Thomas P. Singer

Advances in Our Understanding of the Mechanisms of the Neurotoxicity of MPTP and Related Compounds

The discovery that 1 -methyl4-phenyl1,2,3,6-tetrahydropyndine (MPTP), a contaminant of a synthetic pethidine analogue sold as a street drug, produced a condition resembling Parkinson’s disease has been described in detail (for reviews, see Langston, 1985; Snyder and DAmato, 1986). MPTP was shown to cause a selective destruction of nigrostriatal dopaminergic neurons in primates and some other animal species, and this stimulated a great deal ofwork on the mechanisms involved in its neurotoxic effects and their possible relationship to idiopathic Parkinson’s disease. Earlier work on the mechanism by which MPTP exerts its selective neurotoxic effects has been reviewed before, and the present account will concentrate on more recent discoveries concerning its biochemical actions and the unresolved problems that remain.

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.

Mechanism of the neurotoxicity of MPTP. An update.

This review summarizes advances in our understanding of the biochemical events which underlie the remarkable neurotoxic action of MPTP (1‐methyl‐4‐phenyl‐1‐1,2,3,6‐tetrahydropyridine) and the parkinsonian symptoms it causes in primates. The initial biochemical event is a two‐step oxidation by monoamine oxidase B in glial cells to MPP+ (1‐methyl‐4‐phenylpyridinium). A large number of MPTP analogs substituted in the aromatic (but not in the pyridine) ring are also oxidized by monoamine oxidase A or B, is in some cases faster than any previously recognized substrate. Alkyl substitution at the 2‐position changes MPTP, a predominantly B type substrate, to an A substrate. Following concentration in the dopamine neurons by the synaptic system, which has a high affinity for the carrier, MPP+ and its positively charged neurotoxic analogs are further concentrated by the electrical gradient of the inner membrane and then more slowly penetrate the hydrophobic reaction site on NADH dehydrogenase. Both of the latter events are accelerated by the tetraphenylboron anion, which forms ion pairs with MPP+ and its analogs. Mitochondrial damage is now widely accepted as the primary cause of the MPTP induced death of the nigrostriatal cells. The molecular target of MPP+, its neurotoxic product, is NADH dehydrogenase. Recent experiments suggest that the binding site is at or near the combining site of the classical respiratory inhibitors, rotenone and piericidin A.

Energy-driven uptake of N-methyl-4-phenylpyridine by brain mitochondria mediates the neurotoxicity of MPTP

The oxidation of NAD+-linked substrates by rat brain mitochondria is completely inhibited by pre-incubation with 0.5 mM N-methyl-4-phenylpyridine (MPP+). The effect is dependent on the integrity of the mitochondria because far higher concentrations of MPP+ are required to inhibit NADH oxidation in inverted mitochondria or isolated inner membrane preparations. The reason for this difference in behavior has been traced to a novel system for the uptake of MPP+ into mitochondria against a concentration gradient. The uptake system is energized by the transmembrane potential, as shown by the fact that valinomycin plus K+, which collapses this gradient, abolishes MPP+ uptake, while agents which collapse the proton gradient have no effect on the process. If an uncoupler is added to mitochondria preloaded with MPP+, efflux of the latter occurs with the concentration gradient. The uptake system has been studied in liver, whole brain, cortex, and midbrain preparations from rats. It may be readily distinguished from the synaptic dopamine reuptake system, since the former is blocked by uncouplers and respiratory inhibitors, but not by dopamine or mazindol, whereas the synaptic system is blocked by mazindol and competitively inhibited by dopamine but is not affected by respiratory inhibitors or uncouplers. Energy-driven uptake of MPP+ by brain mitochondria may be a crucial step in the complex sequence of events leading to the neurotoxic actions of its precursor, MPTP.

The reaction sites of rotenone and ubiquinone with mitochondrial NADH dehydrogenase

This article summarizes recent studies in the authors and other laboratories of selective inhibitors acting at the rotenone site and at the Q binding site in the NADH-Q oxidoreductase segment of the respiratory chain. A wide array of inhibitors act at the rotenone site to block electron flux from the enzyme to the Q pool. Using evidence from studies with rotenone, piericidin A, and analogs of the neurotoxic N-methyl-4-phenylpyridinium, we have proposed two binding sites for these inhibitors, both of which must be occupied for complete inhibition of NADH oxidation.

Relation of superoxide generation and lipid peroxidation to the inhibition of NADH-Q oxidoraductase by rotenone, piericidin A, and MPP+

The addition of NADH to submitochondrial particles inhibited by agents which interrupt electron transport from NADH-Q oxidoreductase (Complex I) to Q10 (rotenone, piericidin A, and MPP+) results in superoxide formation and lipid peroxidation. A study of the quantitative relations now shows that oxyradical formation does not appear to be the direct result of the inhibition. Although tetraphenyl boron (TPB) greatly enhances the inhibition by MPP+, it has no effect on O2. formation or lipid peroxidation. When submitochondrial particles completely inhibited by rotenone or piericidin A are treated with bovine serum albumin to remove spuriously bound inhibitor molecules without affecting those bound at the specific inhibition site, NADH-Q activity remains inhibited and lipid peroxidation occurs but superoxide formation ceases. Thus oxyradical formation may be the result of the binding of inhibitors at sites in the membrane other than those related to the inhibition of electron transport.

On the structure of succinic dehydrogenase flavocoenzyme.

Several problems concerning the structure of the so-called covalently bound flavin remain to be resolved [l-3]. This type of flavin is mostly, if not exclusively, connected with succinate dehydrogenase flavoprotein from aerobic cells (“SD-flavin”) and differs from acid-extractable flavin of the same tissue by the following main characteristics: pH-dependence of the fluorescence, optical spectra and alkaline photolysis. SD-flavin is present as “SD-FAD” in the protein [l] but yields “SD-FMN” and “SD-riboflavin” on stepwise acid and/or enzymic hydrolysis. The “SD” prefix means that the flavin is covalently substituted by a group X, which is also covalently connected to the protein backbone or, after proteolysis, to a residual peptide or a final amino acid residue. The following main questions remain unresolved: 1. At which position of the flavin nucleus is the connection to group X? 2. What is the nature of the group X? 3. Which is the C-terminal amino acid and how is it connected to group X? The present communication deals with the first of these questions.

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.

Deficiencies of NADH and succinate dehydrogenases in degenerative diseases and myopathies

This paper examines the experimental foundations of reports in the literature on mitochondrial diseases involving Complexes I and II of the respiratory chain. Many of the reports may be questioned on the basis of the assay conditions used which disregard established knowledge of the precautions required for valid activity measurements. In addition, some findings are open to question because of the experimental material chosen for the study, such as the measurement of NADH oxidase activity in platelets in Parkinsons disease, which affects selectively the dopamine neurons, or the use of autopsy material stored for prolonged periods during which post-mortem changes may have occurred. Deficiencies claimed to involve several components of the respiratory chain may reflect indirect effects, such as defects in the synthesis of iron-sulfur clusters or in the availability of iron, rather than mutations in the genes coding for the deficient enzymes. Nevertheless, there are a few instances reported of Complex II deficiency free from such criticisms. As to Complex I, idiopathic Parkinsonism appears to involve a documentable decline in the activity of this enzyme. Using the model system provided by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces biochemical, pharmacological, and clinical syndromes closely resembling Parkinsonism, the etiology of the disease is examined.

III. Bioactivation of MPTP: Reactive metabolites and possible biochemical sequelae

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.