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]gamma-aminobutyric acid [( 3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5-HT uptake (IC50 value 0.14 microM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 microM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 microM 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 microM) and of [3H]5-HT uptake (IC50 value 0.78 microM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

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 Ki's 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.     

Active uptake of MPP+, a metabolite of MPTP, by brain synaptosomes

Mouse brain synaptosomal preparations were used to study uptake of N-methyl-4-phenylpyridine (MPP+), a metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The uptake of [3H]-MPP+ by striatal synaptosomes was approximately 25 X greater than that of [3H]-MPTP, with a KM of 0.48 microM and a Vmax of 5.3 nmoles/g tissue/min. Uptake was Na+ dependent and inhibited by ouabain, cocaine and dopamine (Ki 0.12 microM). Synaptosomes prepared from the corpus striatum accumulated [3H]-MPP+ at a rate 5-10 times higher than preparations from other brain regions. This selective uptake of MPP+ may contribute to the specificity of the toxic effects of MPTP on nigrostriatal dopaminergic neurons.     

Structure-neurotoxicity trends of analogues of 1-methyl-4-phenylpyridinium (MPP+), the cytotoxic metabolite of the dopaminergic neurotoxin MPTP

The dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) derives from its metabolism to 1-methyl-4-phenyl-pyridinium cation (MPP+), which is then selectively accumulated in dopaminergic neurons. In an effort to assess the structural requirements governing MPP+ cytotoxicity, we evaluated dopaminergic toxicity of MPP+ analogues 3 weeks after their microinfusion into rat substantia nigra. We also evaluated the substrate suitability of MPP+ analogues for high-affinity dopamine uptake in striatal synaptosomes by measuring their ability to induce specific dopamine release. The intranigral neurotoxicity of MPP+ analogues in vivo correlates mainly with their in vitro inhibitory activity on mitochondrial respiration, consistent with a compromise in cellular energy production as the principal mechanism of MPTP-induced cell death. This study extends the structure-neurotoxicity data base beyond that obtainable using MPTP analogues, since many of these are not metabolized to pyridinium compounds. Such information is crucial to assess which possible endogenous or exogenous compounds may exert MPTP/MPP(+)-like toxicity.     

Effect of dopaminergic neurotoxin MPTP/MPP+ on coenzyme Q content

Coenzyme Q10, an endogenous lipophilic antioxidant, plays an indispensable role in ATP synthesis. The therapeutic value of coenzyme Q10 in Parkinson's disease and other neurodegenerative disorders is still being tested and the preliminary results are promising. The 1-methyl-4-phenyl-1, 2, 3, 6 tetrahydropyridine (MPTP)-treated mouse is a valid and accepted animal model for Parkinson's disease. 1-methyl-4-phenylpyridinium (MPP(+)) is an active toxic metabolite of MPTP. MPP(+) and MPTP are known to induce oxidative stress and mitochondrial dysfunction. However, the effect of MPP(+) and MPTP on coenzyme Q is not clearly understood. The present study investigated the in vitro and in vivo effect of MPP(+) and MPTP on coenzyme Q content. Coenzyme Q content was measured using HPLC-UV detection methods. In the in vitro studies, MPP(+) (0-50 microM) was incubated with SH-SY5Y human neuroblastoma cells and NG-108-15 (mouse/rat, neuroblastomaxglioma hybrid) cells. MPP(+) concentration dependently increased coenzyme Q10 content in SH-SY5Y cells. In NG-108-15 cells, MPP(+) concentration dependently increased both coenzyme Q9 and Q10 content. In the in vivo study, mice were administered with MPTP (30 mg/kg, twice 16 h apart) and sacrificed one week after the last administration. Administration of MPTP to mice significantly increased coenzyme Q9 and coenzyme Q10 levels in the nigrostriatal tract. However, MPTP did not affect the coenzyme Q content in the cerebellum, cortex and pons. This study demonstrated that MPP(+)/MPTP significantly affected the coenzyme Q content in the SH-SY5Y and NG-108 cells and in the mouse nigrostriatal tract.     

MPP+ inhibits proliferation of PC12 cells by a p21(WAF1/Cip1)-dependent pathway and induces cell death in cells lacking p21(WAF1/Cip1).

The molecular and biochemical mode of cell death of dopaminergic neurons in Parkinson's disease (PD) is uncertain. In an attempt at further clarification we studied the effects of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on dopaminergic PC12 cells. In humans and nonhuman primates MPTP/MPP+ causes a syndrome closely resembling PD. MPP+ toxicity is thought to be mediated by the block of complex I of the mitochondrial electron transport chain. Treatment of undifferentiated PC12 cells with MPP+ primarily inhibited proliferation of PC12 cells and secondarily led to cell death after the depletion of all energy substrates by glycolysis. This cell death showed no morphological characteristics of apoptosis and was not blocked by treatment with caspase inhibitors. The inhibition of cell growth was not dependent on an inhibition of complex I activity since MPP+ also inhibited cell proliferation in SH-SY5Y cells lacking mitochondrial DNA and complex I activity (p0 cells). As shown by flow cytometric analysis, MPP+ induced a block in the G0/G1 to S phase transition that correlated with increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) and growth arrest. Since treatment with 1 microM MPP+ caused apoptotic cell death in p21(WAF1/Cip1)-deficient (p21(-/-)) but not in parental (p21(+/+)) mouse embryo fibroblasts, our data suggest that in an early phase MPP+-induced p21(WAF1/Cip1) expression leads to growth arrest and prevents apoptosis until energy depletion finally leads to a nonapoptotic cell death.     

Effects of 1-Methyl-4-Phenyl- 1,2,3,6-Tetrahydropyridine and 1 -Methyl-4-Phenylpyridinium Ion on ATP Levels of Mouse Brain Synaptosomes

Mouse brain synaptosomes, essentially devoid of mitochondrial contamination, were used as a model to study the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the levels of ATP of neuronal terminals. Similar to known inhibitors of ATP synthesis, both MPTP and MPP+ caused a dramatic depletion of synaptosomal ATP. This depletion was dose dependent and occurred as a relatively early biochemical event in the absence of any apparent damage to synaptosomal membranes. MPP+ was more effective than its parent compound in decreasing ATP; it induced a significant loss at concentrations (10-100 microM) similar to those it reaches in the brain in vivo. MPTP-induced ATP depletion was completely prevented by the monoamine oxidase B inhibitor deprenyl, which, on the contrary, was ineffective against MPP+. As expected in view of the heterogeneous population of nerve terminals present in our synaptosomal preparations, the catecholamine uptake blocker mazindol did not significantly affect the ATP loss caused by both compounds. Data indicate that (1) administration of MPTP may cause a depletion of ATP within neuronal terminals resulting from the generation of MPP+, and (2) exposure to the levels of MPP+ reached in vivo may cause biochemical changes that are nonselective for dopaminergic terminals.     

Dopamine Agonist 3-PPP Fails to Protect Against MPTP-Induced Toxicity

We investigated the neuroprotective effect of the dopamine agonist, 3-PPP [3-(3-hydroxyphenyl)-N-propylpiperidine] against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP (30 mg/kg, i.p., twice, 16 h apart) causes significant dopamine depletion in nucleus caudatus putamen (NCP) by 1 week. 3-PPP had no effect on the monoamine oxidase-B activity (MAO-B) activity in NCP. 3-PPP did not affect dopamine uptake, whereas mazindol significantly blocked the uptake of dopamine dose dependently. MPTP-induced behavioral changes in mice were not reduced by pretreatment with 3-PPP. This dopamine agonist did not prevent dopamine depletion caused by MPTP. MPP+ (20 microM) significantly inhibited the cell proliferation of SH-SY5Y dopaminergic neuronal cells. 3-PPP had no effect on the SH-SY5Y neuronal cell growth in culture and did not block the MPP(+)-induced cytotoxicity. This study shows that the dopamine agonist 3-PPP failed to protect against MPTP-induced dopaminergic neurotoxicity.     

Distinct Effects of Rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on Cellular Bioenergetics and Cell Death

Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.     

Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes

The relationships between mitochondrial transmembrane potential, ATP concentration, and cytotoxicity were evaluated after exposure of isolated rat hepatocytes to different mitochondrial poisons. Both the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its fully oxidized metabolite, the 1-methyl-4-phenylpyridinium (MPP+) ion, caused a concentration- and time-dependent depolarization of mitochondrial membranes which followed ATP depletion and preceded cytotoxicity. The effect of MPTP, but not that of MPP+, was prevented by deprenyl, an inhibitor of MPTP conversion to MPP+ via monoamine oxidase type B. Addition of fructose to the hepatocyte incubations treated with either MPTP or MPP+ counteracted the loss of mitochondrial transmembrane potential. Fructose was also effective in protecting against the mitochondrial membrane depolarization as well as ATP depletion and cytotoxicity induced by antimycin. A, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and valinomycin. Data confirm the key role played by MPP(+)-induced mitochondrial damage in MPTP toxicity and indicate that (i) ATP produced via the glycolytic pathway can be utilized by hepatocytes to maintain mitochondrial electrochemical gradient, and (ii) a loss of mitochondrial membrane potential may occur only when supplies of ATP are depleted.     

Dopaminergic Neurotoxicity In Vivo and Inhibition of Mitochondrial Respiration In Vitro by Possible Endogenous Pyridinium-Like Substances

Elucidation of the mechanism(s) by which 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+) cause parkinsonism in humans and other primates has prompted consideration of possible endogenous MPTP/MPP(+)-like neurotoxins in the etiology of idiopathic Parkinson's disease. Here we examined inhibition of mitochondrial respiration in vitro and neurotoxicity in rats in vivo produced by beta-carbolinium compounds that are presumed to form following Pictet-Spengler cyclization of serotonin. We also evaluated N-methylisoquinolinium, a putative endogenous neurotoxin, in the same manner. The latter compound exhibited MPP(+)-like mitochondrial respiratory inhibition, whereas the beta-carbolinium compounds, although more potent inhibitors of electron transport, exhibited weak accumulation-dependent enhancement of inhibition in intact mitochondria. It is interesting that the beta-carbolinium compounds inhibited succinate- as well as glutamate-supported respiration, and are best described as inhibitor-uncouplers. The results of partitioning experiments suggest that both the low accumulation potential and the inhibition of succinate respiration may be a consequence of the beta-carboliniums being in equilibrium with neutral "anhydro" bases. Relative to MPP+, all compounds tested had weak dopaminergic uptake activity in vitro and weak dopaminergic toxicity in vivo, consistent with other findings of relatively low neurotoxic potential for presumed endogenous pyridiniums.     

Prevention of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic toxicity in mice by MDL 72145, a selective inhibitor of MAO-B

Pretreatment of mice with the potent and selective monoamine oxidase B (MAO-B) inhibitor MDL 72145 ((E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine) protected against the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice treated with MDL 72145 prior to MPTP did not exhibit the decrement in the neostriatal content of dopamine and its metabolites normally seen after MPTP administration. This observation adds further support to the concept that the oxidation of MPTP by MAO-B to its corresponding pyridinium analog, 1-methyl-4-phenylpyridinium (MPP+), is an important feature of the neurotoxic process.     

Selective Destruction of Cultured Dopaminergic Neurons from Fetal Rat Mesencephalon by 1-Methyl-4-Phenylpyridinium: Cytochemical and Morphological Evidence

Dopaminergic neurons in cultures of dissociated cells from fetal rat mesencephalon were exposed to the principal metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+), and several of its structural analogues. At concentrations between 0.01 and 0.1 microM, MPP+ inhibited catecholamine accumulation as visualized by cytofluorescence. Between 0.1 and 10.0 microM, MPP+ resulted in disappearance of tyrosine hydroxylase immunoreactivity without affecting other cells in the cultures. At concentrations higher than 10 microM, MPP+ was toxic to all cells present in the cultures. The effect of low concentrations of MPP+ on catecholamine cytofluorescence of the dopaminergic neurons was partially reversible. The intermediate concentrations produced irreversible structural changes of tyrosine hydroxylase-positive cells, resulting in complete disappearance of these neurons. The morphological changes were specific to the dopaminergic neurons and were not evident in other cells viewed with phase contrast microscopy. Of the structural analogues tested, the 1-ethyl analogue of MPP+ was effective in selectively destroying dopaminergic neurons in our culture system. The antioxidants L-acetyl-carnitine, beta-carotene, and alpha-tocopherol failed to protect against MPP+ neurotoxicity when co-incubated with the toxin.     

Synthetic analgesics and other phenylpiperidines: effects on uptake and storage of dopamine and other monoamines mouse forebrain tissue

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can induce degeneration of dopamine (DA) and other central monoamine neurons, leading to Parkinson's disease-like effects in man, monkey, and mouse. MPTP and other substituted phenylpiperidines related to synthetic analgesics including alphaprodine and meperidine were evaluated for potency vs. uptake of 0.1 microM tritiated DA, norepinephrine (NE), or serotonin (5HT) in synaptosomal preparations of mouse striatum or cerebral cortex. The most potent inhibitor of the uptake of 3H-DA was N-methyl-4-phenylpyridinium ion (MPP+; IC50 = 1 microM, Ki = 0.4 microM), a metabolite of MPTP; its effect was competitive and reversible. Other analogs of MPTP: the N-ethylindole AHR-1709, N,N-dimethyl-MPTP, and N-methyl-4-phenylpiperidine were all more potent than MPTP against 3H-DA uptake. N-dealkylation and N-propyl substitution, as well as pyridine ring substitution, decreased affinity for DA uptake while 3',4'-dihydroxyphenyl substitution increased potency and selectivity for catecholamine uptake, and quarternarization of the pyridine ring also increased potency against DA uptake. Active compounds showed higher potency against the uptake of NE than of DA. MPP+ was also more potent than MPTP in releasing endogenous DA from striatal synaptosomes (EC50 = 3 vs. 30 microM), but did not release the cytoplasmic markers tyrosine hydroxylase and lactate dehydrogenase (LDH). In contrast to MPTP, synthetic phenylpiperidine analgesics, their potential metabolites and the experimental neuroleptic agent AHR-1709 all failed to deplete striatal DA in vivo, even if active in vitro against DA uptake.     

Neurotoxicity studies with the monoamine oxidase B substrate 1-methyl-3-phenyl-3-pyrroline

The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately leads to the permanently charged 1-methyl-4-phenylpyridinium species MPP(+), a 4-electron oxidation product of MPTP and a potent mitochondrial toxin. The corresponding 5-membered analogue, 1-methyl-3-phenyl-3-pyrroline, is also a selective MAO-B substrate. Unlike MPTP, the MAO-B-catalyzed oxidation of 1-methyl-3-phenyl-3-pyrroline is a 2-electron process that leads to the neutral 1-methyl-3-phenylpyrrole. MPP(+) is thought to exert its toxic effects only after accumulating in the mitochondria, a process driven by the transmembrane electrochemical gradient. Since this energy-dependent accumulation of MPP(+) relies upon its permanent charge, 1-methyl-3-phenyl-3-pyrrolines and their pyrrolyl oxidation products should not be neurotoxic. We have tested this hypothesis by examining the neurotoxic potential of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-chlorophenyl)-3-pyrroline in the C57BL/6 mouse model. These pyrrolines did not deplete striatal dopamine while analogous treatment with MPTP resulted in 65-73% depletion. Kinetic studies revealed that both 1-methyl-3-phenyl-3-pyrroline and its pyrrolyl oxidation product were present in the brain in relatively high concentrations. Unlike MPP(+), however, 1-methyl-3-phenylpyrrole was cleared from the brain quickly. These results suggest that the brain MAO-B-catalyzed oxidation of xenobiotic amines is not, in itself, sufficient to account for the neurodegenerative properties of a compound like MPTP. The rapid clearance of 1-methyl-3-phenylpyrroles from the brain may contribute to their lack of neurotoxicity.     

Interaction of 1-methyl-4-phenylpyridinium ion with human platelets

When uptake of the Parkinson's syndrome inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its major brain metabolite MPP+ (1-methyl-4-phenylpyridinium ion) by human platelets were compared in platelet rich plasma, a much higher rate was observed for the metabolite. The uptake process was saturable (Km = 6.8 microM; Vmax = 0.064 nmole/min/mg protein) and could be blocked by inhibitors of serotonin uptake. The accumulation of MPP+ by the platelets was accompanied by a decrease in intracellular ATP and an inhibition of mitochondrial state 3 respiration. These findings are consistent with earlier reports of the effect of MPP+ on isolated mitochondria as a potential cytotoxic mechanism, but also demonstrate that the dopamine uptake system is not the only means by which this metabolite can be efficiently transported into cells.     

Mesencephalic Dopamine Neurons Become Less Sensitive to 1 -Methyl-4-Phenyl-l,2,3,6-Tetrahydropyridine Toxicity During Development In Vitro

The in vitro development of monoamine oxidase (MAO) activity and [3H]dopamine (DA) uptake capacity of dissociated cell cultures from rat embryo mesencephalon were correlated with the potency of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+) neurotoxicity. Specific activities of both MAO-A and MAO-B increased during in vitro development of the cultures, with MAO-B activity increasing 20-fold between the first and fourth week. Similarly, [3H]DA accumulation increased 2.6-fold between the first and third week in vitro, when it reached a plateau. Unexpectedly, the toxicities of MPTP and MPP+ were substantially decreased in the older cultures. Exposure to MPTP reduced [3H]DA accumulation per culture by 77% in 1-week-old cultures and by 36% in 4-week-old cultures. Similarly, damage caused by MPPT was reduced from 84% of control in the first week to 34% of control in the fourth week. The attenuation of neurotoxicity was not due to an increase in storage of MPP+ in the synaptic vesicles of DA neurons, nor to a change in the distribution of MPP+ between dopaminergic and other cellular components of the cultures. The damage to DA neurons caused by the mitochondrial toxin, rotenone, also showed a similar reduction in the older cultures. These observations coupled with an increase in lactate formation and glucose consumption during the in vitro development of the cultures suggest a shift toward increased glycolysis and decreased dependence on aerobic metabolism. This would render the cells more resistant to the inhibition of mitochondrial function by MPP+.     

Apoptosis inducing factor mediates caspase-independent 1-methyl-4-phenylpyridinium toxicity in dopaminergic cells

Parkinson's disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase-dependent apoptosis in dopaminergic neurons. Using the midbrain-derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over-expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase-9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase-independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large-scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large-scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase-independent cell death in MPP+-treated dopaminergic neurons.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Its Metabolite 1-Methyl-4-Phenylpyridine on Acetylcholine Synthesis in Synaptosomes from Rat Forebrain

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro-striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH-CoQ reductase) of the mitochondrial respiratory chain.