Mitochondria and neuronal glutamate excitotoxicity · 2017-01-03 · At the termination of a...

Mitochondria and neuronal glutamate excitotoxicity

David G. Nicholls *, Samantha L. Budd 1

Neurosciences Institute, Department of Pharmacology and Neuroscience, University of Dundee, Dundee DD1 9SY, UK

Received 5 January 1998; accepted 17 February 1998

Abstract

The role of mitochondria in the control of glutamate excitotoxicity is investigated. The response of cultured cerebellargranule cells to continuous glutamate exposure is characterised by a transient elevation in cytoplasmic free calciumconcentration followed by decay to a plateau as NMDA receptors partially inactivate. After a variable latent period, asecondary, irreversible increase in calcium occurs (delayed calcium deregulation, DCD) which precedes and predictssubsequent cell death. DCD is not controlled by mitochondrial ATP synthesis since it is unchanged in the presence of theATP synthase inhibitor oligomycin in cells with active glycolysis. However, mitochondrial depolarisation (and henceinhibition of mitochondrial calcium accumulation) without parallel ATP depletion (oligomycin plus either rotenone orantimycin A) strongly protects the cells against DCD. Glutamate exposure is associated with an increase in the generation ofsuperoxide anion by the cells, but superoxide generation in the absence of mitochondrial calcium accumulation is notneurotoxic. While it is concluded that mitochondrial calcium accumulation plays a critical role in the induction of DCD wecan find no evidence for the involvement of the mitochondrial permeability transition. ß 1998 Elsevier Science B.V. Allrights reserved.

Keywords: Mitochondrion; Glutamate; NMDA; Excitotoxicity; Calcium; Neuron

1. Introduction

Even in neurones, where the ATP requirement forionic homeostasis is particularly high, the safety mar-gin between cellular ATP supply capacity and de-mand is considerable as long as glucose and oxygenare available in excess. However even a brief inter-ruption in ATP generation, as in transient globalischaemia, can initiate neurodegeneration, which de-pending on the severity of the insult can show ne-

crotic or apoptotic characteristics and can occur witha delay of a few minutes to 1^2 days. Furthermore, aless severe, but chronic, restriction in neuronal ATPgeneration capacity may underlie a range of neuro-degenerative disorders, including Alzheimer's, Hun-tington's and Parkinson's diseases, amyotrophic lat-eral sclerosis and AIDs-related dementia as well asencephalopathies associated with mitochondrial mu-tations (reviewed in [1]). The excitatory neurotrans-mitter glutamate plays a central role in neuronal celldeath associated with these neurodegenerative disor-ders, and the interaction between the NMDA-selec-tive glutamate receptor and the mitochondrion formsthe basis of this review, which will focus on necroticcell death in neuronal culture.

The sequence of events culminating in excitotoxic

0005-2728 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 2 8 ( 9 8 ) 0 0 1 2 3 - 6

* Corresponding author. Fax: +44 (1382) 667120;E-mail : [email protected]

1 Present address: CNS Research Institute, LMRC FirstFloor, 221 Longwood Avenue, Brigham and Women's Hospital,Boston, MA 02115, USA.

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cell death is initiated by the entry of Ca2� throughpredominantly NMDA-selective glutamate receptorsactivated by elevated extracellular glutamate. Gluta-mate is not excitotoxic to anoxic cells as long asNMDA receptors are inactivated prior to the resto-ration of oxygen [2]. The two conditions of greatestrisk are ¢rstly the period when circulation is restoredfollowing transient global ischaemia and before thehighly active glutamate transporters can re-accumu-late the amino acid into neurones and glia, and sec-ondly in the penumbra surrounding a focal ischaemiawhere respiring neurones are exposed to high gluta-mate concentrations di¡using from the ischaemiccore. This latter region is of particular signi¢cance,since it demonstrates that glutamate exposure candestroy the bioenergetic integrity of neurones contin-uously supplied with glucose and oxygen.

It is convenient to divide the excitotoxic processinto two stages: the initial exposure to glutamate;and the subsequent `latent' period, where the contin-ued presence of glutamate is not obligatory andwhich culminates in the failure of cytoplasmic Ca2�

homeostasis, termed by Tymianski `delayed Ca2� de-regulation' [3] and subsequent cell death (Fig. 1). Our

analysis will focus on neuronal bioenergetics and willattempt to deconvolute the interactions between mi-tochondrial ATP synthesis, Ca2� accumulation andthe generation of reactive oxygen species in culturedneurones exposed to excitotoxic concentrations ofglutamate.

2. The initial glutamate exposure

In primary neuronal culture, the excitotoxic cas-cade can be initiated by as little as 5 min exposureto high glutamate concentrations in the absence ofMg2� (to prevent voltage-dependent block of theNMDA receptor). There is overwhelming evidencethat the entry of Ca2� into the cells predominantlythrough NMDA-selective glutamate receptors duringthis period triggers the subsequent neurodegenera-tion; however Ca2� loading via voltage-activatedCa2� channels during KCl-depolarisation is muchless excitotoxic and the reason for this selectivity isthe subject of current controversy. The possibilitiesare that the absolute amount of Ca2� enteringthrough the NMDA receptor may greatly exceedthat through voltage-activated Ca2� channels [4,5],or that Ca2� entering through the NMDA receptoris focussed onto a vulnerable excitotoxic locus withinthe cell [3,6].

When determined with high-a¤nity Ca2� indica-tors, such as fura-2, little di¡erence is seen in theapparent elevation in [Ca2�]c evoked with glutamateor KCl [7^9]; however the use of low a¤nity indica-tors of free Ca2� capable of reporting far higherconcentrations before saturating has indicated thatglutamate-evoked [Ca2�]c elevations may be consid-erably underestimated and may exceed 5 WM, con-siderably in excess of levels achieved with KCl-depol-arisation [7,10].

2.1. Neuronal mitochondria sequester cytoplasmic[Ca2+]c transients

At the termination of a transient glutamate expo-sure, [Ca2�]c tends to return to baseline as the cationis sequestered into internal organelles or is extrudedacross the plasma membrane. Our early studies withisolated mitochondria from liver and brain demon-strated that the activity of the mitochondrial Ca2�

Fig. 1. Phases of acute glutamate excitotoxicity. Neurones ex-posed to glutamate show a transient elevation in cytoplasmicfree Ca2�, [Ca2�]c. Following this initial response, the signal de-cays to a plateau as NMDA receptors partially inactivate. Theplateau is maintained for a variable time (`latent period') untila secondary, irreversible, increase in [Ca2�]c occurs (delayedCa2� deregulation).

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uniporter was highly dependent on the extramito-chondrial Ca2� concentration, [Ca2�]o [11] and ex-ceeded the activity of the independent mitochondrialCa2� e¥ux pathway (coupled to H� and Na� in liverand brain, respectively [12]) when [Ca2�]o rose abovethe `set-point' at which uptake and e¥ux balanced[13]. Mitochondria would therefore be predicted au-tomatically to bu¡er [Ca2�]c above the set-point andto release it back into the cytoplasm when [Ca2�]crecovered to below this value (reviewed in [12]). Witha predicted set-point of 0.3^0.5 WM [13] even theCa2� transients measured with high-a¤nity indica-tors during KCl stimulation rise well above thesevalues and should therefore be in£uenced by mito-chondrial Ca2� sequestration. In 1981, we obtaineddirect evidence of mitochondrial sequestration of de-polarisation-evoked Ca2� loads by measuring mito-chondrial 45Ca2� pools within synaptosomes: Ca2�

loading of these intact isolated terminals by KCl-ac-tivation of voltage-activated Ca2� channels resultedin the large majority of the accumulated Ca2� beingfurther transported into the matrix [14,15].

In 1990, Thayer and Miller [16] showed that briefKCl-mediated depolarisation of dorsal root ganglioncells was followed by a partial recovery in [Ca2�]c toa plateau which varied from 200 to 600 nM, but wasabsent in cells depolarised in the presence of proton-ophore. This plateau was interpreted to be a conse-quence of the slow release from the mitochondrion ofCa2� temporarily accumulated at the peak [Ca2�]c.Addition of the protonophore during the plateauproduced a large transient elevation in [Ca2�]c con-sistent with a rapid release of the mitochondrial pool.A similar plateau was observed in these cells follow-ing trains of s 25 action potentials [17]; again thelevel of the [Ca2�]c plateau during recovery (450^550nM) correlated well with the set-point predicted forisolated brain mitochondria [13]. A comparable pro-tonophore enhancement of the KCl-evoked [Ca2�]ctransient and abolition of the subsequent plateau hasalso been reported for bullfrog sympathetic neurones[18]. These results were consistent with earlier mod-elling with isolated mitochondria [19] and have re-cently been reproduced in whole-cell patch-clampedchroma¤n cells following depolarisation-evoked ac-tivation of voltage-activated Ca2� channels [20].

In 1990, the group of Duchen [21] also reportedenhanced cytoplasmic Ca2� transients in dorsal root

ganglion cells exposed to a range of metabolic in-hibitors, including protonophores, cyanide and glu-cose removal, but noted that these e¡ects could bea consequence of impaired Ca2� extrusion fromthe cells as well as inhibited mitochondrial sequestra-tion.

In a detailed series of papers, White and Reynolds[9,22,23] have analysed the pathways of Ca2� remov-al from the cytoplasm following acute, non-toxic,glutamate exposure. Restoration of baseline [Ca2�]cfollowing as little as 15 s exposure to 3 WM glutamatewas highly dependent upon both vim and extracel-lular Na� ; thus no recovery occurred during wash-out of glutamate by a Na�- free medium containingprotonophore. Interestingly, when the same Ca2�

transient was generated by the combination of highKCl and veratridine (activating voltage-activatedCa2� channels and allowing Na� entry via voltage-activated Na� channels), recovery of [Ca2�]c tobaseline could still occur. This was interpreted asindicating a fundamental di¡erence in the handlingof glutamate-evoked versus KCl/veratridine-evokedCa2� loads. While the reason for this di¡erencewas not directly investigated, a likely explanationwas considered to be a greater uptake of Ca2� intothe cell (and hence the mitochondrion) in the formercondition.

2.2. In situ mitochondrial membrane potential (vim)during acute Ca2+ loading of neurones

The key parameter determining the energetic statusof mitochondria is the proton-motive force, vp,across the inner membrane (reviewed in [24]). Inthe presence of physiological concentrations of Pi

and Mg2�, isolated mitochondria from liver, heartor brain maintain a total vp of some 220 mV ofwhich the membrane potential vim comprises 150^180 mV with a vpH of 30.5 to 31 pH unit contrib-uting the remaining 30^60 mV [25]. The determina-tion of both components of vp for in situ mitochon-dria is exceedingly complex; in collaboration withHoek and Williamson [26], we quanti¢ed vp for insitu hepatocyte mitochondria: vim was determinedfrom the Nernstian distribution between the cyto-plasm and matrix of the lipophilic cation TPMP�,while vpH was measured by weak acid distribution;a value for vp of s 200 mV was consistent with

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isolated mitochondria. Subsequently, we quanti¢edthe vim component for mitochondria within isolatednerve terminals arriving at an estimate of 150 mV forglucose-maintained synaptosomes [27].

Direct determination of the Nernstian gradient be-tween matrix and cytoplasm by confocal microscopyis extremely di¤cult due to the small size of themitochondria and the enormous dynamic range re-quired to quantify a 300-fold gradient. Instead, de-terminations at the population or single-cell level ofresolution membrane-permeant cationic £uorescentindicators report their concentration within the mi-tochondrial matrix either by their £uorescentquenching (e.g. rhodamine-123 [28^34] or tetrameth-ylrhodamine esters [35^37]) or by a change in theiremission spectra (e.g. JC-1 [9]). An essential control,which is not always performed, is to establish thatthe signal is not a¡ected by a change in plasma mem-brane potential, for example high [K�] or glutamatein the absence of external Ca2�, see [9,28]. With thisimportant proviso in mind, £uorescent changes indi-cative of mitochondrial depolarisation have been re-ported following acute glutamate exposure of cul-tured neurones [9,28,30,31,33,34,37], althoughparticularly with JC-1 the pattern of response canbe complex, with both hyperpolarizing and depola-rizing responses being detected [9]. The ATP syn-thase inhibitor oligomycin did not prevent the KCl-evoked vim decrease [28].

The in£uence of mitochondrial Ca2� loading onvim is complex and has been analysed in detailonly for isolated mitochondria: in the presence ofexcess Pi a single addition of Ca2� causes a transientdepression in both the vim and vpH components ofvp, the extent of which is dependent on the Ca2�

concentration [38^40]. When net accumulation ofCa2� is complete both components of vp are restoredto their initial values. The transient depression in vpcan be su¤cient to interrupt ATP synthesis or evento causes reversal of the ATP synthase and hydrol-ysis of ATP ^ consistent with early observations thatCa2� accumulation could take precedence over ATPsynthesis [41]. The signi¢cance of phosphate is fre-quently overlooked: however, the anion is co-accu-mulated into the matrix in parallel with Ca2� whereit forms an osmotically inactive, but rapidly dissoci-able, calcium phosphate complex [12]. In the absenceof added Pi, and particularly following depletion of

endogenous Pi by an ADP/glucose/hexokinase trap[11] the capacity of isolated mitochondria to accumu-late Ca2� is greatly restricted. Thus, while the samereversible drop in vp occurs during Ca2� accumula-tion, the vpH generated by protons extruded by therespiratory chain is no longer neutralised by Pi accu-mulation and a progressive build-up of vpH occurs.This can ultimately result in an equipartition betweenthe two components: a vp of 220 mV being com-prised of 110 mV of vim and almost 2 pH units oftransmembrane gradient [42,43]. Finally, inductionof the mitochondrial permeability transition (MPT,see below) will lead to a collapse in both vim andvp. It is not yet clear which of the above mechanismsunderlies any decrease in vim in glutamate-exposedneurones.

2.3. Deconvolution of vim e¡ects on mitochondrialCa2+ accumulation and ATP synthesis

In intact cells with maintained in situ metabolism,Ca2� homeostasis cannot be separated from cellularmetabolism and bioenergetics and should be consid-ered as part of an integrated picture (Fig. 2). Neuro-nes are almost entirely dependent on exogenous glu-cose, and the consequent supply of pyruvate to themitochondrion and its subsequent oxidation via thetricarboxylic acid cycle drives both ATP synthesisand Ca2� accumulation. Since the two processescompete for the proton circuit they should be con-sidered together in an analysis of the e¡ects of cel-lular Ca2� loading. While Ca2� accumulation by themitochondria may a¡ect ATP synthesis, alterationsin ATP synthesis will in turn a¡ect the activity of ionpumps responsible for removing Ca2� from the cy-toplasm. An analysis of these interactions is one ofthe main goals of our laboratory [31,41].

While protonophore addition has been widely em-ployed to investigate mitochondrial Ca2� pools inneurones (see above), the collapse in vim will notonly inhibit mitochondrial Ca2� accumulation andrelease the cation into the cytoplasm, but shouldalso instantaneously reverse the mitochondrial ATPsynthase resulting in rapid hydrolysis of cytoplasmicATP [41]. Unless the maximal rate of glycolytic ATPsynthesis comfortably exceeds the cellular ATP de-mand during Ca2� loading plus the rate of ATP hy-drolysis by the uncoupled mitochondria, the cells will

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become depleted of ATP and ATP-dependent proc-esses will be compromised.

We have employed rat cerebellar granule cells asour experimental model; these cells have a high den-sity of mitochondria both along their neurites andpacked around the nucleus in the relatively thin an-nulus of cytoplasm [31]. We initially determinedwhether the granule cells showed the same responseto protonophores during KCl-depolarisation as thedorsal root ganglion cells investigated by Thayer andMiller [16]. Cerebellar granule cells maintain a[Ca2�]c of 6 100 nM, well below the estimated set-point for rat brain mitochondria (0.3^0.5 WM [13])and would thus be predicted to be largely depleted ofCa2�. This was con¢rmed by the failure of protono-phore addition to initiate a transient release of Ca2�

into the cytoplasm. In contrast, addition of protono-phore during the plateau of [Ca2�]c following KCl-depolarisation resulted in a transient spike in thefura-2 signal, consistent with release from the mito-chondrial compartment. When protonophore wasadded prior to 50 mM KCl, the size of the depolar-isation-evoked [Ca2�]c spike was greatly enhanced,consistent with earlier studies [18]. While this couldbe interpreted as a direct consequence of a failure ofmitochondrial sequestration, a rapid secondary ele-vation in [Ca2�]c led us to suspect that the protono-phore was causing a collapse in ATP levels, prevent-ing Ca2� extrusion from the cells. This depletion wascon¢rmed by analysis of ATP/ADP ratios within thecells : within 5 min, 2 WM CCCP decreased the ratiofrom a control value of s 7 to 6 3 [41]. There is thusambiguity as to whether the enhanced cytoplasmic[Ca2�]c response is due a failure of mitochondrialCa2� accumulation due to the collapsed vim, or afailure of Ca2� extrusion from the cell in response toa lowered ATP/ADP ratio.

Seven-day in vitro granule cells incubated in thepresence of 15 mM glucose do not have a su¤cientlyactive glycolytic pathway to be able to maintain ahigh ATP/ADP ratio and low basal cytoplasmicCa2� in the presence of protonophore, when glycol-ysis is required to maintain the cell's normal func-tions in the face of the freely reversing mitochondrialATP synthase. However, when the ATP synthase isinhibited by oligomycin, preventing both synthesisand hydrolysis of ATP by the mitochondria, glycol-ysis can maintain high ATP/ADP ratios and pro-

longed cytoplasmic Ca2� homeostasis [31,41]. As inother cells [27,44], the addition of oligomycin causesa slight hyperpolarisation of the in situ mitochondria[45] consistent with a `state 3^state 4' transition [24].Since the entire ATP generation by the cell is glyco-lytic in the presence of oligomycin experiments canbe designed in which vim can be collapsed, for ex-ample by the further addition of a mitochondrialrespiratory chain inhibitor, such as rotenone[31,41], without a priori a¡ecting cellular ATP gen-eration (Fig. 2).

If accumulation of Ca2� into endoplasmic reticu-lum is ignored, the change in free Ca2� concentrationreported by a £uorescent probe is the net resultant ofcytoplasmic Ca2� chelation, accumulation and re-lease from mitochondria and uptake and e¥uxacross the plasma membrane, which in neurones isdue to both the plasma membrane Ca2�-ATPase andNa�/Ca2� exchanger [21,46,47]. In view of the evi-dence discussed above that mitochondria sequestermuch of the Ca2� load imposed by KCl depolarisa-tion, it would be predicted that abolition of the mi-tochondrial pool would enhance the cytoplasmicCa2� transient. This is not, however, what we ob-serve: KCl-depolarisation of granule cells in the pres-ence of rotenone plus oligomycin actually produces asmaller peak [Ca2�]c elevation than in the presence ofoligomycin alone [41]. Thus, mitochondrial depolar-isation by protonophores enhances the KCl-evoked[Ca2�]c transient, while mitochondrial depolarisationby rotenone plus oligomycin decreases the transient.The trivial explanation that rotenone might directlyinhibit voltage-activated Ca2� channels was elimi-nated since when both rotenone and protonophorewere present before KCl-depolarisation, the peakheight was enhanced to the same extent as withFCCP alone [41]. There are two explanations forthis counter-intuitive result : mitochondrial depolar-isation must either reduce Ca2� entry via the VACCsresponsible for the initial transient, or Ca2� extrusionfrom the cell must be enhanced.

Application of the same protocol to granule cellsexposed to glutamate showed that while oligomycindid not signi¢cantly a¡ect the peak and initial pla-teau following maximal stimulation of the NMDAreceptor, the combination of rotenone plus oligomy-cin to depolarise the mitochondria prior to receptoractivation decreased both the initial [Ca2�]c transient

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and the subsequent plateau [31]. In further experi-ments (Budd and Nicholls, unpublished), we havecon¢rmed that metabolic restriction by the complexIII inhibitor antimycin A, protonophore additionand inhibition of succinate dehydrogenase by malo-nate each enhanced Ca2� deregulation followingNMDA receptor activation, while the same inhibi-tors delayed deregulation in the presence of oligomy-cin. Thus, as in the case of KCl depolarisation, in-hibitors which will decrease ATP levels enhance

glutamate-evoked cytoplasmic Ca2� transients, whileremoval of the mitochondrial matrix as a Ca2� sinkwithout depleting ATP result in a decrease in [Ca2�]c.Furthermore, the total 45Ca2� accumulated duringexposure to glutamate was also decreased by rote-none plus oligomycin [31]; thus mitochondrial depol-arisation must restrict Ca2� entry into the cell and/orenhance e¥ux of the cation. The mechanism of thiscontrol is currently being investigated.

One hypothesis is that mitochondria control the

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net Ca2� £ux across the plasma membrane by rapidchanges in ATP availability. ATP depletion of gran-ule cells by simple protonophore addition enhancesthe KCl-evoked peak Ca2� elevation and preventsthe maintenance of a subsequent plateau [21,41].Glucose depletion greatly enhances the [Ca2�]c tran-sient in dorsal root ganglion cells depolarised withKCl [21]. Lowered glucose availability, both in thepresence and absence of oligomycin, enhances boththe peak and subsequent plateau fura-2 signal ofgranule cells during glutamate exposure (Budd,Ward and Nicholls, unpublished). However, sincethe rationale of our experiments in the presence ofoligomycin has been to eliminate e¡ects of mitochon-drial ATP synthesis, how could mitochondria withinhibited ATP synthase activity still in£uence cyto-plasmic ATP/ADP ratios? The experimental observa-tion is that glutamate causes an equivalent decreasein granule cell ATP/ADP ratios in the presence andabsence of oligomycin [31]. One factor which is fre-quently overlooked is the availability of phosphate(Pi), which is co-accumulated into the mitochondrialmatrix together with Ca2� [12] and the rapid accu-mulation of the cation into the mitochondrion fol-lowing NMDA receptor activation could temporarilydeplete the cytoplasm of Pi, which would in turndecrease the ATP/ADP ratio which could be main-tained by glycolysis. Thus it has been observed thatlow extracellular Pi decreases ATP levels in culturedneurones and sensitises cells to glutamate-evokeddeath [48].

3. Delayed Ca2+ deregulation

The initial peak in [Ca2�]c following glutamateaddition and the subsequent recovery to a plateaulargely re£ects the activation and subsequent partialdesensitisation of the NMDA receptors. Once theplateau is established, the cytoplasmic free Ca2� ofglutamate-exposed cells can remain relatively lowand stable [49] until a sudden, essentially irreversible,increase occurs (Fig. 3A). Individual cells survive forvarying periods before this ¢nal Ca2� deregulationand survival ¢ts a single exponential curve, consistentwith a stochastic process [50]. Continued NMDAreceptor activation is not required during this period,since transient exposure of neurones to glutamate foras short a period as 5 min can lead to subsequent celldeath, although prolongation of the glutamate expo-sure does increase toxicity [3]. Plasma membrane in-tegrity is initially maintained, since £uorescentprobes are still retained in the cytoplasm. While theATP/ADP ratio falls in populations of deregulatingcells [31,51] it has not been possible to determine theratio in an individual cell during the process. Thus itis not easy to determine whether there is an upstreamfailure of ATP synthesis or a downstream inhibitionof ATP-dependent ion pumps; for example, the plas-ma membrane Ca2�-ATPase or Na�/K�-ATPase,due to proteolytic or oxidative damage.

Deregulation of [Ca2�]c in our preparation appearsto be a result of failed e¥ux rather than enhancedin£ux, since the rise in [Ca2�]c is not blocked by a

Fig. 2. Schematic inter-relationships between the NMDA receptor, mitochondrial Ca2� transport and cellular bioenergetics. Mitochon-dria in granule cells utilize pyruvate to generate vp, synthesize ATP and accumulate Ca2�. Ca2� extrusion pathways driven by ATP(e.g. the Ca2�-ATPase) maintain a low [Ca2�]c. In each ¢gure, the predicted activity of pathways is represented by the line thickness.In each scheme, the initial response to NMDA receptor activation is depicted. (A) In the absence of inhibitors, ATP synthesis is pre-dominantly via the ATP synthase. Ca2� entering the cell during NMDA receptor activation is largely accumulated into the mitochon-dria. Chronic receptor activation can lead to mitochondrial Ca2� overload. (B) In the presence of oligomycin, glycolysis supports thetotal cellular ATP demand. Both ATP synthesis and hydrolysis by the mitochondria are inhibited. The mitochondria still load withCa2� and despite the presence of the inhibitor cellular ATP falls and Ca2� homeostasis is lost. Thus Ca2�-loading of the mitochondri-on is excitotoxic by a process independent of the oligomycin-sensitive ATP synthase. (C) The complex I inhibitor rotenone preventsthe respiratory chain from pumping protons. Glycolysis now supplies ATP both for the cell and for the ATP synthase which reversesto generate a reduced proton gradient. Even in the absence of glutamate, the cell can only maintain a low [Ca2�]c for a limited peri-od. When glutamate is added the reversal of the ATP synthase accelerates as it attempts to compensate for the inward £ux of Ca2�

into the matrix and the resulting ATP depletion leads to an immediate loss of cytoplasmic Ca2� homeostasis. (D) The combinationof rotenone plus oligomycin allows the proton gradient to decay preventing mitochondrial Ca2� loading. Cytoplasmic ATP is main-tained by glycolysis ; the decreased 45Ca2� uptake into the cells, together with the maintained low cytoplasmic Ca2�, each demonstratethat Ca2� in£ux through the NMDA receptor is balanced by e¥ux across the plasma membrane when mitochondria can no longer se-quester Ca2�, due either to a feed-back inhibition of the NMDA receptor or an enhanced Ca2� e¥ux. (E) The protonophore FCCPcollapses cellular ATP by reversal of the ATP synthase.6

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cocktail of channel inhibitors (Budd and Nicholls,unpublished), while the rate of Mn2� quenching offura-2 £uorescence, indicative of Ca2� entry, doesnot increase as cells deregulate [52]. There may how-ever be a contribution to deregulation from mito-chondrial dumping of Ca2� into the cytoplasm as aresult of mitochondrial depolarisation and/or thepermeability transition.

3.1. The latent period prior to deregulation

The processes which occur during this intermediate`latent' period culminate in cell death which mayhave apoptotic [53,54] necrotic [55^57] or mixed[51,58,59] characteristics depending on the severityof the initial insult. Thus, within a culture of cerebel-lar granule cells transiently exposed to glutamate, asubpopulation were found to depolarise their mito-chondria and undergo necrosis, while the survivingcells subsequently repolarised their mitochondria andregenerated ATP, but underwent later apoptosis [51].Apoptosis is out of the scope of this review, however,while activation of the caspase cascade is a common¢nal pathway for a range of factors inducing granulecell apoptosis [60^62], caspase inhibitors are unableto prevent necrosis [63^66].

3.2. Restricted bioenergetics facilitates delayed Ca2+

deregulation

NMDA receptor activation places a multiple en-

ergy demand upon a neurone. The increased cyto-plasmic Ca2� and Na� concentrations will result inactivation of the plasma membrane Ca2�-ATPaseand Na�/K�-ATPase, respectively. The latter maybe quantitatively more important, since even thoughNMDA receptors display some selectivity for Ca2�

over Na�, the much higher concentration of the lat-ter ion in the extracellular medium means that thein£ux of Na� may exceed that of Ca2� [67]. How-ever, glutamate is still excitotoxic in Na�-free media[3]. In addition to these plasma membrane e¡ects, thepeak values of [Ca2�]c in excess of 5 WM duringglutamate exposure reported in recent experimentswith low a¤nity Ca2� indicators [7,10] would be pre-dicted from isolated mitochondrial studies [40] toresult in such a rapid uptake into the mitochondrialmatrix that vp could be transiently lowered belowthe value required for ATP synthesis.

Depletion of cytoplasmic ATP will lead to a cata-strophic deregulation of [Ca2�]c by inhibiting theplasma membrane Ca2�-ATPase and also limitingthe activity of the two ATP-requiring enzymes inthe glycolytic pathway, hexokinase and phosphofruc-tokinase [68]. In our studies, cultured granule cellstreated with FCCP, rotenone or antimycin A (Fig.3B) show massive Ca2� deregulation upon exposureto glutamate [31]. In this condition, glycolysis is re-quired not only to generate ATP for the activatedplasma membrane ion pumps, but also to supplythe reversed mitochondrial ATP synthase, which at-tempts to regenerate vp which is lowered by the up-

Fig. 3. Delayed Ca2� deregulation following the addition of glutamate/glycine to rat cerebellar granule cells. (A) Addition of 100 WMglutamate plus 10 WM glycine to cells results in a stochastic Ca2� deregulation. (B) In the presence of antimycin A to inhibit the respi-ratory chain, glutamate/glycine addition leads to an immediate deregulation which can be accounted for by insu¤cient ATP synthesis(see Fig. 1C). (C) In the presence of oligomycin plus antimycin A to depolarize the mitochondria without collapsing ATP, glutamate/glycine-evoked Ca2� transients are decreased and no Ca2� deregulation occurs within the timespan of the experiment. Each trace rep-resents [Ca2�]c in an individual cell soma determined with fura-2. For further details see [31].

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take of Ca2� into the matrix. It is evident that theseconditions create an instant ATP de¢cit and imme-diate failure of cytoplasmic Ca2� homeostasis.

There is an extensive literature demonstrating thesynergistic e¡ect of metabolic restriction upon gluta-mate excitotoxicity both in vivo, e.g. [69^72], and invitro [37,59,73^75]. This synergism can also be ob-served in in vivo models of neurodegenerative dis-eases: thus the behavioural and morphological ef-fects of Huntington's disease following injection ofor 3-nitropropionate into the striatum can be dimin-ished by NMDA receptor antagonists [76^78] as canthe motor e¡ects of 1-methyl-4-phenylpyridinium(MPP�) inhibition of mitochondrial complex I inanimal models of Parkinson's disease [71].

3.3. Delayed Ca2+ deregulation is not causallydependent upon a failure of oxidativephosphorylation

While Ca2�-deregulation of glutamate-exposedcultured cerebellar granule cells is an imperfect mod-el for glutamate excitotoxicity, it does allow somesimple hypotheses to be tested. The ¢rst is that afailure of mitochondrial ATP synthesis is necessaryand su¤cient for deregulation. This might be ex-pected in view of the synergistic e¡ects of energyrestriction and NMDA receptor activation discussedabove, as well as the possibility that mitochondriamight be damaged by excessive Ca2� accumulation(discussed below). Since granule cells have su¤-ciently active glycolysis to maintain Ca2� homeosta-sis when mitochondrial oxidative phosphorylation isinhibited from the outset in the presence of oligomy-cin [41], this hypothesis would predict that glutamatewould not be excitotoxic in the presence of the in-hibitor. However, no statistically signi¢cant di¡er-ence is observed in the time or extent of Ca2� dereg-ulation in granule cells exposed to glutamate in thepresence or absence of the inhibitor [31]. In otherwords, delayed Ca2� deregulation occurs in cellswhose mitochondria are polarised, transporting elec-trons and accumulating Ca2�, but are not required tosynthesise (or hydrolyze) ATP. A decline in ATP/ADP ratio is seen in granule cell populations exposedto glutamate for 60 min in the presence of oligomy-cin [31], but it is currently unclear whether this is thecause or e¡ect of the Ca2� deregulation in individual

cells. A further implication of this ¢nding is thatglycolysis can supply su¤cient ATP to supply thecell's energy requirements even when these are en-hanced during NMDA receptor activation.

3.4. Delayed Ca2+ deregulation is not causallydependent upon a failure of glycolysis

The requirement of glycolysis for ATP at two steps(hexokinase and phosphofructokinase) implies that acell whose ATP levels are declining, even transiently,could su¡er an irreversible collapse as glycolysis be-comes limiting, exacerbating a further decrease inATP. In 1986, we found that synaptosomes showedan initial enhancement of glycolysis, consistent witha Pasteur e¡ect, following respiratory inhibition byrotenone, but that this was followed by a progressivefailure [79]. This glycolytic failure has been subse-quently analysed in more detail [68] under conditionswhere the bioenergetic safety margin was eroded byincreased energy demand (e.g. ionophore addition)and inhibited ATP generation (including rotenoneaddition). It was concluded that the ATP require-ment for hexokinase was the limiting factor duringglycolytic failure [68]. Interestingly, this raises thequestion as to how the neurone restarts glycolysison recovery from severe ATP depletion. In this con-text, it has been demonstrated that lactate, whichaccumulates during anoxia, can be utilised by se-verely ATP-depleted cells, generating pyruvate andallowing mitochondrial oxidative phosphorylationto regenerate ATP allowing glycolysis to occur [80].

If delayed Ca2� deregulation occurs in a neuroneat a stage where the capacity of the cell to generateATP is insu¤cient to meet its energy demands, itmight be predicted that an additional substrate sup-ply might be able, at least temporarily, to rescuecells. Lactate and pyruvate are e¡ective neuronalsubstrates [81,82] and Eimerl and Schramm [83]have reported that pyruvate plus phosphate addedafter glutamate exposure decreases cell death. In re-cent experiments (Budd, Ward and Nicholls, unpub-lished), we have found that granule cells incubated inglucose-free media and maintained by lactate or pyr-uvate undergo glutamate-evoked delayed Ca2� de-regulation after a similar delay as cells maintainedby glycolysis. Thus, a time-dependent inhibition ofthe glycolytic pathway following glutamate exposure

BBABIO 44667 30-7-98


is not an inherent component of the delayed Ca2�

deregulation observed in our preparation.

3.5. In situ mitochondria undergo progressivebioenergetic deterioration in glutamate-exposedcells

A number of key catabolic mitochondrial enzymesare readily inhibited by oxidative damage, includingpyruvate dehydrogenase [84] and aconitase [85]. Themitochondrial electron transport chain itself is a fur-ther potential locus for inhibition since complexes I,III and IV each exert some control over the rate ofoxygen consumption of isolated brain mitochondria[86]. There is some disagreement as to the bioener-getic consequences of partial inhibition of these com-plexes: by comparing the titration of respiratorychain inhibitors with intact mitochondria and singlecomplexes Davey and Clark [86] concluded that theindividual complexes had to be inhibited by at least60% before signi¢cant e¡ects were observed on mi-tochondrial respiration and ATP synthesis. Howeverrepopulation of mitochondrially depleted rho-0 cellswith mitochondria from patients with either Parkin-son's disease displaying 26% de¢ciency in complex Iactivity [87] or Alzheimer's disease displaying 52%de¢ciency in complex IV activity [88] both resultedin a slowed recovery of [Ca2�]c following Ca2� loads.

A relatively direct demonstration that in situ mi-tochondria undergo a progressive deterioration ofbioenergetic function following glutamate exposureis the ¢nding by Atlante et al. [89] that mitochondriaisolated from glutamate-exposed cerebellar granulecells display a decrease in state 3 respiration whichwas proportional to the time for which the cells hadbeen exposed to glutamate. After a 5-h exposure, thesubsequently isolated mitochondria displayed no sig-ni¢cant respiratory control. Inhibited state 3 respira-tion implies a metabolic or respiratory chain limita-tion rather than a loss of respiratory control,although the locus of inhibition was not establishedin this study.

3.6. Delayed Ca2+ deregulation may be triggered bymitochondrial Ca2+ accumulation

The total 45Ca2� accumulated by cortical neurones[4] or cerebellar granule cells [5] in the presence of

glutamate has been considered to be a reliable pre-dictor of the extent of subsequent cell death. How-ever, this has recently been challenged by the reportthat NMDA-evoked uptake is still much more toxicthan an equivalent extent of KCl-evoked 45Ca up-take [6], suggesting that the extreme toxicity of glu-tamate-evoked Ca2� uptake may be due to the selec-tive direction of the Ca2� onto an excitotoxic locusin the close vicinity of the intracellular face of theNMDA receptor [3]. However, since the mitochon-drial matrix is the only compartment capable of se-questering this Ca2�, it is of central importance toestablish the role which matrix Ca2� accumulationplays a role in excitotoxicity.

An additional factor, which has not generally beenconsidered, is that the initial rate of mitochondrialCa2� loading, rather than the absolute amount accu-mulated may be critical. Thus, low a¤nity Ca2�

probes indicate very large [Ca2�]c transients in re-sponse to glutamate [7,10] which would be predictedto divert all mitochondrial proton pumping to Ca2�

accumulation [12]. Indeed, the net accumulation of45Ca2� by granule cells is largely complete within5 min (Budd and Nicholls, unpublished). Additionof NMDA or glutamate to granule cells in low K�

medium in the absence of Mg2� (the usual experi-mental protocol) depolarises the plasma membrane[90]; however, glutamate addition to granule cellspredepolarised in high KCl medium has been re-ported to show much decreased excitotoxicity [34].Since NMDA receptors partially desensitise duringprolonged depolarisation, this implies that the initialCa2� entry in polarised cells may be critical in trig-gering excitotoxicity. It is notable that no lag can bedetected between an increase in [Ca2�]c and [Ca2�]mfollowing NMDA receptor activation, whereas somelag is apparent for KCl-evoked Ca2� loading [91].

3.7. Glutamate is not excitotoxic to cells maintaininga high ATP with depolarised mitochondria

In the presence of oligomycin, in situ mitochondriamaintain a high vim and thus retain the ability toaccumulate Ca2�. Consistent with this, 45Ca2� is ac-cumulated in the 20 min following addition of gluta-mate to the same extent as in the absence of theinhibitor [31]. As in the case of depolarisation-evoked Ca2� loading discussed above, inhibition of

BBABIO 44667 30-7-98


the respiratory chain of oligomycin-treated cellsshould collapse vim without a¡ecting ATP synthesisby glycolysis. Respiratory chain inhibitors underthese conditions will, however, collapse vim andabolish mitochondrial Ca2� accumulation [31] in ad-dition to blocking electron transport at de¢ned sitesand thus potentially a¡ecting the generation of reac-tive oxygen species.

The total accumulation of 45Ca2� within culturedcerebellar granule cells exposed to glutamate can ap-proach 20 nmol/Wl of cell volume (equivalent to 20mM!) before the cells die [5]. The mitochondrion isthe only organelle capable of accumulating suchamounts of Ca2�, and loss of the mitochondrialsink for Ca2� entering via the NMDA receptorshould therefore logically result in a rapid saturationof cytoplasmic Ca2� chelators and a massive eleva-tion in [Ca2�]c. It was therefore remarkable thatgranule cells exposed to glutamate in the presenceoligomycin plus rotenone [31] or antimycin A (Fig.3C) show a relatively small peak Ca2� elevation,maintain a low subsequent Ca2� plateau and cansurvive for up to 5 h in a Mg2�-free medium in thecontinuous presence of 100 WM glutamate, 10 WMglycine. Any cell death which occurred in the pres-ence of these two inhibitors was independent of thepresence of glutamate. Thus, mitochondrial Ca2� ac-cumulation may be an essential stage in the chain ofevents associated with glutamate excitotoxicity.

Such a surprising result requires careful controls,the ¢rst is to investigate the possibility that rotenoneor oligomycin have previously unrecognised directinhibitory actions at the NMDA receptor. Oligomy-cin alone is not neuroprotective [31], while as dis-cussed above, rotenone addition in the absence ofoligomycin results in immediate glutamate-evokedCa2� deregulation [31]. This would actually be pre-dicted on bioenergetic grounds: inhibition of the res-piratory chain places a severe demand upon the gly-colytic capacity of the cell : in addition to the normalcellular house-keeping functions which require ATP,respiratory chain inhibition leads to a reversal of themitochondrial ATP synthase as glycolytic ATP isutilised to regenerate vim (Fig. 2).

3.8. Elevated [Ca2+]c is not itself neurotoxic

In this analysis of the role of mitochondria in the

control of glutamate excitotoxicity, it is important todistinguish between a relatively stable and reversibleelevation in [Ca2�]c, which can be a result of en-hanced in£ux and/or restricted e¥ux from the celland the catastrophic and irreversible rise signallingthe onset of Ca2� deregulation. Thus, Khodorov etal. [92] exposed 7^8 DIV cerebellar granule cell cul-tures to the combination of antimycin A and oligo-mycin to depolarise the in situ mitochondria by asimilar mechanism to our earlier experiments [41].These authors found that the combination of inhib-itors resulted in a delayed secondary rise in [Ca2�]cafter glutamate exposure, in contrast to our ¢ndingsthat the same inhibitors decreased both peak andplateau responses to continuous glutamate [31], lead-ing them to conclude that `mitochondria play a dom-inant role in the protection against neuronal Ca2�

overload induced by excitatory amino acids' [92].Apparently opposite ¢ndings with the same prepara-tion require some comment: the initial recovery fol-lowed immediately by a secondary rise in [Ca2�]cafter glutamate found by Khodorov et al. is verysimilar to the time course of [Ca2�]c in our cells inthe presence of protonophore, and suggests an en-ergy limitation; these authors worked at room tem-perature and employed 5 mM glucose (in contrast toour 37³C and 15 mM glucose). Furthermore, no con-trols were performed with glutamate in the presenceof oligomycin alone to control for ATP depletionunder these conditions where the maximal rate ofglycolytic ATP synthesis and Ca2� extrusion will berestricted, but NMDA receptor activity will be vir-tually unchanged. Since di¡erent experimental or cul-ture conditions will a¡ect the complex balance be-tween NMDA receptor activity, Ca2� extrusioncapacity, glycolysis and the capacity for mitochon-drial ATP synthesis, it will clearly not be possibleto separate e¡ects of mitochondrial ATP synthesisand Ca2� sequestration by using conditions whichdeplete ATP as well as depolarizing the mitochon-dria.

The experiments we performed with rotenone/oli-gomycin [31] decreased both the mitochondrial Ca2�

accumulation and the cytoplasmic [Ca2�]c elevationsin response to glutamate. While we interpreted theneuroprotection a¡orded by this combination to theabolition of the matrix accumulation, it was possiblethat the decreased cytoplasmic [Ca2�]c could account

BBABIO 44667 30-7-98


for the neuroprotection. It is therefore of consider-able signi¢cance that the increased [Ca2�]c and de-creased matrix Ca2� produced by protonophores incultured hippocampal neurones [8], cortical neurones[93] and an immortalised hippocampal neuronal cellline [94] is not only not neurotoxic, but a¡ords pro-tection against glutamate excitotoxicity [8,93,94].Putting our results together with these, one can con-clude that mitochondrial Ca2� loading is potentiallyexcitotoxic, but that elevated cytoplasmic Ca2�, whilecontrolling the extent of mitochondrial Ca2� loadingand signalling delayed Ca2� deregulation, is not re-sponsible for the intermediate events leading to aprogressive deterioration in cell function.

3.9. The role of mitochondrially initiated oxidativedamage

Glutamate excitotoxicity requires oxygen; thushippocampal neurones exposed to glutamate underhypoxic conditions show no more cell death thandue to hypoxia alone [2]. Additionally, NMDA an-tagonists, which protect against excitotoxic damageto cortical neurones following chemical ischaemia,need only be present during reperfusion [95]; in otherwords, the return of oxidative metabolism triggers acritical period of toxic NMDA receptor activation.Reoxygenation is associated both with mitochondrialCa2� accumulation and the generation of reactiveoxygen species (ROS) and a central question con-cerns the causal relationships between these two pa-rameters and the ultimate death of the cell. We wishto focus on just one aspect of a complex topic,namely the role of the in situ mitochondrial mem-brane potential in the generation of potentially toxicsuperoxide �Oc3

2 � radicals.The predominant source of �Oc3

2 � in most tissues isthe mitochondrial respiratory chain (reviewed in[96]). Complex III, and in particular the level of re-duction of ubisemiquinone at the Qp site, has gener-ally been considered to be the main source of reactiveoxygen species in mitochondria. Thus, with succinateas substrate, the generation of reactive oxygen spe-cies by isolated heart mitochondria is relatively highin state 4, but virtually abolished when ADP isadded and vim decreases to state 3 levels [97,98].However, in a recent paper, Herrero and Barja [98]failed to detect peroxide generated during succinate

oxidation by non-synaptic rat brain mitochondria,but did detect the reactive oxygen species with sub-strates utilizing complex 1. No decrease in peroxideproduction was found following a state 4^state 3transition; thus it was proposed that complex I couldbe a source of reactive oxygen species in state 3.Continued production of �Oc3

2 � in state 3 by isolatedbrain mitochondria is also indicated by the study ofDykens [99], where Ca2� loading of malate/gluta-mate supplemented mitochondria in the presence ofADP increased production of highly reactive hydrox-yl radicals.

Con£icting consequences of in situ mitochondrialdepolarisation have been reported on the generationof reactive oxygen species in cultured neurones: theproduction of hydrogen peroxide detected by di-chloro£uorescin is inhibited by protonophores[100], in accordance with results with isolated mito-chondria [97,101], although the pH sensitivity of thesignal from the oxidised product, dichloro£uorescein,creates complications during the large cytoplasmicacidi¢cation produced both by protonophores [102]and by NMDA receptor activation [102,103].

The non-£uorescent membrane permeant hydro-ethidine (HEt) is oxidised within neurones to the£uorescent ethidium cation by �Oc3

2 � and has beenwidely used to detect the formation of the radicalin a large number of neuronal and non-neuronalstudies [104^108]. Similar studies have been per-formed with dihydrorhodamine-123 [109], which isoxidised to cationic rhodamine-123 predominantlyby peroxynitrite [110]. Since both cationic productsare membrane permeant, they will accumulate in thematrix of polarised mitochondria, even if they werethe product of non-mitochondrial oxidation; thusthey do not automatically signal the site of free rad-ical production in imaging studies [111]. In addition,generated ethidium undergoes £uorescent quenchingwhen accumulated within the mitochondria matrixand conversely shows a £uorescent dequenchingupon mitochondrial depolarisation [45]. This can bedi¤cult to separate from the inherent radical-inducedoxidation, particularly in experiments involving glu-tamate- or protonophore-mediated mitochondrial de-polarisation. Thus, a number of studies have re-ported that protonophore treatment of neurones todepolarise in situ mitochondria causes an enhancedgeneration of ROS detected by dihydrorhodamine-

BBABIO 44667 30-7-98


123 [109] or hydroethidine [104,108], in contrast toresults with isolated mitochondria. We have re-exam-ined the behaviour of the latter probe in culturedcerebellar granule cells and found that the £uorescentyield of the ethidium generated by hydroethidine ox-idation is enhanced by mitochondrial depolarisation,consistent with £uorescent dequenching as the mito-chondria release ethidium and compounded by fur-ther £uorescent enhancement as the released ethidi-um binds to non-mitochondrial nucleic acids [45].This vim sensitivity could be minimised by the useof very low concentrations of HEt, such that theethidium remains bound within the mitochondriaand is not released on depolarisation, or by extrac-tion and quanti¢cation of the generated ethidium atthe end of the experiment.

With these precautions, we con¢rm reports thatNMDA receptor activation enhances the generationof �Oc3

2 � by granule cells, but ¢nd no enhancementupon protonophore addition, in agreement with theabove studies using dichloro£uorescin but in contra-diction to those using dihydrorhodamine-123 orHEt. It may, therefore, also be necessary to re-exam-ine the sequence of events discussed in the context onapoptotic signalling, where a decrease in vim is pro-posed to initiate an increase in the generation of re-active oxygen species, since this has been, in part,based upon experiments using HEt [112].

3.10. The role of the mitochondrial permeabilitytransition

While a consensus appears to be emerging that theMPT, which can be readily observed with isolatedmitochondria, occurs in intact cells as an obligatorycomponent of apoptotic and necrotic neuronal celldeath, we would wish to counsel caution. While thereis evidence in support of an activation of the MPT inmyocardial reperfusion injury [113^115] and duringoxidative [116] and anoxic [117] stress of hepatocytes,a role in glutamate-evoked neuronal excitotoxicity iscurrently more speculative.

Isolated brain mitochondria incubated in the pres-ence of adenine nucleotides and Pi are able to accu-mulate large amounts of Ca2� and to maintain astable set-point [13]. Cyclosporin A is an e¡ectiveinhibitor of the MPT in isolated liver mitochondriaexposed to Ca2� and Pi (reviewed in [118]) but has

been reported to be much less e¡ective against brainmitochondria [119]. The inhibitor has been employedin an attempt to identify a role for the MPT in intactneurones, but while some measure of protectionagainst glutamate is generally observed [9,36,37]but see [30], it is likely that it is the ability of theagent to inhibit calcineurin, rather than the MPT,which underlies most of the observed e¡ects. Thus,while Ankarcrona et al. [120] found that cyclosporinA protected granule cells against both early necrosisand delayed apoptosis induced by glutamate, a sim-ilar protection was a¡orded by the more selectivecalcineurin inhibitor FK-506, which does not interactwith the mitochondrial permeability transition pore.Calcineurin inhibition by cyclosporin A has multiplee¡ects, including an increase in spontaneous actionpotential ¢ring [121] and an inhibition of NMDAreceptors [122], while chronic cyclosporin A inducesneuronal apoptosis in cortical cultures [123,124]. Inour experiments, cyclosporin A, the more selectivemethylvaline-4-cyclosporin [125] and bongkrekicacid [126] each only a¡orded a slight delay beforethe onset of delayed Ca2� deregulation (Castilho,Budd and Nicholls, unpublished), although a moree¡ective protection has been reported for hippocam-pal neurones [34].

Even if the MPT is an essential stage in both apop-totic and necrotic cell death, there are many ques-tions which remain to be answered. Since increasingexcitotoxic stress causes a shift from apoptosis tonecrosis in a single cell preparation [51], this wouldsuggest that the proportion of mitochondria within aneurone undergoing the transition might de¢ne thefate of the cell : a small proportion swelling and re-leasing the proposed pro-apoptotic factors cyto-chrome c [127,128] and apoptosis-inducing factor[112] might initiate apoptosis with the residual mito-chondria maintaining su¤cient ATP levels for apop-tosis to occur, while a more powerful insult woulddisrupt the majority of mitochondria and lead todeath by ATP depletion and subsequent necrosis.However, delayed Ca2� deregulation still occurs incells where mitochondria are prevented from synthe-sizing ATP by oligomycin [31]. Secondly, with iso-lated mitochondria the MPT occurs within secondsof Ca2� overload; in contrast delayed Ca2� deregu-lation can occur hours after a transient glutamateexposure, when the mitochondria would have un-

BBABIO 44667 30-7-98


loaded their Ca2�. Finally, the MPT as normallyinvestigated in isolated mitochondria is inhibited byMg2�, adenine nucleotides and low pH and is di¤-cult to see with NAD-linked substrates [129]. Theconditions within the cytoplasm of a glutamate ex-posed neurone would on each of these counts bepredicted to be resistant to the MPT and it is cur-rently unclear what factor would induce the transi-tion in situ.

4. Conclusions

The mechanism of glutamate-induced neuronal ne-crosis is far from understood. The mitochondrionoccupies centre stage in three roles: as the primegenerator (and possible dissipator) of cellular ATP;as the main sink for the Ca2� accumulated via theNMDA receptor and as the major source of poten-tially excitotoxic reactive oxygen species. These threeroles are all interconnected, and we may have made acontribution to unravelling the interactions by high-lighting the role of mitochondrial Ca2� accumula-tion.

Acknowledgements

SLB was supported by a Medical Research Coun-cil studentship. The work was supported by a grantfrom the Wellcome Trust.

References

[1] M.F. Beal, N. Howell, I. Bodis-Wollner, Mitochondria andFree Radicals in Neurodegenerative Disease, Wiley-Liss,New York, 1997.

[2] J.M. Dubinsky, B.S. Kristal, M. Elizondo-Fournier, J. Neu-rosci. 15 (1995) 7071^7078.

[3] M. Tymianski, M.P. Charlton, P.L. Carlen, C.H. Tator,J. Neurosci. 13 (1993) 2085^2104.

[4] D.M. Hartley, M.C. Kurth, L. Bjerkness, J.H. Weiss, D.W.Choi, J. Neurosci. 13 (1993) 1993^2000.

[5] S. Eimerl, M. Schramm, J. Neurochem. 62 (1994) 1223^1226.

[6] R. Sattler, M.P. Charlton, M. Hafner, M. Tymianski, Soc.Neurosci. Abstr. 23 (1997) 894.4.

[7] K. Hyrc, S.D. Handran, S.M. Rothman, M.P. Goldberg,J. Neurosci. 17 (1997) 6669^6677.

[8] J.M. Dubinsky, S.M. Rothman, J. Neurosci. 11 (1991) 2545^2551.

[9] R.J. White, I.J. Reynolds, J. Neurosci. 16 (1996) 5688^5697.[10] S. Rajdev, I.J. Reynolds, Neurosci. Lett. 162 (1993) 149^152.[11] F. Zoccarato, D.G. Nicholls, Eur. J. Biochem. 127 (1982)

333^338.[12] D.G. Nicholls, K.E.O. Aî kerman, Biochim. Biophys. Acta

683 (1982) 57^88.[13] D.G. Nicholls, I.D. Scott, Biochem. J. 186 (1980) 833^839.[14] I.D. Scott, K.E.O. Aî kerman, D.G. Nicholls, Biochem. J. 192

(1980) 873^880.[15] K.E.O. Aî kerman, D.G. Nicholls, Biochim. Biophys. Acta

645 (1981) 41^48.[16] S.A. Thayer, R.J. Miller, J. Physiol. (Lond.) 425 (1990) 85^

115.[17] J.L. Werth, S.A. Thayer, J. Neurosci. 14 (1994) 346^356.[18] D.D. Friel, R.W. Tsien, J. Neurosci. 14 (1994) 4007^4024.[19] K.E.O. Aî kerman, D.G. Nicholls, Rev. Physiol. Biochem.

Pharmacol. 95 (1983) 149^201.[20] J. Herrington, Y.B. Park, D.F. Babcock, B. Hille, Neuron 16

(1996) 219^228.[21] M.R. Duchen, M. Valdeolmillos, S.C. O'Neill, D.A. Eisner,

J. Physiol. (Lond.) 424 (1990) 411^426.[22] R.J. White, I.J. Reynolds, J. Neurosci. 15 (1995) 1318^1328.[23] R.J. White, I.J. Reynolds, J. Physiol. (Lond.) 498 (1997) 31^

47.[24] D.G. Nicholls, S.J. Ferguson, Bioenergetics, Vol. 2, Academ-

ic Press, London, 1992.[25] D.G. Nicholls, Eur. J. Biochem. 50 (1974) 305^315.[26] J.B. Hoek, D.G. Nicholls, J.R. Williamson, J. Biol. Chem.

255 (1980) 1458^1464.[27] I.D. Scott, D.G. Nicholls, Biochem. J. 186 (1980) 21^33.[28] M.R. Duchen, Biochem. J. 283 (1992) 41^50.[29] V.P. Bindokas, R.J. Miller, J. Neurosci. 15 (1995) 6999^

7011.[30] N.K. Isaev, D.B. Zorov, E.V. Stelmashook, R.E. Uzbekov,

M.B. Kozhemyakin, I.V. Victorov, FEBS Lett. 392 (1996)143^147.

[31] S.L. Budd, D.G. Nicholls, J. Neurochem. 67 (1996) 2282^2291.

[32] J.H. Prehn, V.P. Bindokas, J. Jordan, M.F. Galindo, G.D.Ghadge, R.P. Roos, L.H. Boise, C.B. Thompson, S. Krajew-ski, J.C. Reed, R.J. Miller, Mol. Pharmacol. 49 (1996) 319^328.

[33] B.I. Khodorov, V. Pinelis, O. Vergun, T. Storozhevykh, N.Vinskaya, FEBS Lett. 397 (1996) 230^234.

[34] J. Keelan, O. Vergun, L. Patterson, M.R. Duchen, Soc. Neu-rosci. Abstr. 23 (1997) 895.1.

[35] L.M. Loew, W. Carrington, R.A. Tuft, F.S. Fay, Proc. Natl.Acad. Sci. U.S.A. 91 (1994) 12579^12583.

[36] A.L. Nieminen, T.G. Petrie, J.J. Lemasters, W.R. Selman,Neuroscience 75 (1996) 993^997.

[37] A.F. Schinder, E.C. Olson, N.C. Spitzer, M. Montal, J. Neu-rosci. 16 (1996) 6125^6133.

[38] K.E.O. Aî kerman, Biochim. Biophys. Acta 502 (1978) 359^366.

BBABIO 44667 30-7-98


[39] G.M. Heaton, D.G. Nicholls, Biochem. J. 156 (1976) 635^646.

[40] D.G. Nicholls, Biochem. J. 170 (1978) 511^522.[41] C.S. Rossi, A.L. Lehninger, J. Biol. Chem. 239 (1964) 3971^

3980.[42] D.G. Nicholls, Biochem. J. 176 (1978) 463^474.[43] D.G. Nicholls, M. Crompton, FEBS Lett. 111 (1980) 261^

268.[44] M.R. Duchen, T.J. Biscoe, J. Physiol. (Lond.) 450 (1992) 33^

61.[45] S.L. Budd, R.F. Castilho, D.G. Nicholls, FEBS Lett. 415

(1997) 21^24.[46] C.D. Benham, M.L. Evans, C.J. McBain, J. Physiol. (Lond.)

455 (1992) 567^583.[47] J.L. Werth, Y.M. Usachev, S.A. Thayer, J. Neurosci. 16

(1996) 1008^1015.[48] M. Glinn, B. Ni, R.P. Irwin, S.W. Kelley, S.-Z. Lin, S.M.

Paul, Soc. Neurosci. Abstr. 23 (1997) 895.11.[49] J.M. Dubinsky, J. Neurosci. 13 (1993) 623^631.[50] J.M. Dubinsky, B.S. Kristal, M. Elizondo-Fournier, Neuro-

pharmacology 34 (1995) 701^711.[51] M. Ankarcrona, J.M. Dypbukt, E. Bonfoco, B. Zhivotov-

sky, S. Orrenius, S.A. Lipton, P. Nicotera, Neuron 15 (1995)961^973.

[52] B.I. Khodorov, D.A. Fayuk, S.G. Koshelev, O.V. Vergun,V.G. Pinelis, N.P. Vinskaya, T.P. Storozhevykh, E.N. Ar-senyeva, L.G. Khaspekov, A.P. Lyzhin, N. Isaev, I.V. Vic-torov, J.M. Dubinsky, Int. J. Neurosci. 88 (1996) 215^241.

[53] T. Nitatori, N. Sato, S. Waguri, Y. Karasawa, H. Araki, K.Shibanai, E. Kominami, Y. Uchiyama, J. Neurosci. 15(1995) 1001^1011.

[54] M. Ankarcrona, B. Zhivotovsky, T. Holmstro«m, A. Diana,J.E. Eriksson, S. Orrenius, P. Nicotera, Neuroreport 7 (1996)2659^2664.

[55] F. Dessi, C. Charriaut-Marlangue, M. Khrestchatisky, Y.Ben-Ari, J. Neurochem. 60 (1993) 1953^1955.

[56] J. Ikeda, S. Terakawa, S. Murota, I. Morita, K. Hirakawa,J. Neurosci. Res. 43 (1996) 613^622.

[57] B.J. Gwag, J.Y. Koh, J.A. Demaro, H.S. Ying, M. Jacquin,D.W. Choi, Neuroscience 77 (1997) 393^401.

[58] E. Bonfoco, D. Krainc, M. Ankarcrona, P. Nicotera, S.A.Lipton, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7162^7166.

[59] Z. Pang, J.W. Geddes, J. Neurosci. 17 (1997) 3064^3073.[60] B.H. Ni, X. Wu, Y.S. Du, Y. Su, E. Hamilton-Byrd, P.K.

Rockey, P. Rosteck Jr., G.G. Poirier, S.M. Paul, J. Neuro-sci. 17 (1997) 1561^1569.

[61] R.P. Xiao, H.H. Valdivia, K. Bogdanov, C. Valdivia, E.G.Lakatta, H.P. Cheng, J. Physiol. (Lond.) 500 (1997) 343^354.

[62] J.B. Schulz, M. Weller, T. Klockgether, J. Neurosci. 16(1996) 4696^4706.

[63] H. Hara, R.M. Friedlander, V. Gagliardini, C. Ayata, K.Fink, Z.H. Huang, M. Shimizu-Sasamata, J.Y. Yuan,M.A. Moskowitz, Proc. Natl. Acad. Sci. U.S.A. 94 (1997)2007^2012.

[64] S.A. Loddick, A. MacKenzie, N.J. Rothwell, Neuroreport 7(1996) 1465^1468.

[65] F.J. Gottron, H.S. Ying, D.W. Choi, Mol. Cell. Neurosci. 9(1997) 159^169.

[66] R.C. Armstrong, T.J. Aja, K.D. Hoang, S. Gaur, X. Bai,E.S. Alnemri, G. Litwack, D.S. Karanewsky, L.C. Fritz,K.J. Tomaselli, J. Neurosci. 17 (1997) 553^562.

[67] L. Kiedrowski, G. Brooker, E. Costa, J.T. Wroblewski, Neu-ron 12 (1994) 295^300.

[68] M. Erecinska, D. Nelson, J. Deas, I.A. Silver, Brain Res. 726(1996) 153^159.

[69] J.G. Greene, J.T. Greenamyre, Prog. Neurobiol. 48 (1996)613^621.

[70] R.C. Henneberry, in: M.F. Beal, N. Howell, I. Bodis-Woll-ner (Eds.), Mitochondria and Free Radicals in Neurodegen-erative Disease, Wiley-Liss, New York, 1997, pp. 111^143.

[71] F. Blandini, R.H.P. Porter, J.T. Greenamyre, Mol. Neuro-biol. 12 (1996) 73^94.

[72] E. Brouillet, P. Hantraye, R.J. Ferrante, R. Dolan, A. LeroyWillig, N.W. Kowall, M.F. Beal, Proc. Natl. Acad. Sci.U.S.A. 92 (1995) 7105^7109.

[73] A. Novelli, J.A. Reilly, P.G. Lysko, R.C. Henneberry, BrainRes. 451 (1988) 205^212.

[74] A.M. Marini, J.P. Schwartz, I.J. Kopin, J. Neurosci. 9(1989) 3665^3672.

[75] P.G. Lysko, J.A. Cox, M.A. Vigano, R.C. Henneberry,Brain Res. 499 (1989) 258^266.

[76] J.G. Greene, J.T. Greenamyre, J. Neurochem. 64 (1995)2332^2338.

[77] R. Henshaw, B.G. Jenkins, J.B. Schulz, R.J. Ferrante, N.W.Kowall, B.R. Rosen, M.F. Beal, Brain Res. 647 (1994) 161^166.

[78] M.F. Beal, E. Brouillet, B. Jenkins, R. Henshaw, B. Rosen,B.T. Hyman, J. Neurochem. 61 (1993) 1147^1150.

[79] R.A. Kauppinen, D.G. Nicholls, J. Neurochem. 47 (1986)1864^1869.

[80] A. Schurr, R.S. Payne, J.J. Miller, B.M. Rigor, Brain Res.744 (1997) 105^111.

[81] M. Villalba, A. Mart|nez-Serrano, P. Gomez-Puertas, P.Blanco, C. Bo«rner, A. Villa, M. Casado, C. Gimenez, R.Pereira, E. Bogonez, T. Pozzan, J. Satrustegui, J. Biol.Chem. 269 (1994) 2468^2476.

[82] C. Vicario, C. Arizmendi, G. Malloch, J.B. Clark, J.M.Medina, J. Neurochem. 57 (1991) 1700^1707.

[83] S. Eimerl, M. Schramm, J. Neurochem. 65 (1995) 739^743.[84] T. Tabatabaie, J.D. Potts, R.A. Floyd, Arch. Biochem. Bio-

phys. 336 (1996) 290^296.[85] M. Patel, B.J. Day, J.D. Crapo, I. Fridovich, J.O. McNa-

mara, Neuron 16 (1996) 345^355.[86] G.P. Davey, J.B. Clark, J. Neurochem. 66 (1996) 1617^1624.[87] J.P. Sheehan, R.H. Swerdlow, W.D. Parker, S.W. Miller,

R.E. Davis, J.B. Tuttle, J. Neurochem. 68 (1997) 1221^1233.[88] J.P. Sheehan, R.H. Swerdlow, S.W. Miller, R.E. Davis, J.K.

Parks, W.D. Parker, J.B. Tuttle, J. Neurosci. 17 (1997)4612^4622.

BBABIO 44667 30-7-98


[89] A. Atlante, S. Gagliardi, G.M. Minervini, E. Marra, S.Passarella, P. Calissano, Neuroreport 7 (1996) 2519^2523.

[90] M.J. Courtney, J.J. Lambert, D.G. Nicholls, J. Neurosci.10 (1990) 3873^3879.

[91] T.-I. Peng, J.T. Greenamyre, Soc. Neurosci. Abstr. 23(1997) 685.19.

[92] B.I. Khodorov, V.G. Pinelis, T. Storozhevykh, O.V. Ver-gun, N.P. Vinskaya, FEBS Lett. 393 (1996) 135^138.

[93] A.K. Stout, H.M. Raphael, J.M. Scanlon, I.J. Reynolds,Soc. Neurosci. Abstr. 23 (1997) 650.6.

[94] S. Tan, Y. Sagara, D. Schubert, Soc. Neurosci. Abstr. 23(1997) 542.1.

[95] J.J. Vornov, J. Neurochem. 65 (1995) 1681^1691.[96] V.P. Skulachev, Q. Rev. Biophys. 29 (1996) 169^202.[97] A. Boveris, N. Oshino, B. Chance, Biochem. J. 128 (1972)

617^630.[98] A. Herrero, G. Barja, J. Bioenerg. Biomembr. 29 (1997)

241^249.[99] J.A. Dykens, J. Neurochem. 63 (1994) 584^591.

[100] I.J. Reynolds, T.G. Hastings, J. Neurosci. 15 (1995) 3318^3327.

[101] M. Cino, R.F. Del Maestro, Arch. Biochem. Biophys. 269(1989) 623^638.

[102] G.J. Wang, R.D. Randall, S.A. Thayer, J. Neurophysiol. 72(1994) 2563^2569.

[103] Z. Hartley, J.M. Dubinsky, J. Neurosci. 13 (1993) 4690^4699.

[104] J.M. Scanlon, E. Aizenman, I.J. Reynolds, Eur. J. Pharma-col. 326 (1997) 67^74.

[105] N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin, A.Macho, T. Hirsch, S.A. Susin, P.X. Petit, B. Mignotte, G.Kroemer, J. Exp. Med. 182 (1995) 367^377.

[106] M. Castedo, A. Macho, N. Zamzami, T. Hirsch, P. Mar-chetti, J. Uriel, G. Kroemer, Eur. J. Immunol. 25 (1995)3277^3284.

[107] A. Macho, M. Castedo, P. Marchetti, J.J. Aguilar, D. De-caudin, N. Zamzami, P.M. Girard, J. Uriel, G. Kroemer,Blood 86 (1995) 2481^2487.

[108] V.P. Bindokas, J. Jordan, C.C. Lee, R.J. Miller, J. Neuro-sci. 16 (1996) 1324^1336.

[109] L.L. Dugan, S.L. Sensi, L.M.T. Canzoniero, S.D. Handran,S.M. Rothman, T.S. Lin, M.P. Goldberg, D.W. Choi,J. Neurosci. 15 (1995) 6377^6388.

[110] N.W. Kooy, J.A. Royall, H. Ischiropoulos, J.S. Beckman,Free Radic. Biol. Med. 16 (1994) 149^156.

[111] L.M. Henderson, J.B. Chappell, Eur. J. Biochem. 217(1993) 973^980.

[112] P.X. Petit, S.A. Susin, N. Zamzami, B. Mignotte, G.Kroemer, FEBS Lett. 396 (1996) 7^13.

[113] R.A. Altschuld, C.M. Hohl, L.C. Castillo, A.A. Garleb,R.C. Starling, G.P. Brierley, Am. J. Physiol. 262 (1992)H1699^H1704.

[114] E.J. Gri¤ths, A.P. Halestrap, J. Mol. Cell Cardiol. 25(1993) 1461^1469.

[115] E.J. Gri¤ths, A.P. Halestrap, Biochem. J. 307 (1995) 93^98.

[116] G.E. Kass, M.J. Juedes, S. Orrenius, Biochem. Pharmacol.44 (1992) 1995^2003.

[117] J.G. Pastorino, J.W. Snyder, J.B. Hoek, J.L. Farber, Am.J. Physiol. 268 (1995) C676^85.

[118] P. Bernardi, V. Petronilli, J. Bioenerg. Biomembr. 28 (1996)131^138.

[119] B.S. Kristal, J.M. Dubinsky, J. Neurochem. 69 (1997) 524^538.

[120] M. Ankarcrona, J.M. Dypbukt, S. Orrenius, P. Nicotera,FEBS Lett. 394 (1996) 321^324.

[121] R.G. Victor, G.D. Thomas, E. Marban, B. O'Rourke,Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6269^6273.

[122] Y.F. Lu, K. Tomizawa, A. Moriwaki, Y. Hayashi, M. To-kuda, T. Itano, O. Hatase, H. Matsui, Brain Res. 729(1996) 142^146.

[123] J.W. McDonald, M.I. Behrens, C. Chung, T. Bhattachar-yya, D.W. Choi, Brain Res. 759 (1997) 228^232.

[124] J.W. McDonald, M.P. Goldberg, B.J. Gwag, S.I. Chi,D.W. Choi, Ann. Neurol. 40 (1996) 750^758.

[125] A. Nicolli, E. Basso, V. Petronilli, R.M. Wenger, P. Ber-nardi, J. Biol. Chem. 271 (1996) 2185^2192.

[126] N. Brustovetsky, M. Klingenberg, Biochemistry 35 (1996)8483^8488.

[127] J. Yang, X.S. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J.Y.Cai, T.I. Peng, D.P. Jones, X.D. Wang, Science 275 (1997)1129^1132.

[128] R.M. Kluck, E. Bossy-Wetzel, D.R. Green, D.D. New-meyer, Science 275 (1997) 1132^1136.

[129] P. Bernardi, K.M. Broekemeier, D.R. Pfei¡er, J. Bioenerg.Biomembr. 26 (1994) 509^518.

BBABIO 44667 30-7-98


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