William J. Nicklas

Studies on the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: inhibition of NAD-linked substrate oxidation by its metabolite, 1-methyl-4-phenylpyridinium

Abstract: The effects of the parkinsonism‐inducing neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its 4‐electron oxidation product 1‐methyl‐4‐phenylpyridinium (MPP+) were studied in isolated mitochondria and in mouse brain striatal slices. ADP‐stimulated oxidation of NAD‐linked substrates was inhibited in a time‐dependent manner by MPP+ (0.1–0.5 mM), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP+ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP+ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.

Dopaminergic toxicity of rotenone and the 1-methyl-4-phenylpyridinium ion after their stereotaxic administration to rats: Implication for the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity

The 1-methyl-4-phenyl-pyridinium ion (MPP+) is the four electron oxidation product of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine (MPTP). MPP+ can be formed by the oxidation of MPTP by monoamine oxidase B to the intermediate dihydropyridinium species, MPDP+, which is spontaneously transformed to MPP+. In the present study, MPP+, like the mitochondrial toxin rotenone, inhibited pyruvate-malate respiration in isolated mitochondrial preparations. Moreover, the stereotaxic administration of both MPP+ and rotenone caused damage to the dopaminergic nigrostriatal pathway. These data clearly demonstrate that a mitochondrial toxin, administered stereotaxically, is extremely neurotoxic. The data lend support to the concept that MPTP-induced neurotoxicity may be due to the detrimental actions of enzymatically formed MPP+ on mitochondrial function.

IV. MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by by isolated mitochondria. 2′-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.

L-dopa cytotoxicity to PC12 cells in culture is via its autoxidation.

Abstract: The mechanism of cytotoxicity of l‐DOPA was studied in the rat pheochromocytoma PC12 cell line. The cytotoxicity of l‐DOPA to PC12 cells was time and concentration dependent. Carbidopa, which inhibited the conversion of l‐DOPA to dopamine, did not protect against l‐DOPA cytotoxicity in PC12 cells. Furthermore, clorgyline, a selective inhibitor of monoamine oxidase type A, and pargyline, an inhibitor of both monoamine oxidase types A and B, both did not have an effect on l‐DOPA toxicity. These findings suggest that cytotoxicity was not due to dopamine formed from l‐DOPA. Catalase or superoxide dismutase each partially protected against l‐DOPA toxicity in PC12 cells. In combination, the effects were synergistic and provided almost total protection against cytotoxicity. 6‐Cyano‐7‐nitroquinoxaline‐2,3‐dione, an antagonist of non‐NMDA receptors, did not protect against l‐DOPA toxicity. These data suggest that toxicity of l‐DOPA is most likely due to the action of free radicals formed as a result of its autoxidation. Furthermore, these findings suggest that patients on long‐term l‐DOPA therapy are potentially at risk from the toxic intermediates formed as a result of its autoxidation.

Evidence that the loss of the voltage-dependent Mg2+ block at the N-methyl-D-aspartate receptor underlies receptor activation during inhibition of neuronal metabolism.

Abstract: In this study, the importance of the Mg2+ blockade of the N‐methyl‐D‐aspartate (NMDA) receptor during metabolic stress was examined in embryonic day 13 chick retina. Retina exposed to mild conditions of metabolic stress (i.e., blockade of glycolysis with 1 mM iodoacetate for 30 min) underwent acute histological somal and neuritic swelling and an increase in γ‐aminobutyric acid (GABA) release into the medium. These acute signs of metabolic stress were eliminated by NMDA antagonists present during pharmacological blockade of glycolysis, occurred in the absence of a net increase in extracellular glutamate or aspartate, and were not affected by the presence or absence of Ca2+ in the incubation medium. One possible explanation for the activation of NMDA receptors in the absence of an increase in extracellular ligand is that NMDA sensitivity during metabolic stress may be governed at the receptor level. Depolarization of membrane potential during metabolic stress may result in the loss of the Mg2+ blockade from the NMDA receptor channel, resulting in an increased potency for glutamate. To test this, the dose‐response characteristics for NMDA, glutamate, and kainate in the presence or absence of extracellular Mg2+ and the effects of Mg2+ on metabolic inhibition were examined. The potency for NMDA‐ or glutamate‐mediated acute toxicity was enhanced two‐ to fivefold in the absence of Mg2+. Omission of Mg2+ greatly decreased the minimal concentration of agonist needed to produce acute excitotoxicity; 25 versus 5 μM for NMDA and 300 versus 10 μM for glutamate in 1.2 or zero Mg2+, respectively. Elevating external Mg2+ to 20 mM completely protected against NMDA‐mediated acute toxic effects. In contrast, varying external Mg2+ had no effect on kainate‐induced toxicity. Acute toxicity caused by inhibition of metabolism was not potentiated in the absence of Mg2+ but was attenuated by elevating extracellular Mg2+. The protective effect of Mg2+ during metabolic inhibition was not additive with NMDA antagonists, suggesting that the action of Mg2+ was at the level of the NMDA receptor. These findings are consistent with the hypothesis that the Mg2+ block is lifted during metabolic inhibition and may be the primary event resulting in NMDA receptor activation.

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.

Dopaminergic toxicity after the stereotaxic administration of the 1-methyl-4-phenylpyridinium ion (MPP+) to rats

The administration of the 1-methyl-4-phenylpyridinium ion (MPP+) stereotaxically into the left neostriatum or left median forebrain bundle of female rats resulted in a very large and highly significant loss of dopamine and of its metabolites in the left neostriatum. The effect of MPP+ on neostriatal dopamine content was in general considerably greater than its effect on serotonin or on several amino acids. These results are consistent with the premise that MPP+, formed from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by the enzyme monoamine oxidase B, may be responsible for the toxicity observed after MPTP administration.

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Abstract: Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1‐14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a >70% decrease in 14CO2 production from [1‐14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.

Excitatory Amino Acid‐Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Abstract: Acute excitotoxicity in embryonic chick retina and the ability of C1− channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N‐methyl‐D‐aspartate resulted in a large dose‐dependent release of γ‐aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glu‐tamate or aspartate. Concentrations inducing maximal γ‐aminobutyric acid release were 50 μM quisqualate, 100 μM kainate, and 100 μM N‐methyl‐D‐aspartate. Treatment with 1 mM glutamate resulted in significant γ‐aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of γ‐aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of C1− completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the C1−/bicarbonate anion channel blocker 4,4′‐di‐isothiocyanatostilbene‐2,2′‐disulfonate at 600 μM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide. which blocks Na+/K+/2C1− cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of C1− is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl−flux may be involved.

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.