View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1366 (1998) 97^112 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 cerebellar granule cells to continuous glutamate exposure is characterised by a transient elevation in cytoplasmic free calcium concentration followed by decay to a plateau as NMDA receptors partially inactivate. After a variable latent period, a secondary, irreversible increase in calcium occurs (delayed calcium deregulation, DCD) which precedes and predicts subsequent cell death. DCD is not controlled by mitochondrial ATP synthesis since it is unchanged in the presence of the ATP synthase inhibitor oligomycin in cells with active glycolysis. However, mitochondrial depolarisation (and hence inhibition of mitochondrial calcium accumulation) without parallel ATP depletion (oligomycin plus either rotenone or antimycin A) strongly protects the cells against DCD. Glutamate exposure is associated with an increase in the generation of superoxide anion by the cells, but superoxide generation in the absence of mitochondrial calcium accumulation is not neurotoxic. While it is concluded that mitochondrial calcium accumulation plays a critical role in the induction of DCD we can find no evidence for the involvement of the mitochondrial permeability transition. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Mitochondrion; Glutamate; NMDA; Excitotoxicity; Calcium; Neuron 1. Introduction crotic or apoptotic characteristics and can occur with a delay of a few minutes to 1^2 days. Furthermore, a Even in neurones, where the ATP requirement for less severe, but chronic, restriction in neuronal ATP ionic homeostasis is particularly high, the safety mar- generation capacity may underlie a range of neuro- gin between cellular ATP supply capacity and de- degenerative disorders, including Alzheimer's, Hun- mand is considerable as long as glucose and oxygen tington's and Parkinson's diseases, amyotrophic lat- are available in excess. However even a brief inter- eral sclerosis and AIDs-related dementia as well as ruption in ATP generation, as in transient global encephalopathies associated with mitochondrial mu- ischaemia, can initiate neurodegeneration, which de- tations (reviewed in [1]). The excitatory neurotrans- pending on the severity of the insult can show ne- mitter glutamate plays a central role in neuronal cell death associated with these neurodegenerative disor- ders, and the interaction between the NMDA-selec- * Corresponding author. Fax: +44 (1382) 667120; tive glutamate receptor and the mitochondrion forms E-mail: [email protected] 1 Present address: CNS Research Institute, LMRC First the basis of this review, which will focus on necrotic Floor, 221 Longwood Avenue, Brigham and Women's Hospital, cell death in neuronal culture. Boston, MA 02115, USA. The sequence of events culminating in excitotoxic 0005-2728 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S0005-2728(98)00123-6 BBABIO 44667 30-7-98 98 D.G. Nicholls, S.L. Budd / Biochimica et Biophysica Acta 1366 (1998) 97^112 cell death is initiated by the entry of Ca2 through analysis will focus on neuronal bioenergetics and will predominantly NMDA-selective glutamate receptors attempt to deconvolute the interactions between mi- activated by elevated extracellular glutamate. Gluta- tochondrial ATP synthesis, Ca2 accumulation and mate is not excitotoxic to anoxic cells as long as the generation of reactive oxygen species in cultured NMDA receptors are inactivated prior to the resto- neurones exposed to excitotoxic concentrations of ration of oxygen [2]. The two conditions of greatest glutamate. risk are ¢rstly the period when circulation is restored following transient global ischaemia and before the highly active glutamate transporters can re-accumu- 2. The initial glutamate exposure late the amino acid into neurones and glia, and sec- ondly in the penumbra surrounding a focal ischaemia In primary neuronal culture, the excitotoxic cas- where respiring neurones are exposed to high gluta- cade can be initiated by as little as 5 min exposure mate concentrations di¡using from the ischaemic to high glutamate concentrations in the absence of core. This latter region is of particular signi¢cance, Mg2 (to prevent voltage-dependent block of the since it demonstrates that glutamate exposure can NMDA receptor). There is overwhelming evidence destroy the bioenergetic integrity of neurones contin- that the entry of Ca2 into the cells predominantly uously supplied with glucose and oxygen. through NMDA-selective glutamate receptors during It is convenient to divide the excitotoxic process this period triggers the subsequent neurodegenera- into two stages: the initial exposure to glutamate; tion; however Ca2 loading via voltage-activated and the subsequent `latent' period, where the contin- Ca2 channels during KCl-depolarisation is much ued presence of glutamate is not obligatory and less excitotoxic and the reason for this selectivity is which culminates in the failure of cytoplasmic Ca2 the subject of current controversy. The possibilities homeostasis, termed by Tymianski `delayed Ca2 de- are that the absolute amount of Ca2 entering regulation' [3] and subsequent cell death (Fig. 1). Our through the NMDA receptor may greatly exceed that through voltage-activated Ca2 channels [4,5], or that Ca2 entering through the NMDA receptor is focussed onto a vulnerable excitotoxic locus within the cell [3,6]. When determined with high-a¤nity Ca2 indica- tors, such as fura-2, little di¡erence is seen in the 2 apparent elevation in [Ca ]c evoked with glutamate or KCl [7^9]; however the use of low a¤nity indica- tors of free Ca2 capable of reporting far higher concentrations before saturating has indicated that 2 glutamate-evoked [Ca ]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 2+ [Ca ]c transients Fig. 1. Phases of acute glutamate excitotoxicity. Neurones ex- At the termination of a transient glutamate expo- posed to glutamate show a transient elevation in cytoplasmic 2 sure, [Ca ]c tends to return to baseline as the cation 2 2 free Ca , [Ca ]c. Following this initial response, the signal de- is sequestered into internal organelles or is extruded cays to a plateau as NMDA receptors partially inactivate. The plateau is maintained for a variable time (`latent period') until across the plasma membrane. Our early studies with 2 isolated mitochondria from liver and brain demon- a secondary, irreversible, increase in [Ca ]c occurs (delayed Ca2 deregulation). strated that the activity of the mitochondrial Ca2 BBABIO 44667 30-7-98 D.G. Nicholls, S.L. Budd / Biochimica et Biophysica Acta 1366 (1998) 97^112 99 uniporter was highly dependent on the extramito- ganglion cells exposed to a range of metabolic in- 2 2 chondrial Ca concentration, [Ca ]o [11] and ex- hibitors, including protonophores, cyanide and glu- ceeded the activity of the independent mitochondrial cose removal, but noted that these e¡ects could be Ca2 e¥ux pathway (coupled to H and Na in liver a consequence of impaired Ca2 extrusion from 2 and brain, respectively [12]) when [Ca ]o rose above the cells as well as inhibited mitochondrial sequestra- the `set-point' at which uptake and e¥ux balanced tion. [13]. Mitochondria would therefore be predicted au- In a detailed series of papers, White and Reynolds 2 2 tomatically to bu¡er [Ca ]c above the set-point and [9,22,23] have analysed the pathways of Ca remov- 2 to release it back into the cytoplasm when [Ca ]c al from the cytoplasm following acute, non-toxic, 2 recovered to below this value (reviewed in [12]). With glutamate exposure. Restoration of baseline [Ca ]c a predicted set-point of 0.3^0.5 WM [13] even the following as little as 15 s exposure to 3 WM glutamate 2 Ca transients measured with high-a¤nity indica- was highly dependent upon both vim and extracel- tors during KCl stimulation rise well above these lular Na ; thus no recovery occurred during wash- values and should therefore be in£uenced by mito- out of glutamate by a Na- free medium containing chondrial Ca2 sequestration. In 1981, we obtained protonophore. Interestingly, when the same Ca2 direct evidence of mitochondrial sequestration of de- transient was generated by the combination of high polarisation-evoked Ca2 loads by measuring mito- KCl and veratridine (activating voltage-activated chondrial 45Ca2 pools within synaptosomes: Ca2 Ca2 channels and allowing Na entry via voltage- 2 loading of these intact isolated terminals by KCl-ac- activated Na channels), recovery of [Ca ]c to tivation of voltage-activated Ca2 channels resulted baseline could still occur. This was interpreted as in the large majority of the accumulated Ca2 being indicating a fundamental di¡erence in the handling further transported into the matrix [14,15]. of glutamate-evoked versus KCl/veratridine-evoked In 1990, Thayer and Miller [16] showed that brief Ca2 loads. While the reason for this di¡erence KCl-mediated depolarisation of dorsal root ganglion was not directly investigated, a likely explanation 2 2 cells was followed by a partial recovery in [Ca ]c to was considered to be a greater uptake of Ca into a plateau which varied from 200 to 600 nM, but was the cell (and hence the mitochondrion) in the former absent in cells depolarised in the presence of proton- condition. ophore. This plateau was interpreted to be a conse- quence of the slow release from the mitochondrion of 2.2. In situ mitochondrial membrane potential (vim) 2 2 2+ Ca temporarily accumulated at the peak [Ca ]c.