Inactivation of tyrosine hydroxylase in rat striatum by 1-methyl-4-phenylpyridinium ion (MPP+)

We report that 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inactivated tyrosine hydroxylase (TH) when MPP+ was directly infused into the striatum. We examined both in vitro TH activity and TH content measured by an enzyme immunoassay in the rat striatum after MPP+ was administered by an in vivo brain microdialysis probe. MPP+ caused the inhibition of TH activity but did not influence TH content in the ipsilateral striatum. These results indicate that MPP+ may cause an acute inactivation of TH after continuous exposure at the high concentrations.     

Central hypothermic effects of some analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+)

Effects of some MPTP or MPP+ analogues on mouse body temperature were studied. Of the analogues tested, 4-phenylpyridine (PPY) and 4-phenyl-1,2,3,6-tetrahydropyridine (PTP) given in single i.p. doses to mice caused marked hypothermia. Intracerebroventricular (i.c.v.) injection of PPY or PTP caused similar hypothermia. Pretreatment with clorgyline or (-)-deprenyl greatly prevented hypothermia induced by i.c.v. PPY, but hypothermia by i.c.v. PTP was prevented only by (-)-deprenyl. These results indicate that, in order to cause central hypothermia, PTP does not seem to require metabolism to PPY and both analogues per se may cause hypothermia.     

Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP+) in isolated mitochondria from mouse brains

Effect of 1-methyl-4-phenylpyridinium ion (MPP+) on adenosine triphosphate (ATP) synthesis was studied using isolated mitochondrial preparations from mouse brains. Oxidation of glutamate + malate in the mitochondria estimated by polarography was significantly inhibited by MPP+, and synthesis of ATP was inhibited to approximately 9% of that of control by 0.06 mM of MPP+. Oxidation of alpha-glycerophosphate and succinate was not inhibited, and ATP was synthesized normally. Energy crisis appears to be one of the most important mechanisms of neuronal degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced experimental parkinsonism.     

Dopaminergic toxicity after the stereotaxic administration of the 1-methyl-4-phenylpyridinium ion (MPP+) to rats

The administration of the 1-methyl-4-phenylpyridinium ion (MPP+) stereotaxically into the left neostriatum or left median forebrain bundle of female rats resulted in a very large and highly significant loss of dopamine and of its metabolites in the left neostriatum. The effect of MPP+ on neostriatal dopamine content was in general considerably greater than its effect on serotonin or on several amino acids. These results are consistent with the premise that MPP+, formed from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by the enzyme monoamine oxidase B, may be responsible for the toxicity observed after MPTP administration.     

Tyrosine hydroxylase immunohistochemistry in the normal and 1-methyl-4-phenyl-tetrahydropyridine (MPP+)-treated retina of goldfish

Dopaminergic interplexiform neurons have been identified in the inner nuclear layer of goldfish retina, with tyrosine hydroxylase (TH) immunocytochemistry in whole-mounted retinae and in cryosections. The neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+), which selectively damages dopaminergic neurons in mammals, caused a marked depletion of TH immunoreactivity in goldfish retina. Three days after intravitreal injection, retinae showed no significant decrease in the number of TH-positive neurons. However most of the TH-immunoreactive cell bodies showed an evident depletion of TH immunoreactivity and their processes, ramified in the inner and outer plexiform layers, disappeared almost completely.     

Imidaprilat suppresses nonylphenol and 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical generation in rat striatum

The present study examined the antioxidant effects of angiotensin-converting enzyme inhibitor (ACE), imidaprilat, on para-nonylphenol and 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical (*OH) formation and dopamine (DA) efflux in extracellular fluid of rat striatum, using a microdialysis technique. para-Nonylphenol clearly enhanced *OH formation and DA efflux induced by MPP+. When imidaprilat was infused in para-nonylphenol and MPP+-treated rats, DA efflux and *OH formation significantly decreased, as compared with that in the para-nonylphenol and MPP+-treated control. Imidaprilat was able to scavenge *OH and DA efflux induced by para-nonylphenol and MPP+. When iron(II) was administered to para-nonylphenol-pretreated animals, iron(II) clearly produced a dose-dependent increase in the levels of 2,3-dihydroxybenzoic acid (2,3-DHBA), as compared with MPP+-only-treated rats. A positive linear correlation was observed between iron(II) and 2,3-DHBA (R2=0.985) in the dialysate. However, in the presence of imidaprilat, a small increase in the levels of 2,3-DHBA products was observed. The results suggest that imidaprilat may protect against para-nonylphenol and MPP+-induced *OH formation via suppressing DA efflux in the rat striatum.     

Activation of protein kinase C enhances 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxylradical generation in rat striatum

The present study examined the effect of chelerlythrine, a protein kinase C (PKC) inhibitor, on 1-methyl-4-phenylpyridine (MPP+)-induced hydroxyl radicals (*OH) in rat striatum. Rats were anesthetized, and sodium salicylate (0.5 mM or 0.5 nmol/microl/min) was infused through a microdialysis probe to detect the *OH generation as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) in the striatum. Dopamine (DA)-selective neurotoxin, MPP+, infusion into the striatum of rats induces *OH formation, trapped as 2,3-DHBA. The application of chelerythrine, a potent and selective protein kinase C (PKC) inhibitor, suppressed MPP+ -induced *OH formation. The results in the present study suggests the protective effect of chelerythrine on *OH generation induced by MPP+.     

The non-competitive NMDA-receptor antagonist MK-801 prevents the massive release of glutamate and aspartate from rat striatum induced by 1-methyl-4-phenylpyridinium (MPP+)

The concentrations of dopamine (DA) and of the excitatory amino acids (EAAs) glutamate (Glu) and aspartate (Asp) were measured in dialysates from the striatum of awake rats in order to study the link between the release of DA and of EAAs induced by the infusion of 1-methyl-4-phenylpyridinium ion (MPP+). DA and EAAs were detected simultaneously by HPLC-EC. The infusion of MPP+ at the concentration of 1 mM elevated DA levels in the perfusates, but did not affect EAA release. However, MPP+ at 10 mM maximally stimulated Glu and Asp release to 230- and 68-fold of baseline, respectively. In this condition, pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (5 mg/kg, i.p.) prevented the MPP(+)-induced EAA release. In contrast, MK-801 had no effect on DA release induced either by 1 or 10 mM MPP+. These results suggest that MPP(+)-induced DA and EAA release are independently regulated processes. In addition, the finding that MK-801 inhibits MPP(+)-induced EAA release suggests that EAAs may act on NMDA receptors to stimulate their own release through a positive-feedback mechanism.     

Conversion of the neurotoxic precursor 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine into its pyridinium metabolite by human platelet monoamine oxidase type B

MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine) causes selective and irreversible degeneration of the substantia nigra of human and non-human primates. In the central nervous system, the oxidative metabolism of MPTP to 1-methyl-4-phenyl-pyridinium (MPP+) by monoamine oxidase type B (MAO-B) seems to be a critical feature in the neurotoxic process. We now report that [3H]MPTP is rapidly converted in vitro into [3H]MPP+ by human platelet MAO-B. The formation of [3H]MPP+ in human platelets is prevented by specific MAO-B but not by MAO-A or by 5-hydroxytryptamine uptake inhibitors.     

The toxic actions of MPTP and its metabolite MPP+ are not mimicked by analogues of MPTP lacking an N-methyl moiety

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenylpyridine (MPP+) and three analogues of MPTP, lacking an N-methyl moiety, namely, 4-phenylpiperidine (I), 4-phenyl-1,2,3,6-tetrahydropyridine (II) and 4-phenylpyridine (III), were infused continuously for a period of 4 days into the rat substantia nigra. Within 12 h of commencing the bilateral infusion of MPTP or MPP+, rats showed marked motor deficits with reduction in locomotor activity, loss of ability to move the forelimbs and grip with forepaws and, following MPP+ infusions, similar loss of movement in the hindlimbs associated with the development of limb and body rigidity. These motor deficits were not induced by the 3 analogues of MPTP on infusion into the substantia nigra. After 4 days of infusion, the motor deficits caused by MPTP and, in particular, MPP+, were still marked, and for MPP+ these correlated with marked loss of striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid. 4-Phenyl-1,2,3,6-tetrahydropyridine caused a small loss in striatal DA and DOPAC, but the other analogues failed to modify the striatal content of DA or its metabolites. Small alterations of chemical structures related to MPTP and its metabolite can critically alter ability to induce behavioural and neurochemical changes reflecting toxicity on the nigrostriatal DA system.     

Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase is not involved in the initiation of apoptosis induced by 1-Methyl-4-phenyl-pyridium iodide (MPP+)

Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in the process of apoptosis. To study the function of GAPDH, we expressed GAPDH C-terminally fused with or without nuclear localization signal (NLS) in SH-SY5Y and NB41A3 cells using a retrovirus expression system. GAPDH carrying NLS (GAPDH-NLS) was expressed mainly in the nucleus. However, expression of GAPDH-NLS did not cause any difference in cell survival rate as compared to that of the vector alone or GAPDH without NLS. Treatment with 1-Methyl-4-phenyl-pyridium iodide (MPP+) caused no difference in the cell survival rate or in the pattern or extent of apoptosis among the three transductants. In the cells expressing GAPDH without NLS, MPP+ did not cause visible translocation of GAPDH into nucleus before the onset of apoptosis. Since GAPDH is known to comprise a CRM1-mediated nuclear export signal, we blocked the nuclear export of GAPDH by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export. The treatment did not cause any difference in apoptosis among the three transductants. An additional treatment with MPP+ induced no apoptotic difference in these cells. Thus, we have concluded that a simple nuclear localization of GAPDH does not induce apoptosis, and that MPP+-induced apoptosis is not caused by nuclear translocation of GAPDH.     

Implication of the c-Jun-NH2-terminal kinase pathway in the neuroprotective effect of puerarin against 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in PC-12 cells

Apoptosis is a widely accepted component of the pathogenesis of Parkinson's disease (PD), a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. In this study, we investigated the neuroprotective effects of puerarin and possible mechanisms by which puerarin acts against MPP(+)-induced toxicity in rat pheochromocytoma PC12 cells. PC12 cells exposed to MPP(+) (500 μM) significantly decreased the viability of PC12 cells when examined by MTT assay, DNA ELISA assay, and Annexin V assays, which was prevented by puerarin in a dose-dependent manner. PC12 cells exposed to MPP(+) (500 μM) elicited phosphorylation of MKK7, c-Jun-NH(2)-terminal kinase (JNK), and c-Jun which followed by the increase in cytochrome c levels, and which was prevented by puerarin. Moreover, puerarin inhibited the activation of caspase-9 and caspase-3 in MPP(+)-exposed PC12 cells. Whereas, the neuroprotective effect of puerarin against MPP(+) insults can be blocked by SP600125 (inhibitor of JNK). Taken together, these results suggest that puerarin protected PC12 cells against MPP(+)-induced neurotoxicity through the inhibition of the JNK signaling pathways. Therefore, puerarin has the possible beneficial effects in PD by attenuating MPP(+)-induced toxicity.     

Different susceptibility to 1-methyl-4-phenylpyridium (MPP+)-induced nigro-striatal dopaminergic cell loss between C57BL/6 and BALB/c mice is not related to the difference of monoamine oxidase-B (MAO-B)

Subcutaneous and intraperitoneal administrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induce selective dopaminergic (DA-ergic) neuronal death in many animal species. After passing through the blood–brain barrier (BBB), MPTP is converted to 1-methy-4-phenylpiridinium (MPP⁺) by astrocytic monoamine oxidase-B (MAO-B). MPP⁺ then induces the dopaminergic neuronal death. In mice, marked strain differences in the susceptibility to MPTP-injection have been reported. To clarify which factor(s) cause the strain differences, MPTP or MPP⁺ was intracerebroventricularly (icv) injected into adult C57BL/6 (highly susceptible to MPTP) and BALB/c (resistant to MPTP) mice. The brain tissues including the striatum and substantia nigra pars compacta (SNpc) were examined immunohistochemically using an antibody to tyrosine hydrocyrase (TH). MPP⁺-injected C57BL/6 mice showed a significant decrease in TH-immunopositive areas in the striatum at Day 3 post injection (p < 0.01), and TH-positive cells in the SNpc at Days 1 and 3 (p < 0.01), respectively, compared to saline-injected control mice. In addition, MPP⁺-injected BALB/c mice showed a significant decrease in TH-positive areas in the striatum at Days 1 and 3, and SNpc TH-positive cells in the SNpc at Day 3, respectively (p < 0.05). However, the decrease rates in the BALB/c mice were lower than that in C57BL/6 mice. MPTP-injected C57BL/6 mice, however, showed no lesions in the striatum and SNpc at Days 1 and 7 after icv injection. All the present findings indicate that factors other than MAO-B can influence the strain susceptibility between C57BL/6 and BALB/c mice after the conversion from MPTP to MPP⁺.     

Neurochemical lesioning in the rat brain with iontophoretic injection of the 1-methyl-4-phenylpyridinium ion (MPP+).

Iontophoretic injections of the 1-methyl-4-phenylpyridinium ion (MPP+) were made in the dopaminergic part of the substantia nigra to see whether this injection technique could be used for inducing localized neurochemical lesions in dopaminergic cell groups and to assess the effects of MPP+ on non-dopaminergic neurons. Three days after the iontophoretic injection of MPP+, a gliosis or necrotic hole was found in the dopaminergic and non-dopaminergic target areas. This effect depended on the injection parameters that were used; iontophoretic injections of short duration (less than or equal to 3 minutes) and low current strength (1.5 microA) caused the gliosis, higher injection parameters gave lesions. The estimated injected amount of MPP+ was between 0.5 and 10.8 nmol. Control injections, with sodium iodide, sodium chloride or N-methylpyridinium iodide showed that the neurodegeneration is not a side-effect of the iontophoretic injection procedure. It is concluded that iontophoretically injected MPP+ is toxic for all neurons, irrespective of the neurotransmitter used, and also for glia cells and fibers of passage. Excessive formation of free radicals, causing induction of lipid peroxidation, may be involved in the neurodegenerative process observed.     

Up-regulation of divalent metal transporter 1 is involved in 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptosis in MES23.5 cells

Apoptosis has been identified as one of the important mechanisms involved in the degeneration of dopaminergic neurons in Parkinson's disease (PD). Our previous study showed increased iron levels in the substantia nigra as well as loss of dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse models. 1-Methyl-4-phenylpyridinium (MPP⁺) is commonly used to establish a cellular model of PD. Although intracellular iron plays a crucial role in MPP⁺-induced apoptosis, the molecular mechanism linking increased iron and MPP⁺-induced neurodegeneration is largely unknown. In the present study, we investigate the involvement of divalent metal transporter 1 (DMT1) that accounts for the ferrous iron transport in MPP⁺-treated MES23.5 cells. In the treated cells, a significant influx of ferrous iron was observed. This resulted in a decreased mitochondrial membrane potential. Additionally, an elevated level of ROS production and activation of caspase-3 were also detected, as well as the subsequent cell apoptosis. These effects could be fully abolished by iron chelator desferal (DFO). Increased DMT1 (−IRE) expression but not DMT1 (+IRE) accounted for the increased iron influx. However, there were no changes for iron regulatory protein 1 (IRP1), despite decreased expression of IRP2. Iron itself had no effect on IRP1 and IRP2 expression. Our data suggest that although DMT1 mRNA contains an iron responsive element, its expression is not totally controlled by this. MPP⁺ could up-regulate the expression of DMT1 (−IRE) in an IRE/IRP-independent manner. Our findings also show that MPP⁺-induced apoptosis in MES23.5 cells involves DMT1-dependent iron influx and mitochondria dysfunction.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuroblastic apoptosis in the subventricular zone is caused by 1-methy-4-phenylpiridinium (MPP+) converted from MPTP through MAO-B

Intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration induces apoptosis of subventricular zone (SVZ) doublecortin (Dcx)-positive neural progenitor cells (migrating neuroblasts, A cells). Actually, a metabolite of MPTP, 1-methy-4-phenylpiridinium (MPP⁺), is responsible for neural progenitor cell toxicity. In the present study, to examine whether the MPTP-induced SVZ cell apoptosis is caused directly by MPP⁺ metabolized through monoamine oxidase B (MAO-B), MPTP or MPP⁺ was intracerebroventricularly (icv) injected into C57BL/6 mice. At Day 1 postinjection, many terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were observed in the SVZ of both low (36 μg) and high (162 μg) dose MPTP- and MPP⁺-injected mice. The number of Dcx-positive A cells showed a significant decrease following high dose of MPTP- or MPP⁺-injection on Days 1 and 3, respectively, whereas that of EGFR-positive C cells showed no change in mice with any treatment. In addition, prior icv injection of a MAO-B inhibitor, R(?)-deprenyl (deprenyl), inhibited MPTP-induced apoptosis, but not MPP⁺-induced apoptosis. MAO-B- and GFAP-double positive cells were detected in the ependyma and SVZ in all mice. It is revealed from these results that icv injection of MPTP induces apoptosis of neural progenitor cells (A cells) in the SVZ via MPP⁺ toxicity. In addition, it is suggested that the conversion from MPTP to MPP⁺ is caused mainly by MAO-B located in ependymal cells and GFAP-positive cells in the SVZ.     

Aged mice are more sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment than young adults

Parkinson's disease is a neurodegenerative disorder characterized mainly by damage to the dopaminergic nigrostriatal system. Recently, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce damage in the nigrostriatal system, accompanied by Parkinson-like symptoms in humans. We present here evidence that MPTP treatment in aged 21-month-old mice produced a marked reduction in the presence and intensity of fluorescence in noradrenergic neurons of the locus coeruleus and in dopaminergic neurons of the ventral tegmental area in addition to extensive damage to the substantia nigra. Aged mice treated with MPTP also showed physical signs of movement disability characterized by marked akinesia, rigidity of the hind limbs, and an initial resting tremor of the entire body. Such symptoms were less evident in young mice treated with MPTP. These remarkable initial behavioral effects of MPTP treatment in aged mice and evidence of reduced catecholamine fluorescence in the locus coeruleus and ventral tegmental area suggest that aged mice are more sensitive to, and more severely affected by MPTP treatment than young mice. We suggest that these MPTP-treated aged mice provide a useful animal model for studying both anatomical and functional characteristics of Parkinson's disease.