Sirtuin 2 (SIRT2) Enhances 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Nigrostriatal Damage via Deacetylating Forkhead Box O3a (Foxo3a) and Activating Bim Protein

Sirtuins are NAD-dependent protein deacetylases that were shown to have beneficial effects against age-related diseases. SIRT2 is a strong deacetylase that is highly expressed in brain. It has been associated with neurodegenerative diseases. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a dopaminergic neurotoxin that replicates most of the clinical features of Parkinson disease (PD) and produces a reliable and reproducible lesion of the nigrostriatal dopaminergic pathway and neurodegeneration after its systemic administration. Chronic administration of MPTP induces lesion via apoptosis. We show here that SIRT2 deacetylates Foxo3a, increases RNA and protein levels of Bim, and as a result, enhances apoptosis in the MPTP model of PD. We also show that neurodegeneration induced by chronic MPTP regimen is prevented by genetic deletion of SIRT2 in mouse. Deletion of SIRT2 leads to the reduction of apoptosis due to an increase in acetylation of Foxo3a and a decrease in Bim levels. We demonstrate that SIRT2 deacetylates Foxo3a, activates Bim, and induces apoptosis only in 1-methyl-4-phenylpyridinium-treated cells. Therefore, designing SIRT2 inhibitors might be helpful to develop effective treatments for PD.     

Duodenal ulcer induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

Experiments in rats revealed that the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) given in multiple daily doses either per os (p.o.) or subcutaneously (s.c.) induced in a dose-dependent manner solitary or double ("kissing") duodenal ulcers in the rat. MPTP also diminished cerebral concentrations of DOPAC and the duodenal ulcers were prevented by pretreatment with dopamine agonists (e.g., bromocriptine, lergotrile) or monoamine oxidase inhibitors (e.g., pargyline, 1-deprenyl). High doses of MPTP also caused gastric erosions and motility changes resembling parkinsonism (e.g., akinesia, rigidity, forward bending of trunk). This chemical decreased gastric secretion of acid and pepsin, as well as pancreatic bicarbonate, trypsin and amylase. Thus, MPTP causes duodenal ulcers that are possibly associated with impaired defense in the duodenal bulb (e.g., decreased availability of duodenal and pancreatic bicarbonate).     

Fructose prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced ATP depletion and toxicity in isolated hepatocytes

The loss of viability of isolated rat hepatocytes exposed to either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) was prevented by addition of fructose to the incubation medium. This protection was dependent on fructose concentration, being complete at 10 mM. Addition of fructose dramatically delayed MPTP- and MPP+-induced depletion of ATP and was accompanied by a significant accumulation of lactate, indicating the occurrence of enhanced glycolytic production of ATP. Glucose was much less effective against MPTP and MPP+ toxicity, probably because it is a relatively poor substrate for glycolysis in liver cells. We conclude that depletion of ATP is a critical event in MPTP cytotoxicity in our in vitro model system, and that the use of alternative sources of ATP production may represent an important protective device against the effects of this toxic agent.     

Potential bioactivation pathways for the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The metabolism of the selective nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been studied in rat brain mitochondrial incubation mixtures. The 1-methyl-4-phenylpyridinium species MPP+ has been characterized by chemical ionization mass spectral and 1H NMR analysis. Evidence also was obtained for the formation of an intermediate product which, with the aid of deuterium incorporation studies, was tentatively identified as the alpha-carbon oxidation product, the 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+. Comparison of the diode array UV spectrum of this metabolite with that of the synthetic perchlorate salt of MPDP+ confirmed this assignment. The oxidation of MPTP to MPDP+ but not of MPDP+ to MPP+ is completely inhibited by 10(-7) M pargyline. MPDP+, on the other hand, is unstable and rapidly undergoes disproportionation to MPTP and MPP+. Based on these results, we speculate that the neurotoxicity of MPTP is mediated by its intraneuronal oxidation to MPDP+, a reaction which appears to be catalyzed by MAO. The interactions of MPDP+ and/or MPP+ with dopamine, a readily oxidizable compound present in high concentration in the nigrostriatum, to form neurotoxic species may account for the selective toxic properties of the parent drug.     

Metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mouse liver preparations

Using a mouse liver microsomal preparation, it was found that the heterocyclic ring system of MPTP underwent an initial α-oxidation to give chemically reactive metabolites that may be associated with the induction of Parkinsonism by MPTP. Subsequent oxidative metabolic transformations of these intermediates were found to give a lactam metabolite and a pyridone metabolite that potentially may interact with the neurotransmitter system.     

Studies on the mechanism of action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Explants of embryonic rat substantia nigra in organotypic culture are sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at concentrations approximating the doses given in vivo to monkeys. Fluorescence microscopy and 3H-dopamine uptake measurements reveal that the toxicity is selective for dopamine neurons, whereas other neurons and cells in the culture appear normal by phase contrast microscopy. Reduced MPTP (piperidine analog) is inactive in the tissue culture model, while fully oxidized MPTP (pyridinium analog) destroys dopamine neurons. Pargyline and deprenyl, two monoamine oxidase inhibitors, inhibit the neurotoxic action of MPTP. Pargyline and deprenyl also protect monkeys in vivo. The results implicate monoamine oxidase in the mechanism of action of MPTP. Two possible mechanisms for protection by monoamine oxidase are discussed.     

New amphibian models for the study of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

We report the development of two animal models in amphibians (frogs and salamanders) in whom 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces the behavioral (neurological) and biochemical equivalents of the human disease and, in addition, a measurable modification in at least one form of pigment-bearing cell from the neural crest, the skin melanocyte. We propose that this new approach can become an inexpensive, easily quantifiable model for the study of the effect of MPTP on the central and peripheral nervous systems. We also demonstrate that the toxic effect of MPTP can be completely abolished in vivo by treatment with a monoamine oxidase inhibitor and potentiated by an inhibitor of catechol-O-methyltransferase. MPTP is catabolised by oxidation into toxic metabolites, but 1-methyl-4-phenylpyridinium ion (MPP+), the proposed end-metabolite, is even more toxic than MPTP in this model, possibly through a different mechanism.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTP treated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.     

Acute effects of a parkinsonism-inducing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mouse body temperature

The parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) given in single systemic doses (i.p.) to mice produced marked hyperthermia, and subsequent long-lasting hypothermia. Administration of MPTP or its oxidized product, 1-methyl-4-phenylpyridinium ion, MPP+, via i.c.v. resulted in only hypothermia. In contrast, i.p. MPP+ administration resulted in only hyperthermia. The MPTP-induced hyperthermia (i.p.) was blocked by quaternary derivatives of anti-cholinergic agents, atropine and scopolamine, but not by the tertiary-derivative of atropine. Duration of this hyperthermic effect was potentiated by neostigmine. Pretreatment with 1-deprenyl did not prevent hypothermia, but nomifensine partially or clorgyline completely prevented the effect without preventing MPTP-induced hyperthermia. The thermic effects by MPTP, unlike its neurotoxicity for the nigrostriatal DA system, may not require metabolism to MPP+. These results indicate that peripheral cholinergic functions are responsible for the MPTP-induced hyperthermia, whereas its hypothermic effect may be centrally mediated via dysregulation of the various neuron systems.     

Hemiparkinsonism in monkeys after unilateral internal carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Infusion of MPTP (0.2-0.8 mg/kg) into the right internal carotid artery of monkeys produces toxin-induced injury to the right nigrostriatal pathway with sparing of other dopaminergic neurones on the infused side and with negligible or little injury to the opposite, untreated side. There are contralateral limb dystonic postures, rigidity, and bradykinesia, but the animals are able to eat and maintain health without drug treatment. Spontaneous motor activity is attended by circling towards the injured side, whereas treatment with L-DOPA/-carbidopa or apomorphine stimulates circling towards the intact side. Dopamine and dopamine metabolite levels are normal in the left caudate and putamen, but markedly depressed on the right (MPTP-treated) side. This animal hemiparkinsonian model will be useful in studies of volitional movement control, drug treatments of Parkinson's disease, and functional efficacy of brain tissue implants.     

Partial protection from the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by Pro-Leu-Gly-NH2(PLG; MIF-1)

The administration of MPTP to man and monkey has been shown to cause a neurotoxic effect on the nigrostriatal dopamine system. MPTP was injected in C57-BL black mice, 36 mg per kg for 7 days, which resulted in permanent reduction of dopamine and serotonin levels in the striatum. In the mice pretreated with PLG, although the striatal dopamine level was also reduced, mean dopamine and serotonin levels were significantly higher than in mice given MPTP alone. It is concluded that PLG could protect at least partially the neurotoxic effect of MPTP.     

Circling behavior in old rats after unilateral intranigral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Old and young adult rats received unilateral injections of MPTP or saline into the substantia nigra. Unilateral injection of MPTP in old rats induced ipsiversive circling on day 1 and day 7 after the injection; contraversive circling behavior was induced in MPTP-treated rats by systemic administration of apomorphine. Young rats showed ipsiversive circling on day 1 but not on day 7 after the injection; administration of apomorphine did not induce contraversive circling. On day 10 after the injection of MPTP, the concentration of D-2 receptors in the striatum of the injected hemisphere of old rats was increased by about 25% compared with the striatum of old rats with saline injection and of young rats with MPTP or saline injections. These results suggest that MPTP exerts neurotoxic effects on the nigrostriatal dopaminergic system of old rats and produces supersensitive dopamine receptors in the ipsilateral denervated striatum.     

Reversible inhibition and mechanism-based irreversible inactivation of monoamine oxidases by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

It has been suggested (Chiba et al., Biochem. Biophys. Res. Communs. (1984) 120, 574) that the neurotoxic effects of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which causes Parkinsonian symptoms in humans and other primates, are due to compounds resulting from the oxidation of MPTP by monoamine oxidase B in the brain. We reported recently that both monoamine oxidase A and B oxidize MPTP to MPDP+, the 2,3-dihydropyridinium form and that the reaction is accompanied by time-dependent, irreversible inactivation of the enzymes. Of the two forms of monoamine oxidase, the B enzyme oxidizes MPTP more rapidly and is also more sensitive to inactivation. We now wish to report that MPTP, as well as its oxidation products, MPDP+ and MPP+, the 4-phenylpyridinium form, are also potent reversible, competitive inhibitors of both monoamine oxidase A and B, particularly the former, and that the order of inhibition for the A enzyme is MPDP+ greater than MPP+ greater than MPTP, while for the B enzyme MPTP greater than MPDP+ greater than MPP+. We further report on the spectral changes and isotope incorporation accompanying the irreversible inactivation.     

Cell membrane transport of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the liver and systemic bioavailability.

Modulation of hepatic disposition of MPTP could influence susceptibility to its neurotoxicity. Therefore, we studied hepatocellular transport of MPTP in the perfused rat liver and isolated rat hepatocytes. The perfused liver extensively extracted MPTP. Amiloride and tubocurarine, inhibitors of OCT1, increased MPTP recovery (253 +/- 78 and 283 +/- 64%, respectively) and reduced PS(influx) (0.69 +/- 0.36 to 0.27 +/- 0.11, and 0.97 +/- 0.50 to 0.23 +/- 0.05 ml/s/g, respectively). P-glycoprotein inhibitor, daunomycin, and Oatp 1 & 2 inhibitor, rifamycin, had no effect. In isolated hepatocytes, amiloride and tubocurarine increased hepatic uptake of MPTP (23 +/- 12 and 6 +/- 2%, respectively). Daunomycin reduced MPTP uptake by 22 +/- 8% and rifamycin had no effect. Only a small proportion of MPTP is taken up into hepatocytes by transporters; however, modulation of these transport mechanisms will influence systemic bioavailability.     

In vitro effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on muscarinic cholinergic receptors in mouse striatum

1. MPTP significantly lowered Kd of the binding of [3H]QNB to muscarine receptor without affecting Bmax values compared with those of control. Hill coefficients (nH) of control and MPTP (250 microM) added group were 1.15 +/- 0.127 and 0.56 +/- 0.202, respectively. 2. Prior addition of pargyline to MPTP did not prevent the decrease of [3H]QNB binding. The patterns of displacement of [3H]QNB by MPTP and MPP+ were similar to those by some muscarinic agonists, such as acetylcholine, carbamyl choline and methacholine. 3. These results suggest that MPTP might be muscarinic agonist and might play a role to produce Parkinsonism through directly affecting the muscarinic cholinergic receptors in vivo.     

Effect of preferential cyclooxygenase-2 (COX-2) inhibitor against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced striatal lesions in rats: behavioral, biochemical and histological evidences

Cyclooxygenase (COX) isoenzyme is known to play an important role in the pathophysiology of Parkinson's disease. The present study evaluated the neuroprotective effect of nimesulide, a preferential COX-2-inhibitor against 1-methyl-4-phenyl-1,2,3,6-tertahydropyridine (MPTP)-model of Parkinson's disease. Intrastriatal administration of MPTP (32 micromol in 2 microl) produced a significant decrease in the locomotor activity. Biochemical investigation of striatal region revealed a significant enhancement in the oxidative stress as evidenced by increased lipid peroxidation levels, nitrite levels and myeloperoxidase activity along with depleted antioxidant pool (reduced glutathione and superoxide dismutase levels) and reduced redox (GSH/GSSG) ratio. MPTP administration also showed significant mitochondrial complex-I inhibition and reduction in the mitochondrial viability. Histological examination of the MPTP-treated brain sections revealed alteration in the histo-architecture as well as undifferentiated bodies of varying contour and lesions. Chronic administration of nimesulide (5 or 10 mg/kg, po) for 12 days, significantly reversed the behavioral, biochemical, mitochondrial and histological alterations induced by MPTP. In conclusion, the findings of the present study implicate the possible neuroprotective potential of nimesulide in MPTP-treated rats and thus highlight the therapeutic potential of COX-inhibitors in treatment of Parkinson's disease.     

Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). Alpha-synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, beta-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.