Gail D. Zeevalk

MK‐801 Fails to Protect Against the Dopaminergic Neuropathology Produced by Systemic 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine in Mice or Intranigral 1‐Methyl‐4‐Phenylpyridinium in Rats

Abstract: Previous studies from this laboratory demonstrated that (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK‐801), an N‐methyl‐D‐aspartate (NMDA) receptor antagonist, did not prevent neurotoxicity to dopaminergic neurons in mice produced by systemic treatment with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). However, Turski et al. [Nature349, 414–418 (1991)] reported that extended treatment of rats with NMDA receptor antagonists (six injections at 4‐h intervals) did prevent the loss of nigral dopaminergic neurons resulting from an intranigral infusion of 1‐methyl‐4‐phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP. The present studies examined if a similar extended treatment with MK‐801 would protect mice from the neurotoxicity of systemically administered MPTP. Six intraperitoneal injections of MK‐801 given at 4‐h intervals did not protect mice against the MPTP‐induced neostriatal dopamine loss measured 1 week after treatment. In other experiments, designed to replicate and expand on the results of Turski et al. (1991), the extended treatment of rats with MK‐801 did not prevent MPP+‐induced cell loss in the infused substantia nigra pars compacta or the dopamine depletion in the ipsilateral neostriatum at 7‐11 days after MPP+ infusion. These results do not support the hypothesis that NMDA receptors are involved with MPTP/MPP+‐induced neurodegeneration.

Role of oxidative stress and the glutathione system in loss of dopamine neurons due to impairment of energy metabolism.

Abstract: Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinsons disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose‐dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high‐affinity uptake of [3H]dopamine and [14C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24‐h pretreatment with 10 µM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin‐trapping agents, α‐phenyl‐tert‐butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate‐induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin‐trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase‐positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin‐trapping agents. NMDA receptors have also been shown to play a role in malonate‐induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1‐h exposure to 50 µM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.

Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: Relevance to Parkinson’s disease

Parkinsons disease (PD) is associated with loss of total glutathione (GSH) which may contribute to progressive cell death. Peripheral GSH administration has been used clinically with reported benefits. Despite this, there is little specific information to characterize its cellular uptake or clearance, brain elevation with peripheral delivery or neuroprotective efficacy in PD models. The current study was carried out to provide this information using in vitro and in vivo approaches. In rat mesencephalic culture, the monoethyl ester of GSH (GEE), but not GSH (1-10 mM, 24 h) produced a dose-dependent elevation in GSH. The half-life for clearance was 10.14 h and was not different in cells depleted of GSH prior to loading. Elevation of GSH with GEE protected neurons from oxidative stress with H2O2 or metabolic stress with the complex I and II inhibitors MPP+ and malonate, respectively. To determine if peripheral administration of GEE could elevate brain GSH levels, rats were administered 0.1-50 mg/kg/day GEE via osmotic minipump either subcutaneously (sc) or via a cannula placed into the left cerebral ventricle (icv) for 28 days. Only central delivery of GEE resulted in significant elevations of brain GSH. Elevation of brain GSH by icv infusion of GEE was examined for its neuroprotective effects against chronic central delivery of MPP+. Infusion of 0.142 mg/kg/day MPP+ for 28 days caused a selective ipsilateral loss of striatal dopamine. Co-infusion of MPP+ with 10 mg/kg/day GEE significantly protected against striatal dopamine loss. These findings show that the ethyl ester of GSH but not GSH per se can elevate intracellular GSH, that brain elevation of GSH requires central delivery of the ethyl ester and that this elevation provides neuroprotection against oxidative stress or chronic mitochondrial impairment.

Inhibition of brain mitochondrial respiration by dopamine: involvement of H2O2 and hydroxyl radicals but not glutathione–protein–mixed disulfides

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 0.5u2003mm DA for 15u2003min 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 (H2O2), hydroxyl radical, oxidized glutathione (GSSG) or glutathione–protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H2O2 (100u2003µm) significantly inhibited state‐3 respiration. In contrast, incubation with GSSG (1u2003mm) had no effect on O2 consumption. Exposure of mitochondria to 1u2003mm GSSG resulted in a 3.3‐fold increase in PrSSG formation compared with 1.4‐ and 1.5‐fold increases in the presence of 100u2003µm H2O2 or 0.5u2003mm 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 membrane‐bound (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, H2O2 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 IC50 approximately 2–3‐fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H2O2. Co‐administration of desferrioxamine with H2O2 had no effect on complex I‐associated inhibition in intact mitochondria, but attenuated inhibition of rotenone‐sensitive NADH oxidase activity by 70% in freeze–thawed mitochondria. The results show that DA‐associated inhibition of respiration is dependent on MAO and that H2O2 and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.

Rat model of Parkinson's disease: Chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+)

Mitochondrial dysfunction is observed in sporadic Parkinsons disease (PD) and may contribute to progressive neurodegeneration. While acute models of mitochondrial dysfunction have been used for many years to investigate PD, chronic models may better replicate the cellular disturbances caused by long-standing mitochondrial derangements and may represent a better model for neurotherapeutic testing. This study sought to develop a chronic model of PD that has the advantages of continuous low level toxin delivery, low mortality, unilateral damage to minimize aphagia and adipsia as well as minimal animal handling to reduce stress-related confounds. Infusion by osmotic minipump of the complex I toxin, 1-methyl-4-phenylpyridinium (MPP+), for 28 days into the left cerebral ventricle in rats caused a selective ipsilateral loss of nigral tyrosine hydroxylase immunoreactive somata (35% loss). In animals that were sacrificed 14 days after the chronic MPP+ administration, there was an even greater loss of nigral tyrosine hydroxylase cells (65% loss). Lewy-body-like structures that stained positive for ubiquitin and alpha-synuclein were found in striatal neurons near the infusion site but were not observed in nigral neurons. At the electron microscope level, however, swollen and abnormal mitochondria were observed in the nigral dopamine neurons, which may represent the early formation of an inclusion body. There were no animal deaths with the chronic treatment regimen that was utilized, and the magnitude of nigrostriatal neuronal loss was relatively consistent among the animals. This model of progressive neurodegeneration of nigrostriatal dopamine neurons may be useful for studying neuroprotective therapeutic agents for PD.

Acute excitotoxicity in chick retina caused by the unusual amino acids BOAA and BMAA: Effects of MK-801 and kynurenate

beta-N-Oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) were tested for their ability to produce acute excitotoxicity in in embryonic chick retina. gamma-Aminobutyric acid (GABA) release and histology were monitored in retina treated with various concentrations of BOAA, BMAA, kainate (KA), N-methyl-D-aspartate (NMDA), or glutamate. BOAA and BMAA caused retinal lesions similar to those produced by the excitatory amino acids. BOAA was slightly less potent than KA, whereas BMAA had a potency similar to glutamate. BOAA, like KA and NMDA, caused a dose-dependent increase in GABA release. Addition of the NMDA antagonist (+)-MK-801, completely blocked acute excitotoxicity caused by NMDA or BMAA but was ineffective against KA or BOAA. Kynurenate, a nonspecific glutamate receptor antagonist, and DIDS, a Cl- channel blocker, were effective in blocking all agonist-induced toxicity. It is concluded that BOAA and BMAA cause excitotoxic damage in retina; BOAA induces toxicity through a non-NMDA type glutamate receptor and BMAA through the NMDA receptor.

Hypothermia, Metabolic Stress, and NMDA‐Mediated Excitotoxicity

Abstract: Isolated embryonic retinas were metabolically stressed by inhibition of glycolysis either with iodoacetate (IOA) or by glucose withdrawal plus 10 mM 2‐deoxy‐D‐glucose, and the effects of hypothermia were examined. Incubation at 30 versus 37°C during 30 min of hypoglycemia with IOA completely reduced the rapid swelling‐related GABA release [6 ± 2 vs. 68 ± 10 nmol/100 mg of protein (mean ± SEM) for 30 and 37°C, respectively]. Histology of the retina immediately following 30 min of metabolic stress at 30°C appeared normal, whereas that at 37°C showed a pattern of acute edema, characteristic of NMDA‐mediated acute excitotoxicity. Coincubation with a competitive or noncompetitive NMDA antagonist, respectively, CGS‐19755 (10 μM) or MK‐801 (1 μM), during 30 min of hypoglycemia at 37°C completely prevented tissue swelling, whereas extracellular GABA content remained at basal levels, indicating that the cytotoxic effects of IOA treatment for 30 min at 37°C were NMDA receptor mediated. Longer periods of hypoglycemia at 37° C produced acute toxicity that was only partially NMDA receptor mediated. Hypothermia delayed the onset of NMDA‐mediated toxicity by 30–60 min. At 30°C, the rate of loss of ATP was slowed during the first several minutes of hypoglycemia (82 and 58% of maximal tissue levels at 30 and 37° C, respectively, at 5 min), but by 10 min, ATP levels were comparably reduced. After a transient exposure of retina to 50 μM NMDA in Mg2+‐free medium, hypothermia significantly attenuated acute GABA release by 30%. At 24 h of recovery, lactate dehydrogenase release was decreased by 37%. Hypothermia had no effect when the exposure was done in medium containing physiological concentrations of Mg2+. The above results suggest that the protective effect of hypothermia during the metabolic insult is predominately directed at the cellular events that lead up to NMDA receptor involvement. Reduction in the rate of loss of ATP, however, does not fully account for the delay in involvement of NMDA receptors during metabolic stress at 30°C. The attenuation of direct NMDA‐mediated toxicity in Mg2+‐free medium further suggests that decreased temperature may result in altered channel properties during situations when the Mg2+ block is lifted.

Developmental differences in antagonism of NMDA toxicity by the polyamine site antagonist ifenprodil

Antagonists of 4 distinct regulatory sites on the N-methyl-D-aspartate (NMDA) receptor were tested for their ability to attenuate NMDA-mediated acute excitotoxicity in isolated chick retina of various embryonic ages between days 11 and 19 in ovo. Acute excitotoxicity was monitored by histology and by release of endogenous gamma-aminobutyric acid (GABA) into the medium during 30 min of incubation with 50 microM NMDA. The uncompetitive PCP channel site antagonist, MK-801, the competitive antagonist, CGS 19755, and the strychnine-insensitive glycine site antagonist, 7-chlorokynurenate, completely blocked NMDA-induced cell swelling and increased GABA release at all ages tested. Potencies versus NMDA were MK-801 greater than CGS 19755 greater than 7-chlorokynurenate with IC50S of 0.02, 0.62, and 15 microM, respectively. NMDA antagonism by the polyamine site antagonist, ifenprodil, differed from other classes of antagonists in several respects. At the earlier embryonic ages tested (E12-13) ifenprodil provided differential protection; completely blocking somal and neuritic swelling in most but not all inner nuclear layer neurons and inner plexiform processes. In dose-response studies, ifenprodil attenuated the NMDA-induced increase in medium GABA at all ages tested with an Imax of 10 microM. Ifenprodil, however, showed a decreased ability to completely protect some NMDA-sensitive neurons. This was reflected both histologically and by GABA release. Maximal attenuation of NMDA evoked GABA release was 83, 80, 62 and 50% at days E12, 13, 15 and 19, respectively. Histologically, differential protection was seen at E12 and 13, in limited areas at E15, and was no longer present at E19.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Parkinsons disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complexu2003II. 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 10u2003µm buthionine sulfoximine for 24u2003h depleted total GSH by 60%, whereas 3u2003h exposure to 5u2003mm 3‐amino‐1,2,4‐triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH‐depleted cells with malonate (40u2003mm) for 6, 12 or 24u2003h 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 400u2003µm ascorbate protected completely against malonate toxicity (50u2003mm, 12u2003h), whereas treatment with 1u2003mm 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 (50u2003mm) for 12u2003h 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 72u2003h 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.

Functional glutaredoxin (thioltransferase) activity in rat brain and liver mitochondria.

Glutaredoxin (Grx) is a specific and efficient catalyst of glutathione-dependent deglutathionylation of protein-SS-glutathione mixed disulfides. Grx has been identified in brain cytosol, but the presence of activity in subcellular organelles has not been reported. Increases in protein glutathionylation are likely to occur in mitochondria during oxidative stress and it is, therefore, important to know if this organelle contains the enzyme activity needed to reverse such protein thiolation. Grx-like activity in the P1 supernatant from rat brain and liver was doubled in the presence of Triton-X 100 suggesting a releasable pool of Grx. Brain and liver homogenates were subfractionated into cytosolic, mitochondrial and microsomal fraction, their purity determined by biochemical assay and EM and assayed for Grx-like activity. The data presented demonstrate that mitochondria contain functional Grx-like activity.