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C Mytilineou

Publications and source records attributed to C Mytilineou.

At least 37 records · Page 2Linked to original sources
PubMed1994

1-Methyl-4-phenylpyridinium-like neurotoxicity of a pyridinium metabolite derived from haloperidol: cell culture and neurotransmitter uptake studies.

It is now generally accepted that the nigrostriatal degenerative properties of the parkinsonian-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine are mediated by the brain monoamine oxidase B generated 1-methyl-4-phenylpyridinium metabolite (MPP+). In this article, the results are described of ongoing efforts to evaluate the MPP(+)-type neurotoxic potential of the haloperidol (HP)-derived pyridinium metabolite HPP+, a 1,4-disubstituted structural analog of MPP+, which is formed in humans and rats treated with HP. Previous studies in the rat have shown that intrastriatal perfusion of HPP+ leads to the irreversible depletion of striatal dopamine and serotonin. Furthermore, HPP+ was a potent inhibitor of NADH-supported mitochondrial respiration. This article reports that HPP+ also is toxic to dopaminergic and serotonergic neurons in cultures of embryonic mesencephalic cells, as measured by loss of the ability of exposed cells to accumulate tritium-labeled dopamine and serotonin and by immunochemical staining techniques. HPP+ also inhibited the uptake of these labeled neurotransmitters by synaptosomes prepared from mouse neostriata (dopamine) and cortical tissues (serotonin). Because HP is unlikely to be a substrate for brain monoamine oxidase B, the production and accumulation of HPP+ in the brain is probably not comparable to that of MPP+. On the other hand, chronic exposure to HP could result in brain levels of this lipophilic quaternary pyridinium species that might coincide with the late-appearing tardive dyskinesias that are observed in some HP-treated patients months and, more often, years after the initiation of HP therapy.

1-Methyl-4-phenylpyridinium↗
PubMed1993

Toxic and protective effects of L-dopa on mesencephalic cell cultures.

The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 microM) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [3H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 microM), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [3H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, L-DOPA prevented cell death by L-BSO.

Animals↗
PubMed1992

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.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Epidermal growth factor-induced survival and proliferation of neuronal precursor cells from embryonic rat mesencephalon.

Neuronal and glial precursor cells were isolated from primary cultures of embryonic rat mesencephalon. The separation of precursor cells from the neurons was accomplished by the resuspension of the primary cells by trypsinization, followed by replating. This procedure resulted in the death of differentiated neurons and the survival of precursor cells. The survival and proliferation of the replated precursor cells required the presence of epidermal growth factor (EGF) in the culture medium. The precursor cells differentiated into neurons and astrocytes, as determined by immunocytochemical staining with antibodies to neuron specific enolase (NSE) and tau protein or glial fibrillary acidic protein (GFAP) respectively.

Animals↗
PubMed1991

EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture.

Epidermal growth factor (EGF) immunoreactive material has been demonstrated to be present in the basal ganglia. In this study, we investigated the effect of EGF on cells cultured from 16-day embryonic rat mesencephalon, which included dopamine neurons that project to the striatum in vivo. EGF receptors were detected in untreated cultures by [125I]-EGF binding. Treatment of the cultures with EGF resulted in up to 50-fold increases in neuronal high-affinity dopamine uptake. Scatchard analysis of uptake kinetics and counting of tyrosine hydroxylase-immunoreactive cells suggest that the effect of EGF on uptake is due to increased survival and maturation of dopaminergic neurons. By contrast, the high-affinity uptake for serotonin was increased only threefold over its controls. There was no significant effect on high-affinity gamma-aminobutyric acid (GABA) uptake. These results suggest that EGF is acting as a neurotrophic agent preferential for dopaminergic neurons in E16 mesencephalic cultures. Immunocytochemistry for glial fibrillary acidic protein demonstrated an increase in astroglia with EGF treatment. Fluorodeoxyuridine, an agent that is toxic to proliferating cells was able to eliminate the effect of EGF on dopamine uptake, suggesting that EGF may be increasing dopaminergic cell survival largely through a population of dividing cells.

Animals↗
PubMed1991

Lewy bodies in parkinsonism share components with intraneuronal protein bodies of normal brains.

Histochemical characteristics of the Lewy bodies, in catecholamine neurons of 10 Parkinsonian patients, were compared to those of the spherical protein bodies, the basic protein-rich markers of catecholamine neurons in man. Special methods for proteins and lipids showed that the core of the Lewy bodies, in the neurons of the locus coeruleus and the substantia nigra, contains basic proteins and lipids normally found in the protein bodies. Acid fuchsin and the lipid-soluble fluorescent dye rhodamine B stained the entire core of the Lewy body in the parkinsonian brains and the entire sphere of the protein body in the control brains. Bromsulfophthalein, another acidic dye, which selectively binds to the enzyme gluthathione-S-transferase, had affinity only for a ring-like lamina at the outer layer of the core of the Lewy body and for the outer rim of the protein body. These results demonstrate that Lewy bodies and protein bodies contain similar macromolecular components, that is lipids and two different types of proteins, which also show similar stratification in the two structures. On the other hand, the presence in several neurons of the Parkinsonian patients, of aggregates representing transitional forms between protein bodies and Lewy bodies, indicates that abnormalities of protein bodies precede, and are somehow linked to Lewy body production.

Brain Stem↗
PubMed1991

Ascorbic acid in mesencephalic cultures: effects on dopaminergic neuron development.

Ascorbic acid exists in high intracellular concentrations in fetal rat brain. In mesencephalic cultures the cellular ascorbic acid content drops sharply to undetectable levels when no ascorbic acid is added to the medium, thus creating a model of scorbutic neuronal tissue and affording the study of ascorbic acid's effects on mesencephalic cell development and function. Cultures treated with 0.2 mM ascorbic acid were compared with controls (scorbutic cultures) by using morphological and biochemical indices. Ascorbic acid cultures at 7 and 14 days in vitro showed a marked increase in glial proliferation on glial fibrillary acidic protein staining and increased neurite growth and number on tyrosine hydroxylase staining. Significantly higher dopamine uptake and levels of dopamine and 3,4-dihydroxyphenylacetic acid were also observed after 7 and 14 days of ascorbic acid treatment. The capacity to accumulate ascorbic acid and the ability to retain the intracellular ascorbic acid developed gradually as the cultures matured. Ascorbic acid reached the embryonal levels by day 14 in vitro. We conclude that although neuronal cultures can survive and grow in the absence of detectable levels of ascorbic acid, its presence exerts a broad effect on dopamine neuron morphology and biochemical functioning either directly or through increased glial proliferation, or possibly both.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1990

Neurochemical and toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridine to rat serotonin neurons in dissociated cell cultures.

Dissociated cell cultures from the pontine area of embryonic rat brain were used to study the sensitivity of serotonin (5-hydroxy-tryptamine (5-HT)) neurons to the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+). Treatment with MPTP (up to 100 microM) for 7 days did not cause degeneration of 5-HT neurons. A 50% inhibition of [3H]5-HT uptake caused by 100 microM MPTP was a direct effect on the 5-HT uptake carrier, reversed by washing for 7 days. Incubation of cultures with MPTP increased the intraneuronal levels of 5-HT and reduced the levels of 5-hydroxyindoleacetic acid, suggesting a reduction in 5-HT metabolism. MPTP reduced monoamine oxidase activity in the cultures, which probably led to the reduction in 5-HT metabolism. Exposure to MPP+ (0.5-10 microM) for 4 to 7 days decreased [3H]5-HT uptake and induced loss of neurons stained with antibodies against 5-HT. Comparison between 5-HT and dopamine (DA) neurons indicated a differential sensitivity to MPP+ toxicity with DA neurons being more susceptible. Analysis of the competition of MPP+ with the natural substrates for uptake sites of 5-HT and DA neurons demonstrated higher affinity of MPP+ for DA compared to 5-HT neurons. The lower affinity of MPP+ for 5-HT neurons could be responsible for the accumulation of lower MPP+ levels observed in pontine cultures and explain the resistance of 5-HT neurons to this toxin.

1-Methyl-4-phenylpyridinium↗
PubMed1989

Mesencephalic dopamine neurons become less sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity during development in vitro.

The in vitro development of monoamine oxidase (MAO) activity and [3H]dopamine (DA) uptake capacity of dissociated cell cultures from rat embryo mesencephalon were correlated with the potency of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+) neurotoxicity. Specific activities of both MAO-A and MAO-B increased during in vitro development of the cultures, with MAO-B activity increasing 20-fold between the first and fourth week. Similarly, [3H]DA accumulation increased 2.6-fold between the first and third week in vitro, when it reached a plateau. Unexpectedly, the toxicities of MPTP and MPP+ were substantially decreased in the older cultures. Exposure to MPTP reduced [3H]DA accumulation per culture by 77% in 1-week-old cultures and by 36% in 4-week-old cultures. Similarly, damage caused by MPPT was reduced from 84% of control in the first week to 34% of control in the fourth week. The attenuation of neurotoxicity was not due to an increase in storage of MPP+ in the synaptic vesicles of DA neurons, nor to a change in the distribution of MPP+ between dopaminergic and other cellular components of the cultures. The damage to DA neurons caused by the mitochondrial toxin, rotenone, also showed a similar reduction in the older cultures. These observations coupled with an increase in lactate formation and glucose consumption during the in vitro development of the cultures suggest a shift toward increased glycolysis and decreased dependence on aerobic metabolism. This would render the cells more resistant to the inhibition of mitochondrial function by MPP+.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1988

Studies on the metabolism and toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in cultures of embryonic rat mesencephalon.

The metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied in dissociated cell cultures prepared from embryonic rat mesencephalon. MPTP was added to the feeding medium and at the end of the incubation period MPTP was separated from the water-soluble metabolite by ether extraction. Our results demonstrate that, in cultures, MPTP is oxidized predominantly by monoamine oxidase B, since deprenyl but not clorgyline had an inhibitory effect on its metabolism. The metabolite of MPTP diffuses freely in the feeding medium (99% of the total) and its concentration increases with time. The concentration of the metabolite can be increased by increasing the number of cells plated into the tissue culture dish and the toxicity to dopamine neurons depends on the amount of metabolite accumulated in the feeding medium. The metabolism of MPTP is reduced by decreasing the number of glial cells present in the cultures.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1987

The toxicity of MPTP to dopamine neurons in culture is reduced at high concentrations.

The toxicity of MPTP to dopamine (DA) neurons was studied in dissociated cell cultures from rat embryo mesencephalon. The cultures were exposed to MPTP (from 0.1 to 200 microM) for 7 days and were analyzed by catecholamine histofluorescence after incubation with alpha-methylnorepinephrine, by tyrosine hydroxylase (T-OH) immunocytochemistry and by [3H]DA uptake. Treatment with MPTP at the lower range of concentrations (between 0.1 and 5 microM) resulted in a dose-dependent reduction of the uptake of [3H]DA, which had a maximum effect at 5 microM. Further increase in the concentration of MPTP (from 10 to 200 microM) resulted in progressive attenuation of the toxic effect. Exposure to 200 microM MPTP did not produce significant reduction in the uptake of [3H]DA. Increased survival of DA neurons at the higher concentrations of MPTP, was documented by catecholamine histofluorescence and by T-OH immunocytochemistry in cultures treated with 10 and 100 microM MPTP. MPTP was shown to be a potent inhibitor of MPP+ uptake by DA neurons in culture. In the presence of 100 or 200 microM MPTP, the uptake of MPP+ was reduced to less than 20% of control levels. Therefore, the reduction of MPTP toxicity at the high concentrations can be explained, at least in part, by the inhibition of the uptake of MPP+ (the toxic metabolite of MPTP oxidation) in the presence of high concentrations of MPTP.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1987

Histochemical evaluation of glutathione in brain.

Glutathione (GSH) is a ubiquitous cellular sulfhydryl compound with a variety of essential functions. A histochemical method that was developed by others for the localization of GSH in tissue sections was used to study the localization of GSH in rodent and primate brain. Sections of freshly frozen tissue were stained for 4 min with Mercury orange dissolved in toluene, and viewed by fluorescence microscopy for the product of the reaction with soluble sulfhydryl compounds. Soluble sulfhydryl compounds are comprised almost exclusively of GSH. Although the brain exhibits strong staining characteristics, reflecting the millimolar levels of GSH that are detected by chemical assay, very little stain is seen in neuronal somata. Pretreatment of animals with diethyl maleate resulted in depletion of GSH from brain (measured by high performance liquid chromatography), as well as decreased Mercury orange staining. The staining pattern observed in the brain may indicate that GSH is primarily localized to non-neuronal elements, such as glia, and/or in axons and nerve terminals.

Animals↗
PubMed1985

1-Methyl-4-phenylpyridine (MPP+) is toxic to mesencephalic dopamine neurons in culture.

1-Methyl-4-phenylpyridine (MPP+) is the product of oxidative metabolism by monoamine oxidase of the Parkinson-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP+ was tested for neurotoxicity on the dopaminergic neurons of embryonic rat midbrain in culture. Compared with MPTP, MPP+ was a more potent neurotoxin for the cultured dopamine (DA) neurons as determined by decrease in [3H]DA uptake by the cultures and decrease in endogenous levels of DA and homovanillic acid. Catecholamine histofluorescence demonstrated the loss of fluorescing fibers after exposure to MPP+. The reduced analogue of MPTP, namely 1-methyl-4-phenylpiperidine, had no significant neurotoxic action on the dopaminergic neurons in culture.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1985

Studies on the mechanism of action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Explants of embryonic rat substantia nigra in organotypic culture are sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at concentrations approximating the doses given in vivo to monkeys. Fluorescence microscopy and 3H-dopamine uptake measurements reveal that the toxicity is selective for dopamine neurons, whereas other neurons and cells in the culture appear normal by phase contrast microscopy. Reduced MPTP (piperidine analog) is inactive in the tissue culture model, while fully oxidized MPTP (pyridinium analog) destroys dopamine neurons. Pargyline and deprenyl, two monoamine oxidase inhibitors, inhibit the neurotoxic action of MPTP. Pargyline and deprenyl also protect monkeys in vivo. The results implicate monoamine oxidase in the mechanism of action of MPTP. Two possible mechanisms for protection by monoamine oxidase are discussed.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1985

Deprenyl protects dopamine neurons from the neurotoxic effect of 1-methyl-4-phenylpyridinium ion.

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗