Nitric Oxide-Mediated Mitochondrial Damage in ... - Wiley Online Library

Journal of Neurorhemistrv Lippincott—Raven Publishers. Philadelphia 1997 International Society for Neurochemistry

Short Review

Nitric Oxide-Mediated Mitochondrial Damage in the Brain: Mechanisms and Implications for Neurodegenerative Diseases Juan P. Bolaños, Angeles Almeida, *Vjctorja Stewart, *Stephan Peuchen, *tJohn M. Land, *John B. Clark, and *~SjmonJ. R. Heales Departainento de Bio quImica *

y

Biologla Molecular, Facultad de Far,‘nacia, Universidad de Salainanca, Salamanca, Spain;

Department of Neurochemistry, institute of Neurology; and t Department of Clinical Biochemistry, National Hospital for Neurology and Neurosurgery, London, England

Abstract: Within the CNS and under normal conditions, nitric oxide (‘NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that ‘No is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances N0 synthesis may be excessive and N0 may become neurotoxic. Excessive glutamatereceptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both ‘No and superoxide (02‘) and hence peroxynitrite (ONOO formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of ‘No, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of ‘No neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy-consuming pathway involving poly(ADPribose) synthetase. Possible mechanisms whereby ONOO impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO production. lt is concluded that neurotoxicity elicited by excessive ‘NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state. Key Words: Nitric oxide— Mitochondria— Neurotoxicity—Glutathione—Neurodegeneration. J. Neurochem. 68, 2227—2240 (1997). )

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Within the CNS, nitric oxide (‘NO) is a highly diffusible, short-lived physiological messenger molecule (Garthwaite et al., 1988) whose metabolism and biological roles have been reviewed extensively (Moncada et al., 1991; Bredt and Snyder, 1994; Vincent, 1994; Hawkins, 1996). Despite its many diverse and

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important physiological functions, under certain circumtances ‘No may become neurotoxic (Dawson and Dawson, 1996). Although the neurotoxic effects of excessive N0 production are well confirmed, the mechanism of toxicity is not well understood. An increasing body of evidence suggests that dysfunction of cellular energy production is an important factor in ‘NO-mediated neurotoxicity (Schulz et al., 1995b). However, the exact mechanism whereby NO brings about this energy impairment is a matter of debate. In the present review, we discuss the effects of ‘No on CNS mitochondrial function, consider potential mechanisms of mitochondrial damage, and evaluate the relevance of such processes for our understanding of the etiology of neurodegenerative disorders. SYNTHESIS AND METABOLISM OF ‘NO IN CNS CELLS ‘NO is synthesized by various isoforms of nitric oxide synthase (NOS) that catalyse the conversion of arginine to citrulline and ‘NO (Bredt and Snyder, 1990; Knowles and Moncada, 1994). AIl CNS cells appear to have the ability to synthesize ‘No in vitro (Murphy et al., 1993; Murphy and Guzybicki, 1996). In general, neurones produce ‘NO mainly by a calcium-dependent activation of neuronal NOS (nNOS or NOS 1), which is expressed constitutively in these cells, whereas glial cells have the ability to synthesize ‘NO in a calciumAddress correspondence and reprint requests to Dr. S. J. R. Heales at Department of Clinical Biochemistry. National Hospital for Neurology and Neurosurgery, Queen Square, London WCIN 3BG. U.K. Abbreviations used: eNOS. endothelial nitric oxide synthase; GSH, glutathione; IFN, interferon: iNOS, inducible nitric oxide synthase; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; ‘NO, nitric oxide; NOS, nitric oxide synthase; O:‘ ‚ superoxide: 0N00, peroxynitrite; TMPD, tetramethylphenylenediamine.

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independent way that requires induction of NOS inducible NOS (iNOS or NOS2)], i.e., de flovo enzyme synthesis (Galea et al.. 1992; Simmons and Murphy. 1992). A third isoform of NOS [endothelial NOS (eNOS or NOS3)] has also been described in the CNS, associated with the brain vasculature (Murphy and Guzybicki, 1996). Activation of nNOS forms part of the cascade pathway triggered by glutamate-receptor activation that leads to intracellular cyclic GMP elevation (Garthwaite et al., 1988: Bredt and Snyder. 1989: Knowles et al., 1989). However, induction of INOS in glial cells (astrocytes and microglial cells) is associated usually with pathological conditions (Murphy and Guzybicki, 1996). However, it should be mentioned that neurones also appear to express iNOS (MuñozFernández et al., 1994; Kifle et al., 1996; Mine-Golomb et al., 1996), although further investigation is needed to establish its role. In addition, astrocyics also appear to synthesize ‘NO in a calcium-dependenl manner (Murphy et al., 1990, 1991; Agulló and Garcia. 1992a,b), leading to the formation of cyclic GMP. although the physiological role for this pathway remains to be established. The metabolism of ‘NO and regulation of the NOSs in CNS cells are summarized in Fig. 1.

FIG. 1. ‘No metabolism. A: Origin and fates of ‘NO. ‘NO is

synthesized by NOS, which forms ‘NO and L-citrulline from L.arginine. ‘NO is degraded rapidly because of its reactions with 02, nitrogen dioxide radical (‘NO 2), or superoxide anion (02‘). Thus, the ‘NO reaction with 02 forms ‘NO2, which is converted further into nitrite (NO2-) and nitrate (NO3 ); the reaction of ‘NO with ‘NO2 forms the anhydride of nitrous acid (N2O3), which is degraded further to N02 finally, ‘NO reacts with 02‘ to form the peroxynitrite anion (0N00), which is a powerful oxidant that may decompose further, on protonation, to the hydroxyl radical (‘OH) and ‘NO2. ‘OH and ‘NO2 can react to form nitric acid, but this occurs at a lower rate than other reactions of ‘OH (Butler et al., 1995). B: Regulation of ‘NO synthesis in CNS cells. 2‘ -dependent Activation the glutamate receptor Neurones producenNOS. ‘NO mainly by theofactivation of constitutive, subtype, N-methyl-D-aspartate (NMDA) triggers Ca2~influx, Ca which binds to calmodulin thereby activating nNOS. This mode of activation stimulates ‘NO formation within seconds, although it is limited because NOS activation ceases when the basal intracellular free Ca2~concentration is restored. Protein kinase C and cyclic AMP-, cyclic GMP (cGMP)-, and Ca27calmodulindependent protein kinases phosphorylate nNOS, leading to enzyme solubilization and inactivation. Astrocytes and microglial cells also synthesize ‘NO either by activation of constitutive nNOS after activation of the quisqualate receptor or by a Ca2 independent means that requires the previous induction of iNOS by lipopolysaccharide ([PS) either alone or in combination with certain cytokines such as interferon-y, tumour necrosis factora, or interleukin-lß (Lee et al., 1993; Simmons and Murphy, 1993; Bolaños et al., 1994; Feinstein et al., 1994a,b). The induction of NOS is regulated in astrocytes by tyrosine kinase, the activity of which is required for induction (Feinstein et al., 1994a; Simmons and Murphy, 1994), and NOS activity is suppressed by noradrenaline, cyclic AMP (Feinstein et al., 1993), interleukin4, interleukin-lO, and dexamethasone (Simmons and Murphy,

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NEUROTOXICITY OF NEURONAL-DERIVED ‘NO The different mechanisms for enhancing ‘NO synthesis in neurones and glial cells have prompted the suggestion that both nNOS and iNOS are involved in pathophysiological processes occurring withi n the CNS (Fig. 2). There is an increasing body of evidence to suggest that excitotoxicity, i.e., neurotoxicity by excessive exposure of neurones to glutamate. is, at least partially, ‘NO-mediated (Dawson et al., 1991. 1993: Schulz et al., 1995b; Dawson and Dawson. 1996). This involvement, which has been implicated in the etiology of neurodegenerative diseases (Turski and Turski, 1993; Ikonomidou and Turski, 1995; Schulz et al., l995a), appears to be a long-term phenomenon. However, brief exposure of neuronal cultures to glutamate-receptor agonists only transiently elevates intracellular calcium, which returns to basal levels within seconds. This short-term calcium elevation may initiate processes that contribute to delayed neurotoxicity. Although some laboratories advocate ‘NO involvement in this process, other laboratories have not been able to reproduce the ‘NO-mediated neurotoxicity of glutamate-receptor activation (Pauwels and Leysen. 1992: Garthwaite and Garthwaite. 1994: Maiese et al., 1995).

1993), suggesting that LPS-mediated iNOS induction requires

cytokine release. lnterleukin-1/1 seems to be essential for iNOS induction (Lee et al., 1993), suggesting an important role for this cytokine in the activation of astrocytes (Lee et al., 1995).

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FIG. 2. Mechanisms of ‘NO-mediated neurotoxicity. Excitotoxicity, i.e., neurotoxicity by excessive exposure of neurones to glutamate, is, at least partially, ‘NO-mediated. A brief exposure of neuronal cultures to glutamate-receptor agonists transiently elevates intracellular calcium, which is returned to basal levels within seconds. This short-term calcium elevation presumably initiates irreversible processes that no longer require a sustained calcium elevation, but cause a delayed neurotoxicity. The possible mechanisms leading to neuronal cell death involve peroxynitrite (ONOO) formation and, in general, it is accepted that energy failure is a key factor. Thus, ONOO initiates unspecific protein and lipid peroxidation, DNA damage with activation of the energyconsuming repair system involving poly(ADP-ribose) synthetase (PARS), intramitochondrial calcium accumulation, inhibition of aconitase activity, and the mitochondrial respiratory chain. iNOS induction in guai cells may also have a relevant contribution to ‘NO-mediated neurotoxicity. Thus, induction of iNOS is triggered by lipopolysaccharide ([PS) and/or certain cytokines in both astrocytes and microglial cells. Either ‘NO or ONOO inhibits mitochondrial respiratory chain in astrocytes, but these cells compensate by switching to glycolysis to maintain energy homeostasis. However, as ‘NO and ONOO are highly diffusible, these compounds may cause cell toxicity in the neighbouring neurones.

thus leaving the universal role of ‘NO in excitotoxicity in question. The mechanism of this discrepancy is a matter of debate. Moreover, the identification of the free radical species implicated in neurotoxicity is also controversial. In contrast to the report of Oury et al. (1992), who suggested that scavenging of superoxide (O2‘ I with superoxide dismutase enhances ‘NO neurotoxicity, evidence is now arising to suggest the contrary, i.e., excitotoxicity requires O2‘ (Patel et al., 1996) or ‘NO plus O:‘ formation (Lafon-Cazal et al., 1993; Gunasekar et al., 1995). As ‘NO avidly reacts with O- to form peroxynitrite (ONOO ) (Beckman et al., 1990), these results lead to the suggestion that ONO() formation in situ is required for excitotoxicity (Lipton et al., 1993) and that ‘NO alone is not neurotoxic. Furthermore, Lipton et al. (1993) suggest that the cell redox status may also determine whether ‘NO formation, following activation of the N-methyl-Daspartate (NMDA) receptor, is neurotoxic or even neuroprotective. An intracellular oxidizing environment favours formation of NO~(nitrosonium ion), which may down-regulate the NMDA receptor by S-nitrosylation (neuroprotection). In contrast, a reducing intracellular environment favours reduction of NO ~ to ‘NO, which does not react with the thiol groups of the redox modulatory site of the NMDA receptor (neurotoxicity). Manipulation of the redox status in a particular cell culture system, therefore, might influence the sensitivity of neurones to ‘NO-mediated excitotoxicity. Discrepancies regarding the role of ‘NO in excitotoxicity may also be related to the number of NOS-positive neurones that form the cell culture population (Dawson et al.. 1993). Thus, NMDA-mediated toxicity is not evident, in cortical neurones, until day 17—21 in culture (Koh and Choi, 1988; Dawson et al., 1993), when

the number of NOS-positive neurones is greatly increased. These neurones are larger than NOS-negative neurones and establish cell contacts with the majority of the neurones in the culture (Dawson et al., 1993). Dawson et al. (1993) suggest that only NOS-positive neurones release high amounts of ‘NO after NMDA stimulation and kill neighbouring NOS-negative neurones, whereas NOS-positive neurones remain undamaged. Although the mechanism of this selective resistance is not yet understood, these authors speculate that a putative higher superoxide dismutase activity in NOS-positive neurones might avoid O2‘ formation and hence limit ONOO formation in situ. However, alternative mechanisms may also be suggested. The intracellular reduced glutathione (GSH) concentration appears to be a key factor at protecting both neurones and astrocytes from ONOO -mediated neurotoxicity (Bolanos et al., 1995, 1996; Barker et al., 1996). Whether NOS-positive neurones possess higher GSH concentrations than NOS-negative neurones remains to be elucidated. NEUROTOXICITY OF GLIAL-DERIVED ‘No A role for glial-derived ‘NO in neurotoxicity has also been suggested. Microglial cells, which express iNOS (Corradin et al., 1993). have been considered to be cytotoxic to other cell types in close proximity (Théry et al., 1991: Banati et al.. 1993). Boje and Arora (1992) demonstrated that microglial-derived ‘NO following induction of iNOS is neurotoxic. Subsequently, Menill et al. (1993) demonstrated that ‘NO mediates microglial cytotoxicity of oligodendrocytes, suggesting a role for ‘NO in the pathogenesis of multiple sclerosis. Astrocytes have also been implicated in ‘NO-mediated neurotoxicity. .1. Neuroshenr, Vol. 68, No. 6, / 997

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TABLE 1. Interference of ‘NO with imtochondrial furtctioit System examined“

Mechanism

t. Derived from CNS tissue Astrocytes Astrocytes and oligodendrocytes Astrocytes Brain mitochondria Synaptosomes Neurones Brain mitochoudria Synaptosomes Astrocytes Synaplosomes Brain submitochondrial particles Astrocyles

iNOS inductioim inluhits complex Il—Ill and lv activities ‘NC) donor inhibits succinate dehydrogenase activity Trolox, a vitamin E analogue, protects complex lv from iNOS—mediated rlanmage ‘NO reversibly inhibits oxygen consumption ‘NO reversibly inhibits oxygen consumption by competing with oxygen at the level of complex lv ONOO~ inhibits complex Il—Ill and IV activities 0N00 inhibits complex Il—Ill activity ‘NO donors decrease oxygen consumption and ATP/ADP ratio iNOS induction reversibly inhibits oxygen consaiaption by competing with oxygen at the level of complex IV ‘NO donor generates EPR-detectable iron-salphur-dinitrosyl complexes without correlation with oxygen consumption ‘NC) and 0N00 inhibit oxygen consnnmption ONOC) inhibits complex I and Il—Ill activities only after GSH depletion NO-releasing astrocytes decrease neuronal ATP in coculture and NC) donor inhibits complex I, Il — Ill, and IV activities in nerirones

Neurones Il. Derived from non-CNS tissue Heart purified comptes or cytochrome u,

tv

Macrophages LlO hepatonsa cells Ll2lO leukaemia cells Adenocarcinoma cells Hepatocytes

Fihroh lasts

vascular smooth muscle cells Heart mitochondria

Macrophages Purified aconitase Skeletal muscle mitochondria Purified acouitase Liver mitochondria Hepatocytes Macrophages Skeletal masele mitochondria Heart purified complex Heart mitochondria Heart submaitochondrial particles Hean mitochondria

tv

Hepatocyte initochondria

vascular smooth mauscle

cells

Perfused lung Hepatoma cells Hepatocytes vascular smooth muscle cells Ehrlich ascites tumour cells

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‘NC) hinds to complex IV, particularly to cytochrome u,

References“

Bolasos et al. 119941 Mitrovic et al. (1994) Heales et al. (1994) Schweizer and Richter 11994) B roan and Coriper 11994) Brilasos et al. 11995) Bolasos et al. 11995) Ereciiiska et al. (1995) Bomwn et al. 11995) Cooper and Brosvn 11995) L.izasoain et al. (1996) Barker et at. (1996) Bolasos et al. 11996)

Keilin and Hartrec 11939): Wainio (1955): Beinen et al. (1962): Kon (1968. 1969): Kon and Kataoka (19691: Blokzijl-Hoinan and Van Gelder 11971): wilson et al. (1976). SIe yens et al. (l979u.h): Brudvig et al. (1980): Bodens et al. 11982. 1983, 1984): Lohrntto et al. (1984) Drapier and Hihbs (1988)

iNOS induction inhibits aconitase activity and oxygen consumption with isocitrate. succinate, and TMPD as sabstrates ‘NC) (or a related compoundi inhibits aconitase activity ‘NC) inhibits oxygen consumption with succinate as sabstrate ‘NO—releasing macrophages inhibit oxygen consumption in the target adenocarcinoma cells ‘NC) inhibits oxygen consuniption with citrate and succi nate as substrates and inhibits mitochondrial. hnt not cytosolic. aconitase activity iNOS indticlion inhibits oxygen consumption with sticcinate as sabstrate iNOS induction inhibits oxygen consumption with pyrnvate. succinate. and TMPD/ascorhate as stibstrates ONOO inhibits oxygen constittiplion with glutamate and sticci sate as substrates and snccinate dehydrogenase, ATPase. and cotaplex IV activities in the intact mitochondria and complex I in the brokensoiticated mitrichondria iNOS induction inhibits oxygen consumption with snccinate as substrate Enzyme activity is inactivated by ONOC) or reversibly inhibited by high ‘NC) concentration ‘NO donor reversibly inhibits complex IV by interaction with cytochroine u and. apparently. with Cd‘ 5 center O,‘~and 0N00 inactivate enzyme activity Nf) reversibly inhibits oxygen consumption ‘NO reversibly inhibits oxygen consamption iNOS induction and ONOO inhibit oxygen consumption c—Arginine inhihits rixygen consumption ‘NO hinds to cytochmme u, competing with oxygen ‘NO inhibits oxygen consumption ‘NO donor inhibits complex I—Ill and IV activities

Haasladcn and Fridos ich (1994) Schsvei,cr and Richter 11994) Richter et al. 11994) Szahd and Salzioan (1995) Kohzik et al. (1995) Torres et al. (1995) Borntaité and Brown (1996) Poderoso et al. I I 996u(

~Nf) reversibly inhibits complex IV and succinate dehydrogenase

Cassina and Radi 11996)

activities and ONOC) inhibits succinate dehydrogenase and ATPase activities In vivo reactieni of ‘NO with hemoglobin avoids detectable impairitment of mitochondrial lanction ONOO inhibits oxygen consumption ‘NC) inhibits oxygen consumption ‘NO—releasing cells inhibit mitochondrial aconitase activity iNOS induction decreases ATP content NOS induction inhibits oxygen consumption ‘NC) inhibits oxygen consumption

Hihbs et ai. 11988) Stnehr and Nathan (19891 Aniher et al. (1991) Stadler et al. (11)1)1)

Dijkmaas and fil han 11991) Geng et al. (1992) Radi et al. (1994)

Sung and Dieter (1994) Castro et al. (1994) Cleeter et al. (19941

Fisch et al. (1996) Szahd et al. (1996«) Sakai et al. (1996) Phillips etat. (1996) Kitade et al. (1996) Szahd et al. (1996« 1 Inai et al. (1996)

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‘NO AND MITOCHONDRIA IN NEURODEGENERATJON

TABLE l—Continued Systema examined“ Ilearl purified complexes Hepatoma cells Alveolar type-II cells

Mechanism

References“ welter et al. (1996) Fnkumura et al. (1996)

Mitochondrial respiratory chain complexes are inhibited by ‘NO ‘NO-releasing Kupffer cells decrease rhodamine-l23 fluorescemmce intensity in cocultured hepatonma cells ‘NO donor reduces oxygeim consumption and ATP levels

Miles et al. (1996)

EPR. electron paramagnetic resonance; TMPD. tetramethylphenylenediamimme. “In order to stress the relevance of the existing literature for netirodegenerative diseases, reporLs have been classified into two main groups: those carried out in CNS tissue (I) and those carried ont in non-CNS tissue (II). Ref erences have been ordered chronologically.

Demerlé-Pallardy et al. (1993) and Hewett et al. (1994) failed to demonstrate neuronal cell death in mixed astrocytic—neuronal co-cultures following iNOS induction, However, it was observed that neurones displayed increased sensitivity to glutamate-receptor activation following coincubation with the activated astrocytes. Further evidence for a role of glialderived ‘NO in neurotoxicity came from the studies of Dawson et al. (1994) and Chao et al. (1996), who demonstrated that expression of iNOS in mixed astrocytic—neuronal cocultures caused neuronal cell death 24—36 h after exposure. Activation of mast cells by myelin basic protein also appears neurotoxic through a mechanism implicating induction of iNOS in astrocytes (Skaper et al., 1996). Finally, Bolaflos et al. (1996) showed that coincubation of iNOS-expressing astrocytes with neurones caused a ‘NO-dependent neuronal loss of ATP, indicating that neuronal energy depletion may be a mechanism for astrocyticderived ‘NO-mediated neuronal toxicity. All the above data Suggest that expression of glial iNOS is neurotoxic and hence may be associated with the neurodegenerative process. MECHANISMS OF ‘NO-MEDIATED NEUROTOXICITY The role of ‘NO in neurotoxicity has been the matter of several reviews (see, for instance, a recent review by Dawson and Dawson, 1996). However, the molecular mechanism explaining neuronal cell death following ‘NO exposure is still a matter of debate. It seems, however, to be a consensus that DNA damage, lipid peroxidation, and energy depletion may contribute to such neurotoxicity. Furthermore, ‘NO may liberate iron from ferritin, which could favour subsequent hydroxyl radical formation and oxidative stress (Gerlach et al., 1994). In this review, we discuss the possible mechanisms whereby ‘NO may lead to brain cell energy failure, with special emphasis on mitochondrial energy metabolism (summarized in Table 1). ‘NO-mediated mitochondrial damage in CNS tissue Despite large and increasing numbers of studies involving the role of mitochondrial dysfunction as a mechanism of cell toxicity, little work has been done

on neural tissue. Evidence that mitochondrial dysfunction may be a mechanism for the ‘NO-mediated neurotoxicity arose independently from the studies of Bolaflos et al. (1994) and Mitrovic et al. (1994). These authors demonstrated that mitochondrial complexes II—III and IV (Bolanos et al., 1994) and succinate dehydrogenase (Mitrovic et al., 1994) are damaged by ‘NO exposure (see Fig. 3 for mitochondrial respiratory chain nomenclature). Inhibition of these enzymes appeared irreversible, because during cell recovery and homogenization procedures no restoration of activity was observed (Bolaflos et al., 1994). These studies were carried out in glial cells with either endogenously synthesized ‘NO (Bolaflos et al., 1994) or an exogenous ‘NO donor (Mitrovic et al., 1994). Mitochondrial damage, at the level of complexes Il—III and IV, may also occur in neurones, because exposure of cultured neurones to ONOO results in apparently irreversible damage to these components of the respiratory chain. Furthermore, such damage appeared to precede cell death (Bolaños et al., 1995). However, a study by Brown and Cooper (1994) showed that, in isolated synaptosomes, ‘NO reversibly inhibited oxygen consumption at the level of complex TV in a manner apparently competitive with oxygen. This reversible inhibition of complex TV was later corroborated by endogenously generated ‘NO in activated astrocytes (Brown et al., 1995) and suggests an additional mechanism for mitochondrial complex TV damage. The contribution, if any, of this reversible inhibition of complex IV to neuronal cell death following ‘NO exposure has yet to be determined. However, such rapid and reversible inhibition of complex IV by ‘NO is proposed to be a physiological mechanism for controlling cellular respiration (Brown, 1995; Darley-Usmar et al., 1995; Takehara et al., 1995; Torres et al., 1995). This hypothesis may be supported by evidence, derived from studies, using an antibody raised to eNOS, suggesting the localization of eNOS in isolated brain mitochondria (Bates et al., 1995). Whether an eNOS-like moiety is localized within mitochondria or indeed displays functional activity remains to be demonstrated. Oxygen consumption and the ATP!ADP ratio have also been shown to be decreased in isolated synaptosomes following exposure to ‘NO donors (Ereciiiska et al., 1995). Furthermore, oxygen consumption is in-

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FIG. 3. Mechanism of mitochondrial ATP synthesis. Although some ATP synthesis is catalysed by soluble glycolytic enzymes, the largest proportion is associated with the respiratory chain, i.e., membrane-bound enzyme complexes located in the mitochondrial inner membrane. The respiratory chain of mammalian mitochondria is an assembly of >20 electron carriers, grouped into four polypeptide complexes (I or NADH-ubiquinone reductase, Il or succinate-ubiquinone reductase, Ill or ubiquinol-cytochrome c reductase, and IV or cytochrome c oxidase). Three of these complexes (I, Ill, and IV) act as oxidation—reduction-driven proton pumps and are associated with the coupling sites of phosphorylation. Respiration consists of the transfer of reducing equivalents from the NADH + H~/NAD~ couple or via flavoprotein-linked dehydrogenases, e.g., succinate dehydrogenase, to the O 2/H2O couple. The transfer of reducing equivalents is coupled to the pumping of protons across the mitochondrial inner membrane, generating an electrochemical gradient of protons, which consists of a membrane potential and a pH gradient (Mitchell, 1961). The electrochemical gradient across the membrane provides the driving force for phosphorylation of ADP to ATP. Thus, the flow of protons back into the mitochondria through the membrane sector of the mitochondrial ATP synthase (complex V) alters the active site of the enzyme leading to the synthesis of ATP (Pedersen, 1994).

hibited by authentic ‘NO gas in isolated brain mitochondria (Schweizer and Richter, 1994) and in brain submitochondrial particles by exposure to a ‘NO donor or ONOO (Lizasoain et al., 1996). ‘NO-mediated mitochondrial damage in non-CNS tissue In addition to studies performed on CNS tissue, other reports have shown damaging effects of ‘NO, and/or one of its derivatives, on mitochondrial function in non-CNS tissue. These studies were initiated by the original studies by Hibbs‘ group, who suggested a mechanism for macrophage-derived ‘NO-mediated cell toxicity. Thus, Drapier and Hibbs (1988) reported that activated macrophages developed a ‘NO-mediated inhibition of DNA synthesis, the Krebs cycle enzyme aconitase (see also Hibbs et al., 1988), and malateand succinate-dependent cellular oxygen consumption (see also Sung and Dietert, 1994), suggesting inhibition at the level of complexes T and TT of the mitochondrial respiratory chain. In addition to ‘NO, ONOO also appears to inhibit overall cellular respiration (Szabó and Salzman, 1995; Szabó et al., 1996a) in macrophages. Similar mechanisms seem to be responsible for ‘NO-mediated macrophage killing of tumour cells (Stuehr and Nathan, 1989; Amber et al., 1991). J. Neuro«hemim., Vol. 68, No. 6. 1997

In hepatocytes, oxygen consumption dependent on citrate, malate, succinate (Stadler et al., 1991; Phillips et al., 1996), and, in isolated liver mitochondria, ascorbate plus tetramethylphenylenediamine (TMPD; an artificial substrate for complex IV-mediated oxygen consumption) (Schweizer and Richter, 1994) was inhibited by a ‘NO-mediated mechanism. In addition. endogenous ‘NO production also inhibited mitochondrial (but not cytosolic) aconitase activity in hepatocytes (Stadler et al., 1991; Phillips et al., 1996) and decreased cellular ATP content (Kitade et al., 1996). Furthermore, hepatoma cell cytotoxicity by activated Kupffer cells also appears to be mediated by a ‘NOmediated mechanism, as revealed by rhodamine 123 fluorescence studies (Kurose et al., 1995; Fukumura et al., 1996). However, the relevance of ‘NO-mediated mitochondrial damage in hepatocytes has been questioned recently (Fisch et al., 1996). In fibroblasts, lipopolysaccharide/interferon-y (IFN-y) treatment inhibited malate- and succinate-dependent cellular respiration by an L-arginine-dependent process (Dijkmans and Billiau, 1991). In vascular smooth muscle cells, endogenously generated ‘NO inhibited pyruvate-, succinate-, and ascorbate/TMPD-dependent cellular respiration (Geng et al., 1992). Overall cell respiratioll is also inhibited by a ‘NO-mediated mechanism in iso-

‘NO AND MITOCHONDRIA IN NEURODEGENERATION

lated perfused lung (Sakai et al., 1996), isolated alveolar type-Il cells (Miles et al., 1996), and Ehrlich ascites tumour cells (Inai et al., 1996). Tn skeletal muscle mitochondria, oxygen consumption is inhibited by a ‘NO-mediated mechanism (Cleeter et al., 1994; Kobzik et al., 1995), apparently through reversible complex IV inhibition (Cleeter et aI., 1994). In heart mitochondria, ONOO inhibits glutamate- and succinate-dependent oxygen consumption (Radi et al., 1994) and succinate dehydrogenase, ATPase, and complex TV activities in the intact mitochondria. However, complex I activity was inhibited only in sonicated mitochondria. Other studies performed with heart mitochondria also revealed that ‘NO inhibits oxygen consumption (Borutaité and Brown, 1996) and reversibly inhibits complex IV and succinate dehydrogenase activities, whereas ONOO inhibits succinate dehydrogenase and ATPase activities (Cassina and Radi, 1996). —

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Mechanisms of ‘NO-mediated mitochondrial respiratory chain inhibition The mechanism of the ‘NO-mediated mitochondrial respiratory chain damage has been the matter of several studies. The initial studies of Hibbs‘ group suggested that ‘NO may attack the iron-sulphur clusters present in complexes T and Il, thus causing inactivation of the activity of these enzymes (Drapier and Hibbs, 1988). However, in isolated synaptosomes, Cooper and Brown (1995) showed no correlation between electron paramagnetic resonance-detectable ïron-sulphur-dinitrosyl complexes and oxygen consumption, ruling out this mechanism, at least in the brain. Inhibition of complex Il—III activity appears to be a consistent feature in neurones or astrocytes exposed to ONOO (Bolaños et al.. 1994, 1995). Tn addition, brief exposure of isolated brain mitochondria to 0N00 results in loss of complex Il—III activity, suggesting direct inactivation of this complex by 0N00. Assay of this complex requires the endogenous quinone pool. Recent observations suggest that ‘NO and possibly ONOO directly react with ubiquinol (Poderoso et al., 1 996b). Such a reaction may be, therefore, a contributing factor to the observed loss of complex II—III activity following ONOO exposure. The ‘NOmediated inhibitory effect on aconitase activity (see above), which is suggested to arise from inactivation of a prosthetic iron-sulphur cluster group, does not appear to be due to ‘NO but to ONOO (Castro et al., 1994; Hausladen and Fridovich, 1994), suggesting an alternative mechanism of aconitase inactivation. However, the relevance of this effect for ‘NO-mediated cell toxicity is not clear, because blocking the activity of aconitase is insufficient to produce macrophage cell death (Messmer and Brune, 1996). Reversible complex IV damage The mechanism of reversible complex IV inhibition by ‘NO has been a matter of intense study for nearly 60 years (Keilin and Hartree, 1939). ‘NO closely resembles dioxygen, and the presence of an unpaired

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electron allows the use of electron paramagnetic resonance to demonstrate that ‘NO forms adducts with complex IV. Consequently. ‘NO has been used as a biochemical probe for the study of oxygen-binding proteins, such as complex IV. Many such studies have been performed on purified proteins from isolated heart mitochondria or heart subniitochondrial particles (Wainio, 1955; Beinert et al., 1962: Kon, 1968, 1969; Kon and Kataoka. 1969; Blokzijl-Homan and Van Gelder, 1971; Wilson et aI., 1976; Stevens et al., l979a,b; Brudvig et al., 1980: Bodens et al., 1982, 1983, 1984; Lobrutto et al., 1984). These studies have contributed in clarifying complex IV structure and catalytic activity (Malmström. 1979). It has also been confirmed that ‘NO reversibly binds to the Fe2~center of cytochrome a 2 ~ center of complex IV (Stevens et 3al., and1979a,b; to the CuBrudvig et al., 1980). However, as the importance of ‘NO in biology was not apparent until 1987 (Palmer et al., 1987), these earlier studies did not appear to have any (patho)physiological relevance. Brudvig et al. (1980) also reported that complex IV catalyzes both the oxidation and reduction of ‘NO, thus leading to ‘NO breakdown. These results have been corroborated recently in isolated heart mitochondria (Borutaité and Brown, 1996). Other studies performed in diverse systems have confirmed that the interaction of ‘NO with complex IV leads to reversible inhibition of oxygen consumption (Brown and Cooper, 1994; Cleeter et al., 1994; Brown et aI.. 1995; Cassina and Radi, 1996; Poderoso et al.. 1996a). The mechanism of the observed complex IV inhibition by ‘NO is, in part, in agreement with studies reported two to four decades ago, i.e.. ‘NO binds to reduced (Fe>) cytochrome a 3 (Stevens et al., 1979a,h; Brudvig et al., 1980; Cleeter et al., 1994; Cassina and Radi. 1996; Poderoso et al., l996a) forming a nitrosyl—haeme complex by donation of one electron to ferne cytochrome a3 (Radi, 1996)(Stevens and then et apparently interacting 2 B center al., 1979a,h; Brudwithettheal..Cu1980; Cleeter et al., 1994). These mechavig nisms explain ‘NO competition with oxygen resulting in reversible inhibition of activity (Brown and Cooper, 1994; Cleeter et al., 1994; Brown et al., 1995). Regardless of the precise mechanism that leads to reversible inhibition, it is interesting to note that complex 1V activity is considered to be an endogenous metabolic marker for neuronal activity (Wong-Riley, 1989). Moreover, its activity in neurones is colocalized with NOS and the NMDA receptor (Zhang and WongRiley, 1996), suggesting that regulation of this respiratory complex by ‘NO may he relevant for neuronal metabolism. Unfortunately, whether reversible modification of complex IV activity compromises neuronal energy metabolism has not yet been determined. Cleeter et al. (1994) have suggested. from work on muscle mitochondria, that, secondary to reversible complex IV inhibition, increased 02‘ formation, via the mitochondrial respiratory chain. may occur resuIting in ONOO production. These authors speculate .1. ‚Vemmmza liemmm., Vol. 68, No. 6, / 997

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that this putative secondary ONOO formation could inhibit complex I (Cleeter et al., 1994). However, this may not occur in the CNS, because brain mitochondrial complex T appears resistant to endogenous or exogenous ONOO (Bolaños et al., 1994, 1995). Moreover, the lack of a direct effect on complex I has also been shown in intact heart mitochondria (Cassina and Radi, 1996). Furthermore, complex I appears only to display sensitivity to ONOO when the intact nature of the mitochondrion is disrupted by sonication (Radi et al., 1994) or the cellular GSH status is severely compromised (Barker et al., 1996). Using heart submitochondrial particles, Poderoso et al. (1996a) reported that ‘NO inhibited the mitochondrial respiratory chain at the level of complex II—III and IV. However, these authors also observed inhibition at the level of complex I—III, suggesting that ‘NO exerts a common effect on both reductases of the electron transfer chain. In addition, it is suggested that ‘NO-mediated inhibition of the ubiquinone-cytochrome b region leads to increased mitochondrial 02‘ production (Poderoso et al., 1996b), hence favouring ONOO formation. Irreversible complex IV damage In contrast to reversible complex IV damage, mediated by ‘NO, irreversible complex IV damage, due to ONOO formation, may contribute to neurotoxicity, ATP depletion, and cell death (Bolaños et al., 1995, 1996). As ONOO causes lipid peroxidation (Radi et al., 1991), it may he suggested that prolonged exposure of cells to ‘NO initiates peroxidation. Cardiolipin, an inner mitochondrial membrane lipid, is susceptible to free radical peroxidation (Radi et al., 1991) and is specifically required for maximal complex IV catalytic activity (Soussi et al., 1990). In support of this, Trolox, a vitamin E analogue and inhibitor of lipid peroxidation, protects complex TV from ‘NO-mediated damage (Heales et al., 1994). However, Radi and colleagues (Radi et al., 1994; Cassina and Radi, 1996) demonstrated that complex IV activity in heart mitochondria was the component of the respiratory chain least sensitive to ONOO -mediated damage. As the mitochondrial membrane cardiolipin concentration in heart is severalfold higher than that in brain (White, 1973), the hypothesis that cardiolipin is a primary target of ONOO-rnediated complex IV damage in CNS gains credibility. Apoptosis or necrosis Whether ‘NO-mediated mitochondrial damage causes neurotoxicity via necrosis or apoptosis is not known. However, it is interesting to note that, following an extreme insult resulting in severe mitochondrial damage, necrosis ensues, possibly because there is rapid and massive energy failure (Ankarcrona et al., 1995; Bonfoco et al., 1995). In contrast, glutamate neurotoxicity proceeds through apoptosis only if there is functional mitochondrial activity (Ankarcrona et al., 1995; Liu et al., 1996), suggesting ATP is required for the apoptotic program to become activated. HowJ. Nc‘urom/menm., Vol. 68, Nmm. 6, /997

ever, this is controversial, because Jacobson et al. (1993) showed that cells lacking mitochondria died via apoptosis, although the possibility that these cells obtained energy from alternative sources, such as glycolysis, cannot be discounted. It is interesting that mild ONOO exposure to neurones is neurotoxic through an apoptotic mechanism in PCI2 cells (Estévez et al.. 1995). This raises the possibility that irreversible inhibition of complex IV activity may be neurotoxic through an apoptotic program. Moreover, mild inhibition of other components of the mitochondrial respiratory chain, by specific inhibitors such as rotenone or antimycin, also causes apoptosis (Hartley et al., 1994; Wolvetang et al., 1994). Differential cell susceptibility to ‘NO and mitochondrial damage It is apparent that there is tissue-specific differential susceptibility of mitochondria to ONOO. Furthermore, it is important to note that, within the brain, there may also be a differential susceptibility of different cell types to ‘NO (Mitrovic et al., 1994; Bolai‘ios et al.. 1995). The factors that are responsible for this might include, as mentioned above, the inner mitochondrial membrane lipid composition and/or intracellular antioxidant defence systems, such as superoxide dismutase activity and GSH concentration. The intracellular concentration of GSH following ONOO exposure has been shown to be dramatically decreased in neurones, but not in astrocytes (Bolaflos et al., 1995). Further support for a key role for 05H in protecting against ONOO came from studies in astrocytes that reveal mitochondrial damage and cell death only in 05Hdepleted astrocytes exposed to ONOO (Barker et al.. 1996). These results strongly suggest that the cellular GSH status may play an important role in the ‘NOmediated neurotoxicity (Barker et al., 1996; Bolaños et al., 1996). Other mechanisms of ‘NO-mediated neurotoxicity There are, of course, additional explanations for ‘NO-mediated energy depletion and cytotoxicity. An interesting mechanism whereby ‘NO may mediate excitotoxicity is by increasing the activity of poly(ADPribose) synthetase, a nuclear enzyme that synthesizes poly(ADP-ribose) from NAD thus potentially lowering cellular energy levels (Radons et al., 1994: Zhang et al., 1994; Zhang and Steiner, 1995). Furthermore, in macrophages or smooth muscle cells, this futile energy-consuming cycle is triggered as a result of ONOO -mediated DNA strand breaks (Szabó et al., l996a,b; Zingarelli et al., 1996). Neuronal glutamate-receptor stimulation also causes mitochondrial depolarization and calcium accumulation within mitochondria (White and Reynolds, 1996). As this is a cyclosporin A-sensitive process, it has been suggested that the opening of the permeability transition pore might be involved. Accordingly, it has been suggested that mitochondria may be a target in excitotoxic neuronal damage (Wang et al., 1994; ‚

‘NO AND MITOCHONDRIA IN NEURODEGENERATION

White and Reynolds, 1996). In the liver. ‘NO-mediated opening of the permeability transition pore has been observed resulting in efflux of calcium from liver mitochondria and providing a putative mechanism of ‘NOmediated cytotoxicity (Packer and Murphy, 1994; Richter et al., 1994; Packer et al.. 1996). Unfortunately, there are no experimental data to demonstrate a similar phenomenon in ‘NO-mediated neurotoxicity. An alternative mechanism for the ‘NO-mediated cytotoxicity has long been proposed to be the nitrosylation and possible inhibition of the activity of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (Molina y Vedia et al., 1992; Zhang and Snyder, 1992). However, this hypothesis has been questioned on the grounds that only under severe anaerobic conditions such inhibition of glycolysis may be relevant (Erecileiska et al., 1995). Furthermore, increased lactate production in macrophages exposed to ‘NO occurs despite inactivation of glyceraldehyde-3-phosphate dehydrogenase (Messmer and Brune, 1996). In addition, induction of iNOS in astrocytes is accompanied by activation of glycolysis (Bolanos et al., 1994), despite possible modification of glyceraldehyde-3-phosphate dehydrogenase. MITOCHONDRIAL DAMAGE, ‘NO, AND NEURODEGENERATION Defects in mitochondrial energy metabolism have long been considered to underlie the pathology of neurodegenerative diseases (Beal et al., 1993). Decreased complex I activity is reported in the substantia nigra of postmortem samples obtained from patients with Parkinson‘s disease (Schapira et al., 1990a,h). Similarly, impaired complex IV activity has been noted in Alzheimer‘s disease (Kish et al., 1992; Mutisya et al., 1994; Chagnon et al., 1995). Although increased free radical production has been associated with the development of such disorders (Halliwell, 1992; Olanow, 1993; Simonian and Coyle, 1996), evidence is now arising to suggest a role for ‘NO (Schulz et aI., 1995(t). Cytokines, such as IFN-y, which are present in normal brain, are elevated in many pathological situations (for reviews, see Chao et al., 1995; Sei et al., 1995). Parkinson‘s disease (Boka et al., 1994), Alzheimer‘s disease (Griffin et al., 1989; Mrak et al., 1995), multiple sclerosis, ischaemia, encephalitis, and central viral infections (Merrill, 1987) are all associated with increased CNS cytokine concentrations. Accordingly, as cytokines promote the induction of iNOS in the brain, a possible role of glial-derived ‘NO in the neurotoxicity of certain neurological diseases has been suggested (Simmons and Murphy, 1993; Bolaños et al.. 1994; Mitrovic et al., 1994). There is now evidence to support cytokine induction of iNOS in human pathological conditions. The stable decomposition products of ‘NO, nitrite (NO,) and nitrate (NO3) (Milstien et al., 1994), are increased, in CSF, in bacterial and viral meningitis, but not in Huntington‘s or Alzheirner‘s dis-

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ease. However, the CSF of patients with multiple sclerosis shows significantly greater NO2 plus NO3 levels when compared with controls (Johnson et al., 1995), suggesting increased ‘NO production in this disorder. Further evidence for induction of iNOS in multiple sclerosis conies from postmortem studies that have revealed elevated levels of mRNA coding for iNOS in postmortem brain samples obtained from patients who died with multiple sclerosis (BC) et aI., 1994; Bagasra et al., 1995). In addition. NADPH-diaphorase activity, which forms the basis of a histochemical stain possibly relating to NOS catalytic activity, has been detected in astrocytes associated with actively demyelinating lesions (Bo et al., 1994). The glial-derived factor SlOOß. which is overexpressed in Alzheimer‘s disease (Sheng et al., 1994), has also been shown to be able to induce iNOS in cultured rat astrocytes (Hu et al.. 1996), suggesting a possible role for an 510041-mediated ‘NO production in the pathology of this disorder. Excessive formation of glial-derived ‘NO has also been implicated in the pathogenesis of Parkinson‘s disease, although the evidence for such a relationship is still weak. One study (Hunot et al., 1996) recently identified NADPH-diaphorase-positive glial cells in the substantia nigra of postmortem brains obtained from individuals with Parkinson‘s disease. As loss of nigral GSH is considered to be an early and key event in the pathogenesis of Parkinson‘s disease (Jenner et al., 1992), decreased scavenging of ONOO may also occur. In addition, depletion of brain OSH appears to increase nNOS activity (Heales et al., 1996). Such perturbations of GSH metabolism may contribute further to increased ONOO generation in Parkinson‘s disease. Although the toxic effects of ‘NO derived from nNOS activity are heavily debated, two recent studies report that the selective inhibition of nNOS prevents I -methyl-4-phenyl- I .2,3,6-tetrahydropyridine (M PTP) -induced parkinsonism in experimental animals (Hantraye et al., 1996; Przedborski et al., 1996). Whether there is a direct relationship between increased ‘NO synthesis, arising from iNOS and/or nNOS activity, and the energy impairment associated with neurodegenerative disorders is still not clear. It is, therefore, necessary to carry out further work tC) clarify the exact role of ‘NO in the development of such diseases. However, if this proves to be the case. clinical studies on the therapeutic value of specific NOS inhibitors in preventing the development of neurological symptoms will ensue. In this light, it is also possible that existing treatnient regimes affect ‘NO metabolism, e.g.. IFN-ß is being used currently as a treatment for certain forms of multiple sclerosis. Recent studies suggest that preparations containing IFN-ß impair the ability of astrocytes to induce NOS following subsequent exposure to IFN-y (Stewart et aI., 1997). In addition, as the sensitivity of cardiolipin to peroxidation increases with age, due to alterations in unsaturated fatty acid composition (Shigenaga et al., 1994), J, A‘m‘mmm‘o«lmm‘iim. . Vol. 6,6, A‘,,. 6. /997

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antioxidants and/or dietary manipulation of fatty acid intake may be beneficial in conditions associated with ‘NO-mediated complex IV damage. POSSIBLE BENEFICIAL AND NEUROPROTECTIVE EFFECTS OF ‘NO Although there is considerable evidence to support a role for ‘NO and 0N00 in the pathogenesis of many neurodegenerative disorders, formation of these species in certain stages of a disease process may actually be beneficial. In this light, it has been proposed that ‘NO might inhibit T-cell activation and cell trafficking across the blood—brain barrier, thereby minimizing the initiation of the autoimmune cascade associated with multiple sclerosis (Merrill and Benveniste, 1996). If this proves to be the case, then inhibition of NOS perhaps should be considered only in the chronic demyelinating stage of multiple sclerosis (Merrill and Benveniste, 1996). Using cultured hamster lung fibroblasts or rat mesencephalic dopaminergic cells, Wink et aI. (1993) demonstrated that, in the presence of a ‘NO donor, cytotoxicity from reactive oxygen species was diminished. Although the exact mechanism for this cellular protection is not known, such a finding implies that ‘NO has a “double-edged“ nature, i.e., depending on cellular environment and cell type, ‘NO formation may be neuroprotective or neurotoxic. CONCLUDING REMARKS A large body of evidence suggests that energy failure, particularly at the level of the mitochondrial respiratory chain, may be an important factor explaining the mechanism of ‘NO-mediated neurotoxicity. Although there are many studies showing the different effects of ‘NO on the mitochondrial respiratory chain in nonCNS tissue, little work has been carried out in CNS tissue. As there are cell-specific biochemical differences, especially with regard to antioxidant status, different cells may have different vulnerability to ‘NO. In the light of the studies carried out in intact neurones, irreversible damage to mitochondrial complexes II—III and IV by the neurotoxic ‘NO derivative, ONOO seems to be a plausible mechanism for ‘NO neurotoxicity.

AguIló L. and GarcIa A. (1992/,) Different receptors mediate slimulation of nitric oxide-dependenl cyclic GMP formation in neurons and astrocytes in culture. Bioche,n. Biophvs. Rex. Cootomiom. 182, 1362—1368. Amber I. J., Hihbs J. B. Jr., Parker C. J., Johnson B. B.. Taintor R. R., and Vavrin Z. (1991) Aclivated macrophage condilioned medium: identification of the soluble factors inducing cytotoxicity and the t.-arginine dependent effector molecule. J. Leukocvte Biol. 49, 610—620. Ankarcrona M., Dyphukt J. M., Bonfoco E., Zhivotovsky B., Orrenius S., Lipton S. A., and Nicotera P. (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961—973. Bagasra O., Michaels F. H., Zheng Y. M., Bobroski L. E., Spilsin S. V., Fu Z. F., Tawadros R.. and Koprowski H. (1995) Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis. Proc. Nati. Ae‘ud. Sei. USA 92, 12041—12045. Banati R. B., Gehrmann J., Schubert P., and Kreulzberg G. W. (1993) Cytotoxicity of microglia. Glia 7, 111—118. Barker J. E., Bolaños J. P.. Land J. M.. Clark J. B., and Heales S. J. R. (1996) Glutathione protects astrocytes from peroxynitrite-mediated mitochondrial damage: implications for neuronal/astrocytic trafficking and neurodegeneration. Der‘. Nettrosci. 18, 391 —396. Bates T. E., Loesch A., Burnstock G.. and Clark J. B. (1995) Immunocytochemical evidence for a mitochondrially located nitric oxmde synthase mn hratn and lmver. Biochem. Biophvs. Res. Coot/nun. 213, 896—900. Beal M. F., Hyman B. T., and Koroshetz W. (1993) Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases‘? Trends Neurosci. 16, 125—131. Beckman J. S., Beckman T. W., Chen J., Marshall P. A.. and Freeman B. A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. NaIl. Acud. Sei. USA 87, 1620—1624. Beinert 1-l., Griffith D. E., Wharton D. C., and Sands R. W. (1962) Properties of the copper associated with cytochrome oxidase as studied by paramagnetic resonance spectroscopy. J. Biol. Chem.

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Acknowledgment: A.A. is a recipient of an “Acciones para la Reincorporación de Doctores y Tecnólogos“ del Ministerio de Educación y Cultura (Spain). S.P. is funded by the Wellcome Trust (U.K.). VS. is funded by the Multiple Sclerosis Society (U.K.). in addition, S.J.R.H. and J.P.B. are in receipt of a Biomedical Collaboration Grant from the Wellcome Trust (U.K.).

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