Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca2+([Ca2+]i) and mitochondrial membrane potential (ΔΨ) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca2+]i. Longer activation (50 min) renders elevation of [Ca2+]i irreversible (Ca2+ overload). Susceptibility to NMDA-induced Ca2+ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca2+]ihomeostasis during excitotoxic challenges. Remarkably, ΔΨ exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of ΔΨ and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.
Excitotoxicity, the process by which overactivation of excitatory neurotransmitter receptors leads to neuronal cell death, must be understood in terms of the intervening cascade of events (Choi, 1987, 1988, 1995). Compelling evidence supports a key role for massive Ca2+ influx through the NMDA receptor (NMDAR) channel as a trigger of glutamate neurotoxicity: (1) selective blockade of NMDARs markedly reduces the extent of excitotoxic damage (Choi et al., 1988; Michaels and Rothman, 1990); (2) neurons survive when challenged in absence of extracellular Ca2+ or presence of intracellular Ca2+([Ca2+]i) chelators (Choi, 1987; Tymianski et al., 1993a,b); and (3) glutamate receptor overstimulation induces elevations of [Ca2+]i that precede cell death (de Erausquin et al., 1990; Michaels and Rothman, 1990; Randall and Thayer, 1992; Tymianski et al., 1993a,b; Witt et al., 1994). Oxidative stress is thought to be a contributing factor in glutamate neurotoxicity through generation of oxygen radicals that disrupt cell functions (Dykens et al., 1987; Dawson et al., 1991; Coyle and Puttfarcken, 1993; Lafon-Cazal et al., 1993; Lipton et al., 1993). However, the link between elevation of [Ca2+]i, free radicals, and neuronal death remains unclear (Choi, 1995).
Mitochondria are unique among cell organelles in their involvement in the concerted consumption of oxygen, production of oxygen radicals, and mobilization of [Ca2+]i(Gunter and Pfeiffer, 1990; Thayer and Miller, 1990; Miller, 1991;Beal, 1992; Coyle and Puttfarcken, 1993; Rizzuto et al., 1993;Jouaville et al., 1995; White and Reynolds, 1995). Given that excessive Ca2+ accumulation in mitochondria uncouples electron transfer from ATP synthesis (Beatrice et al., 1980; Gunter and Pfeiffer, 1990; Bernardi et al., 1994), and considering that impairment of energy metabolism (Wang et al., 1994) increases generation of free radicals (Halliwell and Gutteridge, 1989; Coyle and Puttfarcken, 1993), mitochondria emerge as a plausible link between elevation of [Ca2+]i and glutamate neurotoxicity. The pivotal role of mitochondria in excitotoxicity has been highlighted recently by the deterministic influence of mitochondrial function in the decision between apoptotic or necrotic cell death (Ankarcrona et al., 1995; Bonfoco et al., 1995) and by the protective action of transforming growth factors presumably conferred by enhancing mitochondrial energetics (Mattson et al., 1993; Prehn et al., 1994).
Here, we focus on the following question: is the mitochondrion the sensor that converts elevation of [Ca2+]ifrom a physiological modulator into a trigger for cell death? We show that mitochondria are involved in Ca2+ sequestration during an excitotoxic insult (Schinder et al., 1995) and that this Ca2+ current induces sustained mitochondrial depolarization, which closely parallels the incidence of neuronal death. Our findings suggest that neuronal injury follows from excessive influx of Ca2+ via the NMDAR that, in turn, attenuates the mitochondrial membrane potential (ΔΨ) leading to the opening of the permeability transition pore (PTP), further collapsing ΔΨ. Blockade of the PTP with cyclosporin A (CsA) leads to the recovery of ΔΨ and promotes cell survival. The results outline a novel way of thinking about excitotoxic cell death, shifting the focus of attention from a global event staged by intracellular Ca2+ toward a more compartmentalized question centered on mitochondrial homeostasis.
MATERIALS AND METHODS
Hippocampal cultures. Mixed hippocampal neuronal/glial cultures were established as described (Schinder and Montal, 1993). Briefly, hippocampi from E17–E19 rat embryos were incubated in basic saline solution (BSS) containing (in mm): 137 NaCl, 3.5 KCl, 0.4 KH2PO4, 0.33 Na2HPO4 · 7 H2O, 5 trimethylaminoethanesulfonic acid, pH 7.4, and 10 glucose with 0.25% trypsin for 15 min at 37°C. Tissue was washed and dissociated by trituration with a Pasteur pipette. Cells were centrifuged for 5 min at 200 × g, and the pellet was resuspended in BSS. Cells were plated (105 cells/cm2) and maintained as described.
Cell death assays. In vitro excitotoxicity is commonly investigated using agonist exposures that range from 5 min to 24 hr (Choi, 1987; Weiss et al., 1994). In our conditions, mild and severe types of neuronal death were selectively achieved with exposures of 20 and 50 min, respectively. Culture medium was removed, neurons were rinsed with 10 ml of BSS + 1.1 mm CaCl2 + 10 μm glycine, and treated with 200 μm NMDA (dissolved in the same solution) for either 20 (de Erausquin et al., 1990; Michaels and Rothman, 1990; Lafon-Cazal et al., 1993; Witt et al., 1994) or 50 min (Tymianski et al., 1993a,b) at 23 ± 1°C. Treatments were terminated by addition of culture medium (Schinder and Montal, 1993) supplemented with 20 μm dizolcipine [MK-801] (Research Biochemicals, Natick, MA) to prevent activation of NMDAR attributable to residual NMDA or glutamate released from synapses (Hartley and Choi, 1989). Cultures were returned to the incubator and cell death was assessed 18–24 hr later by trypan blue staining (0.4%). When indicated, CsA (Sandoz, Basel, Switzerland) was dissolved in ethanol or dimethyl sulfoxide (DMSO; 42 mm) and added to solutions held at 37°C under vigorous stirring to a final concentration of 1.6 μm. To mimic those conditions used in imaging experiments, CsA was added 40 min before the NMDA challenge and removed after 2 hr. A minimum of 500 cells were counted blind on each culture dish. For each experiment, two identical culture dishes were treated for control and 20 and 50 min exposure to NMDA, or, in the CsA experiments, control and 20 min NMDA in absence and presence of CsA.
Calcium imaging. Neurons were Fluo-3-loaded for 75 min at a bath concentration of 5.5 μm Fluo-3 AM (Molecular Probes, Eugene, OR) dissolved in DMSO (Kao et al., 1989). Imaging was performed with an argon laser confocal system (Bio-Rad MRC600) equipped with 488 nm excitation/515 nm emission filters on a Zeiss microscope (Thornwood, NY) with a 20× water immersion objective (numerical aperture 0.4). Neurons were constantly superfused at a rate of 3 ml/min with BSS + 1.1 mm CaCl2 + 2 mm MgCl2 + 10 μm glycine (control solution). NMDA was applied in Mg2+-free solution. Images were sampled and collected at 8–15 sec intervals for 2 hr (up to 4 hr for 50 min NMDA experiments) and recorded on videotape. Slower sampling rates (1 image/2 min) and lower laser intensity (1/6 of standard power) yielded identical results, suggesting that phototoxic effects are minimal. Signals obtained during Ca2+ overload were not attributable to saturation of dye, as fluorescence increased further after addition of 5 μm ionomycin (Calbiochem, La Jolla, CA) in the presence of 20 mm CaCl2, pH 8.0. Digital images were acquired either on-line or off-line with a Macintosh computer equipped with a frame grabber (Data Translation) using Image 1.47 software (W. Rasband, National Institutes of Health). A total of 20–25 neurons was studied in each experiment; somatic regions of ∼170 pixels were chosen for quantitation of fluorescence intensity. Regions without neurons or processes were used for background subtraction. For calibration experiments (Kao et al., 1989; Vergara and DiFranco, 1992), [Ca2+]i was calculated as [Ca2+] = K d(F − F min)/(F max − F), where K d was assumed to be 400 nm, Fmax was measured at the end of the experiment by treating neurons with 5 μm ionomycin,F min was derived fromF max, and background fluorescence was obtained after addition of 0.02% saponin.
Imaging of ΔΨ. Neurons were incubated for 30 min in the presence of 0.1 μm tetramethylrhodamine ethylester (TMRE; Molecular Probes). TMRE was added to all solutions used during these experiments, which typically lasted 2 hr. When indicated, 1.6 μm CsA was present in all solutions throughout the experiment, with a preincubation time of 40 min before the NMDA challenge. Three to seven neurons were studied in each experiment. Imaging (Farkas et al., 1989) was as described above, except that samples were collected at 1 image/90 sec and a 514/550 nm filter and a 40× water immersion objective (numerical aperture 0.9) were used. The confocal pinhole was closed to 1/5 to 1/3, and signals were amplified 1.5× with a digital zoom. Fluorescence intensity was quantified in a perinuclear ring (∼1000 pixels) in which mitochondrial clusters are located (see Fig. 4 B). Baseline fluorescence (F 0) was measured as average normalized fluorescence emitted during the initial 5 min of the experiment. Control experiments showed that selective depolarization of the plasma membrane (35 mm KCl, 0 Ca2+) evoked minor changes in mitochondrial fluorescence. Results indicate that decrements of mitochondrial fluorescence primarily reflect mitochondrial depolarization and not reequilibration of dye in response to NMDA-induced plasma membrane depolarization. This assertion was confirmed by the finding that 100 μm carbonyl cyanidem-chlorophenylhydrazone, uncoupler of oxidative phosphorylation, decreases TMRE fluorescence (Farkas et al., 1989) to an extent comparable to that produced by the NMDA treatments.
All experiments reported were performed at 23 ± 1°C on neurons cultured for 14–17 d.
Statistics. Pooled data are presented as mean ± SEM. For comparisons between two groups, a two-tailed Student’st test was used. When three or more groups were compared (Figs. 1 C, 4 C), a single-factor ANOVA, followed by a post hoc Bonferroni t test with correction for multiple comparisons, was used.
NMDAR overstimulation induces neuronal cell death
To assess the effects of NMDAR overstimulation on mitochondrial Ca2+ sequestration and cell death, experimental paradigms that elicit either moderate or severe extents of neuronal death were developed. Neurons were exposed to 200 μm NMDA for variable time intervals, and cell death was determined 18–24 hr later using the trypan blue exclusion assay. Neuronal death in hippocampal cultures increased with the duration of the NMDA challenge: 39 and 67% cell death were elicited by NMDA pulses of 20 and 50 min (Table1), respectively. This finding is consistent with the notion that cellular damage increases with the duration of Ca2+ influx through the NMDAR (Tymianski et al., 1993a).
NMDA produces transient or sustained increases in [Ca2+]i depending on pulse duration
To evaluate the time course of change in [Ca2+]i evoked by NMDAR overstimulation, time-lapse fluorescent imaging was performed on hippocampal neurons loaded with the Ca2+ indicator Fluo-3 AM. As shown in Figure 1 A, 20 min application of NMDA evokes a fast rise in [Ca2+]i to a peak that decays slowly to an intermediate level before rising to a higher plateau. Calcium returns to baseline levels after withdrawal of the stimulus (recovery). However, a 50 min pulse of NMDA induces Ca2+ overload (Fig.1 B), as indicated by the persistence of Ca2+ elevation. The extent of Ca2+ overload increases with duration of the NMDA challenge (Fig. 1 C). Comparison of the incidence of cell death with that of Ca2+overload produced by these treatments, especially at 20 min (see Table1), reveals an apparent disparity: the incidence of cell death is significantly higher than that of Ca2+ overload. This indicates that recovery of [Ca2+]i to baseline levels is not necessarily indicative of cell survival and suggests that additional steps intervene between elevation of [Ca2+]i and cell death.
The susceptibility to NMDA-induced Ca2+ overload is augmented by mitochondrial function inhibitors
To test whether mitochondria lie on the main pathway to excitotoxicity, we studied the effects of specific inhibitors of mitochondrial function on cell death induced by 20 min stimulation with NMDA. Antimycin A and rotenone, specific inhibitors of complex III and complex I of the electron transport chain, were used to block electron transfer and proton extrusion mechanisms, thereby decreasing ΔΨ (see Fig. 3) and ATP synthesis. Antimycin A (10 nm) increased the susceptibility of hippocampal neurons to NMDA-induced cell death, confirming results obtained with cyanide, the specific inhibitor of complex IV (Dubinsky and Rothman, 1991). We next examined its effects on elevation of [Ca2+]i in response to 20 min pulses of NMDA. Antimycin A at 1 nmabolishes the early decrease in [Ca2+]i, hereafter defined as Ca2+ sequestration, yet recovery to baseline is achieved after the stimulus is removed (Fig.2 A). At 100 nm, Ca2+ sequestration is abolished and, moreover, [Ca2+]i remains tonically elevated. Antimycin A inhibits Ca2+ sequestration (•) and recovery (Δ) in a dose-dependent manner, with IC50 = 470 ± 130 pm and IC50 = 15.9 ± 4.5 nm, respectively (Fig. 2 B). IC50 values are 30-fold apart, suggesting that Ca2+ sequestration and recovery are distinct mechanisms, both associated with mitochondria. The primary involvement of mitochondria was confirmed using rotenone (Fig. 2 C,D); pretreatment produces a similar biphasic profile, abolishing Ca2+ sequestration at even lower concentrations (IC50 = 48 ± 19 pm) and blocking recovery at somewhat higher doses (IC50 = 1.5 ± 0.7 μm). These results suggest that although mitochondrial Ca2+ uptake is responsible for Ca2+ sequestration, ATP-dependent plasma membrane transporters may account for recovery by Ca2+ extrusion (Thayer and Miller, 1990; Miller, 1991; White and Reynolds, 1995; Budd and Nicholls, 1996). This conjecture was addressed using oligomycin, which specifically inhibits mitochondrial ATP synthase without altering the redox-generated ΔΨ. In accordance with this prediction, neurons treated with oligomycin before stimulation fail to recover from NMDAR overactivation but show only minor changes in Ca2+sequestration (Fig. 2 E,F). By contrast, thapsigargin (100 nm ≤ thapsigargin ≤ 500 nm), a specific blocker of endoplasmic reticulum (ER) Ca2+ ATPases (Thastrup et al., 1990), does not affect Ca2+ sequestration or recovery, indicating that ER mechanisms are not major determinants of this process.
Effects of mitochondrial function inhibitors on ΔΨ
To examine the effects of mitochondrial function inhibitors on ΔΨ, the fluorescent potentiometric dye TMRE, known to partition specifically into polarized mitochondria, was used for imaging (Farkas et al., 1989). Neurons treated with antimycin A (Fig. 3,top panel) display a prominent decrease in fluorescence, reflecting mitochondrial depolarization (Farkas et al., 1989; Loew et al., 1994). As shown in the trace, a transient increase in relative fluorescence precedes the decay (see also Figs.4 B, 5 B); this may arise from unquenching of the dye before its loss from mitochondria (Budd and Nicholls, 1996). The extent of depolarization (D) was quantified as the ratio of the mitochondrial fluorescence 20 min after antimycin A application to fluorescence before the treatment (D = 0.632 ± 0.043; n = 23 neurons). Oligomycin does not alter ΔΨ (D = 1.008 ± 0.028; n = 19 neurons; bottom panel), however, subsequent application of antimycin A does elicit depolarization of ΔΨ. These results strongly suggest that differences between the effects of blockers of the electron transport chain (antimycin A and rotenone) and the ATP synthase (oligomycin) on Ca2+ sequestration (Fig. 2) are attributable to their distinct actions on ΔΨ.
NMDA collapses ΔΨ
The link between NMDAR overstimulation, Ca2+sequestration, and impairment of mitochondrial function was substantiated by measurements of ΔΨ during the excitotoxic challenge (Fig. 4). Neurons challenged for 20 min with NMDA display a prominent depolarization (Fig. 4 A) that exhibits a time scale coincident with the development of Ca2+ overload (Fig. 1 A). Only partial recovery is observed, suggesting an irreversible process in contrast to the reversibility of [Ca2+]i signals (Fig.1 A). Increasingly longer NMDA stimuli induce more conspicuous depolarizations of ΔΨ, with decreasing extents of recovery (Fig. 4 B,C). A 30 sec stimulus yields a shallow and reversible depolarization of ΔΨ (Fig. 4 C). However, a prominent depolarization is induced by both 20 and 50 min NMDA stimuli, establishing a linkage between NMDAR overactivation and mitochondrial impairment. Moreover, recovery of ΔΨ is only partial for 20 min stimuli, and negligible for 50 min stimuli, suggesting that permanent mitochondrial damage occurred. Heightened extracellular [Ca2+] ([Ca2+]ext) sharply decreases the extent of recovery induced by 20 min NMDA (Fig.4 D), strengthening the connection between NMDA-induced Ca2+ influx and collapse of ΔΨ.
Blockade of the mitochondrial PTP with cyclosporin A promotes recovery of the NMDA-induced collapse of ΔΨ and prevents neuronal death
Depolarization of mitochondrial inner membrane, together with an increase in matrix [Ca2+] may induce opening of the PTP, a mitochondrial channel that allows ionic diffusion and thereby collapses ΔΨ (Petronilli et al., 1993). The immunosuppressant drug CsA is a potent blocker of the PTP (Bernardi et al., 1994; Nicolli et al., 1996) and presumably may prevent the NMDA-induced collapse of ΔΨ. Figure 5 shows that in the presence of 1.6 μm CsA, depolarization of ΔΨ in response to 20 min NMDA is only transient. The rate of decay of ΔΨ appears to be slower in the presence of CsA than in its absence (Fig. 5 B,right panel), suggesting that opening of the PTP may accelerate mitochondrial depolarization. Remarkably, the recovery is practically complete (compare with Fig. 4 B,C), suggesting that persistent opening of the PTP may be the cause for tonic mitochondrial depolarization. To examine the involvement of the PTP in excitotoxic cell death, the survival of neurons challenged in the presence of CsA or its absence was determined. CsA protects neurons from NMDA-induced death by 64.8 ± 6.3% (Fig. 5 C). These findings implicate the PTP in the persistent mitochondrial depolarization in response to NMDAR overstimulation (Fig. 4) and establish a connection between mitochondrial dysfunction and excitotoxic neuronal death.
The results presented here show that (1) overstimulation of NMDARs deregulates homeostasis of [Ca2+]i and induces neuronal death depending on pulse duration; (2) control of [Ca2+]i during an excitotoxic treatment relies primarily on mitochondrial function; (3) NMDA-induced Ca2+ influx is sufficient to induce early and persistent mitochondrial depolarization; and (4) blockade of the mitochondrial PTP promotes recovery of ΔΨ and decreases the incidence of cell death. The complex interplay between elevation of [Ca2+]i and mitochondrial function provides a basis to formulate a model of excitotoxic neuronal death. In essence, this model proposes that cell survival relies on a delicate balance between mitochondrial function and [Ca2+]ihomeostasis. Massive Ca2+ influx induces an imbalance in mitochondrial homeostasis, leading to mitochondrial dysfunction, which in turn triggers neuronal cell death (Fig. 6). We turn now to discuss the experimental foundation of the model.
Time-lapse video imaging of ΔΨ shows that depolarization is a consequence of NMDAR overstimulation (Fig. 4); whereas mitochondrial depolarization evoked by 20 min NMDA does not revert to baseline levels (Fig. 4 B), elevation of [Ca2+]i does (Fig. 1, Table 1). These results have three major implications: (1) sustained mitochondrial depolarization is not attributable to tonically elevated [Ca2+]i; (2) mitochondrial homeostasis is not linked to [Ca2+]i after removal of the excitotoxic stimulus; and (3) persistent mitochondrial depolarization is an expression of mitochondrial dysfunction.
Findings on cell death, [Ca2+]i homeostasis, and ΔΨ are summarized in Table 1. Results from 20 min NMDA treatments reveal an apparent mismatch between the extent of Ca2+ overload and the percentage of dead neurons. This suggests that although existing ATP pools may be sufficient to drive Ca2+ extrusion systems and recover [Ca2+]i homeostasis (Fig.1 A,C), early neuronal damage occurs as revealed by the collapse of ΔΨ (Fig. 4) and the induction of cell death. In contrast, the extent of Ca2+ overload observed for 50 min treatments (Fig. 1 B) parallels the incidence of neuronal death, suggesting exhausted ATP pools. Ca2+overload therefore may be secondary to mitochondrial dysfunction, and not a causal event for cell death (Fig. 6). This conjecture is supported by the results presented in Figure 2, in which inhibition of mitochondrial function leads to Ca2+ overload; for instance, [Ca2+]i responses to 50 min NMDA are mimicked by 20 min NMDA in neurons pretreated with oligomycin. Moreover, in agreement with previous findings (Tymianski et al., 1993a), Ca2+ overload occurs much earlier than loss of plasma membrane integrity, as indicated by the ability of neurons to retain the Ca2+ indicator Fluo-3 in the cytoplasm for more than 4 hr. Thus, Ca2+ overload may be an indicator of cell death, but recovery is not an indicator of cell survival. This notion resolves discrepancies in correlations of [Ca2+]i postinsult and neuronal death (de Erausquin et al., 1990; Michaels and Rothman, 1990; Randall and Thayer, 1992; Witt et al., 1994).
Mitochondria are major buffering compartments for elevations of [Ca2+]i in response to brief exposures to glutamate in cortical neurons (White and Reynolds, 1995). Inhibition of mitochondrial function by picomolar concentrations of antimycin A or rotenone abolishes Ca2+ sequestration in response to prolonged NMDA treatments (Fig. 2), indicating that mitochondria act as a primary buffer of [Ca2+]i also under excessive insults. A unique function of mitochondria (as a main source of ATP) is highlighted by the finding that oligomycin or higher concentrations of antimycin A or rotenone eliminate the recovery phase, presumably by compromising ATP-dependent Ca2+ extrusion systems (Fig. 6). Thus, mitochondria, as intracellular Ca2+buffers, set a point of vulnerability whereby if the boundary between mitochondrial function and dysfunction is crossed, cell death ensues. As discussed below, this crossroad is probably determined by the PTP (Fig. 5).
Our population study suggests a correlation between mitochondrial dysfunction and cell death, but does not establish a causal link (Table1). If mitochondrial impairment were a primary event in the induction of excitotoxic cell death, then neuronal death might be prevented by treatments that protect mitochondrial homeostasis. As shown in Figure5, the NMDA-induced collapse of ΔΨ recovers completely in the presence of CsA. Accordingly, neuronal death is reduced by 65% under these conditions compared with neurons challenged in the absence of CsA (Fig. 5 C). NMDARs are not a target of CsA, because voltage-clamp recordings show that currents elicited by NMDA remain unchanged in the presence of CsA (data not shown). The fact that complete recovery of ΔΨ is accompanied by a substantial, yet partial, reduction of cell death might indicate that pathways that are parallel and/or downstream to mitochondria are also activated by NMDAR overstimulation (Fig. 6). Immunosuppressant drugs, such as CsA, display numerous intracellular binding sites (Kunz and Hall, 1993) and have been shown to reduce excitotoxic neuronal death in cortical neurons, presumably by inhibition of a pathway that activates nitric oxide synthase (NOS) (Dawson et al., 1993). However, because CsA is a potent blocker of the PTP (Petronilli et al., 1993; Bernardi et al., 1994;Nicolli et al., 1996) and because our results show a direct effect of CsA on mitochondrial ΔΨ (Fig. 5 A,B), it appears unlikely that preservation of mitochondrial function and promotion of cell survival are attributable to inhibition of NOS, yet this cannot be ruled out. Our findings suggest that persistent mitochondrial depolarization results from opening of the PTP [presumably as a consequence of combined mitochondrial depolarization and elevation of intramitochondrial Ca2+ (Bernardi et al., 1994)] and that maintenance of mitochondrial homeostasis can protect hippocampal neurons from excitotoxic cell death (Fig. 6).
In view of these findings, glutamate excitotoxicity can be visualized as a cascade of events that starts with overstimulation of NMDARs, which elicit massive Ca2+ influx and abnormally high [Ca2+]i (Fig. 6). Ca2+ is sequestered into the mitochondrial matrix, driven by the proton electrochemical gradient generated by the electron transport chain, depolarizing ΔΨ (Akerman, 1978; Gunter and Pfeiffer, 1990; Loew et al., 1994). This reduction in the electrochemical gradient decreases ATP synthesis at a time of great demand by the plasma membrane Ca2+ pump and, indirectly, by the Na+/Ca2+ exchanger (White and Reynolds, 1995). Concurrent buildup of intramitochondrial [Ca2+] and attenuation of ΔΨ induce opening of the mitochondrial inner membrane PTP, thereby allowing ionic diffusion and collapsing ΔΨ (Beatrice et al., 1980; Gunter and Pfeiffer, 1990; Bernardi et al., 1994; Hoek et al., 1995). The drop of cellular ATP levels, in concert with the generation of free O radicals consequent to high ATP consumption (Halliwell and Gutteridge, 1989; Coyle and Puttfarcken, 1993; Reynolds and Hastings, 1995), is a primary cause of cell death (Siesjö, 1992). The boundary between cell survival and cell death relies on a subtle balance of mitochondrial function/dysfunction, centered on ΔΨ. This model predicts that neuronal lifespan may be prolonged by specific inhibition of mitochondrial Ca2+uptake and that susceptibility to excitotoxic death may be influenced by the energetic state of neurons before and during the insult.
Our findings establish a direct coupling between excitotoxicity and shifts of redox potentials and membrane potentials of mitochondria inside living neurons. This evidence strengthens the involvement of mitochondria in the neurotoxicity associated with mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (Saporito et al., 1992), amyotrophic lateral sclerosis (Brown, 1995; Wong et al., 1995), apoptosis (Newmeyer et al., 1994; Vayssière et al., 1994; Zamzami et al., 1995), and age-related disorders (Luft, 1994; Brouillet et al., 1995). These findings may provide useful guidelines for the development of the next generation of drugs targeted against neurodegeneration and stroke.
This work was supported by grants from National Institutes of Health (GM-49711 to M.M., NS-15918 to N.C.S.) and the U.S. Army Medical Research and Material Command (DAMD 17-93-C-3100 to M.M.). We are indebted to P. Bernardi, F. H. Gage, Y. Mika, C. D. Patten, J. L. Vergara, G. Zlokarnik, and the members of the Montal lab for valuable discussions and comments. We thank Drs. D. Römer, E. Rissi, G. Engel, and H. Widmer from Sandoz (Basel, Switzerland) for kindly providing CsA.
Correspondence should be addressed to Mauricio Montal, Department of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0366.