Neurotoxic effects of papaverine, tetrahydropapaverine and dimethoxyphenylethylamine on dopaminergic neurons in ventral mesencephalic-striatal co-culture

We report neurotoxic effects of papaverine, tetrahydropapaverine, dimethoxyphenylethylamine (DMPEA), and 1-methyl-4-phenylpyridinium ion (MPP+) on dopaminergic neurons in ventral mesencephalic-striatal co-culture. These compounds have been reported as mitochondrial toxins which may be implicated in the etiology and pathogenesis of Parkinson's disease. Tyrosine hydroxylase (TH)-positive neurons were decreased in dose-dependent manner by these compounds. Papaverine and MPP+ were most toxic to TH-positive neurons among the compounds tested. The order of the toxicity on TH-positive neurons was papaverine, MPP+, tetrahydropapaverine and then DMPEA. This order of toxicity was approximately the same as that reported on the inhibitory effect of these compounds on NADH-linked mitochondrial respiration and complex I activity. These findings indicate that the presence of dimethoxy residues in the catechol ring augments toxicity to dopaminergic neurons in culture.     

Mechanism of resistance to NO-induced neurotoxicity in cultured rat dopaminergic neurons

We previously reported that mesencephalic dopaminergic neurons are resistant to cytotoxicity induced by nitric oxide (NO). This study investigated the intracellular mechanism that protects dopaminergic neurons against NO toxicity in rat mesencephalic cultures. Peroxynitrite anion, an active metabolite of NO, caused significant cytotoxic effects against dopaminergic and nondopaminergic neurons, but NO caused cytotoxic effects restricted to nondopaminergic neurons. In addition, we studied the effects of ascorbate, an anti-oxidant, on NO-induced neurotoxicity against dopaminergic neurons and found that coadministration of ascorbate failed to affect resistance against NO-induced neurotoxicity. These findings suggest that the protecting mechanism from NO neurotoxicity in dopaminergic neurons is based on inhibition of conversion of NO to peroxynitrite anion, is independent of the NO redox state, and is possibly due to suppression of superoxide anion production. Furthermore, we investigated NO-induced neurotoxicity with or without pretreatment with sublethal doses of methylphenylpyridium ion (MPP+). Following pretreatment with 1 microM MPP+, which did not show significant cytotoxic effects against dopaminergic neurons, NO demonstrated significant cytotoxicity. Therefore, MPP+ may inhibit the protecting systems from NO neurotoxicity in dopaminergic neurons.     

Rescue of mesencephalic dopamine neurons by anticancer drug cytosine arabinoside

Nanomolar concentrations of cytosine arabinoside (ara-C), a structural analogue of 2'-deoxycytidine (2'dC) used in the chemotherapy of cancer, proved to be highly effective in preventing the death of postmitotic dopaminergic neurons that occurs spontaneously by apoptosis in mesencephalic cultures. The rescued cells were totally functional and highly differentiated. The trophic/neuroprotective effects of ara-C were (1) specific for dopaminergic neurons; (2) long-lived, remaining detectable several days after withdrawal of the nucleoside analogue from the culture medium; (3) still observed when the treatment was delayed after plating; (4) abolished by an excess of 2'dC or dCTP, or by exposure to the neurotoxin 1-methyl-4-phenylpyridinium; and (5) mimicked by ara-CTP, 5-fluoro-2'-deoxyuridine, and aphidicolin. Autoradiographic studies revealed that ara-C was incorporated exclusively into astrocyte nuclei, suggesting that the dopaminotrophic activity was indirect and resulted from the antiproliferative action of the modified nucleoside on glial cells at concentrations that were not neurotoxic. No evidence was found for putative deleterious or trophic molecules secreted by proliferating or ara-C-treated astrocytes, respectively, suggesting that neuroglial contact may play a role. Our results suggest a possible mechanism underlying neurodegeneration in Parkinson's disease, where selective loss of dopaminergic neurons in the mesencephalon is accompanied by astrogliosis.     

Role of oxidative stress and the glutathione system in loss of dopamine neurons due to impairment of energy metabolism

Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose-dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high-affinity uptake of [3H]dopamine and [14C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24-h pretreatment with 10 microM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin-trapping agents, alpha-phenyl-tert-butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate-induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin-trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase-positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin-trapping agents. NMDA receptors have also been shown to play a role in malonate-induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1-h exposure to 50 microM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreases glutamate uptake in cultured astrocytes

The deleterious effect of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic neurons of the substantia nigra is well established. In addition, increased glutamatergic drive to basal ganglia output nuclei is considered a likely contributor to the pathogenesis of Parkinson's disease. One possibility for the increased excitatory tone may be related to an impairment in glutamate uptake. As astrocytes possess efficient transport mechanisms for both MPTP and glutamate, we have examined the effect of this agent on D-aspartate uptake into these cells. Treatment of cultures with 50 microM MPTP for 24 h decreased uptake by 39%. Kinetic analysis revealed that this effect was due to a 35% decrease in Vmax with no change in the Km. Treatment with deprenyl, a monoamine oxidase B inhibitor, produced a complete reversal of MPTP-induced uptake inhibition, but was ineffective following exposure of cells to the MPTP metabolite, 1-methyl-4-phenylpyridinium (MPP+). Removal of MPTP from cultures resulted in a complete restoration of glutamate uptake after 24 h. These results show that MPTP reversibly compromises glutamate uptake in cultured astrocytes, which is dependent on the conversion of MPTP to MPP+. Such findings suggest that the glutamate transporter in astrocytes plays an important role in MPTP-induced neurotoxicity and possibly in parkinsonism.     

Deficits in finite verb morphology: some assumptions in recent accounts of specific language impairment

Multiple aspartate-specific cysteine proteases have been identified and specific members of this family have been implicated in the apoptotic death of many mammalian cell types. Caspase-3-like proteases seem to play a pivotal role in neuronal apoptosis since mice with germline inactivation of the caspase-3 gene manifest profound alterations in neurogenesis. Moreover, inhibitors of caspase-3-related proteases have been shown to inhibit neuronal apoptosis. Here we extend recent work from our laboratory on the mechanisms mediating the neurotoxic actions of 1-methyl-4-phenylpyridinium using ventral mesencephalon cultures containing dopamine neurons. We demonstrate that low concentrations of 1-methyl-4-phenylpyridinium induce apoptosis in dopamine neurons by morphological and biochemical criteria. Moreover, pretreatment of ventral mesencephalon cultures with the tetrapeptide inhibitors of the caspase-3-like proteases zVAD-FMK or Ac-DEVD-CHO specifically inhibit death of dopamine neurons induced by low concentrations of 1-methyl-4-phenylpyridinium, whereas the caspase-1-like inhibitor Ac-YVAD-CHO was without effect. Our data indicate that exposure of cultured ventral mesencephalon dopamine neurons to low concentrations of 1-methyl-4-phenylpyridinium results in apoptotic death and that caspase-3-like proteases may mediate the neurotoxic apoptotic actions of 1-methyl-4-phenylpyridinium.     

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl-coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6-hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase-positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6-hydroxydopamine-injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6-hydroxydopamine-induced toxicity.     

Manganese toxicity in serumless dissociated mesencephalic and striatal primary culture

Exposure to elevated levels of Manganese (Mn) can result in an irreversible brain disease characterized by extrapyramidal signs and symptoms resembling Parkinson's disease. To identify the neuronal target of Mn neurotoxicity, MnCl2 was added to serumless dissociated mesencephalic-striatal cultures from rat embryo on day 4 in vitro. High affinity 3H-dopamine (DA) and 14C-GABA uptakes were assessed as specific functional markers of DAergic and GABAergic cell viability, respectively. After 60-min exposure, MnCl2 at 0-200 microM did not modify the morphologic appearance of the cultures, specific DA and GABA uptakes, or the number of DA neurons visualized by immuno-cytochemical staining with tyrosine hydroxylase. In contrast, culture exposure to 20 microM MnCl2 for 24 h selectively reduced specific GABA uptake without affecting specific DA uptake or the number of DA neurons. The exposure to a higher MnCl2 concentration was accompanied by signs of general toxicity. Striatal GABA neurons seemed to be more susceptible to Mn toxicity than mesencephalic GABA neurons. Overall, our data suggest that striatal neurons rather than mesencephalic DA neurons may be the main target of Mn neurotoxicity.     

Alterations in corticotropin-releasing factor and vasopressin content in rat brain during morphine withdrawal: correlation with hypothalamic noradrenergic activity and pituitary-adrenal response

Oxidative stress is thought to play an important role in the pathogenesis of Parkinson's disease (PD). Glutathione (GSH), a major cellular antioxidant, is decreased in the substantia nigra pars compacta of PD patients. The aim of the present study was to investigate whether deprenyl and its desmethyl metabolite, putative neuroprotective agents in the treatment of PD, could protect cultured rat mesencephalic neurons from cell death caused by GSH depletion due to treatment with L-buthionine-(S,R)-sulfoximine (BSO). BSO (10 microM) caused extensive cell death after 48 hr, as demonstrated by disruption of cellular integrity and release of lactate dehydrogenase into the culture medium. Both deprenyl and desmethylselegiline, at concentrations of 5 and 50 microM, significantly protected dopaminergic neurons from toxicity without preventing the BSO-induced loss in GSH. Protection was not associated with monoamine oxidase type B inhibition in that pargyline, a potent MAO inhibitor, was ineffective and pretreatment with pargyline did not prevent the protective effects of deprenyl. Protection was not associated with inhibition of dopamine uptake by deprenyl because the dopamine uptake inhibitor mazindol did not diminish BSO toxicity. The antioxidant ascorbic acid (200 microM) also protected against BSO-induced cell death, suggesting that oxidative events were involved. This study demonstrates that deprenyl and its desmethyl metabolite can diminish cell death associated with GSH depletion.     

Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.     

L-(-)-desmethylselegiline, a metabolite of selegiline [L-(-)-deprenyl], protects mesencephalic dopamine neurons from excitotoxicity in vitro

Selegiline [L-(-)-deprenyl], a monoamine oxidase B inhibitor, has been used in the treatment of Parkinson's disease as a putative neuroprotective agent. Selegiline is metabolized rapidly in the gastrointestinal tract and liver to desmethylselegiline (DMS) and methamphetamine. We have previously shown that selegiline protects dopamine neurons in mesencephalic cultures from toxicity resulting from activation of glutamate receptors. In the present study we examined whether DMS has similar neuroprotective effects. Our data show that DMS protects dopamine neurons from N-methyl-D-aspartate receptor-mediated excitotoxic damage. The efficacy of DMS is greater than that of selegiline, as it can cause protection at lower concentrations and provide significantly greater levels of protection at the same concentrations. Our results suggest that DMS might be the active compound responsible for the neuroprotective properties of selegiline.     

Excitotoxicity and oxidative stress during inhibition of energy metabolism

Glutamate receptor involvement and oxidative stress have both been implicated in damage to neurons due to impairment of energy metabolism. Using two different neuronal in vitro model systems, an ex vivo chick retinal preparation and dopamine neurons in mesencephalic culture, the involvement and interaction of these events as early occurring contributors to irreversible neuronal damage have been examined. Consistent with previous reports, the early acute changes in the retinal preparation, as well as irreversible loss of dopamine neurons due to inhibition of metabolism, can be prevented by blocking NMDA receptors during the time of energy inhibition. Oxidative stress was suggested to be a downstream consequence and contributor to neuronal cell loss due to either glutamate receptor overstimulation or metabolic inhibition since trapping of free radicals with the cyclic nitrone spin-trapping agent MDL 102,832 (1 mM) attenuated acute excitotoxicity in the retinal preparation or loss of mesencephalic dopamine neurons due to either metabolic inhibition by the succinate dehydrogenase inhibitor, malonate, or exposure to excitotoxins. In mesencephalic culture, malonate caused an enhanced efflux of both oxidized and reduced glutathione into the medium, a significant reduction in total reduced glutathione and a significant increase in total oxidized glutathione at time points that preceded those necessary to cause toxicity. These findings provide direct evidence for early oxidative events occurring following malonate exposure and suggest that the glutathione system is important for protecting neurons during inhibition of energy metabolism. Consistent with this, lowering of glutathione by buthionine sulfoxamine (BSO) pretreatment greatly potentiated malonate toxicity in the mesencephalic dopamine population. In contrast, BSO pretreatment did not potentiate glutamate toxicity. This latter finding indicates dissimilarities in the type of oxidative stress that is generated by the two insults and suggests that the oxidative challenge during energy inhibition is not solely a downstream consequence of glutamate receptor overstimulation.     

Characterization of the expression and steroid hormone control of sialomucin complex in the rat uterus: implications for uterine receptivity

Oxidative stress, a process in which neurotoxic oxygen free radicals cause dopaminergic neuronal degeneration, has been implicated in the degenerative process in Parkinson's disease. Glutamate-induced neurotoxicity is a model of oxidative stress. We demonstrated that preincubation with D2-type dopamine agonists bromocriptine and quinpirole provides neuroprotection against glutamate-induced neurotoxicity in cultured rat mesencephalic neurons. Simultaneous administration of D2 agonists, however, did not provide neuroprotection. The protective effects were dependent on the duration of preincubation and were blocked by a D2 antagonist and a protein synthesis inhibitor. Furthermore, preincubation with D2 agonists provided neuroprotection against toxicity induced by calcium overload and exposure to superoxide anions. Confocal microscopic analysis, using 2,7-dichlorofluorescin diacetate, revealed that bromocriptine preincubation suppressed the action of radicals on neurons. These findings indicate that dopamine D2 agonists provide protection mediated not only by the inhibition of dopamine turnover but also via D2-type dopamine receptor stimulation and the subsequent synthesis of proteins that scavenge free radicals.     

Growth/differentiation factor 5 and glial cell line-derived neurotrophic factor enhance survival and function of dopaminergic grafts in a rat model of Parkinson's disease

Growth/differentiation factor 5 is a member of the transforming growth factor beta superfamily, which has neurotrophic and neuroprotective effects on dopaminergic neurons both in vitro and in vivo. Here we investigate the effects of growth/differentiation factor 5 on foetal mesencephalic grafts transplanted into a rat model of Parkinson's disease, and compare them with those of glial cell line-derived neurotrophic factor. Mesencephalic tissue was suspended in solutions containing either growth/differentiation factor 5 or glial cell line-derived neurotrophic factor prior to transplantation into the left striatum of rats with 6-hydroxydopamine lesions of the left medial forebrain bundle. Both proteins enhanced graft-induced compensation of amphetamine-stimulated rotations. Positron emission tomography studies showed that both neurotrophins increased graft-induced recovery of striatal binding of [11C]RTI-121, a marker for dopaminergic nerve terminals. Post mortem analysis at 8 weeks after transplantation showed that both neurotrophins significantly increased the survival of grafted dopaminergic neurons. This study shows that growth/differentiation factor 5 is at least as effective as glial cell line-derived neurotrophic factor in enhancing the survival and functional activity of mesencephalic grafts, and thus is an important candidate for use in the treatment of Parkinson's disease.     

Isoquinoline derivatives as endogenous neurotoxins in the aetiology of Parkinson's disease

The cause of neurodegeneration in Parkinson's disease (PD) remains unknown. However, isoquinoline derivatives structurally related to the selective dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite, 1-methyl-4-phenylpyridinim (MPP+), have emerged as candidate endogenous neurotoxins causing nigral cell death in Parkinson's disease. Isoquinoline derivatives are widely distributed in the environment, being present in many plants and foodstuffs, and readily cross the blood-brain barrier. These compounds occur naturally in human brain where they are synthesized by non-enzymatic condensation of biogenic amines (e.g. catecholamines and phenylethylamine) with aldehydes, and are metabolized by cytochrome P450s and N-methyltransferases. In addition, isoquinoline derivatives are oxidized by monoamine oxidases to produce isoquinolinium cations with the concomitant generation of reactive oxygen species. Neutral and quaternary isoquinoline derivatives accumulate in dopaminergic nerve terminals via the dopamine re-uptake system, for which they have moderate to poor affinity as substrates. Several isoquinoline derivatives are selective and more potent inhibitors of NADH ubiquinone reductase (complex I) and alpha-ketoglutarate dehydrogenase activity in mitochondrial fragments than MPP+, and lipophilicity appears to be important for complex I inhibition by isoquinoline derivatives. However, compared with MPP+, isoquinoline derivatives are selective but less potent inhibitors of NADH-linked respiration in intact mitochondria, and this appears to be a consequence of their rate-limiting ability to cross mitochondrial membranes. Although both active and passive processes are involved in the accumulation of isoquinoline derivatives in mitochondria, inhibition of respiration is determined by steric rather than electrostatic properties. Compared with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or MPP+, isoquinoline derivatives show selective but relatively weak toxicity to dopamine-containing cells in culture and following systemic or intracerebral administration to experimental animals, which appears to be a consequence of poor sequestration of isoquinoline derivatives by mitochondria and by dopamine-containing neurones. In conclusion, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-like cytotoxic characteristics of isoquinoline derivatives and the endogenous/environmental presence of these compounds make it conceivable that high concentrations of and/or prolonged exposure to isoquinoline derivatives might cause neurodegeneration and Parkinson's disease in humans.     

A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion

An early and highly specific decrease in glutathione (GSH) in the substantia nigra is associated with Parkinson's disease, and low levels of GSH lead to the degeneration of cultured dopaminergic neurons. Using immature cortical neurons and a clonal nerve cell line, it is shown that a decrease in GSH triggers the activation of neuronal 12-lipoxygenase (12-LOX), which leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The supporting evidence includes: 1) inhibitors of arachidonate metabolism and 12-LOX block cell death induced by GSH depletion; 2) there is an increase in 12-LOX activity and a membrane translocation in HT22 cells, and an induction of the enzyme in primary cortical neurons following the reduction of GSH; 3) 12-LOX is directly inhibited by GSH; and 4) exogenous arachidonic acid potentiates cell death. These data show that the LOX pathway is a critical intermediate in at least some forms of neuronal degeneration.     

A conformational epitope in the N-terminus of the Escherichia coli heat-stable enterotoxins is involved in receptor-ligand interactions

The relationship between the central dopaminergic and the immune system is poorly understood. Experimental work suggest that damage of the nigrostriatal system may influence immunity. Immunological abnormalities have been described in Parkinson's disease and in a mouse model of this disorder induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this report, we present evidence that reduced numbers of L3T4 T cells in blood, and diminished primary antibody response to sheep erythrocytes in MPTP treated mice can be restored by pargyline pretreatment. Since pargyline prevents dopamine depletion in the striatum in MPTP treated animals, our data extend previous experimental observations and support a possible role for dopamine in immune regulation.