Although the etiology for many neurodegenerative diseases is unknown, the common findings of mitochondrial defects and oxidative damage posit these events as contributing factors. The temporal conundrum of whether mitochondrial defects lead to enhanced reactive oxygen species generation, or conversely, if oxidative stress is the underlying cause of the mitochondrial defects remains enigmatic. This review focuses on evidence to show that either event can lead to the evolution of the other with subsequent neuronal cell loss. Glutathione is a major antioxidant system used by cells and mitochondria for protection and is altered in a number of neurodegenerative and neuropathological conditions. This review also addresses the multiple roles for glutathione during mitochondrial inhibition or oxidative stress. Protein aggregation and inclusions are hallmarks of a number of neurodegenerative diseases. Recent evidence that links protein aggregation to oxidative stress and mitochondrial dysfunction will also be examined. Lastly, current therapies that target mitochondrial dysfunction or oxidative stress are discussed.
Examination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mM DA for 15 min at 30°C inhibited pyruvate/glutamate/malate-supported state-3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H 2 O 2 ), hydroxyl radical, oxidized glutathione (GSSG) or glutathione-protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H 2 O 2 (100 lM) significantly inhibited state-3 respiration. In contrast, incubation with GSSG (1 mM) had no effect on O 2 consumption. Exposure of mitochondria to 1 mM GSSG resulted in a 3.3-fold increase in PrSSG formation compared with 1.4-and 1.5-fold increases in the presence of 100 lM H 2 O 2 or 0.5 mM DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membranebound (2-fold) and intramatrix (3.5-fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze-thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H 2 O 2 showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze-thawed mitochondria were inhibited with IC 50 approximately 2-3-fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H 2 O 2 . Co-administration of desferrioxamine with H 2 O 2 had no effect on complex I-associated inhibition in intact mitochondria, but attenuated inhibition of rotenone-sensitive NADH oxidase activity by 70% in freezethawed mitochondria. The results show that DA-associated inhibition of respiration is dependent on MAO and that H 2 O 2 and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.
Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exempli®ed the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further de®nes the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 mM buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mM 3-amino-1,2,4-triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH-depleted cells with malonate (40 mM) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3-amino-1,2,4-triazole in the presence of malonate signi®cantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these ®ndings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These ®ndings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a ®rst line of defense in its removal. Pre-treatment of cultures with 400 mM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein±GSH mixed disul®de formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mM) for 12 h resulted in a modest increase in mixed disul®de formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein±GSH mixed disul®des three-fold. Mixed disul®de levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These ®ndings also suggest that protein±GSH mixed disul®de formation under these circumstances may play a protective role.
Glutamate receptor involvement and oxidative stress have both been implicated in damage to neurons due to impairment of energy metabolism. Using two different neuronal in vitro model systems, an ex vivo chick retinal preparation and dopamine neurons in mesencephalic culture, the involvement and interaction of these events as early occurring contributors to irreversible neuronal damage have been examined. Consistent with previous reports, the early acute changes in the retinal preparation, as well as irreversible loss of dopamine neurons due to inhibition of metabolism, can be prevented by blocking NMDA receptors during the time of energy inhibition. Oxidative stress was suggested to be a downstream consequence and contributor to neuronal cell loss due to either glutamate receptor overstimulation or metabolic inhibition since trapping of free radicals with the cyclic nitrone spin-trapping agent MDL 102,832 (1 mM) attenuated acute excitotoxicity in the retinal preparation or loss of mesencephalic dopamine neurons due to either metabolic inhibition by the succinate dehydrogenase inhibitor, malonate, or exposure to excitotoxins. In mesencephalic culture, malonate caused an enhanced efflux of both oxidized and reduced glutathione into the medium, a significant reduction in total reduced glutathione and a significant increase in total oxidized glutathione at time points that preceded those necessary to cause toxicity. These findings provide direct evidence for early oxidative events occurring following malonate exposure and suggest that the glutathione system is important for protecting neurons during inhibition of energy metabolism. Consistent with this, lowering of glutathione by buthionine sulfoxamine (BSO) pretreatment greatly potentiated malonate toxicity in the mesencephalic dopamine population. In contrast, BSO pretreatment did not potentiate glutamate toxicity. This latter finding indicates dissimilarities in the type of oxidative stress that is generated by the two insults and suggests that the oxidative challenge during energy inhibition is not solely a downstream consequence of glutamate receptor overstimulation.
A decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein-glutathione mixed disulfide (Pr-SSG) levels. The present study further examined ascorbate-glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate-induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH-depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr-SSG formation during mitochondrial impairment in normal and GSH-depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate-treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr-SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.
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