Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum

The impact of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in the pathology of Parkinson's disease (PD) and in MPTP neurotoxicity remains unclear. Here, male TNF-alpha (-/-) deficient mice and C57bL/6 mice were treated with MPTP (4 x 15 mg/kg, 24 h intervals) and in one series, thalidomide was administered to inhibit TNF-alpha synthesis. Real-time RT-PCR revealed that the striatal mRNA levels of TNF-alpha, of the astrocytic marker glial fibrillary acidic protein (GFAP) and of the marker for activated microglia, macrophage antigen complex-1 (MAC-1), were significantly enhanced after MPTP administration. Thalidomide (50 mg/kg, p.o.) partly protected against the MPTP-induced dopamine (DA) depletion, and TNF-alpha (-/-) mice showed a significant attenuation of striatal DA and DA metabolite loss as well as striatal tyrosine hydroxylase (TH) fiber density, but no difference in nigral TH and DA transporter immunoreactivity. TNF-alpha deficient mice suffered a lower mortality (10%) compared to the high mortality (75%) seen in wild-type mice after acute MPTP treatment (4 x 20 mg/kg, 2 h interval). HPLC measurement of MPP(+) levels revealed no differences in TNF-alpha (-/-), wild-type and thalidomide treated mice. This study demonstrates that TNF-alpha is involved in MPTP toxicity and that inhibition of TNF-alpha response may be a promising target for extending beyond symptomatic treatment and developing anti-parkinsonian drugs for the treatment of the inflammatory processes in PD.     

Dopamine turnover is upregulated in the caudate/putamen of asymptomatic MPTP-treated rhesus monkeys

In Parkinson's disease (PD) and experimental parkinsonism, losses of up to 60% and 80%, respectively, of dopaminergic neurons in substantia nigra, and dopamine (DA) in striatum remain asymptomatic. Several mechanisms have been suggested for this functional compensation, the DA-mediated being the most established one. Since this mechanism was recently challenged by striatal DA analysis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, we present data on several DAergic parameters in three groups of rhesus monkeys: MPTP-treated asymptomatic animals; symptomatic MPTP-treated animals with stable parkinsonism; and untreated sex and age matched controls. We determined ratios of striatal and nigral 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA levels and tyrosine hydroxylase (TH) enzyme activity to DA levels, in addition to the commonly used homovanillic acid (HVA)/DA ratios which, as such, might be less reliable under the conditions of partial denervation. We found that in the asymptomatic MPTP monkeys the DOPAC/DA ratios in putamen and caudate nucleus were shifted with high statistical significance 1.9-5.8-fold, as compared to controls, the shifting of the ratios being in the same range as the 2.6-5.4-fold shifts in the symptomatic animals. Also TH/DA ratios were significantly increased in both, the asymptomatic and the symptomatic MPTP-treated monkeys, with shifts in the putamen and caudate nucleus of 3- and 2.7-7.0-fold, respectively. In the substantia nigra, DOPAC levels and TH activity were strongly decreased after MPTP (-77 to -97%), but the ratios DOPAC/DA and TH/DA were not changed in this brain region. Collectively, our findings support the concept of DAergic compensation of the progressive striatal DA loss in the presymptomatic stages of the parkinsonian disease process.     

Prevention of the Nigrostriatal Toxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Inhibitors of 3,4-Dihydroxyphenylethylamine Transport

The 3,4-dihydroxyphenylethylamine (DA, dopamine) uptake inhibitors GBR 13,069, amfonelic acid, WIN-35,065-2, WIN-35,428, nomifensine, mazindol, cocaine, McN-5908, McN-5847, and McN-5292 were effective in preventing [3H]DA and [3H]1-methyl-4-phenylpyridinium (MPP+) uptake in rat and mouse neostriatal tissue slices. These DA uptake inhibitors also were effective in attenuating the MPP+-induced release of [3H]DA in vitro. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to mice (6 X 25 mg/kg i.p.) resulted in a large (70-80%) decrement in neostriatal DA. WIN-35,428 (5 mg/kg), GBR 13,069 (10 mg/kg), McN-5292 (5 mg/kg), McN-5908 (2 mg/kg), and amfonelic acid (2 mg/kg), when administered intraperitoneally 30 min prior to each MPTP injection, fully protected against MPTP-induced neostriatal damage. Other DA uptake inhibitors showed partial protection in vivo at the doses selected. Desmethylimipramine did not prevent [3H]MPP+ uptake or MPP+-induced release of [3H]DA in vitro, and did not protect against MPTP neurotoxicity in vivo. These results support the hypothesis put forth previously by others that the active uptake of MPP+ by dopaminergic neurons is necessary for toxicity.     

Behavioral and biochemical changes following acute administration of MPTP and MPP+

The acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on mouse locomotor activity and striatal dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were investigated. A single dose of either MPTP (10-30 mg/kg, i.p.) or MPP+ (5-20 ug/mouse, i.c.v.) decreased locomotor activity 10-40 min after injection: this locomotor effect was significantly suppressed by either pretreatment with nomifensine or 1-deprenyl alone, or by the combination of desmethylimipramine and 6-hydroxydopamine. Pretreatment with clorgyline did not suppress this behavior and a single dose of haloperidol enhanced the effect. The striatal levels of DA, 3-methoxytyramine and 5-HT increased in parallel with the decrease in locomotor activity caused by MPTP or MPP+. In contrast, levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were decreased by injection of either MPTP or MPP+. Possible mechanism(s) of the behavioral and biochemical changes caused by the acute actions of MPTP and MPP+ with respect to their neurotoxic effects on the nigrostriatal DA system are discussed.     

1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Metabolism and l-Methyl-4-Phenylpyridinium Uptake in Dissociated Cell Cultures from the Embryonic Mesencephalon

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. It also induces alterations of the dopamine (DA) pathways in rodents. MPTP neurotoxicity requires its enzymatic transformation into 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons. Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium. MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum-free conditions). However, mesencephalic neurons in culture revealed a high-affinity uptake mechanism for the metabolite MPP+, similar to that for DA. The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 microM, respectively), whereas the number of uptake sites for MPP+ is double (Vmax = 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively). Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions. Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons. Small concentrations of MPP+ are also taken up by striatal neurons in vitro. The amount taken up represented less than 10% of the MPP+ uptake in mesencephalic neurons. Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trace dosages of the neurotoxins MPTP and MPP+ may affect brain dopamine in vivo.

The present study has examined the effects of systemically administered MPTP and MPP+ upon striatal DA and Dopac of C57 mice, also treated concurrently with either saline or reserpine. MPTP followed by saline did not affect DA level but decreased that of Dopac only at 5.0 mg/kg and higher dosages. The potency of MPTP affecting DA increased greatly when the neurotoxicant was followed by either 5.0 or 10.0 mg/kg reserpine; MPTP at 0.10 mg/kg and higher dosages significantly reversed the DA depleting effects of reserpine. MPP+ (1.0 or 10.0 mg/kg) with saline did not affect either DA or Dopac. In contrast, MPP+ at 0.10 mg/kg and higher dosages, when followed by 10.0 mg/kg reserpine, dose-dependently enhanced the DA depleting effects of reserpine. In agreement with the earlier results obtained in vitro, the present study indicates that MPTP administration at trace level dosages may lead to an inhibition of MAO in vivo. The effect of systemically given MPP+ on DA, however, appears to be more complex in nature, conceivably comprised of actions at the striatal neurones including the intraneuronal vesicles and, possibly, at the substantia nigra which may affect striatum in turn. That MPP+ may have reached brain areas in these experiments is also indicated by the observation of a significant striatal level of 3H-MPP+ after its systemic administration. In conclusion, irrespective of MPTP and MPP+ action mechanisms, trace levels of these neurotoxicants appear to affect brain dopamine neurons.     

4-Phenylpyridine (4PP) and MPTP: the relationship between striatal MPP+ concentrations and neurotoxicity

Because of the chemical and structural similarity between 4-phenylpyridine (4PP) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the effects of 4PP alone and in combination with MPTP on striatal dopamine (DA) concentrations were studied in mice. 4PP did not deplete striatal DA, even when given in maximally tolerated doses (five times that required for MPTP neurotoxicity). However, when 4PP was administered prior to MPTP, it provided significant protection against the DA-depleting effects of MPTP. Additional experiments showed that 4PP pretreatment reduced striatal concentrations of 1-methyl-4-phenylpyridinium ion (MPP+) - the putative toxic biotransformation product of MPTP, and that the concentration of this metabolite closely mirrored striatal DA depletion in MPTP-treated mice. In vitro studies established that 4PP probably lowers MPP+ concentrations by inhibiting the biotransformation of MPTP to MPP+. These observations could be of clinical interest in view of the lower incidence of cigarette smoking among Parkinson's disease patients, and the fact that 4PP is known to be present in cigarettes.     

Treatment with GM1 Ganglioside Restores Striatal Dopamine in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mouse

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an approximately 50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc-GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose- and time-response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for approximately 23 days resulted in an approximately 80% restoration of the DA level and complete restoration of the 3,4-dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1-treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.     

Extracellular Concentrations of Dopamine and Metabolites in the Rat Caudate After Oral Administration of a Novel Catechol-O-Methyltransferase Inhibitor Ro 40–7592

The effect of the systemic administration of a novel, orally active, catechol-O-methyltransferase (COMT) inhibitor, Ro 40-7592, on the in vivo extracellular concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), was studied by transcerebral microdialysis in the dorsal caudate of freely moving rats. Ro 40-7592 (at doses of 3.0, 7.5, and 30 mg/kg p.o.) elicited a marked and long-lasting reduction of HVA, and at doses of 7.5 and 30 mg/kg, an increase of DOPAC output, but it failed to increase DA output. The administration of L-beta-3,4-dihydroxyphenylalanine (L-DOPA, 20 and 50 mg/kg p.o.) with a DOPA decarboxylase inhibitor (benserazide) increased both HVA and DOPAC output, but failed to modify significantly extracellular DA concentrations in dialysates; in contrast, combined administration of L-DOPA+benserazide with Ro 40-7592 (30 mg/kg p.o.) resulted in a significant increase in DA output. Ro 40-7592 prevented the L-DOPA-induced increase in HVA output and markedly potentiated the increase in DOPAC output. To investigate to what extent the increase in extracellular DA concentrations was related to an exocitotic release, tetrodotoxin (TTX) sensitivity was tested. Addition of TTX to Ringer, although abolishing DA output in the absence of L-DOPA, partially reduced it in the presence of L-DOPA+Ro 40-7592 and even more so after L-DOPA without the COMT inhibitor. The results of the present study suggest that metabolism through COMT regulates extracellular concentrations of DA formed from exogenously administered L-DOPA but not of endogenous DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible role of nitric oxide in the regulation of dopamine transporter function in the striatum.

Brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) on striatal dopamine (DA) release in the anesthetized rat. Systemic administration of L-NAME (10 mg/kg, i.p.) significantly decreased the resting release of DA. The peak effect (23% decrease) was reached 45 min after injection. The inactive enantiomer D-NAME (10 mg/kg, i.p.) or the vehicle (saline, 5 ml/kg i.p.) had no effect on the striatal DA level. Neither treatment altered significantly the concentration of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). To investigate the possible involvement of the DA uptake system L-NAME was injected also in the presence of the DA uptake inhibitor nomifensine. Local application of nomifensine (10 microM in the dialysate medium) increased the extracellular concentration of DA to about eight-fold of the basal value and stabilized it at this higher level. Under these conditions L-NAME (10 mg/kg, i.p.) was not able to alter the striatal DA level. Neither nomifensine nor L-NAME caused any change in the level of DOPAC and HVA. Our data suggest that endogenously produced nitric oxide may influence the activity of the DA transporter which effect may have special importance in the regulation of extracellular transmitter concentration in the striatum.     

Effects of l-Methyl-4-Phenyl-l,2,3,6-Tetrahydropyridine in the Dog: Effect of Pargyline Pretreatment

Adult beagle dogs of either sex were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-HCl (2.5 mg/kg, i.v.) alone or after pretreatment with pargyline (5.0 mg/kg, s.c., twice), with pargyline alone, or were uninjected. Groups were killed 2 h, 3 weeks, or 3 months after injection, and several brain areas were assayed for biogenic amines and their synthetic and degradative enzymes. MPTP caused a massive and permanent loss of striatal dopamine, tyrosine hydroxylase, and 3,4-dihydroxyphenylalanine decarboxylase activities and the loss of cells within the substantia nigra pars compacta. Dopamine and norepinephrine also were depleted to various degrees in cortex, olfactory bulb, and hypothalamus; however, dopamine beta-hydroxylase activity in cortex was normal. There was no cell loss in the ventral tegmental area or locus ceruleus. The activities of monoamine oxidase (MAO)-A and MAO-B in cortex and caudate were not affected by MPTP. Despite a permanent loss of the nigrostriatal system, the dogs exhibited only a transient hypokinesia lasting 1-2 weeks. Pargyline pretreatment prevented the loss of striatal dopamine and cells from the substantia nigra, but did not prevent a prolonged but reversible decrease in the concentration of dopamine metabolites. It is argued that this apparent inhibition of MAO is due not to suicide inactivation of the enzyme by MPTP, but to reversible inhibition by accumulation of the pyridinium metabolite, 1-methyl-4-phenylpyridinium, selectivity in aminergic terminals.     

Neurotoxicity,drugs of abuse,and the CuZn-superoxide dismutase transgenic mice

Administration of methamphetamine (METH) to animals causes loss of DA terminals in the brain. The manner by which METH causes these changes in neurotoxicity is not known. We have tested the effects of this drug in copper/zinc (CuZn)-superoxide dismutase transgenic (SOD Tg) mice, which express the human CuZnSOD gene. In nontransgenic (non-Tg) mice, acute METH administration causes significant decreases in DA and dihydroxyphenylacetic acid (DOPAC) in the striata of non-Tg mice. In contrast, there were no significant decreases in striatal DA in the SOD Tg mice. The effects of METH on DOPAC were also attenuated in SOD Tg mice. Chronic METH administration caused decreases in striatal DA and DOPAC in the non-Tg mice, but not in the SOD-Tg mice. Similar studies were carried out with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), which also causes striatal DA and DOPAC depletion. As in the case of METH, MPTP causes marked depletion of DA and DOPAC in the non-Tg mice, but not in the SOD Tg mice. These results suggest that the mechanisms of toxicity of both METH and MPTP involve superoxide radical formation.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of Drug-Induced Changes in Dopamine and Serotonin Metabolite Concentrations in the CSF of the Rat

Cerebrospinal fluid (CSF) was removed at a constant flow rate of 1 microliter/min from the third ventricle of anesthetized rats. Every 15 min, CSF dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined by direct injection of CSF into a liquid chromatographic system coupled with electrochemical detection. Mean CSF concentrations of DOPAC, HVA, and 5-HIAA were 1.29 microM, 0.88 microM, and 2.00 microM, respectively. In order to determine the turnover rates of dopamine (DA) and serotonin, experiments using monoamine oxidase (MAO) inhibition were performed. Tranylcypromine (20 mg/kg i.p.) induced a sharp exponential decrease of CSF DOPAC, HVA, and 5-HIAA, with respective half-lives of 15.60 min, 16.91 min, and 77.23 min. Their respective turnover rates were 3.74, 2.22, and 1.18 nmol X ml-1 X h-1. m-Hydroxybenzylhydrazine (NSD-1015, 100 mg/kg i.p.) and monofluoromethyl-DOPA (100 mg/kg i.p.), two decarboxylase inhibitors, induced a slow exponential decrease of all three CSF metabolites. alpha-Methyl-p-tyrosine (250 mg/kg i.p.) also induced a slow exponential decrease of DOPAC and HVA. These decreases of CSF DOPAC and HVA induced by DA synthesis inhibitors may reflect the turnover of DA in vivo. Haloperidol (0.5 mg/kg i.p.) considerably enhanced CSF DOPAC and HVA without affecting 5-HIAA, confirming that dopaminergic receptors modulate DA neurotransmission in vivo. Haloperidol administered 1.5 h after NSD-1015 did not increase DOPAC and HVA, in contrast to reserpine (5 mg/kg i.p.) injected under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity in high-fat diet-induced obesity

Currently, obesity is considered a systemic inflammation; however, the effects of obesity on the vulnerability of dopaminergic neurons to oxidative stress are not fully defined. We evaluated the effects of high-fat diet-induced obesity (HF DIO) on neurotoxicity in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Eight weeks after a HF or matched normal diet, a severe decrease in the levels of striatal dopamine and of nigral microtubule-associated protein 2, manganese superoxide dismutase, and tyrosine hydroxylase was observed in obese mice treated with subtoxic doses of MPTP (20 mg/kg) compared with the matched lean group. In addition, the levels of nitrate/nitrite and thiobarbituric acid-malondialdehyde adducts in the substantia nigra of obese mice were reciprocally elevated or suppressed by MPTP. Interestingly, striatal nNOS phosphorylation and dopamine turnover were elevated in obese mice after MPTP treatment, but were not observed in lean mice. The nitrotyrosine immunoreactivity for evaluation of nigral nitrogenous stress in obese mice with MPTP was higher than that in matched lean mice. At higher doses of MPTP (60 mg/kg), the mortality was higher in obese mice than in lean mice. These results suggest that DIO may increase the vulnerability of dopaminergic neurons to MPTP via increased levels of reactive oxygen and nitrogen species, and the role of nNOS phosphorylation in the MPTP toxicities and dopamine homeostasis should be further evaluated.     

Clonidine Suppresses 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Reductions of Striatal Dopamine and Tyrosine Hydroxylase Activity in Mice

Abstract: Recent findings have shown that excitatory amino acid may be involved in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. At the same time, evidence is accumulating that the endogenous nor-adrenergic system plays a protective role in MPTP-induced striatal dopamine (DA) depletion and nigral dopaminergic cell death. Recently, α₂-adrenoceptors located on glutamatergic axons have been shown to inhibit glutamate overflow. In this study, we evaluated the effects of an α₂-agonist (clonidine) and an α₂-antagonist (yohimbine) on MPTP-induced striatal DA depletion and tyrosine hydroxylase activity reduction. We show that clonidine is able to prevent the neurotoxicity of MPTP in mice. To exert this effect, clonidine (0.5 mg/kg) must be administered at least twice (30 min before and 30 min after MPTP). Administration of another α₂-agonist (detomidine, 0.3 mg/kg) attenuated the neurotoxicity induced by MPTP. We provide evidence that the protective effect obtained with clonidine was not due to decreased striatal content of 1-methyl-4-phenylpyridinium (MPP⁺). We also show that yohimbine, which is a classic α₂-adrenoceptor antagonist with low affinity for imidazoline receptors, produced by itself an enhancement of MPTP toxicity and was able to block the protective effect of clonidine. These data raise the possibility that α₂-adrenoceptor may modulate the susceptibility of the nigrostriatal dopaminergic pathway to neurotoxicity.     

Acute effects of a parkinsonism-inducing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mouse body temperature

The parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) given in single systemic doses (i.p.) to mice produced marked hyperthermia, and subsequent long-lasting hypothermia. Administration of MPTP or its oxidized product, 1-methyl-4-phenylpyridinium ion, MPP+, via i.c.v. resulted in only hypothermia. In contrast, i.p. MPP+ administration resulted in only hyperthermia. The MPTP-induced hyperthermia (i.p.) was blocked by quaternary derivatives of anti-cholinergic agents, atropine and scopolamine, but not by the tertiary-derivative of atropine. Duration of this hyperthermic effect was potentiated by neostigmine. Pretreatment with 1-deprenyl did not prevent hypothermia, but nomifensine partially or clorgyline completely prevented the effect without preventing MPTP-induced hyperthermia. The thermic effects by MPTP, unlike its neurotoxicity for the nigrostriatal DA system, may not require metabolism to MPP+. These results indicate that peripheral cholinergic functions are responsible for the MPTP-induced hyperthermia, whereas its hypothermic effect may be centrally mediated via dysregulation of the various neuron systems.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice is enhanced by ethanol or acetaldehyde

Persistent neurochemical changes consistent with parkinsonism have been reported in brains of mice treated with repeated high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We now report that ethanol or acetaldehyde potentiate MPTP-induced damage to mouse striatum. One hour after the combined treatments (ethanol and MPTP or acetaldehyde and MPTP), the animals exhibited a marked and long-lasting catatonic posture and then returned gradually to apparently normal locomotion. Seven days after MPTP administration, depletion of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mouse striatum were further potentiated in the group of animals treated with ethanol. This effect was more evident when the treatment was repeated twice and was dose-dependent. Acetaldehyde was more potent than ethanol in enhancing MPTP neurotoxicity. A single exposure to acetaldehyde before and during MPTP treatment produced a very consistent fall of DA, DOPAC and HVA but not serotonin (5HT) or 5-hydroxyindoleacetic acid (5HIAA) in the striatum. This suggests that ethanol effects on MPTP neurotoxicity might be related to acetaldehyde formation.     

Mechanisms of Toxicity and Cellular Resistance to 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and 1-Methyl-4-Phenylpyridinium in Adrenomedullary Chromaffin Cell Cultures

Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19-6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4-1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration-dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose-free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days' exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic "buffer."(ABSTRACT TRUNCATED AT 400 WORDS)     

Systemic Administration of Proteasome Inhibitor Protects Against MPTP Neurotoxicity in Mice

Dysfunction of the proteasome has been suggested to contribute in the degeneration of nigrostriatal dopaminergic neurons. Here, we investigated to determine whether systematic administration of proteasome inhibitor, carbobenzoxy-l-γ-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI) protects against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in mice. Three administrations of MPTP at 1-h intervals to mice reduced significantly the concentration of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) in the striatum after 5 days. In contrast, PSI (0.3 and 1.0 mg/kg) prevented a significant decrease in dopamine, DOPAC and HVA contents of the striatum 5 days after MPTP treatment. In our Western blot analysis study, PSI at a dose of 1.0 mg/kg prevented a significant decrease in TH (tyrosine hydroxylase) protein and a significant increase in glial fibrillary acidic protein 5 days after MPTP treatment. Furthermore, our immunohistochemical study showed that PSI at a dose of 1.0 mg/kg prevented a significant loss in TH immunopositive neurons in the striatum and substantia nigra 5 days after MPTP treatment. In contrast, PSI caused a significant increase in the number of intense ubiquitin immunopositive cells in the striatum and substantia nigra 5 days after MPTP treatment. These results indicate that proteasome inhibitors can protect against MPTP neurotoxicity in mice. The neuroprotective effect of PSI against dopaminergic cell damage may be mediated by the elevation of ubiquitination. Thus, our findings provide further valuable information for the pathogenesis of Parkinson’s disease. Takuya Oshikawa and Hayato Kuroiwa contributed equally to this work.