Energy Stress-Induced Dopamine Loss in Glutathione Peroxidase-Overexpressing Transgenic Mice and in Glutathione-Depleted Mesencephalic Cultures

Abstract: The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 µ M buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 µmol) into the left side had significantly less loss of striatal dopamine than their hGPE-negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines 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 microM 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 significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre-treatment of cultures with 400 microM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein-GSH mixed disulfide 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 disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein-GSH mixed disulfides three-fold. Mixed disulfide 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 findings also suggest that protein-GSH mixed disulfide formation under these circumstances may play a protective role.     

Striatal Damage and Oxidative Stress Induced by the Mitochondrial Toxin Malonate Are Reduced in Clorgyline-Treated Rats and MAO-A Deficient Mice

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.     

Malonate-Induced Generation of Reactive Oxygen Species in Rat Strium Depends on Dopamine Release but Not on NMDA Receptor Activation

Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions similar to that seen in cerebral ischemia and Huntington's disease. The mechanisms of neuronal cell death involve secondary excitotoxicity and the generation of reactive oxygen species. Here, we investigated the effects of dopamine on malonate-induced generation of hydroxyl radicals and striatal lesion volumes. Using in vivo microdialysis, we found that malonate induced a 94-fold increase in extracellular striatal dopamine concentrations. This was paralleled by an increase in the generation of hydroxyl radicals. Prior unilateral lesioning of the nigrostriatal dopaminergic pathway by focal injection of 6-hydroxydopamine blocked the malonate-induced increase in dopamine concentrations and the generation of hydroxyl radicals and attenuated the lesion volume. In contrast, the NMDA receptor antagonist MK-801 attenuated malonate-induced lesion volumes but did not block the generation of hydroxyl radicals. Thus, the dopaminergic and glutamatergic pathways are essential in the pathogenesis of malonate-induced striatal lesions. Our results suggest that the malonate-induced release of dopamine but not NMDA receptor activation mediates hydroxyl radical formation.     

Cooperative interaction between ascorbate and glutathione during mitochondrial impairment in mesencephalic cultures

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.     

Neuronal Loss and Cytoskeletal Disruption Following Intrahippocampal Administration of the Metabolic Inhibitor Malonate: Lack of Protection by MK-801

Abstract: Impaired energy metabolism may contribute to the pathogenesis of late-onset neurodegenerative disorders such as Alzheimer's disease by increasing neuronal vulnerability to excitotoxic damage through the NMDA receptor. The effects of metabolic impairment on the striatum have been extensively examined, but relatively little is known regarding the vulnerability of the hippocampus. To examine the effect of metabolic impairment on the hippocampal formation, malonate (0.25–2.5 µmol), a reversible inhibitor of succinate dehydrogenase, was administered by stereotaxic injection into the hippocampus of male Sprague-Dawley rats. Neuronal loss was assessed by Nissl stain, and immunocytochemistry was used to examine cytoskeletal disruption. Malonate produced a dose-dependent lesion in which CA1 pyramidal neurons were most vulnerable, followed by CA3 and dentate gyrus. Cytoskeletal alterations included the loss of microtubule-associated protein 2 (MAP2) and dendritic MAP1B immunoreactivity, whereas axonal MAP1B and τ proteins were relatively spared. Spatially and temporally correlated with the loss of MAP2 was an increase in the immunoreactivity of calpain-cleaved spectrin. A similar pattern of neuronal damage and cytoskeletal disruption was produced by intrahippocampal injection of quinolinate (0.1 µmol), an NMDA agonist. Although these results are consistent with the hypothesis that metabolic impairment results in excitotoxic death, MK-801 (dizocilpine maleate), a noncompetitive NMDA receptor antagonist, did not attenuate the lesions produced by malonate but was effective against quinolinate. The results suggest that NMDA receptor activation is not required for malonate-induced damage in the hippocampal formation.     

The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis,rather than enhancing striatal damage,protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

The cannabinoid CB₂ receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington''s disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E₂ glyceryl ester (PGE₂-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE₂-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE₂-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE₂-G, formed from COX-2-mediated oxygenation of 2-AG. Accordingly, MAGL inhibition or the administration of PGE₂-G aggravates the malonate toxicity.     

Malonate-Induced Degeneration of Basal Forebrain Cholinergic Neurons: Attenuation by Lamotrigine, MK-801, and 7-Nitroindazole

Abstract: Previously, we have reported that intranigral infusions of malonate, an inhibitor of mitochondrial function, lead to the degeneration of the dopaminergic neurons of the nigrostriatal pathway that is mediated, at least in part, through NMDA receptor activation and nitric oxide formation. In the present study, unilateral focal infusions of malonate into the nucleus basalis magnocellularis (nbM) of male Sprague-Dawley rats (weighing 250–300 g) resulted in a dose-related depletion in ipsilateral cortical and amygdaloid choline acetyltransferase (ChAT) activity. Infusion of a 3 µmol dose of malonate into the nbM of vehicle-treated animals resulted in a 41 and 54% decrease in cortical and amygdaloid ChAT activity, respectively. Systemic pretreatment with lamotrigine (16 mg/kg, i.p.) and MK-801 (5 mg/kg, i.p.) attenuated the depletions in cortical and amygdaloid ChAT activity that resulted from an infusion of this dose of malonate into the nbM. Acetylcholinesterase (AChE) histochemistry of the nbM following focal infusion of malonate (3 µmol) showed a marked decrease in the number of AChE-positive neurons that was partially prevented by MK-801 pretreatment. Before examining the role of nitric oxide formation in malonate-induced toxicity, the ability of systemic administration of N^ω-nitro-l -arginine (l -NA) to inhibit nitric oxide synthase (NOS) activity in the nbM and cerebellum was investigated. l -NA (2, 10, and 20 mg/kg, i.p.) produced a dose-related inhibition of nbM and cerebellar NOS activity that was maximal following a dose of 10 mg/kg l -NA. This level of NOS inhibition persisted for at least 13 h following l -NA (10 mg/kg) administration. Subsequently, the effect of l -NA pretreatment on malonate toxicity was evaluated. Following pretreatment with l -NA (2 and 10 mg/kg, i.p.), the toxic action of malonate on cortical and amygdaloid ChAT activity was not altered. In addition, infusion of a lower dose of malonate (2 µmol) into the nbM resulted in decreases in cortical and amygdaloid ChAT activity that were not altered by pretreatment with l -NA (2 and 10 mg/kg, i.p.). In 7-nitroindazole (7-NI; 25 and 50 mg/kg, i.p.)-pretreated animals, malonate (3 µmol) produced decreases in cortical and amygdaloid ChAT activity that were attenuated by both doses of 7-NI. Thus, malonate-induced destruction of the basal forebrain cholinergic neurons was attenuated by systemic pretreatment with lamotrigine, MK-801, and 7-NI but not by l -NA.     

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µ M ) nor dopamine (2.5 µ M ) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µ M ) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 µ M , reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µ M dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.     

Metabolic Changes Detected by Ex Vivo High Resolution 1H NMR Spectroscopy in the Striatum of 6-OHDA-Induced Parkinson’s Rat

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons; however, its crucial mechanism of the metabolic changes of neurotransmitters remains ambiguous. The pathological mechanism of PD might involve cerebral metabolism perturbations. In this study, ex vivo proton nuclear magnetic resonance (¹H NMR) was used to determine the level changes of 13 metabolites in the bilateral striatum of 6-hydroxydopamine (6-OHDA)-induced PD rats. The results showed that, in the right striatum of 6-OHDA-induced PD rats, increased levels of glutamate (Glu) and γ-aminobutyric acid (GABA) concomitantly with decreased level of glutamine (Gln) were observed compared to the control. Whereas, in the left striatum of 6-OHDA-induced PD rats, increased level of Glu with decreased level of GABA and unchanged Gln were observed. Other cerebral metabolites including lactate, alanine, creatine, succinate, taurine, and glycine were also found to have some perturbations. The observed metabolic changes for Glu, Gln, and GABA are mostly likely the result of a shift in the steady-state equilibrium of the Gln-Glu-GABA metabolic cycle between astrocytes and neurons. The altered Gln and GABA levels are most likely as a strategy to protect neurons from Glu excitotoxic injury after striatal dopamine depletion. Changes in energy metabolism and tricarboxylic acid cycle might be involved in the pathogenesis of PD.     

Chronic mitochondrial inhibition induces selective motoneuron death in vitro: a new model for amyotrophic lateral sclerosis

Evidence is increasing that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis, a neurodegenerative disease characterized by selective motoneuron death. To study the role of mitochondrial dysfunction in the pathways leading to motoneuron death, we developed an in vitro model of chronic motoneuron toxicity, based on malonate-induced inhibition of complex II in the mitochondrial electron transport chain. Treatment with malonate resulted in a dose-dependent decrease in cellular ATP levels. We observed that motoneurons were significantly more vulnerable to mitochondrial inhibition than control neurons in the dorsal horn. We could reproduce this dose-dependent phenomenon with the complex IV inhibitor sodium azide. The free radical scavenger alpha-phenyl-N-tert-butylnitrone, the AMPA/kainate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione, and riluzole, a drug that is currently used for the treatment of amyotrophic lateral sclerosis, were protective against malonate-induced motoneuron death. Furthermore, the caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and z-Asp-Glu-Val-Asp-fluoromethyl ketone were both protective against malonate toxicity. Our model shows that chronic mitochondrial inhibition leads to selective motoneuron death, which is most likely apoptotic.     

<Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">D</Emphasis>-aspartate Receptors are Involved in the Quinolinic Acid,but not in the Malonate Pro-oxidative Activity <Emphasis Type="Italic">in vitro</Emphasis>

Oxidative stress plays a significant role in the neurotoxicity of a variety of agents that interact with the N-methyl-D-aspartate (NMDA) receptors. Here we investigated in a comparative way the pro-oxidative effects of quinolinic acid (QA) and malonate, two neurotoxic substances that act through distinct primary molecular mechanisms on the production of thiobarbituric acid reactive species (TBARS) by brain homogenates. In fact, QA is thought to activate directly the NMDA receptor, whereas malonate seems to act primarily by inhibiting oxidative metabolism. The malonate-induced TBARS formation was not modified by cyanide (CN^–) or 2,4-dinitrophenol. MK-801 did not reduce basal or malonate induced-TBARS production in fresh tissues preparations. However, in heat-treated preparations a significant effect of MK-801 against basal TBARS production was observed, but not on the malonate induced-TBARS production. QA induced-TBARS production was significantly prevented by MK-801 either in fresh or heat-treated preparations. The antioxidant effect of MK-801 on basal and QA-induced TBARS production increased as the temperatures used to treat S1 were increased. Succinate dehydrogenase (SDH) was inhibited by malonate but not by QA. Malonate was able to chelate iron(II) and the malonate-iron complex(es) is(are) active as measured by its(their) activity on deoxyribose degradation assay. These findings indicate that direct interactions of malonate with NMDA receptors are not involved in malonate pro-oxidative activity in vitro. QA pro-oxidative activity in vitro was related, at least in part, to its capability in stimulate NMDA receptors. Taken together, these findings indicated that malonate pro-oxidative activity in vitro could be attributed to its capability of changing the ratio Fe²⁺/ Fe³⁺, which is essential to TBARS production.     

Cellular iron depletion weakens induction of heme oxygenase-1 by cadmium

Heme oxygenase-1 is an inducible cytoprotective gene, although its induction by environmental factors is not completely understood. This study aimed to ascertain if specific nutritive factors or related compounds influence heme oxygenase-1 expression. In HCT-116 cells, cadmium increased heme oxygenase-1 enzymatic activity. This effect of cadmium was weaker in cells made iron-deficient with the iron chelator, desferrioxamine, which was associated with repression of heme oxygenase-1 protein and mRNA expression. The repression by desferrioxamine of cadmium-induced heme oxygenase-1 upregulation was reversed upon iron replenishment of the cells. Additionally, it was found that thiol antioxidants inhibited the heme oxygenase-1 upregulation caused by cadmium and also by ethacrynic acid, which each decreased intracellular glutathione as did buthionine sulfoxamine. Interestingly, cadmium and ethacrynic acid increased nuclear translocation of Nrf2 and subsequent heme oxygenase-1 expression, but buthionine sulfoxamine did not. Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin, and a superoxide scavenger (Tiron) inhibited cadmium-induced upregulation of heme oxygenase-1. Diphenyleneiodonium was the most potent and inhibited NADPH-cytochrome P450 reductase as well, whereas apocynin and Tiron did not. It is concluded that adequate amounts of iron, which at the atomic level can serve as the pivotal element of heme in NADPH oxidase, must be present in cells to permit what appears to be thiol redox-sensitive, NADPH oxidase-dependent upregulation of heme oxygenase-1. Thus, these findings are significant because they suggest that cells without adequate iron would be unable to fully express the stress gene, heme oxygenase-1, when confronted with the toxic metal, cadmium.     

Manipulation of Membrane Potential Modulates Malonate-Induced Striatal Excitotoxicity In Vivo

Abstract: Malonate is a reversible inhibitor of succinate dehydrogenase (SDH) that produces neurotoxicity by an N -methyl- d -aspartate (NMDA) receptor-dependent mechanism. We have examined the influence of pharmacological manipulation of membrane potential on striatal malonate toxicity in rats in vivo by analysis of lesion volume. Depolarization caused by coinjection of the Na⁺,K⁺-ATPase inhibitor ouabain or a high concentration of potassium greatly exacerbated malonate toxicity; this combined toxicity was blocked by the noncompetitive NMDA antagonist MK-801. The toxicity of NMDA was also exacerbated by ouabain. The overt toxicity of a high dose of ouabain (1 nmol) was largely prevented by MK-801. Coinjection of the K⁺ channel activator minoxidil (4 nmol) to reduce depolarization attenuated the toxicity of 1 µmol of malonate by ∼60% without affecting malonate-induced ATP depletion. These results indicate that membrane depolarization exacerbates malonate neurotoxicity and that membrane hyperpolarization protects against malonate-induced neuronal damage. We hypothesize that the effects of membrane potential on malonate toxicity are mediated through the NMDA receptor as a result of its combined agonist- and voltage-dependent properties.     

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.     

Locally infused taurine, GABA and homotaurine alter differently the striatal extracellular concentrations of dopamine and its metabolites in rats

Summary We studiedin vivo the effects of locally infused taurine (50, 150, and 450 mM) on the striatal dopamine and its metabolites in comparison with those of GABA and homotaurine, a GABA_A receptor agonist, in freely moving rats. The extracellular dopamine concentration was elevated maximally 2.5-, 2- and 4-fold by taurine, GABA and homotaurine, respectively. At 150 mM concentration, at which the maximum effects occurred, homotaurine increased the extracellular dopamine more than taurine or GABA. When taurine and GABA were infused simultaneously with tetrodotoxin the output of dopamine did not differ from that in the presence of tetrodotoxin alone. In comparison, tetrodotoxin did not inhibit the increase in extracellular dopamine caused by homotaurine. Furthermore, omission of calcium from the perfusion fluid inhibited the increase of extracellular dopamine caused by GABA. However, it did not block the increase of dopamine caused by taurine or homotaurine. The present study suggests that the effects of intrastriatal taurine, GABA and homotaurine on the striatal extracellular dopamine differ. Thus, these amino acids seem to affect the striatal dopaminergic neurons via more than one mechanism.     

Nigrostriatal Dopaminergic Neurons Remain Undamaged in Rats Given High Doses of l-DOPA and Carbidopa Chronically

Rats were fed maximally tolerated doses of L-3,4-Dihydroxyphenylalanine (L-DOPA) and carbidopa daily for 120 days in order to achieve a sustained elevation in brain dopamine levels. Some animals were also given buthionine sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, in an unsuccessful effort to reduce brain glutathione contents. L-DOPA- and carbidopa-treated animals displayed no behavioral changes suggestive of nigrostriatal dopaminergic neuronal loss. When sacrificed 60 days after L-DOPA treatment ended, all rats had normal tyrosine hydroxylase activities and dopamine contents in their striata, and cell counts were normal in the substantia nigra. It therefore seems unlikely that a model of Parkinson's disease, suitable for exploring the etiological importance of glutathione deficiency, can be produced in rats merely by administering the largest tolerable doses of L-DOPA.     

Role of glutamate in neurodegeneration of dopamine neurons in several animal models of parkinsonism

Summary Although controversial, studies with methamphetamine and MPTP suggest a link between glutamate-mediated excitotoxicity and degeneration of dopamine cells. Both compounds are thonght to create a metabolic stress. To further explore glutamate actions in DA degeneration, we investigated the effects of other metabolic inhibitors. In mesencephalic cultures, DA cell loss produced by 3-NPA or malonate was potentiated by NMDA and prevented by MK-801. In vivo, striatal DA loss produced by intranigral infusions of malonate was also potentiated by intranigral NMDA and prevented by systemic MK-801. In contrast, systemic MK-801 did not prevent DA loss produced by intrastriatal malonate. Intrastriatal MK-801 or CGS 19755 did attenuate DA loss in METH-treated mice, but was confounded by the findings that METH-induced hyperthermia, an important component in toxicity, was also attenuated. Taken together, the data support the hypothesis of NMDA receptor involvement in degeneration of DA neurons. Furthermore, the data also suggest that this interaction is likely to occur in the substantia nigra rather than in the striatum.