Nigrostriatal Toxin Research Articles

Manganese-enhanced magnetic resonance imaging detects VIPR2-mediated immune transformation in a model of Parkinson’s disease

Abstract Progressive nigrostriatal degeneration and neuroinflammation underlie the pathobiology of Parkinson’s disease (PD). Biomarkers to detect such pathogenic events remain in development. We report that manganese-enhanced magnetic resonance imaging (MEMRI) can be used to assess inflammatory activities related to deficits in neural connectivity in an experimental PD model. Vasoactive intestinal peptide (VIP), an endogenous neuropeptide that binds to VIPR1 and VIPR2 inducing a phenotypic shift of neurotoxic effector memory to neuroprotective regulatory T cells was modified (referred to as LBT-3627) to improve its half-life and limit proteolytic cleavage. LBT-3627 was used to investigate its ability to elicit neuroprotection in mice treated with the nigrostriatal toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Treatment with LBT-3627 lead to neuronal sparing when compared to MPTP-treated mice. Seven days post MPTP intoxication, mice were scanned using MEMRI, and images were registered to an MEMRI-based brain atlas for comparison. Manganese-induced MRI signal enhancement was calculated. MEMRI detected MPTP-induced inflammation as well as a corresponding reduction in signal with low and high doses of LBT-3627. MPTP-treated mice showed a significantly higher signal enhancement within the hippocampus (p = 0.04), with a trend towards significance in both the substantia nigra and striatum when compared to saline controls. The higher signal enhancement was reversed by LBT-3627 treatment. These findings indicate the ability of MEMRI to detect MPTP-induced brain pathology and its reversal following LBT-3627 treatment, supporting MEMRI as a method to assess changes in nigrostriatal-induced neuroinflammation.

Protective role of group-II metabotropic glutamate receptors against nigro-striatal degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice

To examine how mGlu2/3 metabotropic glutamate receptors affect nigro-striatal degeneration, we used the agonist, LY379268, and the antagonist, LY341495, in mice challenged with the nigro-striatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In control mice, high doses of MPTP (20 mg/kg, i.p., injected four times with 2 h of interval) induced a nearly total degeneration of the nigro-striatal pathway, as shown by measurements of striatal dopamine (DA) levels and by immunohistochemical analysis of tyrosine hydroxylase, high affinity dopamine transporter, and glial fibrillary acidic protein in the corpus striatum and substantia nigra. Lower cumulative doses of MPTP (30 mg/kg, i.p., injected only once) produced a partial lesion of the nigro-striatal pathway (about 50% reduction of striatal DA content). Systemic injection of LY379268 (1 mg/kg, i.p., 30 min prior to each injection of MPTP) partially reduced the extent of nigro-striatal degeneration induced by high doses of MPTP. Similar results were obtained by continuously delivering LY379268 (1 mg/kg/d for 7 d) by means of a subcutaneous osmotic minipump. The protective effect of LY379268 was antagonized by LY341495 (also delivered by the osmotic minipump). In mice challenged with the lower cumulative dose of MPTP, injection of LY379268 did not produce a significant neuroprotective effect. In contrast, the lesion was amplified by the antagonist, LY341495. Neither LY379268 nor LY341495 influenced the central bioavailability and the local half-life of MPTP, as shown by measurements of the toxin and its active metabolite, MPP +, in the striatum. We conclude that mGlu2/3 receptors play a protective role against MPTP toxicity, and that the efficacy of the agonist, LY379268, critically depends on the extent of the nigro-striatal lesion.

1-Methyl-4-phenyl-2,3-dihydropyridinium is transformed by ubiquinone to the selective nigrostriatal toxin 1-methyl-4-phenylpyridinium

We have studied the interaction of coenzyme Q with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP +) and 1-methyl-4-phenylpyridinium (MPP +), the real neurotoxin to cause Parkinson’s disease. Incubation of MPTP or MPDP + with rat brain synaptosomes induced complete reduction of endogenous ubiquinone-9 and ubiquinone-10 to corresponding ubiquinols. The reduction occurred in a time- and MPTP/MPDP + concentration-dependent manner. The reduction of ubiquinone induced by MPDP + went much faster than that by MPTP. MPTP did not reduce liposome-trapped ubiquinone-10, but MPDP + did. The real toxin MPP + did not reduce ubiquinone in either of the systems. The reduction by MPTP but not MPDP + was completely prevented by pargyline, a type B monoamine oxidase (MAO-B) inhibitor, in the synaptosomes. The results indicate that involvement of MAO-B is critical for the reduction of ubiquinone by MPTP but that MPDP + is a reductant of ubiquinone per se. It is suggested that ubiquinone could be an electron acceptor from MPDP + and promote the conversion from MPDP + to MPP + in vivo, thus accelerating the neurotoxicity of MPTP.

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.

Synthesis of novel MPTP analogs as potential monoamine oxidase B (MAO-B) inhibitors.

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetahydropyridine (MPTP) is an excellent substrate and a weak inactivator of the flavoenzyme monoamine oxidase B (MAO-B). In an attempt to develop novel mechanism-based inactivators of MAO-B, we have synthesized analogs of MPTP bearing a variety of functional groups at either the N or the C(4) position and have examined their interactions with a purified MAO-B preparation isolated from beef liver. The substituents selected include allyl, propargyl, ethenyl, ethynyl, and cyclobutyl, that is, functionalities which were considered potential sources of enzyme generated electrophilic or radical intermediates that might alkylate and inactivate the enzyme. None of the C(4)-substituted compounds displayed significant enzyme inhibitor properties although some proved to be good substrates. In the N-substituted MPTP series only the 4-phenyl-1-propargyl analog was a good inhibitor. The time- and concentration-dependent inhibition of MAO-B displayed by this compound is consistent with a mechanism-based inactivation pathway and the catalytic mechanism currently held for monoamine oxidases. The results of these studies provide additional insights into the steric features of the active site of MAO-B and predict that the area in which the C(4) substituent of the tetrahydropyridine ring resides lacks a reactive nucleophilic group.

Studies on the cytochrome P-450 catalyzed ring .alpha.-carbon oxidation of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

In vitro metabolic studies have established that rat liver cytochromes P-450IIB1 and P-450IA1 but not rabbit liver cytochrome P-450IIB4 catalyze the oxidation of the Parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the corresponding dihydropyridinium (MPDP+) and pyridinium (MPP+) species. Kinetic experiments with the most effective isozyme, cytochrome P-450IA1, indicate that the reaction proceeds at a moderate velocity [Vmax = 20.1 nmol/(min.nmol of P-450IA1)] and high Km (0.87 mM). Furthermore, kinetic deuterium isotope effect measurements provided DV and D(V/K) values of 2.99 and 1.04, respectively. A comparison with the corresponding values for the monoamine oxidase B (MAO-B) catalyzed reaction (4.37 and 9.35, respectively) suggests that either these enzymes catalyze the ring alpha-carbon oxidation of MPTP by different pathways or that the initial one-electron transfer to generate an aminium radical intermediate previously proposed for both enzyme systems is reversible in the case of MAO-B and irreversible in the case of cytochrome P-450IA1.

Distribution of 1‐(3H)methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (3H‐MPTP) in the Frog: Uptake in Neuromelanin

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective destruction of pigmented monoaminergic neurons of the brain, mainly in the substantia nigra. Primates and amphibians, whose nerve cells contain melanin, have shown a higher sensitivity for the toxic effects of MPTP than species which are lacking neuromelanin, e.g. rodents. In the present study the distribution after intraperitoneal injection of 3H-MPTP in frogs (Rana temporaria) was studied by whole-body autoradiography. Histochemical staining methods for melanin were used in order to identify the pigment in various tissues. Melanin-containing nerve cells were present bilaterally in the ventral motor parts of the frog brain. Melanin was also found in the meninges, around the cerebral ventricles and the aqueducts, and in the eyes, skin and liver. The results from the autoradiographic study of 3H-MPTP revealed a high accumulation and retention in all melanin-containing structures up to 15 days after administration (the longest survival time). The pigmented tissues showed the highest concentration of radioactivity in the body at all survival times. The MPTP-induced destruction of pigmented nerve cells may be related to the binding and storage of MPTP and/or its metabolites in neuromelanin, causing toxic cytoplasmic concentrations through the continuous release of substance from the melanin depot.

Characterization of a product derived from the 1-methyl-4-phenyl-2,3-dihydropyridinium ion, a metabolite of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Characterization of a product derived from the 1-methyl-4-phenyl-2,3-dihydropyridinium ion, a metabolite of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Studies on semirigid tricyclic analogues of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The tetrahydro-beta-carboline derived from the condensation of N-methyltryptamine and formaldehyde, a semirigid tricyclic analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) tha has been detected in the brains of normal laboratory rats, is biotransformed in a monoamine oxidase B (MAO-B) catalyzed reaction to the corresponding dihydro compound at a rate that is approximately 0.5% of that observed with MPTP. The corresponding tetrahydroindenopyridine in which the double bond beta,gamma to the nitrogen atom retains allylic character is a somewhat better MAO-B substrate. The steric bulk of the nitrogen and methylene bridges in addition to ring strain present in the proposed carbon-centered radical intermediates derived from these types of tricyclic structures may contribute to their relatively poor MAO-B substrate properties. Although no MPTP-like neurotoxic properties were observed following acute administration of the test compounds to mice, we speculate that the chronic accumulation of beta-carbolinium type metabolites could contribute to the rate of nigrostriatal cell loss associated with idiopathic Parkinson's disease.

Metabolic studies on the nigrostriatal toxin MPTP and its MAO B generated dihydropyridinium metabolite MPDP+.

The metabolic fates of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its two-electron oxidation product, the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), have been examined in mouse brain and liver tissue fractions. Incubations of MPTP (50 microM and 1 mM) with mouse brain preparations result in the expected MAO B catalyzed formation of MPDP+. The four-electron oxidation product, the 1-methyl-4-phenyl-pyridinium species (MPP+), was the only other metabolite detected. The oxidation of 50 microM MPDP+ to MPP+ in the same preparations appears to be mediated by unidentified components present in membrane containing structures. The behavior of MPTP in mouse liver subcellular fractions is considerably more complex. NADPH-supplemented liver microsomes convert MPTP to the desmethyl and N-oxide metabolites. At high (1 mM) initial concentrations of MPTP there is evidence that NADPH-dependent, pargyline-insensitive liver microsomal enzymes also catalyze the oxidation of MPTP to MPDP+. Incubations of MPDP+ with mouse liver preparations containing the cytosolic fraction led to the rapid disappearance of the substrate and the quantitative formation of a metabolic product with mass spectral and diode array UV characteristics expected for a lactam structure. Menadione, an inhibitor of the cytosolic enzyme aldehyde oxidase, inhibits the formation of this product. Unambiguous characterization of this metabolite as 1-methyl-4-phenyl-5,6-dihydro-2-pyridone was achieved by comparison of its high-resolution 1H NMR and high-resolution EI mass spectra with the corresponding spectra of a synthetic standard.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of reactive intermediates in the MAO-catalyzed oxidation of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Oxidation of MPTP by monoamine oxidase (MAO), leading to the formation of reactive metabolites, is a critical step in the expression of the nigrostriatal toxicity of this molecule. A catalytic mechanism for the 2-electron oxidation of MPTP to MPDP + and for the further 2-electron oxidation of MPDP + to MPP + is proposed, involving the formation of carbon-centered radical intermediates. These radical species appear to be involved in the mechanism-based inactivation of MAO by MPTP, possibly by generating 1,4-dihydropyridine adducts with the enzyme apoprotein or its coenzyme FAD. The pathways of metabolism of MPTP in brain and peripheral tissues and the active accumulation of metabolites of MPTP in dopaminergic neurons are discussed in terms of their possible contribution to the selective cytotoxicity of the compound.

Interactions of the 1-methyl-4-phenyl-2, 3-dihydropyridinium species with synthetic dopamine-melanin

This paper describes interactions between the 1-methyl-4-phenyl-2, 3-dihydropyridinium (MPDP +) metabolite of the nigrostriatal toxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) with synthetic dopamine-melanin, a polymeric pigment which is similar to the neuromelanin found in the nigrostriatal cell bodies of humans and primates. Although MPTP and its 1-methyl-4-phenylpyridinium (MPP +) metabolite bind to the synthetic pigment at physiological pH, both compounds are recovered quantitatively upon treatment with acid. Unlike MPTP and MPP +, MPDP + proved to be unstable in the presence of synthetic dopamine-melanin which promoted its conversion to the fully oxidized pyridinium product MPP +. The possible biological significance of this interaction is discussed.

ChemInform Abstract: METABOLISM OF THE NIGROSTRIATAL TOXIN 1‐METHYL‐4‐PHENYL‐1,2,3,6‐TETRAHYDROPYRIDINE BY LIVER HOMOGENATE FRACTIONS

Chemischer InformationsdienstVolume 16, Issue 48 Other Subjects ChemInform Abstract: METABOLISM OF THE NIGROSTRIATAL TOXIN 1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE BY LIVER HOMOGENATE FRACTIONS J. WEISSMAN, J. WEISSMANSearch for more papers by this authorA. TREVOR, A. TREVORSearch for more papers by this authorK. CHIBA, K. CHIBASearch for more papers by this authorL. A. PETERSON, L. A. PETERSONSearch for more papers by this authorP. CALDERA, P. CALDERASearch for more papers by this authorN. JUN. CASTAGNOLI, N. JUN. CASTAGNOLISearch for more papers by this authorT. BAILLIE, T. BAILLIESearch for more papers by this author J. WEISSMAN, J. WEISSMANSearch for more papers by this authorA. TREVOR, A. TREVORSearch for more papers by this authorK. CHIBA, K. CHIBASearch for more papers by this authorL. A. PETERSON, L. A. PETERSONSearch for more papers by this authorP. CALDERA, P. CALDERASearch for more papers by this authorN. JUN. CASTAGNOLI, N. JUN. CASTAGNOLISearch for more papers by this authorT. BAILLIE, T. BAILLIESearch for more papers by this author First published: December 3, 1985 https://doi.org/10.1002/chin.198548342AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume16, Issue48December 3, 1985 RelatedInformation

Studies on the 1-methyl-4-phenyl-2,3-dihydropyridinium species 2,3-MPDP+, the monoamine oxidase catalyzed oxidation product of the nitrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is biotransformed by brain monoamine oxidase (MAO) to an unstable dihydropyridinium intermediate that reacts with cyanide ion to form an alpha-cyano-tetrahydropyridine adduct and, in the absence of cyanide ion, undergoes disproportionation to the 1-methyl-4-phenylpyridinium species MPP+ and MPTP. Comparison of the HPLC retention times, diode array UV, and chemical ion mass spectral characteristics of these products with those of synthetic standards led us to propose the 1-methyl-4-phenyl-2,3-dihydropyridinium species 2,3-MPDP+ and 6-cyano-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as tentative structure assignments for the dihydropyridinium metabolite and the cyano adduct, respectively. Results presented in this paper confirm the first assignment and establish that, although the proposed 6-cyano adduct is initially formed, the product that was isolated from the mitochondrial incubation mixtures of MPTP and sodium cyanide actually is the isomeric 2-cyano-1-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine. On the basis of the selective incorporation of deuterium into these products, we provide rational mechanistic interpretations of the disproportionation reaction and the rearrangement of the cyano adducts. These results establish that the MAO-catalyzed bioactivation of MPTP leads to the formation of a variety of reactive molecules that are potentially cytotoxic to nigrostriatal cells.

Metabolism of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by liver homogenate fractions.

The metabolic fate of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been examined in rat and rabbit liver mitochondrial and rabbit liver microsomal preparations. The mitochondrial preparations rapidly oxidized MPTP, in a pargyline-sensitive reaction, to a polar material that was shown to contain the 1-methyl-4-phenylpyridinium species as the principal product. NADPH-supplemented microsomal preparations converted MPTP to two principal products: 4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine N-oxide. Carbon monoxide and SKF 525A selectively inhibited the oxidation of MPTP to the nor compound, indicating that this N-demethylation reaction is cytochrome P-450 catalyzed. Attempts to trap possible unstable iminium metabolites of MPTP in microsomal incubation mixtures with sodium cyanide led to the isolation of a monocyano adduct that proved to be the N-cyanomethyl derivative. Thus, hepatic mitochondrial and microsomal enzyme systems catalyze the oxidation of MPTP by different pathways, the former leading to the generation of species that may possess neurotoxic properties.

V. 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 α-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 otherhand, 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.