Richard E. Heikkila

Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine.

1-methyl-4-phenylpyridine (MPP+), a major metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) inhibited the ADP-stimulated and uncoupled oxidation of NADH-linked substrates by brain mitochondrial preparations. MPTP itself was ineffective. The apparent Kis for MPP+ inhibition of pyruvate or glutamate oxidation by purified rat brain mitochondria were approximately 300 and 400 microM, respectively; with mouse brain mitochondria the values were lower, 60 and 150 microM, respectively. Succinate oxidation was unaffected by either compound. Compromise of mitochondrial oxidative capacity by MPP+ could be an important factor in mechanisms underlying the toxicity of MPTP.

Behavioral properties of GBR 12909, GBR 13069 and GBR 13098: specific inhibitors of dopamine uptake

Two aryl 1,4-dialkylpiperazines (GBR 12909 and GBR 13098) and one aryl 1,4-dialkenylpiperazine (GBR 13069) were very potent inhibitors of [3H]dopamine uptake in vitro in tissue slices obtained from rat neostriatum (IC50 values between 40 and 51 nM). Each compound was considerably weaker as an inhibitor of [3H]norepinephrine uptake in tissue slices obtained from rat occipital cortex (IC50 values between 560 and 2600 nM). These compounds thus are relatively specific inhibitors of [3H]dopamine uptake in vitro. The three compounds caused ipsilateral circling in rats with unilateral lesions of the nigrostriatal pathway as well as increased locomotor activity in naive mice, both of which could be greatly attenuated by pretreatment of the rodents with the dopamine receptor antagonist haloperidol. It thus follows that the compounds have dopaminergic activity in vivo. Ex vivo experiments with GBR 13069 (drug administration in vivo, uptake in vitro) suggested that these compounds may have the same relative specificity as dopamine uptake blockers in vivo. These compounds should prove to be useful pharmacological agents.

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.

The influence of dose and dosing interval on MPTP-induced dopaminergic neurotoxicity in mice

The effects of different dosing paradigms for the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were investigated in C57-black and CF-W albino mice. Several groups of mice received a total dose of 80 mg/kg of MPTP administered at different doses per injection and/or at different time intervals. In C57-black mice, the effects of MPTP administration on the neostriatal content of dopamine ranged from a 91% depletion (4 injections of 20 mg/kg per injection at 1 h intervals) to a non-significant effect (4 injections of 10 mg/kg per injection at 2 h intervals on each of 2 successive days). There was also considerable influence of the MPTP dose per injection and the dosing interval in CF-W mice, although the extent of neostriatal dopamine depletion in CF-W mice was not as great as that observed in C57-black mice. In addition, MPTP produced variable effects on neostriatal dopamine levels in different strains of mice as well as in Swiss-Webster mice obtained from different sources. Some of the strains were affected to a great extent while others were only marginally affected.

Behavioral correlations of dopamine receptor activation

According to the classification scheme of Kebabian and Calne,2 there are two types of dopamine (DA) receptors: D1 (activation of which causes increased cyclic AMP formation) and D2 (activation of which causes no increment in cyclic AMP). It is not clear what role the different receptors play in mediating motor behavior. Using drugs that act selectively at only one receptor site, we studied the effects of D1 and D2 receptor activation in two different models of parkinsonism—the rotating rat and the reserpinized mouse. Neither the D1 agonist nor the D2 agonist, given alone, could overcome reserpine akinesia, but together they restored locomotor activity. In rats with unilateral nigrostriatal lesions, both drugs induced a rotational response, each with a distinct temporal pattern. Pretreatment with alpha-methyl-paratyrosine (an inhibitor of DA synthesis) led to decrements in the rotational response induced by D2 agonists, but not that induced by D1 agonists. The mechanism by which these DA agonists induce motor activity is different; activation of both types of DA receptors seems to be necessary for normal motor behavior.

Some features of the nigrostriatal dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse

The discovery that a rather simple chemical substance can produce such a highly selective neuronal degeneration in the substantia nigra, the brain area most affected in Parkinson’s disease, has resulted in a vast amount of research with MPTP. We and others have hoped that by gaining an understanding of its mechanism of action, we might come closer to discovering the cause(s) of idiopathic Parkinson’s disease. Indeed, we have learned some fascinating things regarding the roles of monoamine oxidase B and the dopamine transport system in mediating the actions of MPTP. It is clear that the MPTP-treated mouse is a good model for Parkinson’s disease. As such, it may help to define the role of dopamine deficiency in the pathophysiology of Parkinson’s disease as well as provide a model in which potential anti-Parkinsonian therapeutic agents can be tested.

Prevention of the Nigrostriatal Toxicity of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine by Inhibitors of 3,4‐Dihydroxyphenylethylamine Transport

Abstract The 3,4‐dihydroxyphenylethylamine (DA, dopamine) uptake inhibitors GBR 13,069, amfonelic acid, WIN‐35,065‐2, WIN‐35,428, nomifensine, mazindol, cocaine, McN‐5908, McN‐5847, and McN‐5292 were effective in preventing [3H]DA and [3H]l‐methyl‐4‐phenylpyridinium (MPP+) uptake in rat and mouse neostriatal tissue slices. These DA uptake inhibitors also were effective in attenuating the MPP+‐induced release of [3H]DA in vitro. 1‐Methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) administration to mice (6 ± 25 mg/kg i.p.) resulted in a large (70–80%) decrement in neostriatal DA. WIN‐35,428 (5 mg/kg), GBR 13,069 (10 mg/kg), McN‐5292 (5 mg/kg), McN‐5908 (2 mg/ kg), and amfonelic acid (2 mg/kg), when administered intraperitoneally 30 min prior to each MPTP injection, fully protected against MPTP‐induced neostriatal damage. Other DA uptake inhibitors showed partial protection in vivo at the doses selected. Desmethylimipramine did not prevent [3H]MPP+ uptake or MPP+‐induced release of [3H]DA in vitro, and did not protect against MPTP neurotoxicity in vivo. These results support the hypothesis put forth previously by others that the active uptake of MPP+ by dopaminergic neurons is necessary for toxicity.

The relationship between loss of dopamine nerve terminals, striatal [3H]spiroperidol binding and rotational behavior in unilaterally 6-hydroxydopamine-lesioned rats.

The relationship between the destruction of dopamine-containing nerve terminals, specific binding of [3H]spiroperidol and contralateral rotation in response to L-DOPA, was studied in rats with unilateral lesions of the nigrostriatal dopamine (DA) system, induced by intracerebral injections of the neurotoxin 6-hydroxydopamine (6-OHDA). Animals with significant rotational behavior in response to L-DOPA had both a greater amount of specific binding of [3H]spiroperidol in the lesioned striatum compared to the non-lesioned striatum, and at least 90% destruction of DA terminals in the lesioned striatum (less than 10% of control uptake). The nonrotators to L-DOPA had considerably less destruction of DA terminals and no significant increase in specific binding on the lesioned side. The data from this study suggest that before L-DOPA is effective as an inducer of contralateral rotational behavior, there must be both unilateral damage to the DA terminals greater than 90%, and increased specific binding.