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1.
Abstract: Recent reports suggest that NMDA receptor antagonists when administered in vivo can protect dopaminergic neurons from the toxic actions of MPP+. In the present study the possible neuroprotective effects against MPP+ toxicity of the noncompetitive NMDA receptor antagonist MK-801 was studied in primary cultures of fetal rat mesencephalic dopamine neurons. MK-801 failed to protect dopaminergic neurons from MPP+ toxicity at concentrations that completely block NMDA-induced toxicity of these same neurons. In contrast to work carried out in cerebellar granule cells, MPP+ toxicity of mesencephalic dopamine neurons was unaffected by preexposure to subtoxic concentrations of either NMDA or cycloheximide. Our findings suggest that the toxic effects of MPP+ on dopaminergic neurons are not mediated through a direct interaction with the NMDA subtype of glutamate receptor.  相似文献   

2.
Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP+ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP+ intoxication and melatonin protection. We found that MPP+ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP+-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP+-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro . Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP+, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP+ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.  相似文献   

3.
Abstract: The effects of the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its 4-electron oxidation product 1-methyl-4-phenylpyridinium (MPP+) were studied in isolated mitochondria and in mouse brain striatal slices. ADP-stimulated oxidation of NAD-linked substrates was inhibited in a time-dependent manner by MPP+ (0.1–0.5 m M ), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP+ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP+ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.  相似文献   

4.
Abstract: The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of ∼33–45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is ∼16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at ∼40 pg/ml, or 1 p M , and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.  相似文献   

5.
Abstract: Exposure of various neuronal cells or cell lines to high concentrations of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), results in cell death. Recently, it has been reported that low concentrations of MPP+ induce apoptosis in susceptible neurons. We have further characterized MPP+-mediated toxicity of cultured cerebellar granule neurons (CGNs) and found that exposure of CGNs to relatively low concentrations of MPP+ results in apoptosis, whereas higher concentrations result in necrosis. Cotreatment of CGNs with MPP+ and the tetrapeptide inhibitor of caspase-3-like proteases, acetyl-DEVD-CHO, markedly attenuates apoptotic but not necrotic death of these neurons. The more specific inhibitor of caspase-1-like proteases, acetyl-YVAD-CHO, however, was ineffective against MPP+ neurotoxicity. Moreover, cytoplasmic extracts prepared from MPP+-treated CGNs contain markedly increased protease activity that cleaves the caspase-3 substrate acetyl-DEVD- p -nitroaniline. Finally, the cytoplasmic concentration of the apoptogenic protein cytochrome c was increased in a time-dependent fashion in MPP+-treated CGNs before the onset of apoptosis. Our data confirm that the neurotoxicity of MPP+ is due to both necrosis and apoptosis and suggest that the latter is mediated by activation of a caspase-3-like protease.  相似文献   

6.
Abstract: The ionic species 1-methyl-4-phenylpyridinium (MPP+) seems to be the metabolite responsible for the damage to dopaminergic neurons occurring after administration of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In the present study we show that the unilateral stereotaxic microinjection of MPP+ into the substantia nigra pars reticulata in rats produces immediately intense and long-lasting (up to 96 h) contralateral turning behavior in a dose-dependent manner. This behavioral effect was correlated with a dose- and time-dependent decrease (up to 90%) of glutamate decarboxylase activity and with a notable loss of neurons in the injected nigra reticulata. GABA levels in the injected nigra were also decreased, whereas the dopamine concentration in the ipsilateral striatum was not affected at 24 h, when maximal behavioral effects were observed. The circling behavior was prevented by the dopamine carrier blocker nomifensine only during the first 2 h, whereas the dopamine receptor antagonist haloperidol was ineffective. The results indicate that MPP+ is toxic for inhibitory GABAergic neurons in the nigra pars reticulata and, furthermore, suggest that disruption of the function of these GABAergic neurons may be involved in the abnormal motor behavior produced by the injection of MPP+ in the substantia nigra.  相似文献   

7.
Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6-hydroxydopamine-induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival-promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson's disease.  相似文献   

8.
Abstract: The c- ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 µ M K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.  相似文献   

9.
Abstract: Dibutyryl cyclic AMP (dbcAMP), a permeant analogue of cyclic AMP (cAMP), prevented, for at least 3 weeks, the death of tyrosine hydroxylase (TH)-immunopositive dopaminergic neurons, which occurred spontaneously by apoptosis in mesencephalic cultures. Treatment with the cyclic nucleotide analogue also led to a significant increase in the uptake of [3H]dopamine, attesting that the rescued TH+ neurons were fully functional and differentiated. dbcAMP was most effective when added immediately after plating, but delayed treatment could still arrest the ongoing degenerative process. Trophic/survival effects were long-lasting, declining only progressively after withdrawal of dbcAMP from the culture medium. They were independent of cell density and still detectable in the absence of serum proteins. The effects of dbcAMP were mimicked by depolarizing concentrations of potassium and by agents that increase endogenous production of cAMP, such as forskolin or 3-isobutyl-1-methylxanthine, but not by native cAMP, which cannot cross cell membranes. Elimination of glial cells by arabinoside-C did not reduce the activity of dbcAMP. GABAergic neurons, also present in these cultures, were much less dependent on the cyclic nucleotide analogue for their survival, and serotoninergic cells were not dependent at all. Therefore, cAMP-dependent signaling may be particularly crucial for the maturation and long-term survival of mesencephalic dopaminergic neurons.  相似文献   

10.
Abstract: Microdialysis was used to evaluate the effect of desferrioxamine (DES) against 1-methyl-4-phenylpyridinium (MPP+) toxicity. The presence of DES (40 fmol-40 nmol/15 min for a total of 90 min) in the Ringer solution, coperfused with MPP+ (40 nmol/15 min) on day 1, produced on day 2 a higher extracellular dopamine output after perfusion of MPP+ than in control MPP+ perfusion experiments, in which no DES was administered on day 1. Both Ringer perfusion alone (control Ringer) and coperfusion of 40 nmol DES with 40 nmol MPP+ on day 1 produced on day 2 similar increases in extracellular dopamine output after a second MPP+ perfusion. In the control Ringer experiment, note that the MPP+ on day 2 is the first MPP+ perfusion. Perfusion of 800 fmol FeCl3/15 min along with 40 nmol MPP+ and 400 fmol DES on day 1 completely abolished on day 2 the neuroprotective effect found with 40 nmol MPP+ and 400 fmol DES; 800 fmol FeCl3 did not increase the neurotoxic effect of 40 nmol MPP+ perfusion. The ability of DES to protect against MPP+ toxicity may indicate a therapeutic strategy in the treatment of diseases when iron is implicated.  相似文献   

11.
Human embryonic stem (hES) cells have the ability to renew themselves and differentiate into multiple cell types upon exposure to appropriate signals. In particular, the ability of hES cells to differentiate into defined neural lineages, such as neurons, astrocytes, and oligodendrocytes, is fundamental to developing cell-based therapies for neurodegenerative disorders and studying developmental mechanisms. However, the utilization of hES cells for basic and applied research is hampered by the lack of well-defined methods to maintain their self-renewal and direct their differentiation. Recently we reported that neural precursor (NP) cells derived from mouse ES cells maintained their potential to differentiate into dopaminergic (DA) neurons after significant expansion in vitro . We hypothesized that NP cells derived from hES cells (hES-NP) could also undergo the same in vitro expansion and differentiation. To test this hypothesis, we passaged hES-NP cells and analyzed their proliferative and developmental properties. We found that hES-NP cells can proliferate approximately 380 000-fold after in vitro expansion for 12 weeks and maintain their potential to generate Tuj1+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes as well as tyrosine hydroxylase-positive (TH+) DA neurons. Furthermore, TH+ neurons originating from hES-NP cells expressed other midbrain DA markers, including Nurr1, Pitx3, Engrail-1, and aromatic l -amino acid decarboxylase, and released significant amounts of DA. In addition, hES-NP cells maintained their developmental potential through long-term storage (over 2 years) in liquid nitrogen and multiple freeze–thaw cycles. These results demonstrate that hES-NP cells have the ability to provide an expandable and unlimited human cell source that can develop into specific neuronal and glial subtypes.  相似文献   

12.
The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP+-induced cytosolic phospholipase A2 (cPLA2) activation of dopaminergic neurons. MPP+ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca2+]i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA2, and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF3), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA2 on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA2 phosphorylation at Ser505. Inhibitors of cPLA2 and PKG increased viability and reduced MPP+-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA2 phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA2.  相似文献   

13.
Abstract: Incubation of 10 m M I-methyl-4-phenylpyridinium (MPP+) with sonicated beef heart mitochondria caused an irreversible time-dependent decrease in NADH-ubiquinone-l (CoQ1) reductase activity (52% inhibition after 1 h). Inclusion of glutathione, ascorbate, or catalase in the incubation mixture protected the NADH-CoQ1 reductase activity. These results suggest that the interaction of MPP+ with complex I induces free radical generation, which in turn leads to the irreversible inhibition of complex I activity. The generation of free radicals by neurotoxin-induced inhibition of complex I has important implications for our interpretation of the increased oxidative stress observed in Parkinson's disease substantia nigra and for our understanding of the cause(s) of dopaminergic cell death in this disorder.  相似文献   

14.
Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP+) on the oxygen consumption, ATP production, H2O2 production, and mitochondrial NADH-CoQ1 reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP+ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP+ produced a concentration-dependent decrease in H2O2 production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP+ caused no loss of complex I activity after washing of MPP+ from the mitochondrial membranes. These data are consistent with MPP+ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H2O2 by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H2O2 in the presence of Cu2+ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP+ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.  相似文献   

15.
Abstract: 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 µ M MPTP for 24 h decreased uptake by 39%. Kinetic analysis revealed that this effect was due to a 35% decrease in V max with no change in the K m. 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.  相似文献   

16.
17.
Strong evidence indicates that oxidative stress may be causally involved in the pathogenesis of Parkinson's disease. We have employed human dopaminergic neuroblastoma cells and rat primary mesencephalic neurons to assess the protective potential of three novel bisarylimine antioxidants on dopaminergic cell death induced by complex I inhibition or glutathione depletion. We have found that exceptionally low concentrations (EC50 values ∼20 nM) of these compounds (iminostilbene, phenothiazine, and phenoxazine) exhibited strong protective effects against the toxicities of MPP+, rotenone, and l -buthionine sulfoximine. Investigating intracellular glutathione levels, it was found that MPP+, l -buthionine sulfoximine, and rotenone disrupted different aspects of the native glutathione equilibrium, while the aromatic imines did not further influence glutathione levels or redox state on any baseline. However, the imines independently reduced protein oxidation and total oxidant flux, saved the mitochondrial membrane potential, and provided full cytoprotection under conditions of complete glutathione depletion. The unusually potent antioxidant effects of the bisarylimines could be reproduced in isolated mitochondria, which were instantly protected from lipid peroxidation and pathological swelling. Aromatic imines may be interesting lead structures for a potential antioxidant therapy of Parkinson's disease and other disorders accompanied by glutathione dysregulation.  相似文献   

18.
Abstract: We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24-h treatment with fibroblast growth factor-1, −2, or −9 elevated secreted GDNF protein levels five- to 10-fold. The proinflammatory cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α, and lipopolysaccharide elevated GDNF release 1.5- to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13-didecanoate (10 n M ) activation of protein kinase C, the Ca2+ ionophore A23187 (1 µ M ), okadaic acid (10 n M ) inhibition of type-2A protein phosphatases, nitric oxide donors (1 m M ), and H2O2 (1 m M )-induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 µ M ) or dibutyryl cyclic AMP (1 m M ) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 µ M ). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.  相似文献   

19.
20.
Abstract: The neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite 1-methyl-4-phenylpyridinium (MPP+), parkinsonism in humans, monkeys, and mice but not in rats. When incubated with mouse brain homogenates, [3H]-MPP+ is recovered in relatively large concentrations in the brain cell nucleus. Although isolated cell nuclei from rat and mouse brain contain uptake systems for dopamine (DA), only brain cell nuclei from mice avidly take up [3H]MPP+. This nuclear uptake is ATP dependent and can be blocked by ouabain and Nethylmaleimide. It is not, however, affected by neuronal and vesicular blockers such as benztropine. mazindol, and reserpine. Selective uptake of MPP+ into brain cell nuclei may provide a new avenue for future investigation into the complex modes of action of the neurotoxin MPTP.  相似文献   

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