this hippocampal area express higher levels of Cx43 than cells that are more resistant [39]. These data would suggest that unlike focal ischemia, excitotoxic neuronal death after global ischemia might be augmented by Cx43 gap junctions that facilitate the spreading of abnormal neuron–astrocyte signaling and ionic imbalance. These various findings suggest that gap junctions serve both neuroprotective and neurotoxic roles after ischemia, and that under different conditions the balance between these two roles could shift. Whether net neuroprotection or tissue damage occurs probably depends on cell type, the persistence of junctional coupling and which specific connexins are expressed. More recently, it has been shown that besides connexins, another whole family of proteins called pannexins also might significantly contribute to hemichannel physiology in mammalian nervous systems [40]. In the context of neuronal death, a recent study demonstrated that one particular pannexin, pannexin 1 (Px1), might significantly contribute to neuronal vulnerability after cerebral ischemia

Review [41]. Hippocampal neuronal cultures subjected to oxygen–glucose deprivation showed massive activation of Px1-hemichannel currents that tended to be far larger than those mediated via NMDA currents. Opening of these Px1 hemichannels also appeared to allow a loss of internal ATP stores that would trigger anoxic depolarization and rapid and necrotic cell death. Further dissection and validation of these hemichannel pathways are warranted before we can truly assess these new therapeutic targets for stroke and brain injury. Volume-regulated anion channels VRACs are thought to be involved in astrocytic release of excitatory amino acids and ATP after astrocytic swelling [42–44]. Once VRACs are abnormally activated, many subsequent pathologic mechanisms also might be triggered, including additional fluid loading via aquaporin channels; inhibition of NCX due to ATP depletion; pH-dependent activation of the sodium, chloride and bicarbonate exchangers; pH lowering due to dysregulation of glutamate metabolism and ammonia buildup; and buildup of potentially toxic arachidonic acid levels [42]. The VRAC inhibitor tamoxifen has been shown to be protective when administered within 3 hr of stroke in a rat model, supporting the hypothesis that astrocytic swelling and subsequent VRAC activation contribute to neurotoxicity [43]. The multiplicity of downstream effectors following VRAC activation would suggest that these channels might be parsimonious targets for neuroprotection. Besides VRACs per se, another group of volume-activated channels–known as maxianion channels–also recently have been characterized in cultured astrocytes [45]. These channels appear to release glutamate under conditions of swelling even more efficiently than VRACs. A reasonable therapeutic strategy would be to simultaneously inhibit both VRACs as well as maxianion channels. At least in a cell-culture system, this combination approach strongly suppresses astrocytic glutamate efflux after chemical ischemia [45]. Undoubtedly, many feedback loops exist between astrocytic dysfunction and the broad spectrum of anion channels that are activated in response [44]. But, given how rapidly and consistently astrocytes swell after stroke, these events will surely comprise important targets for future dissection and analysis. Acid-sensing channels Ischemia is accompanied by acidosis that worsens neurotoxicity [46–48]. Until recently, the mechanism of this neuronal injury was unknown. ASICs now have been characterized in the brain and are believed to be involved in acidosis-induced injury. ASICs are ligand-gated multimeric channels that belong to the epithelial sodium channel superfamily. ASICs can be activated by low pH, membrane stretching, lactate, arachidonic acid and decreased extracellular calcium [46,49,50]. Because all of these conditions can be caused by ischemia, this makes ASICs a potential therapeutic target for the inhibition of ischemic neuronal death. Although a main function of ASICs is probably to gate amiloride-sensitive Na+ currents, some channels, notably the homomeric 1a channel, enable an influx of Ca2+ during

Trends in Pharmacological Sciences

Vol.29 No.5

acidosis [51]. During acidosis, some Ca2+ influx occurs even in the presence of combinations of a variety of glutamate and calcium-channel blockers. This Ca2+ influx can be blocked by the addition of ASIC inhibitors, indicating that the ASIC-mediated neurotoxicity is perpetuated at least partially via glutamate-independent mechanisms [52]. In vitro experiments have shown that oxygen–glucose deprivation increases both the amplitude of ASIC currents and ASIC desensitization, thereby increasing time of Ca2+ influx [51,52]. Knockout mice for ASIC1 do not experience acidosis-related injury, infarct volume is decreased in mice after middle cerebral artery occlusion when ASIC blockers are given, and cultured cortical neurons experience less neurotoxicity when exposed to low pHs in the presence of ASIC inhibitors [52]. ASICs also might be activated indirectly by glutamate. NMDA activation has been shown to enhance ASIC activation by upregulating CaMKII [53]. This feedback loop between ASICs and proximal NMDA receptors could make this approach especially important in terms of cutting off rate-limiting steps in the neuronal injury cascade. Most importantly, recent findings now suggest that the therapeutic time window for interrupting ASIC currents might be particularly prolonged, with treatments still showing benefit up to 5 hr after ischemic onset in rodent models [54]. Whether these temporal profiles translate into clinical meaning remains to be determined. Transient receptor potential channels TRP channels broadly comprise six subfamilies termed TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin) and TRPA (ankyrin). The TRPM subset of nonspecific cation channels might be especially important in neuronal death after ischemia. TRPM2 and TRPM7 have been implicated in the delayed calcium deregulation that follows the initial calcium spike during excitotoxic conditions [55,56]. In cellculture preparations, TRPM2 can be activated by arachidonic acid, ROS and nitric oxide [55,57–60]. In vivo, TRPM2 mRNA is upregulated in rats after cerebral artery occlusion [61]. TRPM2 also has been detected in human microglia and can be upregulated by interleukin1-b, leading some to speculate that TRPM2 contributes to the microglial response during ischemia [61]. TRPM7 also can be activated by many mediators known to be involved in stroke, including free radicals and second messengers such as phosphatidylinositol (4,5)-bisphosphate (PIP2) [55]. In vitro studies have shown that blockade of TRPM7 channels can prevent calcium influx and decrease cell death after oxygen–glucose deprivation [55,57]. Suppression of TRPM7 by RNAi (RNA interference) methods blocked calcium influx and neuronal death after oxygen–glucose deprivation [62] and also reduced the generation of ROS and reactive nitrogen species (RNS) [55]. The precise underlying mechanisms of TRPM-mediated neurotoxicity remain to be fully dissected. TRPM channels are permeable to calcium, so calcium overload is one possible mechanism [55]. But TRPM7 also is permeable to other ions, such as zinc, so accumulation and overall imbalance of other ions might also be involved [55]. Furthermore, potential interactions between different 271

Review TRP channel subfamilies might also participate in the neuronal response to injury. A recent study suggested that TRPC channels are neuroprotective, by sustaining BDNF levels in cerebellar neurons [63]. How these various TRP channels can be selectively targeted for stroke and neurodegeneration warrants further investigation, especially in in vivo model systems. Other cation channels Besides ASICs and TRP channels, another nonselective cation channel that might be a potential neuroprotective target is the NCCa–ATP channel [64], which appears to be linked to the formation of cerebral edema. The NCCa–ATP channel is a nonselective channel for monovalent cations in neurons and astrocytes. Its pore-forming subunits are not yet fully characterized. However, a regulatory subunit consists of the sulfonylurea receptor 1 (SUR1) that is opened by ATP depletion and accumulation of nanomolar concentrations of intracellular calcium [65]. Because these two events (energy depletion and calcium overload) are central to neuronal death, this class of channels should play crucial roles in the pathophysiology of stroke and brain injury. The discovery of the NCCa–ATP channel and its regulation of SUR1 could provide a new therapeutic approach to stroke by targeting the cerebral edema due to ischemia. SUR1 is upregulated in neurons, astrocytes and capillary endothelial cells in the core and in the peri infarct area after ischemia [66]. This transcriptional upregulation appears to result, at least in part, from the activation of transcription factor Sp1 [66]. In a rodent model of stoke, blockade of this channel with the SUR1 inhibitor glibenclamide reduced cytotoxic edema, infarct volume and mortality by 50%. In a rodent model of spinal cord injury, glibenclamide ameliorated the development of hemorrhagic necrosis [67]. Interestingly, glibendamide is a drug that has been used extensively in humans for treatment of type 2 diabetes. A recent study suggested that these sulfonylurea compounds might in fact improve stroke outcomes in type 2 diabetes patients [68]. Besides perturbations in calcium and sodium, there is a large literature implicating a wide range of potassium channels as well. A full review here is not possible, but two examples deserve mention. At the plasma membrane, a class of channels termed ‘maxi-K’ or ‘BK’ channels could play crucial roles in neuronal injury. Agents that open these maxi-K channels were neuroprotective in neuronal cultures as well as rodent models of focal stroke [69]. At the mitochondrial surface activation of another class of channels called ‘mito-K’ channels prevented oxyradical generation and the release of proapoptotic cytochrome c and effectively reduced infarction in rat cerebral ischemia [70]. Ultimately, an emerging consensus is that all calcium, sodium and potassium currents are intimately linked and multiple methods to ameliorate ionic imbalance after neuronal injury will be required to prevent death and salvage function. Signaling and crosstalk In retrospect, earlier attempts at targeting the NMDA and AMPA channels for neuroprotection were perhaps doomed 272

Trends in Pharmacological Sciences Vol.29 No.5

by the assumption that these were single, free-standing targets. Excitotoxicity and ionic imbalance comprise a central framework for understanding brain cell injury in stroke, trauma and neurodegeneration. But even as new mechanisms are revealed that go beyond traditional models of glutamate receptor overactivation, it is also increasingly recognized that there are multiple opportunities for crosstalk between excitotoxicity, oxidative stress and apoptosis [71]. Redundant and interacting signals of cell death are likely to emerge, such that targeting a single point in a single pathway might not yield maximal neuroprotection. For example, TRPM channel currents are known to be amplified by free radicals. As activated TRPM channels allow the influx of calcium leading to an increase in oxygen radical production, a positive feedback loop will be induced [55]. Oxidative injury also can damage reuptake transporters [72], thus further increasing glutamate in extracellular space and leading to more excitotoxicity (Figure 3a). During initial phases of neuronal injury, NCX activity could help normalize ionic perturbations. But as intracellular calcium accumulates, the family of calcium-dependent neutral cysteine proteases, or calpains, becomes activated [73,74]. A positive feedback loop is formed wherein calcium activates calpains that then cleave NCX, resulting in a further dysregulation in intracellular calcium and further cell damage [75] (Figure 3b). Calpains themselves are involved in many pro-death pathways including ROS damage of mitochondria, activation of Bad and subsequent release of cytochrome c, and activation of caspases [73,75]. In turn, calpain activity is regulated by the caspases via cleavage and inactivation of the calpain inhibitor calpastatin [75,76]. Recently, it has been proposed that some NCX subtypes can be cleaved by caspases as well, thus setting up a highly complex interplay between calcium, calpain and caspases [77]. Caspasemediated DNA damage might overactivate the repair enzyme poly(ADP-ribose) polymerase (PARP), which can further deplete energy stores and thus lead to neuronal depolarization and even more glutamate release (Figure 3c). In the final analysis, the ionic mechanisms discussed here might only represent proximal triggers, after which a multifactorial cascade of intracellular signals and proteases become activated that leads to cell death. Some of these signals will include intracellular kinases such as p38, JNK, ERK and Akt; a detailed discussion of these pathways is beyond the scope of this minireview. But it is obvious that the entire network of intracellular signals should be a potential treasure trove of new targets. The downside is that the multiplicity of connections, both good and bad, within these signaling networks could end up making it difficult to develop purely protective targets. Nevertheless, the complexity of downstream prolife and prodeath signaling will have to be carefully considered as we attempt to target the various ionic channels and discussed here. Caveats and conclusions Much has been learned about additional mechanisms of cell death in the central nervous system beyond the overactivation of NMDA and AMPA glutamate receptors. However,

Review

Trends in Pharmacological Sciences

Vol.29 No.5

Figure 3. Interactions and feedback loops between multiple mechanisms of ionic imbalance. (a) Crosstalk between TRPM-mediated excitotoxicity and oxidative stress. TRPM-mediated calcium currents induce the generation of ROS and RNS, which in turn further activate the TRPM channels. (b) Crosstalk between NCX, calpain and caspases. Excitotoxic glutamate signals lead to elevations in calcium levels that activate calpains and caspases, which in turn degrade NCX function and induce more calcium imbalance. (c) Crosstalk between glutamate, apoptosis and energetic stress. Excitotoxicity might induce apoptotic-like pathways that damage DNA. Overactivation of the endogenous repair enzyme PARP leads to further energetic stress and secondary perturbations in glutamate release-reuptake.

with this additional knowledge comes the challenge of determining the interplay between these many mechanisms, as well as interactions between excitotoxic ionic imbalance and other forms of brain cell death such as apoptosis, necroptosis or autophagy. Key points for intervention will have to be defined that are situated at points of crosstalk or convergence between multiple mechanisms. In addition, future research must include an appreciation of the entire neurovascular unit comprising neuroglial and neurovascular interactions rather than focusing on the neuron alone [78,79]. Our previous failures with NMDA and AMPA might be related to limited time windows for treatment, side effects of some of these receptor antagonists, inefficient drug delivery through the blood–brain barrier and, perhaps, even crucial

differences between humans and experimental animal models. The present minireview has surveyed emerging new targets comprising NCX hemichannels, VRACs, ASICs, TRPs and other nonselective cation channels. The accumulating data are promising but some are only derived from cell-culture systems, so in vivo validation of these hypotheses will have to be rigorously pursued. Will the targets be clinically accessible? Will side effects overwhelm efficacy? Will both gray and white matter be protected? It also will be essential to determine how these new approaches might influence both cell death as well as repair and recovery over time [80]. Translating any experimental target into a clinically effective pharmacology is extremely challenging. But with an integrative approach that addresses multiple mechanisms in multiple cell types, we could yet reveal new 273

Review therapies for stroke and neurodegeneration in the coming years. Acknowledgements Supported in part by a Bugher award from the American Heart Association and National Institutes of Health grants R01-NS37074, R01-NS48422, R01-NS53560, R01-NS56458, P50-NS10828 and P01NS55104.

References 1 Lee, J.M. et al. (1999) The changing landscape of ischaemic brain injury mechanisms. Nature 399, A7–A14 2 Wahlgren, N.G. and Ahmed, N. (2004) Neuroprotection in cerebral ischaemia: facts and fancies––the need for new approaches. Cerebrovasc. Dis. 17 (Suppl. 1), 153–166 3 Cheng, Y.D. et al. (2004) Neuroprotection for ischemic stroke: two decades of success and failure. NeuroRx 1, 36–45 4 Dirnagl, U. (2006) Bench to bedside: the quest for quality in experimental stroke research. J. Cereb. Blood Flow Metab. 26, 1465–1478 5 Gladstone, D.J. et al. (2002) Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. Stroke 33, 2123–2136 6 Hsu, C.Y. et al. (2000) The therapeutic time window–theoretical and practical considerations. J. Stroke Cerebrovasc. Dis. 9, 24–31 7 O’Collins, V.E. et al. (2006) 1,026 experimental treatments in acute stroke. Ann. Neurol. 59, 467–477 8 Ikonomidou, C. and Turski, L. (2002) Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury? Lancet Neurol. 1, 383–386 9 Hardingham, G.E. and Bading, H. (2003) The yin and yang of NMDA receptor signalling. Trends Neurosci. 26, 81–89 10 Hetman, M. and Kharebava, G. (2006) Survival signaling pathways activated by NMDA receptors. Curr. Top. Med. Chem. 6, 787–799 11 LoPachin, R.M. and Lehning, E.J. (1997) Mechanism of calcium entry during axon injury and degeneration. Toxicol. Appl. Pharmacol. 143, 233–244 12 Li, S. et al. (1999) Novel injury mechanism in anoxia and trauma of spinal cord white matter: glutamate release via reverse Na+dependent glutamate transport. J. Neurosci. 19, RC16 13 Li, S. and Stys, P.K. (2000) Mechanisms of ionotropic glutamate receptor-mediated excitotoxicity in isolated spinal cord white matter. J. Neurosci. 20, 1190–1198 14 Monnerie, H. and Le Roux, P.D. (2006) Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro. J. Neurosci. Res. 83, 944–956 15 Monnerie, H. et al. (2003) Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: involvement of NMDA receptors. J. Neurosci. Res. 74, 688–700 16 Bruno, V. et al. (2001) Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J. Cereb. Blood Flow Metab. 21, 1013–1033 17 Rothstein, J.D. et al. (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433, 73–77 18 Rossi, D.J. et al. (2000) Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 403, 316–321 19 Gribkoff, V.K. and Winquist, R.J. (2005) Voltage-gated cation channel modulators for the treatment of stroke. Expert Opin. Investig. Drugs 14, 579–592 20 Anderson, C.M. and Swanson, R.A. (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32, 1–14 21 Matute, C. et al. (2002) Excitotoxicity in glial cells. Eur. J. Pharmacol. 447, 239–246 22 Karadottir, R. et al. (2005) NMDA receptors are expressed in oligodendrocytes and activated in ischaemia. Nature 438, 1162–1166 23 Deng, W. et al. (2004) Role of metabotropic glutamate receptors in oligodendrocyte excitotoxicity and oxidative stress. Proc. Natl. Acad. Sci. U. S. A. 101, 7751–7756 24 Krebs, C. et al. (2003) Functional NMDA receptor subtype 2B is expressed in astrocytes after ischemia in vivo and anoxia in vitro. J. Neurosci. 23, 3364–3372

274

Trends in Pharmacological Sciences Vol.29 No.5 25 Wong, R. (2006) NMDA receptors expressed in oligodendrocytes. Bioessays 28, 460–464 26 Pignataro, G. et al. (2004) Evidence for a protective role played by the Na+/Ca2+ exchanger in cerebral ischemia induced by middle cerebral artery occlusion in male rats. Neuropharmacology 46, 439–448 27 Matsuda, T. et al. (2001) SEA0400, a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models. J. Pharmacol. Exp. Ther. 298, 249– 256 28 Boscia, F. et al. (2006) Permanent focal brain ischemia induces isoformdependent changes in the pattern of Na+/Ca2+ exchanger gene expression in the ischemic core, periinfarct area, and intact brain regions. J. Cereb. Blood Flow Metab. 26, 502–517 29 Contreras, J.E. et al. (2004) Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res. Brain Res. Rev. 47, 290–303 30 Contreras, J.E. et al. (2002) Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proc. Natl. Acad. Sci. U. S. A. 99, 495–500 31 Retamal, M.A. et al. (2006) S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: induction by oxidant stress and reversal by reducing agents. Proc. Natl. Acad. Sci. U. S. A. 103, 4475–4480 32 Farahani, R. et al. (2005) Alterations in metabolism and gap junction expression may determine the role of astrocytes as ‘‘good samaritans’’ or executioners. Glia 50, 351–361 33 Nakase, T. et al. (2004) Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes. Am. J. Pathol. 164, 2067–2075 34 Oguro, K. et al. (2001) Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice. J. Neurosci. 21, 7534– 7542 35 Nakase, T. et al. (2006) Enhanced connexin 43 immunoreactivity in penumbral areas in the human brain following ischemia. Glia 54, 369– 375 36 Lin, J.H. et al. (1998) Gap-junction-mediated propagation and amplification of cell injury. Nat. Neurosci. 1, 494–500 37 Frantseva, M.V. et al. (2002) Ischemia-induced brain damage depends on specific gapjunctional coupling. J. Cereb. Blood Flow Metab. 22, 453–462 38 de Pina-Benabou, M.H. et al. (2005) Blockade of gap junctions in vivo provides neuroprotection after perinatal global ischemia. Stroke 36, 2232–2237 39 Rami, A. et al. (2001) Effective reduction of neuronal death by inhibiting gap junctional intercellular communication in a rodent model of global transient cerebral ischemia. Exp. Neurol. 170, 297–304 40 Barbe, M.T. et al. (2006) Cell-cell communication beyond connexins: the pannexin channels. Physiology (Bethesda) 21, 103–114 41 Thompson, R.J. et al. (2006) Ischemia opens neuronal gap junction hemichannels. Science 312, 924–927 42 Kimelberg, H.K. (2005) Astrocytic swelling in cerebral ischemia as a possible cause of injury and target for therapy. Glia 50, 389–397 43 Kimelberg, H.K. et al. (2003) Neuroprotective activity of tamoxifen in permanent focal ischemia. J. Neurosurg. 99, 138–142 44 Kimelberg, H.K. et al. (2006) Anion channels in astrocytes: biophysics, pharmacology, and function. Glia 54, 747–757 45 Liu, H.T. et al. (2006) Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress. Glia 54, 343–357 46 Allen, N.J. and Attwell, D. (2002) Modulation of ASIC channels in rat cerebellar Purkinje neurons by ischaemia-related signals. J. Physiol. 543, 521–529 47 Diarra, A. et al. (1999) Anoxia-evoked intracellular pH and Ca2+ concentration changes in cultured postnatal rat hippocampal neurons. Neuroscience 93, 1003–1016 48 Obrenovitch, T.P. et al. (1990) A rapid redistribution of hydrogen ions is associated with depolarization and repolarization subsequent to cerebral ischemia reperfusion. J Neurophysiol 64, 1125–1133 49 Immke, D.C. and McCleskey, E.W. (2001) Lactate enhances the acidsensing Na+ channel on ischemia-sensing neurons. Nat. Neurosci. 4, 869–870

Review

Trends in Pharmacological Sciences

50 Immke, D.C. and McCleskey, E.W. (2003) Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade. Neuron 37, 75–84 51 Yermolaieva, O. et al. (2004) Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a. Proc. Natl. Acad. Sci. U. S. A. 101, 6752–6757 52 Xiong, Z.G. et al. (2004) Neuroprotection in ischemia: blocking calciumpermeable acid-sensing ion channels. Cell 118, 687–698 53 Gao, J. et al. (2005) Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death. Neuron 48, 635– 646 54 Pignataro, G. et al. (2007) Prolonged activation of ASIC1a and the time window for neuroprotection in cerebral ischaemia. Brain 130, 151–158 55 Aarts, M.M. and Tymianski, M. (2005) TRPMs and neuronal cell death. Pflugers Arch. 451, 243–249 56 Chinopoulos, C. et al. (2004) Inhibition of glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical neurones. J. Neurochem. 91, 471–483 57 MacDonald, J.F. et al. (2006) Paradox of Ca2+ signaling, cell death and stroke. Trends Neurosci. 29, 58–75 58 Kaneko, S. et al. (2006) A critical role of TRPM2 in neuronal cell death by hydrogen peroxide. J. Pharmacol. Sci. 101, 66–76 59 Wehage, E. et al. (2002) Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose. J. Biol. Chem. 277, 23150–23156 60 Hara, Y. et al. (2002) LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell 9, 163–173 61 Fonfria, E. et al. (2006) TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia. J. Recept. Signal Transduct. Res. 26, 179–198 62 Aarts, M. et al. (2003) A key role for TRPM7 channels in anoxic neuronal death. Cell 115, 863–877 63 Jia, Y. et al. (2007) TRPC channels promote cerebellar granule neuron survival. Nat. Neurosci. 10, 559–567 64 Chen, M. and Simard, J.M. (2001) Cell swelling and a nonselective cation channel regulated by internal Ca2+ and ATP in native reactive astrocytes from adult rat brain. J. Neurosci. 21, 6512–6521 65 Chen, M. et al. (2003) Functional coupling between sulfonylurea receptor type 1 and a nonselective cation channel in reactive astrocytes from adult rat brain. J. Neurosci. 23, 8568–8577

Vol.29 No.5

66 Simard, J.M. et al. (2006) Newly expressed SUR1-regulated NC(CaATP) channel mediates cerebral edema after ischemic stroke. Nat. Med. 12, 433–440 67 Simard, J.M. et al. (2007) Endothelial sulfonylurea receptor 1regulated NC Ca-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury. J. Clin. Invest. 117, 2105–2113 68 Kunte, H. et al. (2007) Sulfonylureas improve outcome in patients with type 2 diabetes and acute ischemic stroke. Stroke 38, 2526–2530 69 Gribkoff, V.K. et al. (2001) Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels. Nat. Med. 7, 471–477 70 Liu, D. et al. (2002) Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release. J. Cereb. Blood Flow Metab. 22, 431–443 71 Lo, E.H. et al. (2003) Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 4, 399–415 72 Ouyang, Y.B. et al. (2007) Selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain ischemia. J. Neurosci. 27, 4253–4260 73 Chong, Z.Z. et al. (2005) Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease. Prog. Neurobiol. 75, 207–246 74 Ishihara, I. et al. (2000) Activation of calpain precedes morphological alterations during hydrogen peroxide-induced apoptosis in neuronally differentiated mouse embryonal carcinoma P19 cell line. Neurosci. Lett. 279, 97–100 75 Bano, D. et al. (2005) Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity. Cell 120, 275–285 76 Araujo, I.M. and Carvalho, C.M. (2005) Role of nitric oxide and calpain activation in neuronal death and survival. Curr. Drug Targets CNS Neurol. Disord. 4, 319–324 77 Bano, D. et al. (2007) The plasma membrane Na+/Ca2+ exchanger is cleaved by distinct protease families in neuronal cell death. Ann. N. Y. Acad. Sci. 1099, 451–455 78 Lo, E.H. (2008) Experimental models, neurovascular mechanisms and translational issues in stroke research. Br. J. Pharmacol. 153, S396– S405 79 Lok, J. et al. (2007) Cell-cell Signaling in the Neurovascular Unit. Neurochem. Res. 32, 2032–2045 80 Lo, E.H. A new penumbra: transitioning from injury into repair after stroke. Nat. Med. (in press)

Free journals for developing countries In 2002, the WHO and six medical journal publishers launched the Health InterNetwork Access to Research Initiative, which enabled nearly 70 of the world’s poorest countries to gain free or reduced-cost access to biomedical literature through the internet. Currently more than 70 publishers are participating in the program, providing access to over 2000 journals. Gro Harlem Brundtland, former director-general for the WHO, said that this initiative was ‘‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’’.

For more information, visit www.who.int/hinari 275