Inhibition of the neuronal isoform of nitric oxide synthase significantly attenuates 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in vitro.

The possible protection against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)) afforded by inhibitors of nitric oxide synthase (NOS) and the antagonist of N-methyl-D-aspartate receptor function, MK-801, was studied in a brain-slice superfusion system. Significant decreases in levels of dopamine and its metabolites 3,4-dihyroxyphenylacetic acid (DOPAC) and homovanillic acid were observed following incubation of slices with 25 microM MPP(+). The activity of intracellular lactate dehydrogenase (LDH), a marker of cell viability, was also significantly decreased. These effects were attenuated by preincubation with I mM 7-nitroindazole (7NI), a selective inhibitor of the neuronal isoform of nitric oxide synthase (NOS). In contrast, the nonspecific NOS inhibitor N(omega)-nitro-L-arginine, also at 1 mM, had no effect on levels of dopamine metabolites but did show a small attenuation of the levels of dopamine. 7NI alone caused some increase in levels of dopamine and a decrease in the metabolite DOPAC, which is consistent with it also acting as an inhibitor of monoamine oxidase-B. MK-801 afforded no significant protection of aminergic cells, although changes in LDH activity suggested that there may have been some protection of non-aminergic neurons affected by this, relatively high concentration of MPP(+).

Pergolide protects dopaminergic neurons in primary culture under stress conditions.

Dopamine agonists are an important therapeutic strategy in the treatment of Parkinson's disease. They postpone the necessity for and reduce the required dose of L-3,4-dihydroxyphenylalanine (L-DOPA) medication thus protecting against the development of motor complications and potential oxidative stress due to L-DOPA metabolism. In primary cultures from mouse mesencephalon we show that pergolide, a preferential D(2) agonist enhanced the survival of healthy dopaminergic neurons at low concentrations of 0.001 microM. About 100 fold higher concentrations (0.1 microM) were necessary to partially reverse the toxic effects of 10 microM 1-methyl-4-phenylpyridinium (MPP(+)). Pergolide was equally effective in preventing the reduction of dopamine uptake induced by 200 microM L-DOPA. Furthermore, between 0.001-0.1 microM it also reduced lactate production thus promoting aerobic metabolism. The present findings suggest that pergolide protects dopaminergic neurons under conditions of elevated oxidative stress.

Neuroprotective effect of acute and chronic administration of copper (II) sulfate against MPP+ neurotoxicity in mice.

Neurodegenerative effects of MPP+, the main metabolite of MPTP include dopamine (DA) depletion and enhanced lipid peroxidation (LPO) in mice striata, both associated to free radicals overproduction. Since copper is related to several antioxidant enzymes, we tested its neuroprotective effect against MPP+-induced neurotoxicity (20 microg/3 microl). CuSO4 pretreatment was administrated by either acute (2.5 mg/kg, i.p.) or chronic (350 or 700 mg/l doses through drinking water, for 30 days) schemes. Acute administration blocked MPP+-induced striatal LPO only when administered 16 or 24 hours before MPP+, and prevented the DA-depleting effect only at 24 hours. Chronic CuSO4 prevented the LPO increase, and blocked the DA depletion only at the higher dose used (700 mg/l). Neuroprotective effect of CuSO4 was dependent on the dose and the time of pretreatment, which suggest that this lag could be related with mechanisms of activation or synthesis of copper-dependent proteins responsible of cellular defense against MPP+.

MPP(+) increases the vulnerability to oxidative stress rather than directly mediating oxidative damage in human neuroblastoma cells.

MPP(+), an active metabolite of MPTP, causes a dopaminergic neuronal degeneration similar to that observed in Parkinson's disease. Current data suggest that MPP(+)-induced cytotoxicity may be mediated by oxygen free radicals. To evaluate this hypothesis, we first investigated whether MPP(+) could cause oxidative stress by producing oxygen free radicals in the SH-SY5Y, human neuroblastoma cell line. MPP(+) was toxic to the cells dose-dependently but did not increase the level of lipid peroxidation at toxic concentrations. Second, we examined the effects of various antioxidants and an inhibitor of nitric oxide synthase (NOS) on the development of MPP(+) cytotoxicity. Pretreatment with antioxidants such as ascorbic acid, Trolox, phenyl-tertiary-butyl-nitrone (PBN), which show protective effects on tert-butyl hydroperoxide (tBOOH) toxicity did not attenuate MPP(+) cytotoxicity. Similarly, the combination of antioxidant enzymes, SOD and catalase (50 U/ml, respectively), did not protect the cells from the toxic action of MPP(+). Also N-nitro-l-arginine methyl ester (NAME), a competitive inhibitor of NOS, and combined incubation with NAME and antioxidant enzymes failed to attenuate MPP(+) cytotoxicity. On the other hand, a sublethal dose of MPP(+) potentiated iron and H(2)O(2)-induced cytotoxicity. These results suggest that oxygen free radicals may not be a primary cause of MPP(+)-induced cell death but that MPP(+) increases the vulnerability of cells to oxidative stress.

Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in Parkinson's disease.

Vasoactive intestinal peptide (VIP) provides neuroprotection against beta-amyloid toxicity in models of Alzheimer's disease. A superactive analogue, stearyl-Nle17-VIP (SNV) is a 100-fold more potent than VIP. In primary neuronal cultures, VIP protective activity may be mediated by femtomolar-acting glial proteins such as activity-dependent neurotrophic factor (ADNF), activity-dependent neuroprotective protein (ADNP), peptide derivatives ADNF-9 (9aa) and NAP (8aa), respectively. It has been hypothesized that beta-amyloid induces oxidative stress leading to neuronal cell death. Similarly, dopamine and its oxidation products were suggested to trigger dopaminergic nigral cell death in Parkinson's disease. We now examined the possible protective effects of VIP against toxicity of dopamine, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion (MPP+) in neuronal cultures [rat pheochromocytoma (PC12), human neuroblastoma (SH-SY5Y) and rat cerebellar granular cells]. Remarkably low concentrations of VIP (10(-16)-10(-8) M), ADNF-9 and NAP (10(-18)-10(-10) M) protected against dopamine and 6-OHDA toxicity in PC12 and neuroblastoma cells. VIP (10(-11)-10(-9) M) and SNV (10(-13)-10(-11) M), protected cerebellar granule neurons against 6-OHDA. In contrast, VIP did not rescue neurons from death associated with MPP+. Since dopamine toxicity is linked to the red/ ox state of the cellular glutathione, we investigated neuroprotection in cells depleted of reduced glutathione (GSH). Buthionine sulfoximine (BSO), a selective inhibitor of glutathione synthesis, caused a marked reduction in GSH in neuroblastoma cells and their viability decreased by 70-90%. VIP, SNV or NAP (over a wide concentration range) provided significant neuroprotection against BSO toxicity. These results show that the mechanism of neuroprotection by VIP/SNV/NAP may be mediated through raising cellular resistance against oxidative stress. Our data suggest these compounds as potential lead compounds for protective therapies against Parkinson's disease.

Parkinsonism-inducing neurotoxin MPP+: uptake and toxicity in nonneuronal COS cells expressing dopamine transporter cDNA.

Expression of a cloned dopamine transporter complementary DNA in COS cells allows these primate kidney cells to accumulate the parkinsonism-inducing neurotoxin metabolite MPP+ (1-methyl-4-phenylpyridinium) avidly, and MPP+ toxicity results. By documenting that the dopamine transporter can confer MPP+ sensitivity to nonneural cells, these results highlight the key role that this transporter could play in mechanisms underlying parkinsonism.

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.

Toxicity of 1-methyl-4-phenylpyridinium for rat dopaminergic neurons in culture: selectivity and irreversibility.

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 microM 1-methyl-4-phenylpyridinium (MPP+), a concentration shown earlier to result in loss of greater than 85% of tyrosine hydroxylase (TH)-positive neurons without affecting the total number of cells observed by phase-contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 microM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to less than 4% of control values, whereas the uptake of gamma-[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high-affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 microM MPP+ for 48 h and then kept for up to 6 days in MPP(+)-free medium. After exposure to 10 microM MPP+, the number of TH-positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 microM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin-free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.

The neurotoxicity of 1-methyl-4-phenylpyridinium in cultured cerebellar granule cells.

Cerebellar granule cells in enriched primary culture are susceptible to the neurotoxic effects of 1-methyl-4-phenylpyridinium (MPP+). Relatively high MPP+ concentrations are required to elicit neurotoxic effects at early culture times, but lower concentrations of MPP+ produce comparable neurotoxic effects at later culture times. Under identical culture conditions 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is not neurotoxic. Preincubation with the glutamate uptake blockers, DL-threo-3-hydroxyaspartic acid or dihydrokainate, or the dopaminergic uptake blocker mazindol, protects the granule cells from the cytotoxic effects of MPP+. Although MPTP is not neurotoxic in an enriched granule cell culture, in coculture with cerebellar astrocytes MPTP is toxic to granule cells, presumably because it is converted in astrocytes to MPP+. Cerebellar astrocytes remain confluent and viable. The addition of pargyline to the coculture abolishes the neurotoxicity consistent with a role of MAO B in bioactivation of MPTP. The concentration of MPP+ in the coculture medium (13 microM) was less than that required for the toxic effect in enriched neuronal cultures at earlier culture times, suggesting that an astroglial-neuronal interaction, perhaps by proximity, enhances the neurotoxicity of MPP+. These results might explain reported effects of MPTP on some cerebellar cells in mice.