Dennis W. Choi

Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage

Abstract An influx of extracellular Ca 2+ , with subsequent cellular Ca 2+ overload, can clearly cause certain types of cell death, and has been hypothesized to be a primary etiological event in hypoxic-ischemic neuronal injury. Recently, this hypothesis has acquired new specificity, as hypoxic-ischemic neuronal injury has been linked to the excessive activation of postsynaptic glutamate receptors, and glutamate neurotoxicity itself has been linked to a lethal influx of extracellular Ca 2+ through cell membrane channels. Available data suggest that channels gated by the N-methyl-d-aspartate (NMDA) subclass of glutamate receptors may be the predominant route of glutamate- or hypoxia-induced lethal Ca 2+ entry; however, other routes, including L- and N-type voltage-gated Ca 2+ channels, the Na + /Ca 2+ exchanger, and non-specific membrane leak, may also participate. Current efforts to develop an effective therapy for hypoxic-ischemic neuronal injury are appropriately focused on NMDA antagonists; however, it is possible that additional benefit might be gained by combining NMDA antagonists with pharmacological manipulations designed to attenuate Ca 2+ entry through these other routes.

Ionic dependence of glutamate neurotoxicity

The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation (“excitotoxicity” hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly “excitotoxic.” The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage- dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium- free solution, supports the notion that the activation of voltage- dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.

Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay.

Measurement of lactate dehydrogenase (LDH) activity released to the extracellular bathing media has been found to be a simple yet quantitative method for assessing glutamate mediated central neuronal cell injury in cortical cell culture. Extracellular LDH is both chemically and biologically stable; the magnitude of LDH efflux in the cultures correlates in a linear fashion with the number of neurons damaged by glutamate exposure.

The changing landscape of ischaemic brain injury mechanisms.

Thrombolysis has become established as an acute treatment for human stroke. But despite multiple clinical trials, neuroprotective strategies have yet to be proved effective in humans. Here we discuss intrinsic tissue mechanisms of ischaemic brain injury, and present a perspective that broadening of therapeutic targeting beyond excitotoxicity and neuronal calcium overload will be desirable for developing the most effective neuroprotective therapies.

The role of zinc in selective neuronal death after transient global cerebral ischemia

Zinc is present in presynaptic nerve terminals throughout the mammalian central nervous system and likely serves as an endogenous signaling substance. However, excessive exposure to extracellular zinc can damage central neurons. After transient forebrain ischemia in rats, chelatable zinc accumulated specifically in degenerating neurons in the hippocampal hilus and CA1, as well as in the cerebral cortex, thalamus, striatum, and amygdala. This accumulation preceded neurodegeneration, which could be prevented by the intraventricular injection of a zinc chelating agent. The toxic influx of zinc may be a key mechanism underlying selective neuronal death after transient global ischemic insults.

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures. Addition of 1- 3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate. As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate- induced neuronal cell loss markedly. Lower concentrations of APV or D- APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity. This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the “late” addition of 10–1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage. If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations. Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino- 7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L- glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity. The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists. Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.

Very delayed infarction after mild focal cerebral ischemia : a role for apoptosis ?

The temporal evolution of cerebral infarction was examined in rats subjected to transient occlusion of both common carotid arteries and the right middle cerebral artery. After severe (90-min) ischemia, substantial right-sided cortical infarction was evident within 6 h and fully developed after 1 day. After mild (30-min) ischemia, no cortical infarction was present after 1 day. However, infarction developed after 3 days; by 2 weeks, infarction volume was as large as that induced by 90-min ischemia. These data suggest that infarction after mild focal ischemia can develop in a surprisingly delayed fashion. Some evidence of neuronal apoptosis was present after severe ischemia, but only to a limited degree. However, 3 days after mild ischemia, neurons bordering the maturing infarction exhibited prominent TUNEL staining, and DNA prepared from the periinfarct area of ischemic cortex showed internucleosomal fragmentation. Furthermore, pretreatment with 1 mg/kg cycloheximide markedly reduced infarction volume 2 weeks after mild ischemia. These data raise the possibility that apoptosis, dependent on active protein synthesis, contributes to the delayed infarction observed in rats subjected to mild transient focal cerebral ischemia.

Ischemia-induced neuronal apoptosis

Hypoxic-ischemic neuronal death has long been considered to represent necrosis, but it now appears that many brain neurons undergo apoptosis after either global or focal ischemic insults. This event is probably substantially distinct from ischemia-triggered excitotoxicity, which tends to produce necrosis. While many questions remain unanswered, the concept of ischemic apoptosis has raised exciting prospects of combining anti-apoptotic with anti-excitotoxic treatments to achieve heightened therapeutic benefits in the brains of patients traumatized by cardiac arrest or stroke.

Chapter 6 Glutamate receptors and the induction of excitotoxic neuronal death

Publisher Summary The participation of glutamate receptors in excitotoxic induction is discussed in this chapter with an emphasis on some recent developments with regard to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and metabotropic receptors. Ca 2+ influx triggered by glutamate receptor activation is a critical mediator of excitotoxic injury. The presence of AMPA/kainate receptors gating Ca 2+ -permeable channels should confer enhanced vulnerability to death induced by AMPA or kainate. Specifically, neurons bearing substantial numbers of such atypical AMPA/ kainate receptors should be destroyed by exposures to AMPA or kainate too brief to destroy neurons lacking these receptors. To test this idea, the chapter utilizes kainate-activated Co 2+ uptake as a histochemical marker for cells bearing Ca 2+ -permeable AMPA receptors. Another feature of AMPA/kainate receptor behavior that may influence participation in excitotoxicity is desensitization.

Brain tissue responses to ischemia

The brain is particularly vulnerable to ischemia. Complete interruption of blood flow to the brain for only 5 minutes triggers the death of vulnerable neurons in several brain regions, whereas 20–40 minutes of ischemia is required to kill cardiac myocytes or kidney cells. In part, the prominent vulnerability of brain tissue to ischemic damage reflects its high metabolic rate. Although the human brain represents only about 2.5% of body weight, it accounts for 25% of basal metabolism, a metabolic rate 3.5 times higher even than that of the brains of other primate species. In addition, central neurons have a near-exclusive dependence on glucose as an energy substrate, and brain stores of glucose or glycogen are limited. However, over the last 15 years, evidence has emerged indicating that energetics considerations and energy substrate limitations are not solely responsible for the brain’s heightened vulnerability to ischemia. Rather, it appears that the brain’s intrinsic cell-cell and intracellular signaling mechanisms, normally responsible for information processing, become harmful under ischemic conditions, hastening energy failure and enhancing the final pathways underlying ischemic cell death in all tissues, including free radical production, activation of catabolic enzymes, membrane failure, apoptosis, and inflammation. Since these common pathways are explored in other accompanying JCI Perspectives, we will emphasize the role of injury-enhancing signaling mechanisms specific to the central nervous system (CNS) and discuss potential therapeutic approaches to interrupting these mechanisms. Refinement of glutamate receptor antagonist approaches. A major limitation in past clinical trials of glutamate receptor antagonists has been dose ceilings imposed by drug side effects. Not unexpectedly, interfering with the brain’s major excitatory transmitter system can lead to alterations in motor or cognitive function (prominent with NMDA antagonists), or sedation (prominent with AMPA antagonists). It seems plausible that the therapeutic index of NMDA antagonist therapy might be improved by the utilization of subtype-selective agents, such as ifenprodil, an antagonist selective for the NR2B subtype of NMDA receptors. NR2B receptors are preferentially expressed in forebrain relative to hindbrain, so blocking these receptors may produce greater neuroprotection in forebrain with less interference with motor function than subtype-unselective NMDA antagonists. In addition, ifenprodil inhibition of NR2B receptors increases with increasing agonist stimulation, a “use dependency” that might increase drug effect at overactivated synapses relative to normal synapses (46). The neuroprotective efficacy of NMDA antagonist therapy might also be enhanced by combination with AMPA or kainate receptor antagonists, both to increase overall antiexcitotoxic efficacy on ischemic neurons, as well as specifically to extend protection to GABAergic neurons expressing Ca2+-permeable AMPA receptors, and oligodendrocytes. Indeed, failure to rescue GABAergic neurons while successfully rescuing nearby excitatory neurons might lead to an increase in local circuit excitation and seizure activity in stroke survivors. High-level pan-blockade of both NMDA and AMPA receptors could have problematic side effects, for example, respiratory depression, but these difficulties might be surmountable through the use of subtype-selective drugs. An alternative approach to blocking NMDA and AMPA receptors concurrently might be to reduce glutamate release, for example, through hypothermia or reduction of circuit excitability with GABA agonists or blockers of voltage-gated Na+ channels. Zinc-directed therapies. While current putative antiexcitotoxic therapies have focused on glutamate receptor activation and resultant Ca2+ overload, the pathological role of neuronal Zn2+ overload suggests additional targets for therapeutic intervention. Indeed, variable reduction of toxic Zn2+ influx may underlie some of the inconsistent beneficial effects of voltage-gated Ca2+-channel antagonists observed in animal models of transient global ischemia (47). Further delineation of the precise routes responsible for toxic Zn2+ may permit greater reduction in this toxic Zn2+ overload. Another possible approach would be to reduce Zn2+ release from nerve terminals. In settings where ischemia is anticipated, it may even prove possible to accomplish this via acute dietary zinc reduction, as anecdotal evidence in humans has suggested that such reduction profoundly disturbs brain function, likely due to reduction of transmitter Zn2+ release (48). Further off, one can envision strategies for modifying neuronal Zn2+ transporters to improve the extrusion or sequestration of intracellular Zn2+, or for upregulating intracellular Zn2+-binding proteins such as metallothioneins. Combination therapies. Recent implication of apoptosis in the pathogenesis of ischemic neuronal death raises an unsettling possibility that current efforts to block NMDA receptor-mediated Ca2+ influx may go too far, achieving the desired reduction of toxic calcium overload and excitotoxicity in some neurons, but then promoting apoptosis in other neurons through Ca2+ starvation (4). It is plausible that different neurons might sustain different levels of [Ca2+]i at different times, with neurons further from the ischemic core or at later time points after ischemia onset sustaining less calcium influx than counterparts in the acute ischemic core. These neurons may be damaged badly enough to trigger apoptosis, but their [Ca2+]i levels may fall below the “set point” optimal for promoting survival (49), such that broad and sustained NMDA receptor blockade promotes apoptosis, reducing the benefits to be had by attenuating calcium overload in other neurons. If this scenario proves valid, it may be possible to enhance the benefits and reduce the dangers of NMDA antagonists by concurrently administering antiapoptotic treatments. Dual inhibition of excitotoxic necrosis and ischemic apoptosis has shown promise in two experimental studies to date. Coadministration of the NMDA antagonist dextrophan with cycloheximide produced greater than 80% reduction in infarct volume following transient focal ischemia in rats, better than either agent alone (50); and Ma et al. (51) observed neuroprotective synergy between MK-801 and the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.FMK) on both infarct size and therapeutic window. The combination of antiexcitotoxic strategies with thrombolysis has also been shown to provide additive protection in a rodent model of embolic stroke (52). On theoretical grounds, antioxidant drugs might be especially valuable in reducing reperfusion-induced injury, for example in association with thrombolytic therapy, or the deleterious component of certain growth factor actions.