M W Ward

University of Dundee, Dundee, Scotland, United Kingdom

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Publications (7)45.39 Total impact

  • A C Rego, M W Ward, D G Nicholls
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    ABSTRACT: Although mitochondria mediate the delayed failure of cytoplasmic Ca(2+) homeostasis [delayed Ca(2+) deregulation (DCD)] in rat cerebellar granule cells resulting from chronic activation of NMDA receptors, their role in AMPA/KA-induced DCD remains to be established. The mitochondrial ATP synthase inhibitor oligomycin protected cells against KA- but not NMDA-evoked DCD. In contrast to NMDA-evoked DCD, no additional protection was afforded by the further addition of rotenone. The effects of KA on cytoplasmic Ca(2+) homeostasis, including the protection afforded by oligomycin, could be reproduced by veratridine. KA exposure induced a partial mitochondrial depolarization that was enhanced by oligomycin, indicating ATP synthase reversal. The nonglycolytic substrates pyruvate and lactate were unable to maintain Ca(2+) homeostasis in the presence of KA. In contrast to NMDA, KA exposure did not cause mitochondrial Ca(2+) loading. The data indicate that Na(+) entry via noninactivating AMPA/KA receptors or voltage-activated Na(+) channels compromises mitochondrial function sufficiently to cause ATP synthase reversal. Oligomycin may protect by preventing the consequent mitochondrial drain of cytoplasmic ATP.
    Journal of Neuroscience 04/2001; 21(6):1893-901. · 6.91 Impact Factor
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    ABSTRACT: The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.
    Journal of Neuroscience 10/2000; 20(19):7208-19. · 6.91 Impact Factor
  • D G Nicholls, M W Ward
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    ABSTRACT: In the past few years it has become apparent that mitochondria have an essential role in the life and death of neuronal and non-neuronal cells. The central mitochondrial bioenergetic parameter is the protonmotive force, Deltap. Much research has focused on the monitoring of the major component of Deltap, the mitochondrial membrane potential Deltapsim, in intact neurones exposed to excitotoxic stimuli, in the hope of establishing the causal relationships between cell death and mitochondrial dysfunction. Several fluorescent techniques have been used, and this article discusses their merits and pitfalls.
    Trends in Neurosciences 05/2000; 23(4):166-74. · 13.58 Impact Factor
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    ABSTRACT: 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.
    Journal of Neurochemistry 05/1999; 72(4):1394-401. · 3.97 Impact Factor
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    ABSTRACT: 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.
    Biochemical Society Symposium 02/1999; 66:55-67. · 2.74 Impact Factor
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    ABSTRACT: The bioenergetic properties of the in situ mitochondria play a central role in controlling the susceptibility of neurons to acute or chronic neurodegenerative stress. The mitochondrial membrane potential, delta psi m is the parameter that controls three interrelated mitochondrial functions of great relevance to neuronal survival: namely, ATP synthesis, Ca2+ accumulation, and superoxide generation. The in vitro model we study is the rat cerebellar granule cell in primary culture and its susceptibility to NMDA receptor-mediated necrosis, which is preceded by a delayed failure of cytoplasmic Ca2+ homeostasis ("delayed Ca2+ deregulation," DCD). DCD is not caused by a failure of mitochondrial ATP synthesis since it also occurs in cells maintained purely by glycolysis. The in situ mitochondria maintain a delta psi m sufficient for ATP synthesis throughout the exposure of the cells to glutamate until DCD occurs. Even at that stage it appears that mitochondrial depolarization may be an effect of DCD rather than a primary cause. This somewhat unorthodox view resolves a number of apparent paradoxes, such as observations of enhanced superoxide generation by in situ mitochondria during excitotoxic exposure, since isolated mitochondria generate superoxide only under conditions of high delta psi m. Mitochondrial depolarization by selective inhibitors that do not deplete cellular ATP is acutely neuroprotective.
    Annals of the New York Academy of Sciences 02/1999; 893:1-12. · 4.38 Impact Factor
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    ABSTRACT: Mitochondria within cultured rat cerebellar granule cells have a complex influence on cytoplasmic free Ca2+ ([Ca2+]c) responses to glutamate. A decreased initial [Ca2+]c elevation in cells whose mitochondria are depolarized by inhibition of the ATP synthase and respiratory chain (conditions which avoid ATP depletion) was attributed to enhanced Ca2+ extrusion from the cell rather than inhibited Ca2+ entry via the NMDA receptor. Even in the presence of elevated extracellular Ca2+, when [Ca2+]c responses were restored to control values, such cells showed resistance to acute excitotoxicity, defined as a delayed cytoplasmic Ca2+ deregulation (DCD) during glutamate exposure. DCD was a function of the duration of mitochondrial polarization in the presence of glutamate rather than the total period of glutamate exposure. Once initiated, DCD could not be reversed by NMDA receptor inhibition. In the absence of ATP synthase inhibition, respiratory chain inhibitors produced an immediate Ca2+ deregulation (ICD), ascribed to an ATP deficit. In contrast to DCD, ICD could be reversed by subsequent ATP synthase inhibition with or without additional NMDA receptor blockade. DCD could not be ascribed to the failure of an ATP yielding metabolic pathway. It is concluded that mitochondria can control Ca2+ extrusion from glutamate-exposed granule cells by the plasma membrane in three ways: by competing with efflux pathways for Ca2+, by restricting ATP supply, and by inducing a delayed failure of Ca2+ extrusion. Inhibitors of the mitochondrial permeability transition only marginally delayed the onset of DCD.
    Journal of Neuroscience 01/1999; 18(24):10277-86. · 6.91 Impact Factor