MPTP, MPDP+ and MPP+ cause decreases in dopamine content in mouse brain slices

MPTP causes a Parkinson's disease-like syndrome in which the dopamine content of the nigrostriatal system decreases. We have studied the relationship between physiological changes and dopamine content using a brain slice preparation developed for electrophysiological studies of corticostriate and nigrostriatal synaptic transmission. We report that MPTP, MPDP+ and MPP+ cause significant decreases in dopamine content of mouse brain slices. We also report that compounds (pargyline and GBR-12909) which block MPTP's toxicity in vivo and prevent non-reversible changes in synaptic transmission are not able to alter MPTP's ability to decrease slice dopamine contents. This indicates that the dopamine content in slices may not be causally related to the non-reversible decrease in synaptic transmission or in vivo neurotoxicity.     

Histochemistry of MPTP oxidation in the rat brain: sites of synthesis of the parkinsonism-inducing toxin MPP+

Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is toxic to the nigrostriatal dopaminergic neurons and produces a syndrome similar to Parkinson's disease. Conversion of MPTP to 1-methyl-4-phenylpyridine (MPP+) by monoamine oxidase-B (MAO-B) appears necessary for this neurotoxicity. When MPTP was used as the substrate for the histochemical localization of monoamine oxidase activity on sections of the rat brain, only a few specific sites were found in which MPTP oxidation to MPP+ occurs. These include the noradrenergic and serotoninergic neurons of the brainstem and the histamine neurons of the caudal hypothalamus. Dopamine neurons themselves do not display the capacity to oxidize MPTP. It is proposed that the conversion of MPTP to MPP+ occurs via MAO-B within serotonin and histamine neurons which may innervate the substantia nigra where the toxin MPP+ could be released and then taken up into the dopamine neurons.     

MPTP toxicity in the mouse brain and vitamin E

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused transient alterations in vitamin E levels in every brain region examined. However, vitamin E returned to normal levels within a few hours in all brain regions but the substantia nigra, where at 2 days vitamin E levels first rose above normal levels. Vitamin E deficient mice were much more susceptible to MPTP toxicity than controls, in terms of lethality and DOPAC depletion in the substantia nigra. However, in the same vitamin E deficient mice, the striatum was partially protected from neurotransmitter and metabolite depletion by MPTP. The mechanism of toxicity of MPTP may differ in the striatum and the midbrain.     

Selective retention of MPP+ within the monoaminergic systems of the primate brain following MPTP administration: an in vivo autoradiographic study

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively destroys dopaminergic neurons of the substantia nigra pars compacta in humans and other primates, producing a parkinsonian condition. MPTP is metabolized to the toxin 1-methyl-4-phenylpyridine (MPP+) which is taken up by dopamine terminals. The subsequent events culminating in cell death in the substantia nigra pars compacta are not understood. To examine these events we first produced a chronic hemiparkinsonian condition in monkeys by administering a toxic dose of MPTP via the right carotid artery. One year later, these monkeys were given a trace dose of [14C]MPTP intravenously and allowed to survive 1, 3, or 10 days. In two acute conditions, monkeys were either given the radiolabeled trace dose intravenously immediately following the toxic intracarotid dose, or were given a single toxic intracarotid radiolabeled dose, and allowed to survive 1, 3, or 10 days. We show by histology and autoradiography that the chronic hemiparkinsonian condition is characterized by selective unilateral loss of nigrostriatal dopamine neurons and absence of MPP+ retention in the caudate-putamen. In the acute conditions, MPP+ is accumulated and selectively retained in high concentrations in the caudate-putamen bilaterally and throughout the nigrostriatal pathway only on the side receiving the toxic dose. In the substantia nigra pars compacta. MPP+ is accumulated in very low concentrations in the dopamine cell bodies and is not selectively retained there. At 10 days survival, the caudate-putamen on the side receiving the toxic dose loses its ability to retain MPP+. The apparent degeneration of the dopamine axon terminals in the caudate-putamen and the development of Parkinson-like behavioral signs seen at 10 days survival were observed to precede the loss of cell bodies in the substantia nigra, which appeared normal by the criteria of Nissl staining and neuromelanin content at all time points in the acute conditions. Other areas of dense MPP+ retention in all cases include noradrenergic and serotonergic cell groups and noradrenergic pathways. MPP+ in the locus coeruleus and other caudal catecholaminergic cell groups is apparently retrogradely transported there after uptake in terminal regions, and although it is retained in high concentrations, no cell loss occurs. These findings suggest that experimentally induced Parkinsonism results from molecular events initiated in the neostriatum and selectively elaborated in the nigrostriatal pathway, ultimately resulting in the death of substantia nigra pars compacta dopamine neurons. They do not support a significant role for neuromelanin binding in the toxicity of MPP+.     

The biodisposition of MPP+ in mouse brain

These studies assessed the role of biotransformation in the rapid elimination of MPP+ from the central nervous system (CNS) compartment of mice. Mice were given either MPP+ via the intracerebroventricular (i.c.v.) route, or MPTP intraperitoneally. The elimination of MPP+ from the brain over 10 h was similar in both groups and followed exponential kinetics. Using liquid scintillation counting, high-pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analysis, no compounds other than MPP+ were detected in brain 2-10 h after the i.c.v. administration of radiolabelled [14C/3H]MPP+. MPP+ was also unchanged after incubation with brain homogenates. These data indicate that MPP+ is not removed from the CNS compartment via biotransformation. Because of its positive charge and kinetics of elimination, the possibility of an active transport system is suggested.     

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.     

线粒体转录终止因子蛋白家族的表达以及在MPTP/MPP~+诱导下的表达变化

目的探讨线粒体转录终止因子(MTERFs)蛋白家族在组织和细胞中的表达以及在神经毒素MPTP/MPP+诱导下的表达变化。方法取正常C57BL小黑鼠的脑、肝、肾组织,以及人神经母细胞瘤细胞SH-SY5Y、人心肌细胞HCM、人肝细胞L-02,检测MTERFs的蛋白表达情况;通过MPTP/MPP+构建帕金森病(PD)动物/细胞模型,检测MTERFs的蛋白表达变化情况。结果 MTERFs在小鼠脑中和SH-SY5Y细胞中蛋白表达水平较高;MPTP/MPP+诱导的PD模型中,MTERF3蛋白表达水平明显降低。结论在小鼠脑和SH-SY5Y细胞中,MTERFs表达较高;MPTP/MPP+诱导下MTERF3蛋白表达明显降低,影响了线粒体转录水平,这可能参与了帕金森病发生的过程。     

Involvement of monoamine oxidase-B in the acute neurotoxicity of MPTP in embryonic and newborn mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces damage to the nigrostriatal system and subventricular zone (SVZ) of mice. While there have been many researches on the neurotoxicity of MPTP in adult mice, there have been few reports concerning that in embryonic and newborn mice. Very recently, we revealed that such neurotoxicity of MPTP and 1-methyl-4-phenylpyridinium (MPP⁺), a metabolite of MPTP, is observed not only in adult mice but also in embryonic and newborn mice; however, the mechanism of acute toxicity is not well elucidated. In the present study, we attempted to reveal the involvement of monoamine oxidase B (MAO-B) in the metabolism of MPTP to MPP⁺ and dopamine transporter (DAT) in the neuronal cellular uptake of MPP⁺ during the acute toxicity of MPTP in both embryonic and newborn mice. Immunohistochemistry and double-labeling immunofluorescent staining demonstrated an increase of MAO-B-positive glial cells in the brain only in MPTP-treated mice, indicating the involvement of MAO-B in the metabolism of MPTP to MPP⁺ during the acute neurotoxicity of MPTP in both embryonic and newborn mice. The expression of DAT was not observed in the nigrostriatal zone of embryonic mice and in the zone and SVZ of newborn mice. The mechanism of how MPP⁺ is taken up into those neuronal cells remains unknown. In conclusion, MAO-B is involved in the acute neurotoxicity of MPTP in embryonic and newborn mice.     

6-OHDA、DA和MPTP（MPP＋）神经毒性作用机理

帕金森病为中枢老年退行性疾病，发病原因尚未阐明。6-OHDA、多巴胺（DA）和MPTP（MPP＋）为DA能神经元相对选择性神经毒物质。研究6-OHDA、DA和MPTP（MPP＋）的DA能神经毒性作用机理对于理解帕金森病的发病机理具有重要意义。利用DA能神经毒物质在体内和体外模拟帕金森病为有效防治帕金森病提供了实验依据。     

Monoamine oxidase activity and distribution in marmoset brain: implications for MPTP toxicity

This study describes the histochemical localisation of monoamine oxidase (MAO) in marmoset brain. No MAO A staining was observed but MAO B was found in many areas, with medium intensity of staining in the substantia nigra, and high intensity staining in the striatum and nucleus accumbens, among other regions. The high activity in the nigrostriatal tract, compared with the rat, may partially explain the greater sensitivity of the marmoset to MPTP toxicity. As in the rat, high activity was present in the raphe nuclei. However, unlike the rat, no enrichment of MAO B was observed in blood vessels or ventricular linings.     

Retinal dopamine sensitivity to MPP+ toxicity: electrophysiological and biochemical evaluation

This study concerns the effect of intravitreal injection of 1-methyl-4-phenylpyridinium ion (MPP+) on the electroretinograms (ERG) and on the levels of retinal dopamine (DA) in rabbits. The right eye was injected intravitreously with MPP+ while the other received only the vehicle and served as control. The administration of 7, 40, 70 or 700 micrograms MPP+ resulted in a dose-related decrease of the amplitude of the a and b-waves as well as the oscillatory potentials (OPs) of the ERG, down to extinction. In contrast, the retinal DA content was decreased only with the 700 micrograms MPP+ dose. Fluorescein angiography demonstrated abnormalities in the retinal circulation of all MPP+-treated eyes. These observations indicate that MPP+ causes lesions to the retinal vessels at doses non-toxic to the retinal dopaminergic neurons. These data suggest that intravitreal injection of MPP+ cannot be used to study the physiological role of retinal DA.     

过表达VMAT2的CHO细胞可抵抗rotenone和MPP+的毒性作用

目的为研究毒物鱼藤酮(rotenone)和1-甲基-4-苯基吡啶离子(MPP )对野生型及过表达囊泡单胺转运蛋白(VMAT2)的中国仓鼠卵巢细胞(CHO)的毒性作用。方法将不同浓度的MPP r、otenone与野生型(WT-CHO)和过表达VMAT2的CHO细胞(VMAT2-CHO)共培养,通过形态学改变和MTT比色法检测细胞活力来观察毒物的毒性作用。结果VMAT2-CHO细胞活力明显增强,第5~7天VMAT2-CHO数量约为WT-CHO的3倍。0.2~2.0 mmol/L的MPP (P<0.05)和0.05~1.0μmol/L rotenone(P<0.01)作用72 h,VMAT2-CHO的存活率高于WT-CHO,细胞中毒后的形态改变不如WT-CHO明显。结论过表达VMAT2的CHO细胞对MPP 和rotenone的毒性作用有抵抗。     

Acute and subacute toxicity study of 1,8-cineole in mice

The effects of acute and subacute toxicity of 1,8-cineole in Kunming mice were studied. After acute oral administration, the LD₅₀ value (95% CL) was 3849 mg/kg (3488.8~4247.1 mg/kg). In the subacute toxicity study, there were no significant differences in body weight and relative organ weight between the control group and 1,8-cineole treatment groups. The histopathological examinations showed that granular degeneration and vacuolar degeneration appeared in liver and kidney tissue after administration of high dose of 1,8-cineole. Under electron microscopy, a series of ultrastructural changes were observed: The electron microscopy assays indicated that the influence of 1,8-cineole on the target organ at the subcellular level were mainly on the mitochondria, endoplasmic reticulum and other membrane type structure of liver and kidney.     

Acute toxicity of an anti-Fas antibody in mice.

By histopathologic examination of various organs in 3 normal strains, C3H/HeN, ICR, and DBA/1J, of mice treated intravenously once with anti-Fas antibody (Jo2), we failed to determine any target organ, except the liver, responsible for the acute lethality induced by the Fas/anti-Fas antibody interaction. However, we could show the presence of Fas-mediated apoptosis in other organs aside from the liver and normal mouse strain differences in susceptibility to anti-Fas antibody. Among these strains, C3H/HeN was the most susceptible to the antibody, followed by ICR and DBA/1J. We observed Fas-mediated apoptosis in the liver, spleen, thymus, lymph nodes, Peyer's patch, intestine, skin, coagulation glands, ovary, uterus, and vagina in all 3 strains and additionally in the epididymides and seminal vesicles in the DBA/1J strain. We also demonstrated that Fas-mediated apoptosis of small lymphocytes in the mantle zone of splenic lymphatic follicles preceded that of the hepatocytes or thymic cells. Since cellular damage was most severe in the liver among all the apoptotic organs in the 3 mouse strains, liver injury induced by anti-Fas antibody is speculated to play a significant role in the death.     

The acute convulsant effect of MPTP is dependent on intracerebral MPP+.

The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57 black mice causes an acute seizure syndrome the severity of which is dose dependent; there is also a good correlation between the seizure inducing potential of MPTP and the neostriatal dopamine (DA) depletion caused by MPTP. The simultaneous administration of MPTP and MAO B inhibitors attenuates both epileptiform phenomena and neostriatal DA depletion. On the contrary diethyldithiocarbamate (DDC) exacerbates both responses. All these pharmacological manipulations are known to affect the accumulation of 1-methyl-4-phenylpyridinium ion (MPP+) the main metabolite of MPTP. Thus the present data support the hypothesis of a strict dependence of the epileptiform phenomena on the presence of MPP+. Furthermore the tight correlation existing between the severity of epileptic events and DA depletion suggests that the acute excitotoxic syndrome may contribute to the long-term toxicity of MPTP. *On leave from the Department of Neurology, University of Pisa, Pisa, Italy.     

Intracerebral microdialysis neurotoxicity studies of quinoline and isoquinoline derivatives related to MPTP/MPP

The in vivo dopaminergic neurotoxicity of a series of quinoline and isoquinoline derivatives was assessed in rats using an intrastriatal microdialysis technique that measures the release of dopamine. The N-methyl quaternary salts of these two heterocyclic aromatic systems displayed about 10% of the potency of MPP⁺ in this assay. Furthermore, tetrahydroisoquinoline, which has been reported to be present in human brain, and N-methyltetrahydroisoquinoline were found to be MAO-B substrates, being oxidized at about 3% the rate of MPTP. Thus, although tetrahydroisoquinoline and N-methyltetrahydroisoquinoline are not neurotoxic, it is conceivable that the chronic endogenous formation of quaternary species could cause neuronal lesions that contribute to the etiology of idiopathic Parkinson's disease.     

Overexpression of CYP2D6 attenuates the toxicity of MPP+ in actively dividing and differentiated PC12 cells

Clonal pheochromocytoma cell lines overexpressing cytochrome P450 2D6 (CYP2D6) were established. CYP2D6 was localized in the endoplasmic reticulum, and its enzymatic activity in the microsomal fraction was confirmed by using high performance liquid chromatography analysis with [guanidine-14C]debrisoquine as a substrate. Overexpression of CYP2D6 protected both actively dividing and differentiated cells against the toxic effects of 1-methyl-4-phenylpyridinium ion at the concentration range of 20-40 microM, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The production of reactive oxygen species in the mitochondria was suppressed. The cytotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was unchanged in both actively dividing and differentiated cells overexpressing CYP2D6 versus mock-transfected controls at concentrations up to 500 microM. These results suggest that the lowered enzyme activity of CYP2D6 in individuals termed "poor metabolizers" may represent a risk factor from exposure to select neurotoxicants.     

胚胎及新生大鼠脑组织神经干细胞巢蛋白表达的发育改变

目的：检测不同发育时期脑组织神经干细胞巢蛋白(nestin)的表达和发育变化规律,为分离、富集和增殖神经干细胞提供实验依据。方法：将Wistar大鼠按胎龄和日龄分组,剥离脑组织,制成单细胞悬液,经间接荧光法标记nestin抗原,用流式细胞术检测并分析。结果：E13至P90整个发育过程中都有nestin抗原的表达;从E13到E19表达逐渐升高,E19表达水平最高;出生后表达急剧下降,出生后7d便接近于出生后90d(成年鼠)表达水平。结论：胚胎发育近中期后,脑神经干细胞nestin表达水平随胎龄逐渐升高,E19达到高峰,出生后表达迅速下降,一周内趋于成鼠表达量。     

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.