Potential environmental neurotoxins related to 1-methyl-4-phenylpyridinium: selective toxicity of 1-methyl-4-(4'-acetamidophenyl)-pyridinium and 1-methyl-4-cyclohexylpyridinium for dopaminergic neurons in culture

Mesencephalic cells in culture were exposed to various compounds which we hypothesized to be selective toxins for dopaminergic neurons. The culture system was previously shown suitable for assessing selective dopaminergic neurotoxicity, since 1-methyl-4-phenyl-pyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium, destroyed dopaminergic neurons without affecting other cells. Some compounds tested were selected to fulfill two criteria believed to underly the selective dopaminergic neurotoxicity of MPP+, i.e., to be a potential substrate for the uptake carrier for dopamine and to possess a strong delocalized positive charge to inhibit the mitochondrial respiratory system. Other compounds were chosen on the basis of clinical or anecdotal evidence linking them to Parkinson's disease. Among the tested compounds two pyridinium analogs, 1-methyl-4-(4'-acetamidophenyl)pyridinium (MACPP+) and 1-methyl-4-cyclohexylpyridinium (MCP+) were found to be selectively toxic toward dopaminergic neurons. Incubation of cultures with both MACPP+ and MCP+ produced a dramatic reduction in the number of tyrosine hydroxylase-positive cells and the uptake of [3H]dopamine without reducing the number of cells visualized by phase-contrast microscopy or the uptake of [3H]aminobutyric acid. Besides MACPP+ and MCP+ none of the tested compounds exhibited any selective dopaminergic neurotoxicity. Together with earlier findings, these data suggest that the structural requirements are rather strict for a chemical to be a selective dopaminergic neurotoxin and make it unlikely that there is a wide spectrum of environmental dopaminergic toxins.     

Effect of GDNF on differentiation of cultured ventral mesencephalic dopaminergic and non-dopaminergic calretinin-expressing neurons

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for ventral mesencephalic (VM) dopaminergic neurons. Subpopulations of dopaminergic and non-dopaminergic VM neurons express the calcium-binding proteins calbindin (CB) and calretinin (CR). Characterization of the actions of GDNF on distinct subpopulations of VM cells is of great importance for its potential use as a therapeutic molecule and for understanding its role in neuronal development. The present study investigated the effects of GDNF on the survival and morphological differentiation of dopaminergic and non-dopaminergic neurons in primary cultures of embryonic day (E) 18 rat VM. As expected from our results obtained using E14 VM cells, GDNF significantly increased the morphological complexity of E18 CB-immunoreractive (CB-ir), tyrosine hydroxylase (TH)-ir, and CR-ir neurons and also the densities of CB-ir and TH-ir neurons. Interestingly, densities of E18 CR-ir neurons, contrarily to our previous observations on E14 CR-ir neurons, were significantly higher after GDNF treatment (by 1.5-fold). Colocalization analyses demonstrated that GDNF increased the densitiy of dopaminergic neurons expressing CR (TH+/CR+/CB-), while no significant effects were observed for TH-/CR+/CB- cell densities. In contrast, we found that GDNF significantly increased the total fiber length (2-fold), number of primary neurites (1.4-fold), number of branching points (2.5-fold), and the size of neurite field per neuron (1.8-fold) of the non-dopaminergic CR-expressing neurons (TH-/CR+/CB-). These cells were identified as GABA-expressing neurons. In conclusion, our findings recognize GDNF as a potent differentiation factor for the development of VM dopaminergic and non-dopaminergic CR-expressing neurons.     

Protection from 1-methyl-4-phenylpyridinium (MPP) toxicity and stimulation of regrowth of MPP-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP+ toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP+ was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP+, after they had become confluent with dividing glial cells, the MPP(+)-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2'-deoxyuridine, the protective effects of EGF and bFGF against MPP+ toxicity were abolished. Continuous treatment of MPP(+)-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP+ and promotion of recovery of MPP(+)-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.     

Transplacental effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on brain dopaminergic neurons in the mouse

Summary Immunohistochemical studies of monoamme neurons werè performed to evaluate toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on young adult mice and compare them with chose of their offspring. Mice, 9–11 weeks old (C57BL/6J), injected subcutaneously with a large dose of MPTP (17 mg/kg per day) during pregnancy on Day 9 and 12 of gestation (G9 and G12) miscarried and were examined at 13 weeks of age. Conversely, mice treated during pregnancy with sequential low dose of MPTP (2.8 mg/kg per day at G9–G17 for 8 days) successfully delivered their babies and were examined at the age of 15 weeks. Baby mice were examined at 1 and 6 weeks of age. The tyrosine hydroxylase-, aromatic l-amino acid decarboxylase-and dopamine (DA)-immunoreactive density of caudoputamen was reduced in 13-week-old mice treated with high dose of MPTP but not in the 15-week-old mothers exposed to a low dose of MPTP as compared to their respective controls. The DA-immunoreactive density of the caudoputamen was the only staining that was reduced in both 1- and 6-week-old baby mice. In conclusion, these results demonstrate that MPTP injected to pregnant mice causes a DA depletion in the striatum of their offspring indicating a transplacental effect of MPTP. The findings also indicate that fetal brain is more susceptible to MPTP toxicity than the brain of young pregnant mice.Supported by Grant-in-Aid for Scientific Research on Priority Areas, Ministry of Education, Science and Culture, Japan (62623002, 62480226), and by a Fujita-Gakuen Health University Grant, Japan     

Rag-1-dependent cells are necessary for 1,25-dihydroxyvitamin D(3) prevention of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a demyelinating disease involving genetic and environmental risk factors. Geographic, genetic, and biological evidence suggests that one environmental risk factor may be lack of vitamin D. Here, we investigated how 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) inhibits experimental autoimmune encephalomyelitis (EAE), an MS model. The experiments used adoptive transfer of TCR-transgenic (TCR1) cells specific for myelin basic protein (MBP) peptide into unprimed recipients. When unprimed TCR1 splenocytes were transferred, and the recipients were immunized with peptide, the mock-treated mice developed EAE, but the 1,25-(OH)(2)D(3)-treated recipients remained disease-free. Both groups had TCR1 T cells that proliferated in response to MBP Ac1-11 and produced IFN-gamma but not IL-4 in the lymph node. In the central nervous system (CNS), the mock-treated mice had activated TCR1 T cells that produced IFN-gamma but not IL-4, while the 1,25-(OH)(2)D(3)-treated mice had TCR1 T cells with a non-activated phenotype that did not produce IFN-gamma or IL-4. When activated TCR1 T cells producing IFN-gamma were transferred into unprimed mice, the mock-treated and the 1,25-(OH)(2)D(3)-treated recipients developed EAE. Likewise, the 1,25-(OH)(2)D(3) did not inhibit Th1 cell IFN-gamma production or promote Th2 cell genesis or IL-4 production in vitro. Finally, the 1,25-(OH)(2)D(3) inhibited EAE in MBP-specific TCR-transgenic mice that were Rag-1(+), but not in animals that were Rag-1-null. Together, these data refute the hypothesis that the hormone inhibits Th1 cell genesis or function directly or through an action on antigen-presenting cells, or promotes Th2 cell genesis or function. Instead, the evidence supports a model wherein the 1,25-(OH)(2)D(3) acts through a Rag-1-dependent cell to limit the occurrence of activated, autoreactive T cells in the CNS.     

Neuroprotective effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline on cultured rat mesencephalic neurons in the presence or absence of various neurotoxins

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous brain amine and its content in parkinsonian brain is decreased compared with that in control brain. There is some evidence that 1MeTIQ protects dopaminergic neurons against dysfunction such as that seen in Parkinson's disease. In this study, we examined the neuroprotective effect of 1MeTIQ against four dopaminergic neurotoxins, 1-methyl-4-phenylpyridinuim ion, 6-hydroxydopamine, rotenone, and l-benzyl-1,2,3,4-tetrahydroisoquinoline, in cultured rat mesencephalic neurons. 1MeTIQ exerted neuroprotective action against all these toxins. Furthermore, (R)-1MeTIQ was neuroprotective, while (S)-1MeTIQ had little effect, indicating that the effect is stereoselective. The protective action of 1MeTIQ was most effective in mesencephalic neurons, especially in tyrosine hydroxylase-positive neurons. 1MeTIQ showed no affinity for dopamine receptors and did not influence the inhibition of mitochondrial respiratory complex I by rotenone, 1-methyl-4-phenylpyridinuim ion, or 1-benzyl-1,2,3,4-tetrahydroisoquinoline. These results raise the possibility that 1MeTIQ indirectly acts as an anti-oxidant such as the induction of anti-oxidative enzymes, because all these four neurotoxins can burden oxidative stress in common. This is the first report to confirm a protective effect of 1MeTIQ at the cultured neuron level, and it may have potential as a lead compound for the development of new agents to treat Parkinson's disease.     

Dopamine and 7-OH-DPAT may act on D(3) receptors to inhibit tuberoinfundibular dopaminergic neurons

Whether the tuberoinfundibular dopaminergic (TIDA) neurons resided in the dorsomedial arcuate nucleus (dmARN) can respond to dopamine and a dopamine D(3) receptor agonist, 7-hydroxydipropylaminotetralin (7-OH-DPAT), was the focus of this study. In studies using extracellular single-unit recording of dmARN neurons in brain slices obtained from ovariectomized rats, dopamine and 7-OH-DPAT inhibited 60.1% (n = 141) and 80.9% (n = 47) of recorded dmARN neurons, respectively. Other dopamine D(1) or D(2) receptor agonists were not as effective. Intracerebroventricular injection of 7-OH-DPAT (10(-9) mol/3 microl) in ovariectomized, estrogen-primed rats significantly lowered the TIDA neuronal activity as determined by 3, 4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence. Co-administration of a putative D(3) receptor antagonist, U-99194A, could prevent the effect of 7-OH-DPAT. Unilateral microinjection of 7-OH-DPAT or dopamine itself (10(-11)-10(-9) mol/0.2 microl) into the right dmARN exhibited the same inhibitory effect on TIDA neurons. In all, dopamine may act on D(3) receptors to exhibit an inhibitory effect on its own release from the TIDA neurons.     

Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Mechanisms responsible for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine (DA) neuronal death remain unknown and in mice it is even unclear whether neuronal death does occur. In vitro studies suggest that 1-methyl-4-phenylpyridinium ion (MPP⁺), the active metabolite of MPTP, kills neurons by apoptosis. Herein, we investigated whether MPTP induces DA neuronal death in vivo in mice and whether the mechanism is that of apoptosis. C57/bl Mice received different doses of MPTP administered in four intraperitoneal injections every 2 hours and were sacrificed at different time points for analyses of tyrosine hydroxylase (TH) immunohistochemistry, silver staining, and Nissl staining within the mesencephalon. We found that MPTP induces neuronal destruction in the substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA). The active phase of degeneration began at 12 h postinjection and continued up to 4 days. During this period, there was a greater decrease in TH-defined neurons than in Nissl-stained neurons suggesting that MPTP can cause a loss in TH without necessarily destroying the neuron. Thereafter, neuronal counts by both techniques equalized and there was no further loss of DA neurons. Dying neurons showed shrunken eosinophilic cytoplasm and shrunken darkly stained nuclei. Double staining revealed degenerating neurons solely among TH positive neurons of SNpc and VTA. At no time point and at no dose of MPTP was apoptosis observed. In addition, in situ labelling revealed no evidence of DNA fragmentation. This study demonstrates that the MPTP mouse model replicates several key features of neurodegeneration of DA neurons in PD and provides no in vivo evidence that, using this specific paradigm of injection, MPTP kills DA neurons by apoptosis.     

Effect of vitamin D Deficiency and 1,25-dihydroxyvitamin D3 on metabolism and D-glucose transport in rat cerebral cortex

We previously demonstrated that feeding rats Steenbock and Black's rickets-inducing diet produces remarkable changes in the metabolic pattern of the in testinal mucosa, kidney, and liver and in some membrane transport Systems of intestinal mucosa and kidney. 1,25-Dihydroxyvitamin D₃ administration to rachitic rats did not always prove to be effective in restoring normal values. We have now investigated the effect of 1,25-dihydroxyvitamin D₃ on the levels of some metabolites in rat cerebral cortex, on the activity of some enzymes, and on the transport of 2-deoxy-D-glucose and D-glucose in synaptosomes. Our experiments were carried out on three rat groups: control, rachitic, and rachitic treated with 1,25-dihydroxyvitamin D₃. The decrease in phosphorus content and the increase in citrate concentration observed in rachitic rat cerebral cortex were corrected by 1,25-dihydroxyvitamin D₃ treatment. The activity of acetylcholinesterase, glucose-6-phosphate dehydrogenase, and acyl phosphatase significantly increased in rachitic rat synaptosomes, as well as NAD⁺-dependent isocitrate dehydrogenase in cerebral cortex mitochondria; the administration of 1,25-dihydroxyvitamin D₃ to rachitic rats restored enzyme levels to normal. The transport of 2-deoxy-D-glucose and D-glucose in rachitic rat synaptosomes was lower than in the control group and returned to control values in consequence of 1,25-dihydroxyvitamin D₃ treatment. The results reported here support the hypothesis of a participation of 1,25-dihydroxyvitamin D₃ in some aspects of cerebral cortex metabolism.     

Neurotrophic and neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (pACAP) on mesencephalic dopaminergic neurons

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is present in many regions of the adult and developing brain as are receptors for PACAP. PACAP stimulates different signalling cascades in neurons, involving cAMP, MAP kinase, and calcium. These characteristics suggest that PACAP may influence neuronal development. Here we have studied the effects of PACAP on mesencephalic dopaminergic neurons using primary cultures from embryonic rats. PACAP increased the number of tyrosine hydroxylase (TH)-immunoreactive neurons, elevated TH protein, and enhanced tritiated dopamine uptake in these cultures. Moreover, PACAP counteracted the effects of 6-hydroxydopamine treatments, which induce cell death of dopaminergic neurons. In situ hybridisation showed that both PACAP and PACAP receptor type 1 are present in developing and adult rat mesencephalon. These results show that PACAP has a neurotrophic action on dopaminergic neurons and partially protects them against 6-OHDA induced neurotoxicity. J. Neurosci. Res. 54:698–706, 1998. © 1998 Wiley-Liss, Inc.     

Death by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP) and protection by EGF in GH3 cells

In this study we investigated the uptake and effect of a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP⁺) on a clonal strain, GH3 cells, established from rat anterior pituitary. Although the level was very low compared with that in PC12 cells, a clonal rat pheochromocytoma cell line, there was a detectable amount of tyrosine hydroxylase protein in GH3 cells. The levels of monoamines including dopamine in GH3 cells were also very low compared with those in PC12 cells.

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was incorporated to GH3 cells in a concentration-dependent manner and the uptake was inhibited by nomifensine, an inhibitor of dopamine transporter. Addition of 200 μM MPP⁺ stimulated the leakage of lactate dehydrogenase (LDH) after a lag of 24 h. Pretreatment with 50 ng/ml of epidermal growth factor (EGF), but not nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), protected against MPP⁺-induced cell death. These findings show that: (1) MPP⁺ uptake to GH3 cells was via an effective dopamine transport system and causes delayed cell death, and (2) EGF protects against MPP⁺-induced cell death. A possible role for GH3 cells as dopaminergic neurons is discussed.     

Aging and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of dopaminergic neurons in the substantia nigra

This study assessed the influence of aging on substantia nigra degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive neuronal degeneration was found in the substantia nigra of older (8–12 months of age) but not younger (6–8 weeks of age) mice given MPTP. Older mice did not have higher brain concentrations of either MPTP or 1-methyl-4-phenylpyridinium (MPP⁺), the putative toxic metabolite of MPTP, to account for the greater toxicity. In fact, older mice metabolized MPTP more rapidly than younger mice, probably because of the increase in monoamine oxidase activity that occurs with aging. Striatal synaptosomes from older mice did not accumulate more [³H]MPP⁺ than synaptosomes from younger mice. Thus, it is concluded that the greater neurodegenerative effect of MPTP in older animals is not due to greater levels or uptake of MPP⁺, but rather is related to a true increase in sensitivity of older dopaminergic cells to MPTP. For comparative purposes, the toxic effect of another dopaminergic neurotoxin, methamphetamine, was tested. Older animals were not more sensitive than young mature animals to the toxic effect of methamphetamine. This finding indicates that the increased sensitivity of older dopaminergic neurons to MPTP is selective. The link established here between aging and the neurodegenerative effect of MPTP, a toxin which produces parkinsonism in humans, provides a mechanism by which an age-related neurodegenerative disorder such as Parkinson's disease could be caused by an MPTP-like toxin in the environment.     

Expression of c-Jun in dopaminergic neurons of the substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice

The expression of c-Jun in the brains of young (8-week-old) and older (52-week-old) mice following administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was investigated immunocytochemically. Both age groups exhibited reduction in the number of dopaminergic neurons in the substantia nigra after administration of MPTP. There was a significant difference in the magnitude of decrease in the number of dopaminergic neurons between the two groups, as has previously been reported, and the older mice exhibited more extensive loss of dopaminergic neurons in the substantia nigra after MPTP administration than did the young mice. Prolonged c-Jun expression was induced in the substantia nigra following administration of MPTP, and this induction was more prominent in the older mice than in the young mice. Maximum expression of c-Jun occurred on day 7 after the administration of MPTP in both groups. Double staining for tyrosine hydroxylase (TH; a dopaminergic neuron marker) and c-Jun revealed their co-localization indicating that the cells expressing c-Jun were dopaminergic neurons. Cytoplasmic volumes of strongly c-Jun positive cells were reduced, suggesting that they may have been degenerating. In situ end labeling revealed no apoptotic neurons after MPTP administration. These results suggest the existence of some cascade mechanism of nonapoptotic death of dopaminergic neurons following administration of MPTP.     

The glutamate antagonist, MK-801, does not prevent dopaminergic cell death induced by the 1-methyl-4-phenylpyridinium ion (MPP) in rat dissociated mesencephalic cultures

The neuroprotective effects of MK-801, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor/channel, were assessed in a culture model which reproduces in vitro the selective degeneration of mesencephalic dopaminergic neurons seen in parkinsonian brains. Dissociated mesencephalic cells derived from rat embryonic brains were subjected for 24 h to intoxication by the 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP+ at 3 and 10 microM produced selective and dose-dependent damages to dopaminergic neurons as quantified by the loss of the number of TH immunoreactive cells and the loss of [3H]DA uptake whereas other cell types remained unaffected. MK-801 at 3 and 10 microM failed to rescue degenerating dopaminergic neurons in presence of MPP+. At 50 microM, i.e. the highest concentration that is not toxic by itself in this culture system, MK-801 was also found ineffective. Furthermore, degree of dopaminergic cell damage was not reduced when repeated additions of the glutamate antagonist (10 microM/6 h for 24 h) were performed during exposure to MPP+ or when mesencephalic cultures were left after intoxication for up to 2 days in a culture medium still supplemented with MK-801 but free of toxin. In accordance with these results, MK-801 did not affect significantly the uptake of [3H]DA in control cultures, thereby suggesting that this compound cannot prevent intracellular accumulation of MPP+ within dopaminergic neurons. At higher concentrations of MPP+ (100 microM) tested, toxic effects were seen toward dopaminergic neurons and non-dopaminergic cells as quantified by Trypan blue dye accumulation and loss of [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cigarette smoke and nicotine protect dopaminergic neurons against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Parkinsonian toxin

Epidemiological studies have found a negative association between cigarette smoking and Parkinson's disease (PD). In order to analyze the putative neuroprotective effect of cigarette smoke and nicotine, one of its major constituents, we examined their effects in an animal model of PD provoked by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. Two groups of mice were chronically exposed to cigarette smoke (a low exposure subgroup and a high exposure subgroup; 5 exposures per day at 2-h intervals), two other groups received nicotine treatment (two doses tested 0.2 and 2 mg/kg, 5 injections i.p. per day at 2-h intervals) and one group placebo. On day 8 after the beginning of the treatment, 4 injections of MPTP hydrochloride (15 mg/kg, i.p., at 2-h intervals) or saline were administered to these animals. Nicotine and cotinine plasmatic concentration was quantified by the HPLC method, and degeneration of the nigrostriatal system was assessed by tyrosine hydroxylase (TH) immunohistochemistry. The loss of dopaminergic neurons induced by MPTP in the substantia nigra was significantly less severe in the chronic nicotine treatment groups (at 0.2 and 2 mg/kg) and the low exposure to cigarette smoke group than in the high exposure to cigarette smoke subgroup and the placebo treated subgroup. In contrast, no preservation of TH immunostaining of nerve terminals was observed in the striatum in any group. This suggests that nicotine and low exposure to cigarette smoke may have a neuroprotective effect on the dopaminergic nigrostriatal system by an as yet unknown mechanism.     

Synthetic bovine proline-rich-polypeptides generate hydroxyl radicals and fail to protect dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity in mice

Proline-rich-polypeptides (PRPs) isolated from bovine hypothalamus have been shown to render protection against neuronal injury of the brain and spinal cord. We examined two PRPs containing 15 and 10 amino acid residues (PRP-1 and PRP-4 synthetic polypeptide) for their effect, if any, on dopaminergic neuronal damage caused by the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Effects of these PRPs on hydroxyl radical ((*)OH) generation in a Fenton-like reaction as well as from isolated mitochondria were monitored, employing a sensitive salicylate hydroxylation procedure. Balb/c mice treated (i.p., twice, 16 h apart) with MPTP (30 mg/kg) or PRP-1 (1.6 mg/kg), but not PRP-4 (1.6 mg/kg) showed significant loss of striatal dopamine and norepinephrine as assayed by an HPLC-electrochemical procedure. Pretreatment with the PRPs, 30 min prior to the neurotoxin administration failed to attenuate MPTP-induced striatal dopamine or norepinephrine depletion, but significantly attenuated the MPTP-induced decrease in dopamine turnover. A significant increase in the generation of (*)OH by the PRPs in a Fenton-like reaction or from isolated mitochondria suggests their pro-oxidant action, and explains their failure to protect against MPTP-induced parkinsonism in mice.     

Dopamine-releasing action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP) in the neostriatum of the rat as demonstrated in vivo by the push-pull perfusion technique: dependence on sodium but not calcium ions

This study examined the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+) on the levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in push-pull perfusates of the striatum in chloral hydrate-anaesthetized rats. In control animals the levels of DA and DOPAC remained stable for at least 6 h and responded rapidly to a depolarizing stimulus of 25 mM K+. This K+-induced DA release was Ca2+-dependent since no stimulation was observed when the striatal sites were perfused with high K+ in a Ca2+-free medium containing 2 mM EGTA thus verifying that the striatal sites were functionally active. MPTP (0.025 and 0.05 microgram/microliter) stimulated DA release and inhibited DOPAC output in a dose-related manner. MPP+ (0.01, 0.025 and 0.05 microgram/microliter) produced a more robust dose-dependent increase in DA levels in the perfusates; however, the level of suppression of DOPAC was similar to that in response to MPTP. The effect of MPP+ on DA release was attenuated by 10(-6) M benztropine, the DA re-uptake blocker and completely inhibited by 10 micrograms/kg i.p. benztropine and 10(-4) M ouabain, the Na+, K+-ATPase (Na pump) inhibitor. However, although these substances prevented the MPP+-induced release of DA, the levels of DOPAC in the perfusates did not recover and remained completely suppressed suggesting that MPP+ may inhibit extraneuronal rather than intraneuronal monoamine oxidase (MAO). Perfusion of the striatal sites with a Ca2+-free medium containing 2 mM EGTA did not prevent the MPP+-induced DA release indicating that MPP+ does not release DA from the striatal DA terminals by the Ca2+-dependent process of exocytosis. The responses of DA and DOPAC to 25 mM K+ were markedly suppressed in animals treated with MPTP and MPP+, these effects being most severe with the highest dose of MPP+. Moreover, this suppression of the K+-induced responses persisted in animals perfused with MPP+ in the presence of benztropine or ouabain, thus suggesting that MPP+ may have potent deleterious membrane effects. These studies have provided the first direct in vivo demonstration of the action of MPTP and MPP+ and the neuropharmacological basis of this action on DA metabolism in the rat striatum. The results show that the elevated levels of DA in the striatal perfusates are due to a direct action of MPTP and MPP+ on the nigrostriatal DA terminals and cannot be fully accounted for solely by their inhibition of MAO activity and/or inhibition of DA re-uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Astrocytic responses to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in cat and mouse brain

Glial fibrillary acidic protein immunohistochemistry was used as a selective marker for regional reactive gliosis in the striatum and ventral mesencephalon in cats and mice exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Thirty mice (C-57 black strain) were injected with 30 mg/kg intraperitoneally (IP) MPTP.HCl for seven days. Five adult cats were injected with 10 mg/kg IP MPTP.HCl for seven days. Animals were killed five to seven days after the last MPTP injection. Reactive gliosis was observed throughout the mouse striatum but not in the substantia nigra. In contrast, reactive gliosis was topographically represented in the cat caudate nucleus with a dorsal-ventral and medial-lateral gradient evident. Gliosis was also observed in the putamen and the substantia nigra, pars compacta. Tyrosine hydroxylase immunocytochemistry revealed a loss of dopamine in the mouse striatum but no loss of substantia nigra neurons. Nigral neurons were destroyed in the cat. These results suggest that MPTP may destroy nigrostriatal dopamine cell bodies and terminals in the cat while destruction in the mouse is at least initially confined to striatal terminals.