Lawrence Manzino

Some features of the nigrostriatal dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse

The discovery that a rather simple chemical substance can produce such a highly selective neuronal degeneration in the substantia nigra, the brain area most affected in Parkinson’s disease, has resulted in a vast amount of research with MPTP. We and others have hoped that by gaining an understanding of its mechanism of action, we might come closer to discovering the cause(s) of idiopathic Parkinson’s disease. Indeed, we have learned some fascinating things regarding the roles of monoamine oxidase B and the dopamine transport system in mediating the actions of MPTP. It is clear that the MPTP-treated mouse is a good model for Parkinson’s disease. As such, it may help to define the role of dopamine deficiency in the pathophysiology of Parkinson’s disease as well as provide a model in which potential anti-Parkinsonian therapeutic agents can be tested.

Parallel increases in lipid and protein oxidative markers in several mouse brain regions after methamphetamine treatment

The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH‐treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5‐HT] in striatum, cortex and hippocampus by 60–90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non‐toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or 5HT oxidation.

Effects of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine on neostriatal dopamine in mice

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) causes a destruction of the nigrostriatal dopamine pathway in humans as well as in monkeys. However, it has been reported that MPTP is inert in several small animal species. We now report that MPTP, given to mice at 30 mg/kg intraperitoneally, causes severe and long-lasting depletions of dopamine and its major metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the neostriatum.

Studies on the Oxidation of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine by Monoamine Oxidase B

Abstract: 1‐Methyl‐4‐phenyl‐ 1,2,5,6 ‐tetrahydropyridine (MPTP) is a chemical that, after injection into experimental animals, including mice and monkeys, causes a degeneration of the nigrostriatal pathway. We carried out experiments designed to study the in vitro oxidation of MPTP by mouse brain mitochondrial preparations. MPTP was actively oxidized by the mitochondrial preparations, with Km and Vmax values very similar to those of benzyl amine, a typical substrate for MAO‐B. MPTP was oxidized considerably better than many of its analogs, even those with relatively minor structural changes. Several monoamine oxidase inhibitors (MAOI) were potent inhibitors of MPTP oxidation, and there was a highly significant correlation between the capacity of the MAOI tested to inhibit MPTP oxidation and benzylamine oxidation. There was no correlation between the capacity of the MAOI to inhibit MPTP oxidation and their capacity to inhibit the oxidation of tryptamine, a substrate for MAO‐A. In other experiments, MPTP was an excellent substrate for pure MAO‐B, prepared from bovine liver. All of these data. combined with the fact that MAO‐B inhibitors can protect against MPTP‐induced dopami nergic neurotoxicity in vivo. point to an important role for MAO‐B in MPTP metabolism in vivo.

Effects of 1‐Methyl‐4‐Phenyl‐1,2,5,6‐Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4‐Dihydroxyphenylethylamine and [3H]5‐Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1‐Methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4‐dihydroxyphenylethylamine‐([3H]DA), [3H]5‐hydroxytryptamine ([3H]5‐HT), and [3H]γ‐aminobutyric acid ([3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5‐HT uptake (IC50 value 0.14 μM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 μM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 μM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5‐HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4‐electron oxidation product of MPTP, i.e., 1‐methyl‐4‐phenyl‐pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 μM) and of [3H]5‐HT uptake (IC50 value 0.78 μM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and several of its analogues on the dopaminergic nigrostriatal pathway in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a recently discovered neurotoxin, caused extensive losses of dopamine and its major metabolites after its administration to male Swiss-Webster mice. In contrast, under identical conditions, several MPTP analogues, even those with relatively minor structural changes, were without toxicity. These include compounds with a 1-ethyl and 1-propyl substituent rather than the 1-methyl, the compound lacking the double bond in the tetrahydropyridine ring, as well as the compound with no phenyl substituent. It follows that each part of the MPTP molecule is important in determining its neurotoxic activity.

Stereospecific binding of 3H-Dopamine in neostriatal membrane preparations: Inhibitory effects of sodium ascorbate

It has been pointed out by several different groups of investigators in the past several years that ascorbic acid was a potent inhibitor of the binding of dopamine (DA) agonists including 3H-DA itself and 3H-ADTN, 3H-apomorphine and 3H-norpropylapomorphine to neostriatal membrane preparations. However, the significance of this effect of ascorbic acid has been controversial. For example, it has recently been claimed that the stereospecific binding of DA agonists is facilitated by ascorbic acid and can be measured only in its presence. In the present study in neostriatal membrane preparations in the absence of ascorbic acid, the binding of 3H-DA was very potently inhibited by potent DA agonists (DA, ADTN, apomorphine). Considerably weaker effects were obtained with norepinephrine, isoproterenol, serotonin, catechol and pyrogallol. Stereospecific effects were clearly observed in that the binding of 3H-DA was inhibited to a much greater extent by several biologically active enantiomers than by their less active counterparts. For example, (-)-2-hydroxyapomorphine and (-)-norpropylapomorphine were much more potent inhibitors than their corresponding (+) isomers. This binding of 3H-DA was also very strongly inhibited by sodium ascorbate and several other reducing agents. In control experiments in the neostriatal membrane preparation in the absence of ascorbic acid, there was no detectable decomposition of 3H-DA. The data suggest that 3H-DA can, in the absence of sodium ascorbate, bind stereospecifically to a site that has the properties of a DA receptor. Furthermore, sodium ascorbate is a potent inhibitor of this stereospecific binding.

NMDA Receptors Modulate Dopamine Loss due to Energy Impairment in the Substantia Nigra but not Striatum

Defects in energy metabolism have been detected in patients with Parkinsons disease and have been proposed as a contributing factor in the disease. Previous in vitro studies showed that NMDA receptors contribute to the loss of dopamine neurons caused by the metabolic inhibitor malonate. In vivo, it is not known whether this interaction occurs through a postsynaptic action on the cell body in the substantia nigra or through a presynaptic action at the dopamine terminal in the striatum. So we could discern the anatomical level of NMDA receptor involvement, rats were infused with malonate, either into the left striatum or into the left substantia nigra. NMDA receptors were locally blocked by an intranigral or intrastriatal coinfusion of malonate plus MK-801 followed by a second infusion of MK-801 3 h later. Animals were examined at 1 week for striatal and nigral dopamine and GABA levels. Intranigral infusion of malonate (0.5 micromol) produced an approximate 50% loss of both nigral dopamine and GABA. MK-801 (0.1 micromol) provided significant protection against both nigral dopamine and GABA loss and against anterograde damage to dopamine terminals in the striatum. Intrastriatal administration of malonate (2 micromol) produced a 68 and 35% loss of striatal dopamine and GABA, respectively. In contrast to intranigral administration, intrastriatal blockade of NMDA receptors did not protect against striatal dopamine loss, although GABA loss was significantly attenuated. Core body temperature monitored several hours throughout the experiment was unchanged. Consistent with a lack of effect of NMDA antagonists on malonate-induced toxicity to dopamine neurons in striatum, intrastriatal infusion of NMDA had a pronounced effect on long-term GABA toxicity with little effect of dopamine loss. These findings are consistent with a postsynaptic action of NMDA receptors on mediating toxicity to dopamine neurons during impaired energy metabolism.

Protection of malonate-induced GABA but not dopamine loss by GABA transporter blockade in rat striatum.

Previous work has shown that overstimulation of GABA(A) receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA(A) antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinsons and Huntingtons diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 micromol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA(A) receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 micromol) plus or minus the GABA(A) receptor agonist muscimol (1 micromol), the GABA(A) Cl- binding site antagonist picrotoxin (50 nmol) or the GABA(B) receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntingtons disease. These findings also point to fundamental differences between immature and adult neurons in the downstream involvement of GABA receptors during metabolic insult.