Uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the N-methyl-4-phenylpyridinium ion (MPP+) into fetal mouse brain through the placenta

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to be taken up into fetal mice through the placenta from a maternal mouse. C57 black mice were mated and MPTP (30 mg/kg) was given intramuscularly at the 18th day of gestation. Definite amounts of MPTP were detected in fetal brains by assay using high-performance liquid chromatography (HPLC) at 1 h after MPTP injection, and much higher contents of MPTP were found in maternal brains, too. An oxidative product of MPTP, the N-methyl-4-phenylpyridinium ion (MPP+), was also detected in brains of both mother and fetus and its concentrations in their brains were followed at 1, 3, 6, 12, 24 h after MPTP injection. The time to reach the maximal MPP+ concentration in brains was different between mother and fetus; 1 h and 3 h respectively. In addition to brain, considerable amounts of MPP+ were found in fetal liver, maternal liver and kidney, and in the placenta.     

Paraquat and two endogenous analogues of the neurotoxic substance N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine do not damage dopaminergic nigrostriatal neurons in the mouse

C57 black mice were injected repeatedly with maximal tolerated doses of 4 different chemical analogues of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or its metabolite N-methyl-4-phenylpyridinium ion (MPP+), in order to assess their possible neurotoxicity for dopaminergic nigrostriatal neurons and their potential for causing idiopathic Parkinson's disease. The 4 analogues were the herbicide paraquat, reduced paraquat (having two N-methyl-tetrahydropyridine moieties), N-methyl-1,2,3,4-tetrahydroisoquinoline, and 2-methyl-1,2,3,4-tetrahydro-beta-carboline, the latter two compounds being possible endogenous neurotoxins. Contents of striatal dopamine, measured by high-performance liquid chromatography with electrochemical detection one month after injections were completed, were not depleted by any of these 4 compounds in mice. They might conceivably prove more neurotoxic in primates.     

Neuroprotective effects of (±)-catechin against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity in mice

The neuroprotective effects of (±)-catechin against toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were investigated in mice. MPTP caused the death of dopaminergic neurons in the substantia nigra and decreased the level of striatal dopamine. Additionally, MPTP increased the level of phospho-c-Jun, a known substrate of c-Jun N-terminal kinase (JNK) and caused a rapid activation of GSK-3β, evidenced by the decrease in the level of phospho-Ser9 of GSK-3β. However, pretreatment with (±)-catechin was found to protect dopaminergic neurons in the substantia nigra against MPTP toxicity, and restore the depletion of striatal dopamine in mice. (±)-Catechin attenuated the phosphorylation of c-Jun and recovered the phosphorylation of GSK-3β (Ser9). These results suggested that the suppression of JNK and GSK-3β signaling cascades might contribute to the neuroprotective effect of (±)-catechin against toxicity of MPTP.     

Partial protection from the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by four different antioxidants in the mouse

C57 black mice given a single injection of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 40 mg/kg, developed marked reduction of striatal dopamine content and loss of dopaminergic neurons in the zona compacta of the substantia nigra. However, pretreatment with any one of four different antioxidants, alpha-tocopherol, beta-carotene, ascorbic acid or N-acetylcysteine, significantly decreased MPTP-induced striatal dopamine loss, and alpha-tocopherol prevented neuronal loss in the substantia nigra. Four chemical analogues of MPTP (cinnamaldehyde, N,N-dimethylcinnamylamine, arecoline and 2-methyl-1,2,3,4-tetrahydro-6,7-isoquinolinediol) were all found to lack dopaminergic nigrostriatal neurotoxicity in the mouse.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage of striatal dopaminergic fibers attenuates subsequent astrocyte response to MPTP

Acute administration of the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the C57BL/6 mouse caused a rapid decrease in the amount of striatal tyrosine hydroxylase (TH), a marker of the nigrostriatal dopaminergic pathway, followed by a large increase in the astrocyte protein, glial fibrillary acidic protein (GFAP). The astrocyte (GFAP) response declined to baseline three weeks after administration of MPTP. Administration of a second dosage of MPTP at this time evoked a second GFAP response. The magnitude of the second response, however, was decreased in comparison to the response seen after only a single exposure to MPTP. Increasing the initial dosage of MPTP resulted in greater reductions of the second GFAP response. These data indicate that MPTP-induced damage or loss of striatal dopaminergic neurons reduces the signal available for initiating astrogliosis and thereby reduces the astrocyte response to a second exposure to MPTP.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) accelerates the accumulation of lipofuscin in mouse adrenal gland

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes degeneration of nigrostriatal dopaminergic neurons. Recently, we reported that it also destroys dopaminergic neurons in retina and it induces the accumulation of lipofuscin. We now present morphologic and biochemical evidence that MPTP causes the accumulation of lipofuscin in the adrenal cortex. We speculate, that generation of free radicals during the transformation of MPTP to metabolites might be responsible for lipofuscin formation. MPTP-induced accumulation of lipofuscin may be a useful model for studying the biochemistry of the aging process.     

Riluzole (2-amino-6-trifluoromethoxy benzothiazole) attenuates MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in mice

The protective effects of 2-amino-6-trifluoromethoxy benzothiazole (riluzole), a Na(+) channel blocker with antiglutamatergic activity were investigated in the model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced depletion of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in mice. The mice were injected intraperitoneally (i.p.) with four administrations of MPTP (10 mg/kg) at 1 h intervals and then the brains were analyzed at 3 and 7 days after the treatments. Dopamine, DOPAC and HVA levels were significantly decreased in the striatum 3 days after MPTP treatments. Riluzole dose-dependently antagonized the MPTP-induced decrease in dopamine, DOPAC and HVA levels in the striatum. MPTP treatment also caused a severe decrease in the amount of nigral tyrosine hydroxylase protein (TH) and microtuble-associated protein 2 (MAP 2) and produced a marked increase in the striatal glial fibrillary acidic protein (GFAP). Our immunohistochemical study with TH and MAP 2 staining showed that riluzole can protect against MPTP-induced neuronal damage in the substantia nigra. Furthermore, riluzole markedly increased the striatal GFAP-positive astrocytes 3 days after MPTP treatments. These results suggest that riluzole is effective against MPTP-induced neurodegeneration of the nigrostriatal dopaminergic neuronal pathway. Our findings also may provide a rationale for the identification of astrocytes as a prominent target for the development of new therapies of Parkinson's disease.     

LRRK2 G2019S transgenic mice display increased susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurotoxicity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common causes of late onset autosomal dominant form of Parkinson disease (PD). Gain of kinase activity due to the substitution of Gly 2019 to Ser (G2019S) is the most common mutation in the kinase domain of LRRK2. Genetic predisposition and environmental toxins contribute to the susceptibility of neurodegeneration in PD. To identify whether the genetic mutations in LRRK2 increase the susceptibility to environmental toxins in PD models, we exposed transgenic mice expressing human G2019S mutant or wild type (WT) LRRK2 to the environmental toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP treatment resulted in a greater loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta (SNpc) in LRRK2 G2019S transgenic mice compared to the LRRK2 WT overexpressing mice. Similarly loss of dopamine levels were greater in the striatum of LRRK2 G2019S mice when compared to the LRRK2 WT mice when both were treated with MPTP. This study suggests a likely interaction between genetic and environmental risk factors in the PD pathogenesis and that the G2019S mutation in LRRK2 increases the susceptibility of dopamine neurons to PD-causing toxins.     

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces cytoskeletal alterations on 'Swiss 3T3' mouse fibroblasts

The effect of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) on Swiss 3T3 cells was investigated. Cell morphology alterations were observed when 3T3 cell cultures were exposed for 6 h to 1.5 mM MPTP. Using indirect immunofluorescence technique, cytoskeletal elements' organization of microfilaments and microtubules, has been analysed. MPTP modified both actin and tubulin networks, stress fibers appeared less sharp and microtubules were disorganized. The effect of MPTP was completely reversible and cell viability was unaffected. These results suggest that changes in cytoskeletal organization may be the first visible effect related to biochemical alterations induced by MPTP.     

Depletion of glutathione in brainstem of mice caused by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is prevented by antioxidant pretreatment

C57 black mice showed significantly decreased glutathione (GSH) content in the brainstem 24 h after a single s.c. injection of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 40 mg/kg. This loss of GSH could be prevented by pretreating animals with large amounts of alpha-tocopherol or beta-carotene. Increasing dopamine turnover of nigrostriatal neurons in mice by dietary administration of L-dihydroxy-phenylalanine and carbidopa did not accentuate the neurotoxic effects of MPTP. It seems likely that MPTP metabolites directly damage dopaminergic nigrostriatal neurons and that GSH is consumed in attempts to detoxify these metabolites in the substantia nigra.     

Immunohistochemical evaluation of the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic nigrostriatal neurons of young adult mice using dopamine and tyrosine hydroxylase antibodies

The recovery of dopamine (DA) neurons in young adult mice from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damage was analyzed at various times after MPTP treatment with DA and tyrosine hydroxylase (TH) immunohistochemistry and also by chemical DA assay. A remarkable discrepancy in the recovery rate of DA and TH reactivities of the nigral neurons was observed: the TH immunoreactivities of both cell bodies in the substantia nigra and terminals in the neostriatum were markedly reduced 4 days after MPTP. However, these reactivities progressively improved and almost fully recovered after 25 days, while the DA immunoreactivities were maximally depleted 10 days after, and the depletion continued even through the 25th day. The alteration of DA levels was correlated with that of DA immunoreactivity. These findings suggest that a major effect of MPTP on the DA neurons of young adult mice is a transient neurotoxicity, and that the TH content improves more promptly than that of DA.     

Acute ultrastructural and behavioral effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice

Acute actions of MPTP on behavior and on neostriatal ultrastructure were examined in young C57 Black mice. Autonomic, motor, and toxic effects of MPTP exhibited dependence on dose (20-40 mg/kg) and time during the first 4 h after subcutaneous injection. The ultrastructure of the neostriatum was altered very quickly (2-24 h) after single injections of MPTP. Darkened glial processes were found within 2-8 h, followed by dark degeneration of synaptic boutons, especially those making small symmetric synapses. More rarely, swollen axons and postsynaptic degeneration were also observed.     

Systemic administration of antioxidants does not protect mice against the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)

We examined whether DA neurotoxicity of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) can be prevented by combined systemic administration of antioxidants. C57 black mice were injected s.c. with (1) MPTP (30 mg/kg), once daily for two days, alone, or with ascorbic acid (1 g/kg), α-tocopherol (100 mg/kg), or dimethylsufoxide (50 μl) i.p. for two days before, two days with and two days after MPTP, and decapitated 30 days later. (2) MPTP once (30 mg/kg), alone, or with ascorbic acid (200 mg/kg) or cysteamine (75 mg/kg), two days before, one day with and 4 days after, and decapitated 10 days post-MPTP. (3) MPTP once (15 mg/kg), alone, or with ascorbic acid (500 mg/kg), α-tocopherol (100 mg/kg), cysteamine (50 mg/kg) or sodium selenite (2.5 mg/kg), 90 min before and again 90 min after MPTP, and decapitated 7 days later. In all experiments, the marked striatal DA depletions produced by MPTP alone (by 40–70% from controls) were unchanged by cotreatments with the various antioxidants. Findings do not favor intraneuronal generation of superoxides and related cytotoxic free radicals as a major factor in the DA neurotoxicity of MPTP. They suggest that if natural Parkinson's disease is caused by an MPTP-like neurotoxin, early treatment with antioxidants is unlikely to protect nigrostriatal neurons and prevent disease progression.     

Time-dependent inhibition of rat brain monoamine oxidase by an analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine

Inhibitory effects of some MPTP and MPP+ analogues on rat brain MAO activity were studied to further clarify the structure-activity relationships of MPTP neurotoxicity. Of the analogues tested, 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (CPTP), 4-(4-chlorobenzyl)-pyridine (CBP), 4-benzylpyridine (BPY) and 4-benzylpiperidine (BPIP) dose-dependently inhibited both MAO-A and -B activities. CPTP, BPY and BPIP showed a higher MAO-A selectivity, while CBP was a selective MAO-B inhibitor. In preincubation studies, only CPTP greatly enhanced the degree of inhibition of MAO-B when the preincubation time was increased, but inhibition of MAO-A was not enhanced. Together with our previous MPTP and MPP+ analogue findings, the present results indicate that, in these chemical structures, a 4-phenyl-1,2,3,6-tetrahydropyridine ring is most essential for time-dependent inhibition of MAO. This chemical requirement is consistent with the ability to cause nigrostriatal dopaminergic neurotoxicity.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on ultrastructure of nigral neuromelanin in Macaca fascicularis

In neurons of the substantia nigra (SN) of Macaca fascicularis the administration of parkinsongenic doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused morphological changes of the neuromelanic granules. Under light microscopy, the granules appeared more dispersed and larger. Electron microscopy revealed coalescence of granules in large masses with loss of the electrodense component. Phagocytosis of neuromelanin by glial cells was also observed. In several neurons the neuromelanic changes were evident in the presence of morphologically intact mitochondria. These data suggest an interaction between MPTP and neuromelanin that may have relevance to the nigrotropic toxicity of MPTP and are in agreement with observations on neuromelanin in parkinsonian patients.     

Variability and functional recovery in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism in monkeys

Fourteen macaque monkeys were injected intravenously with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. All developed the cardinal signs of parkinsonism (akinesia, rigidity, etc.) in varying degrees; some required repeated series of injections of the drug, while others developed the syndrome readily after the first series. Most of the subjects that were kept for longer than 4 weeks after the first dose of the drug showed complete or partial recovery after that time. Measurement, in some of the subjects, of the neostriatal levels of dopamine and dihydroxyphenylacetic acid showed the expected depletion of these substances at the peak of the behavioral action of the drug, but no recovery when the animals had returned to, or near, pre-drug behavioral status. No firm conclusion can be reached at this time as to the reasons for the behavioral recovery or the variability of the effects of the drug across subjects.     

Potential neuroprotective effect of low dose whole-body gamma-irradiation against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic toxicity in C57 mice

Low dose whole-body γ-irradiation is recently reported to confer neuroprotection against optic nerve crush and contusive spinal cord injury. Here, we extended the study and investigated whether the pretreatment of a single low dose whole-body γ-irradiation may have a preventive effect in MPTP-induced model of PD. One week after the last MPTP treatment, HPLC determination of striatal dopamine and immunostaining for tyrosine hydroxylase (TH), CD11b and GFAP to detect dopamine neurons and associated glial reaction in the substantia nigra pars compacta (SNpc) were performed. MPTP treatment reduced striatal DA levels significantly; nigral TH immunoreactivity was reduced to a lower extent; robust gliosis was also observed in SNpc. We found that 3.5 Gy irradiation but not 5.5 Gy restores the level of DA and its metabolites decreased by MPTP. However, there was no difference in the number of TH positive neurons between 3.5 Gy irradiated and saline treated mice after MPTP treatment. Irradiation also did not have obvious influence on microgliosis and astroglial reaction induced by MPTP treatment. In conclusion, the results presented here demonstrated that low dose whole-body γ-irradiation renders neuroprotection against MPTP-mediated damage of striatal dopaminergic nerve fibers, though it does not seem to influence the MPTP-induced reduction of SNpc dopaminergic neurons and associated glial responses.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced astrogliosis does not require activation of ornithine decarboxylase.

Mechanical injury to the brain results in enhanced immunostaining for glial fibrillary acidic protein (GFAP) that is markedly inhibited by difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. In the current study, systemic exposure of mice to the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), also increased GFAP but, unlike mechanical injury, this increase was not prevented by DFMO pretreatment. These results indicate that de novo polyamine biosynthesis is not obligatory for the MPTP-induced increase in GFAP. MPTP administration, unlike mechanical injury, does not disrupt the blood-brain barrier; thus, a role for polyamine biosynthesis in the astrocyte response to injury may be restricted to insults involving a compromised blood-brain barrier.     

Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

BACKGROUND. Intracerebral transplantation of fetal dopaminergic neurons is a promising new approach for the treatment of Parkinson's disease. Patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have a relatively stable lesion limited to the nigrostriatal system, rendering them ideal candidates for transplantation. Improvement of motor function after neural grafting has previously been observed in nonhuman primates with MPTP-induced parkinsonism. METHODS. We grafted human fetal tissue from the ventral mesencephalon (obtained six to eight weeks after conception) bilaterally to the caudate and putamen in two immunosuppressed patients with severe MPTP-induced parkinsonism, using a stereotaxic technique. The patients were assessed regularly with clinical rating scales, timed tests of motor performance, and [18F]fluorodopa positron-emission tomography during the 18 months before the operation and the 22 to 24 months after the operation. RESULTS. Both patients had substantial, sustained improvement in motor function and became much more independent. Postoperatively, the second patient's maintenance dose of levodopa was decreased to 150 mg daily, which was 30 percent of the original dose. Striatal uptake of fluorodopa was unchanged 5 to 6 months postoperatively but was markedly and bilaterally increased at 12 to 13 and 22 to 24 months in both patients, closely paralleling the patients' clinical improvement. There were no serious complications. CONCLUSIONS. Bilateral implantation of fetal mesencephalic tissue can induce substantial long-term functional improvement in patients with parkinsonism and severe dopamine depletion and is accompanied by increased uptake of fluorodopa by the striatum. The results in these patients resemble those obtained in MPTP-treated primates and suggest that this will be a useful model for the assessment of transplantation therapies in Parkinson's disease.