Stephen K. Youngster

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.

Interaction of 1-Methyl-4-Phenylpyridinium Ion (MPP+) and Its Analogs with the Rotenone/Piericidin Binding Site of NADH Dehydrogenase

Abstract: Nigrostriatal cell death in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinsons disease results from the inhibition of mitochondrial respiration by 1‐methyl‐4‐phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4′‐analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4′‐heptyl‐MPP+, being 6 μM. The 4′‐analogs compete with rotenone for the binding site in a concentration‐dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4′‐propyl‐MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.

Evaluation of the Biological Activity of Several Analogs of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: Several analogs of l‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO‐A or MAO‐B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO‐A but many of the compounds were found to be good substrates for MAO‐B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1‐methyl‐4‐cyclohexyl‐1,2,3,6‐tetrahydropyridine, 1 ‐methyl‐4‐(2′‐methylphenyl)‐1,2,3,6‐tetrahydropyridine and 1‐methyl‐4‐(3′‐methoxyphenyl)‐1,2,3,6‐tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO‐B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO‐B. The capacity of the compounds studied to be oxidized by MAO‐B appears to be an important aspect of the neurotoxic process.

Effects of 1‐Methyl‐4‐Phenyl‐1,2,5,6‐Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4‐Dihydroxyphenylethylamine and [3H]5‐Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1‐Methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4‐dihydroxyphenylethylamine‐([3H]DA), [3H]5‐hydroxytryptamine ([3H]5‐HT), and [3H]γ‐aminobutyric acid ([3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5‐HT uptake (IC50 value 0.14 μM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 μM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 μM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5‐HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4‐electron oxidation product of MPTP, i.e., 1‐methyl‐4‐phenyl‐pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 μM) and of [3H]5‐HT uptake (IC50 value 0.78 μM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.

Oxidation of Analogs of l‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine by Monoamine Oxidases A and B and the Inhibition of Monoamine Oxidases by the Oxidation Products

Abstract: Twenty analogs of l‐methyl‐4‐phenyl‐l,2,3,6‐tet‐rahydropyridine (MPTP) were tested for their capacity to be xidized by pure monoamine oxidase‐A (MAO‐A) prepared from human placenta and pure monoamine oxidase‐B (MAO‐B) prepared from beef liver. Several of the MPTP analogs were very good substrates for MAO‐A, for MAO‐B, or for both and had low Km values and high turnover numbers. These values were similar to or even better than those of kynuramine and benzylamine, good substrates for MAO‐A and MAO‐B, respectively. MPTP had relatively low Km values for oxidation by both MAO‐A and MAO‐B. In contrast, the turnover number for MPTP oxidation by MAO‐B was considerably higher than the value for MAO‐A. The corresponding pyridinium species of MPTP and several of the MPTP analogs inhibited MAO‐A competitively with Ki values at micromolar concentrations; in contrast the pyridinium species inhibited MAO‐B competitively at considerably higher concentrations (i.e., 100 μM or greater Ki values). The data provide information concerning the structural requirements for the oxidation of tetrahydropyridines by MAO‐A and MAO‐B and the inhibition of these enzymes by pyridini‐ums

Studies on the Characterization of the Inhibitory Mechanism of 4′‐Alkylated 1‐Methyl‐4‐Phenylpyridinium and Phenylpyridine Analogues in Mitochondria and Electron Transport Particles

Abstract: 1‐Methyl‐4‐phenylpyridinium (MPP+), the toxic agent in MPTP‐induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′‐alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4′‐alkylated phenylpyridine derivatives to inhibit NADH‐linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1‐octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ∼600‐fold, versus only a 36‐fold, increase in the IC50 of MPP+ versus 4′‐pentyl‐MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′‐decyl‐MPP+ was nearly 80‐fold more potent than phenylpyridine although the latter compound partitions twice as much into 1‐octanol. In addition, the lipophilic anion TPB− was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.

1-Methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine (MCTP): an alicyclic MPTP-like neurotoxin

1-Methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine (MCTP), an analog of MPTP, was found to be an MPTP-like neurotoxin. MCTP administration caused extensive losses of neostriatal dopamine and its major metabolites in male Swiss-Webster mice. Under similar experimental conditions, MCTP was approximately as potent as MPTP. Like MPTP, MCTP was a good substrate for monoamine oxidase-B (MAO-B) and its neurotoxicity was prevented in mice by AGN-1135, a selective inhibitor of MAO-B. The neurotoxicity of MCTP and of MPTP was also prevented by the dopamine uptake inhibitor mazindol. 1-Methyl-4-cyclohexylpyridinium ion (MCP+), the 4-electron oxidation product of MCTP, caused release of previously accumulated [3H]dopamine from mouse neostriatal synaptosomes. This release was blocked by mazindol, which indicates that MCP+, like 1-methyl-4-phenylpyridinium ion (MPP+), the 4-electron oxidation product of MPTP, is a substrate for the dopamine transport system. Like MPP+, MCP+ was found to inhibit the mitochondrial oxidation of NADH-linked substrates. It appears that conjugation between the tetrahydropyridine ring and a 4-substituent is not a requirement for an MPTP analog to possess neurotoxicity.

Studies with the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and several of its analogs

The nigrostriatal dopaminergic neurotoxicity of MPTP was prevented in mice in a dose-dependent manner by the monoamine oxidase-B (MAO-B) inhibitor deprenyl. This finding, combined with other observations, points out the important role of MAO-B in the bioactivation of MPTP. In the present study, some comparisons between MPTP and several of its structural analogs will be presented.