Methylmalonate inhibits succinate-supported oxygen consumption by interfering with mitochondrial succinate uptake.

The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.

Oxidative stress in Ca(2+)-induced membrane permeability transition in brain mitochondria.

Mitochondrial permeability transition (PT) is a non-selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca(2+) ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca(2+)-induced brain mitochondrial PT was stimulated by Na(+) (10 mM) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca(2+) from the mitochondrial suspension by EGTA or inhibition of Ca(2+) uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca(2+)-induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca(2+) uptake. Ca(2+)-induced dichlorodihydrofluorescein oxidation was enhanced by Na(+) and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca(2+)-induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca(2+)-induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.

Suramin inhibits respiration and induces membrane permeability transition in isolated rat liver mitochondria.

Suramin, a polysulfonated naphthylamine, caused a dose dependent inhibition of carbonyl cyanide p-(tri-fluoromethoxy)phenylhydrazone-stimulated respiration supported either by succinate or a cocktail of alphaketoglutarate, malate and isocitrate in isolated rat liver mitochondria. The half-maximum effect was obtained at 40 and 140 microM suramin for NADH- or FADH(2)-linked substrates, respectively. The respiration supported by N,N,N'N'-tetramethyl-p-phenylenediamine oxidation was unaffected by suramin (

Mice transgenic for exon 1 of the Huntington's disease gene display reduced striatal sensitivity to neurotoxicity induced by dopamine and 6-hydroxydopamine.

Huntington's disease is an autosomal dominant hereditary neurodegenerative disorder characterized by severe striatal cell loss. Dopamine (DA) has been suggested to play a role in the pathogenesis of the disease. We have previously reported that transgenic mice expressing exon 1 of the human Huntington gene (R6 lines) are resistant to quinolinic acid-induced striatal toxicity. In this study we show that with increasing age, R6/1 and R6/2 mice develop partial resistance to DA- and 6-hydroxydopamine-mediated toxicity in the striatum. Using electron microscopy, we found that the resistance is localized to the cell bodies and not to the neuropil. The reduction of dopamine and cAMP regulated phosphoprotein of a molecular weight of 32 kDa (DARPP-32) in R6/2 mice does not provide the resistance, as DA-induced striatal lesions are not reduced in size in DARPP-32 knockout mice. Neither DA receptor antagonists nor a N-methyl-d-aspartate (NMDA) receptor blocker reduce the size of DA-induced striatal lesions, suggesting that DA toxicity is not dependent upon DA- or NMDA receptor-mediated pathways. Moreover, superoxide dismutase-1 overexpression, monoamine oxidase inhibition and the treatment with the free radical scavenging spin-trap agent phenyl-butyl-tert-nitrone (PBN) also did not block DA toxicity. Levels of the antioxidant molecules, glutathione and ascorbate were not increased in R6/1 mice. Because damage to striatal neurons following intrastriatal injection of 6-hydroxydopamine was also reduced in R6 mice, a yet-to-be identified antioxidant mechanism may provide neuroprotection in these animals. We conclude that striatal neurons of R6 mice develop resistance to DA-induced toxicity with age.

Resistance to NMDA toxicity correlates with appearance of nuclear inclusions, behavioural deficits and changes in calcium homeostasis in mice transgenic for exon 1 of the huntington gene.

Transgenic Huntington's disease (HD) mice, expressing exon 1 of the human HD gene (lines R6/1 and R6/2), are totally resistant to striatal lesions caused by the NMDA receptor agonist quinolinic acid (QA). Here we show that this resistance develops gradually over time in both R6/1 and R6/2 mice, and that it occurred earlier in R6/2 (CAG-155) than in R6/1 (CAG-115) mice. The development of the resistance coincided with the appearance of nuclear inclusions and with the onset of motor deficits. In the HD mice, hippocampal neurons were also resistant to QA, especially in the CA1 region. Importantly, there was no change in susceptibility to QA in transgenic mice with a normal CAG repeat (CAG-18). R6/1 mice were also resistant to NMDA-, but not to AMPA-induced striatal damage. Interestingly, QA-induced current and calcium influx in striatal R6/2 neurons were not decreased. However, R6/2 neurons had a better capacity to handle cytoplasmic calcium ([Ca2+]c) overload following QA and could avoid [Ca2+]c deregulation and cell lysis. In addition, basal [Ca2+]c levels were increased five-fold in striatal R6/2 neurons. This might cause an adaptation of R6 neurons to excitotoxic stress resulting in an up-regulation of defense mechanisms, including an increased capacity to handle [Ca2+]c overload. However, the increased level of basal [Ca2+]c in the HD mice might also disturb intracellular signalling in striatal neurons and thereby cause neuronal dysfunction and behavioural deficits.

Partial resistance to malonate-induced striatal cell death in transgenic mouse models of Huntington's disease is dependent on age and CAG repeat length.

Transgenic Huntington's disease (HD) mice, expressing exon 1 of the HD gene with an expanded CAG repeat, are totally resistant to striatal lesion induced by excessive NMDA receptor activation. We now show that striatal lesions induced by the mitochondrial toxin malonate are reduced by 70-80% in transgenic HD mice compared with wild-type littermate controls. This occurred in 6- and 12-week-old HD mice with 150 CAG repeats (line R6/2) and in 18-week-old, but not 6-week-old, HD mice with 115 CAG repeats (line R6/1). Therefore, we show for the first time that the resistance to neurotoxin in transgenic HD mice is dependent on both the CAG repeat length and the age of the mice. Importantly, most HD patients develop symptoms in adulthood and exhibit an inverse relationship between CAG repeat length and age of onset. Transgenic mice expressing a normal CAG repeat (18 CAG) were not resistant to malonate. Although endogenous glutamate release has been implicated in malonate-induced cell death, glutamate release from striatal synaptosomes was not decreased in HD mice. Malonate-induced striatal cell death was reduced by 50-60% in wild-type mice when they were treated with either the NMDA receptor antagonist MK-801 or the caspase inhibitor zVAD-fmk. These two compounds did not reduce lesion size in transgenic R6/1 mice. This might suggest that NMDA receptor- and caspase-mediated cell death pathways are inhibited and that the limited malonate-induced cell death still occurring in HD mice is independent of these pathways. There were no changes in striatal levels of the two anti cell death proteins Bcl-X(L) and X-linked inhibitor of apoptosis protein (XIAP), before or after the lesion in transgenic HD mice. We propose that mutant huntingtin causes a sublethal grade of metabolic stress which is CAG repeat length-dependent and results in up-regulation over time of cellular defense mechanisms against impaired energy metabolism and excitotoxicity.

Ca2+ induces a cyclosporin A-insensitive permeability transition pore in isolated potato tuber mitochondria mediated by reactive oxygen species.

Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis-trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.

Mitochondrial permeability transition and oxidative stress.

Mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that may precede necrotic and apoptotic cell death. Although this process has a specific inhibitor, cyclosporin A, little is known about the nature of the proteinaceous pore that results in MPT. Here, we review data indicating that MPT is not a consequence of the opening of a pre-formed pore, but the consequence of oxidative damage to pre-existing membrane proteins.

The iron chelator pyridoxal isonicotinoyl hydrazone inhibits mitochondrial lipid peroxidation induced by Fe(II)-citrate.

Pyridoxal isonicotinoyl hydrazone (PIH) is able to prevent iron-mediated hydroxyl radical formation by means of iron chelation and inhibition of redox cycling of the metal. In this study, we investigated the effect of PIH on Fe(II)-citrate-mediated lipid peroxidation and damage to isolated rat liver mitochondria. Lipid peroxidation was quantified by the production of thiobarbituric acid-reactive substances (TBARS) and by antimycin A-insensitive oxygen consumption. PIH at 300 microM induced full protection against 50 microM Fe(II)-citrate-induced loss of mitochondrial transmembrane potential (deltapsi) and mitochondrial swelling. In addition, PIH prevented the Fe(II)-citrate-dependent formation of TBARS and antimycin A-insensitive oxygen consumption. The antioxidant effectiveness of 100 microM PIH (on TBARS formation and mitochondrial swelling) was greater in the presence of 20 or 50 microM Fe(II)-citrate than in the presence of 100 microM Fe(II)-citrate, suggesting that the mechanism of PIH antioxidant action is linked with its Fe(II) chelating property. Finally, PIH increased the rate of Fe(II) autoxidation by sequestering iron from the Fe(II)-citrate complex, forming a Fe(III)-PIH, complex that does not participate in Fenton-type reactions and lipid peroxidation. These results are of pharmacological relevance since PIH is a potential candidate for chelation therapy in diseases related to abnormal intracellular iron distribution and/or iron overload.

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore.

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.

FK506 and cyclosporin A enhance the survival of cultured and grafted rat embryonic dopamine neurons.

We examined the effects of the immunophilin ligands and calcineurin inhibitors FK506 and cyclosporin A on the survival of rat embryonic dopamine (tyrosine hydroxylase (TH)-immunoreactive) neurons. The protective effects of FK506 and cyclosporin A were first studied in dissociated mesencephalic cell cultures subjected to serum deprivation. Significant increases in both the total number of surviving mesencephalic cells and the number of surviving TH-immunoreactive neurons were observed when FK506 or cyclosporin A was present following withdrawal of serum from the culture medium. In a second series of experiments, FK506 increased the survival of dopamine neurons when added only to a hibernation medium in which donor tissue pieces were stored for 7 days prior to preparation of the cultures. In a third set of experiments, we investigated the effects of FK506 and cyclosporin A on the survival of grafted rat embryonic dopamine neurons. When FK506 or cyclosporin A was present during tissue preparation and in the final mesencephalic cell suspension used for grafting, the survival of TH-immunoreactive neurons implanted in the striatum increased to around 185% of control values. In contrast, treatment of graft recipient rats, but not the graft suspension itself, with immunosuppressive doses of FK506 or cyclosporin A did not augment the survival of grafted TH-immunoreactive neurons. We conclude that administration of FK506 during storage of embryonic mesencephalic tissue and FK506 or cyclosporin A during preparation of nigral cell suspensions used for grafting can increase the survival of grafted embryonic dopamine neurons.

Additive effects of caspase inhibitor and lazaroid on the survival of transplanted rat and human embryonic dopamine neurons.

Major practical constraints on neural grafting in Parkinson's disease are the shortage of human donor tissue and the great loss of dopamine neurons during the grafting procedure. The vast majority of implanted embryonic dopamine neurons are believed to die within a few days of transplantation surgery, at least in part through apoptosis. We have previously found that survival of nigral grafts in rodents can be significantly augmented by pretreatment with the caspase inhibitor Ac-YVAD-cmk or by lazaroids (lipid peroxidation inhibitors). We now report that pretreatment with the caspase inhibitor Ac-DEVD-cmk, but not z-VAD-fmk, results in a significantly improved survival of transplanted dopamine neurons of similar magnitude to that achieved in this study using Ac-YVAD-cmk (both 220-230% of control). In addition, we found that treatment of the graft tissue with tirilazad mesylate (a lazaroid allowed for clinical use) almost doubled the survival of grafted dopamine neurons. When Ac-YVAD-cmk and tirilazad mesylate treatments were combined, the number of surviving dopamine neurons increased significantly further to 280% of control. Importantly, the same combination of neuroprotectants enhanced the survival of human dopamine neurons xenotransplanted to immunosuppressed rats (to 240% of control). In conclusion, these results suggest that combining treatments that counteract oxidative stress and caspase activation is a valuable strategy to enhance nigral graft survival that should be considered for clinical application.

Improving the survival of grafted dopaminergic neurons: a review over current approaches.

Neural transplantation is developing into a therapeutic alternative in Parkinson's disease. A major limiting factor is that only 3-20% of grafted dopamine neurons survive the procedure. Recent advances regarding how and when the neurons die indicate that events preceding actual tissue implantation and during the first week thereafter are crucial, and that apoptosis plays a pivotal role. Triggers that may initiate neuronal death in grafts include donor tissue hypoxia and hypoglycemia, mechanical trauma, free radicals, growth factor deprivation, and excessive extracellular concentrations of excitatory amino acids in the host brain. Four distinct phases during grafting that can involve cell death have been identified: retrieval of the embryo; dissection and preparation of the donor tissue; implantation procedure followed by the immediate period after graft injection; and later stages of graft maturation. During these phases, cell death processes involving free radicals and caspase activation (leading to apoptosis) may be triggered, possibly involving an increase in intracellular calcium. We review different approaches that reduce cell death and increase survival of grafted neurons, typically by a factor of 2-4. For example, changes in transplantation procedure such as improved media and implantation technique can be beneficial. Calcium channel antagonists such as nimodipine and flunarizine improve nigral graft survival. Agents that counteract oxidative stress and its consequences, such as superoxide dismutase overexpression, and lazaroids can significantly increase the survival of transplanted dopamine neurons. Also, the inhibition of apoptosis by a caspase inhibitor has marked positive effects. Finally, basic fibroblast growth factor and members of the transforming growth factor-beta superfamily, such as glial cell line-derived neurotrophic factor, significantly improve the outcome of nigral transplants. These recent advances provide hope for improved survival of transplanted neurons in patients with Parkinson's disease, reducing the need for human embryonic donor tissue and increasing the likelihood of a successful outcome.

Oxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187-induced death of cultured striatal neurons.

Disruption of intracellular calcium homeostasis is thought to play a role in neurodegenerative disorders such as Huntington's disease (HD). To study different aspects of putative pathogenic mechanisms in HD, we aimed to establish an in vitro model of calcium-induced toxicity in striatal neurons. The calcium ionophore A23187 induced a concentration- and time-dependent cell death in cultures of embryonic striatal neurons, causing both apoptosis and necrosis. Cell death was significantly reduced by the cell-permeant antioxidant manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP). Cyclosporin A and its analogue N-MeVal-4-cyclosporin also reduced the incidence of cell death, suggesting the participation of mitochondrial permeability transition in this process. Furthermore, addition of either of two types of caspase inhibitors, Ac-YVAD-CHO (acetyl-Tyr-Val-Ala-Asp-aldehyde) and Ac-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde), to the striatal cells blocked A23187-induced striatal cell death in a concentration-dependent manner. These results suggest that oxidative stress, opening of the mitochondrial permeability transition pore and activation of caspases are important steps in A23187-induced cell death.

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids.

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used (< or = 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.

Transgenic mice expressing a Huntington's disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder presenting with chorea, dementia, and extensive striatal neuronal death. The mechanism through which the widely expressed mutant HD gene mediates a slowly progressing striatal neurotoxicity is unknown. Glutamate receptor-mediated excitotoxicity has been hypothesized to contribute to the pathogenesis of HD. Here we show that transgenic HD mice expressing exon 1 of a human HD gene with an expanded number of CAG repeats (line R6/1) are strongly protected from acute striatal excitotoxic lesions. Intrastriatal infusions of the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid caused massive striatal neuronal death in wild-type mice, but no damage in transgenic HD littermates. The remarkable neuroprotection in transgenic HD mice occurred at a stage when they had not developed any neurological symptoms caused by the mutant HD gene. At this stage there was no change in the number of striatal neurons and astrocytes in untreated R6/1 mice, although the striatal volume was decreased by 17%. Moreover, transgenic HD mice had normal striatal levels of NMDA receptors, calbindin D28k (calcium buffer), superoxide dismutase activity (antioxidant enzyme), Bcl-2 (anti-apoptotic protein), heat shock protein 70 (stress-induced anti-apoptotic protein), and citrate synthase activity (mitochondrial enzyme). We propose that the presence of exon 1 of the mutant HD gene induces profound changes in striatal neurons that render these cells resistant to excessive NMDA receptor activation.

Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells.

On exposure to glutamate, cultured rat cerebellar granule cells undergo a delayed Ca2+ deregulation (DCD), which precedes and predicts cell death. We have previously shown that mitochondria control the sensitivity of the neurons to DCD. Mitochondrial depolarization by rotenone/oligomycin before glutamate addition is strongly neuroprotective, and the indication is therefore that mitochondrial Ca2+ loading leads to a delayed loss of bioenergetic function culminating in DCD and cell death. In this report it is shown that superoxide (O2.-) generation in intact cells, monitored by oxidation of hydroethidine to ethidium, was enhanced by glutamate only when mitochondria were polarized. Production of superoxide was higher in the subset of cells undergoing DCD. In the presence of rotenone and oligomycin, addition of glutamate did not result in increased superoxide generation. Menadione-generated superoxide enhances the DCD of cells exposed to glutamate; in contrast, glutamate-induced DCD was potently inhibited by the presence of the cell-permeant antioxidant manganese(III) tetrakis(4-benzoic acid) porphyrin. An inverse correlation is observed between the cytoplasmic free Ca2+ maintained in individual cells in the presence of glutamate and the ability of these cells to restore basal Ca2+ when NMDA receptors are inhibited and mitochondrial Ca2+ is released. It is concluded that mitochondrial Ca2+ accumulation and reactive oxygen species each contribute to DCD, probably related to damage to a process controlling Ca2+ efflux from the cell.

Inhibition of mitochondrial permeability transition by low pH is associated with less extensive membrane protein thiol oxidation.

Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.

Excitotoxicity and mitochondria.

Excitotoxicity is the process whereby a massive glutamate release in the central nervous system in response to ischaemia or related trauma leads to the delayed, predominantly necrotic death of neurons. Excitotoxicity is also implicated in a variety of slow neurodegenerative disorders. Mitochondria accumulate much of the post-ischaemic calcium entering the neurons via the chronically activated N-methyl-D-aspartate receptor. This calcium accumulation plays a key role in the subsequent death of the neuron. Cultured cerebellar granule cells demonstrate delayed calcium de-regulation (DCD) followed by necrosis upon exposure to glutamate. DCD is unaffected by the ATP synthase inhibitor oligomycin but is inhibited by the further addition of a respiratory chain inhibitor to depolarize the mitochondria and inhibit mitochondrial calcium accumulation without depleting ATP [Budd and Nicholls (1996) J. Neurochem. 67, 2282-2291]. Mitochondrial depolarization paradoxically decreases the cytoplasmic calcium elevation following glutamate addition, probably due to an enhanced calcium efflux from the cell. Cells undergo immediate calcium de-regulation in the presence of glutamate if the respiratory chain is inhibited; this is due to ATP depletion following ATP synthase reversal and can be reversed by oligomycin. In contrast, DCD is irreversible. Elevated cytoplasmic calcium is not excitotoxic as long as mitochondria are depolarized; alternative substrates do not rescue cells about to undergo DCD, suggesting that glycolytic failure is not involved. Mitochondria in situ remain sufficiently polarized during granule cell glutamate exposure to continue to generate ATP and show a classic mitochondrial state 3-state 4 hyperpolarization on inhibiting ATP synthesis; mitochondrial depolarization follows, and may be a consequence of rather than a cause of DCD. In addition, our studies show no evidence of the mitochondrial permeability transition prior to DCD. The mitochondrial generation of superoxide anions is enhanced during glutamate exposure and a working hypothesis is that DCD may be caused by oxidative damage to calcium extrusion pathways at the plasma membrane.