Ikuko Miyazaki

Dopamine- or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson's disease.

Dopamine (DA)- or L-dihydroxyphenylalanine- (L-DOPA-) induced neurotoxicity is thought to be involved not only in adverse reaction induced by longterm L-DOPA therapy but also in the pathogenesis of Parkinsons disease. Numerousin vitro andin vivo studies concerning DA- or L-DOPA-induced neurotoxicity have been reported in recent decades. The reactive oxygen or nitrogen species generated in the enzymatical oxidation or auto-oxidation of an excess amount of DA induce neuronal damage and/or apoptotic or non-apoptotic cell death; the DA-induced damage is prevented by various intrinsic and extrinsic antioxidants. DA and its metabolites containing two hydroxyl residues exert cytotoxicity in dopaminergic neuronal cells mainly due to the generation of highly reactive DA and DOPA quinones which are dopaminergic neuron-specific cytotoxic molecules. DA and DOPA quinones may irreversibly alter protein function through the formation of 5-cysteinyl-catechols on the proteins. For example, the formation of DA quinone-α-synuclein consequently increases cytotoxic protofibrils and the covalent modification of tyrosine hydroxylase by DA quinones. The melanin-synthetic enzyme tyrosinase in the brain may rapidly oxidize excess amounts of cytosolic DA and L-DOPA, thereby preventing slowly progressive cell damage by auto-oxidation of DA, thus maintaining DA levels. Since tyrosinase also possess catecholamine-synthesizing activity in the absence of tyrosine hydroxylase (TH), the double-edged synthesizing and oxidizing functions of tyrosinase in the dopaminergic system suggest its potential for application in the synthesis of DA, instead of TH in the degeneration of dopaminergic neurons, and in the normalization of abnormal DA turnover in long-term L-DOPA-treated Parkinsons disease patients.

Dopamine D2 receptor-mediated antioxidant and neuroprotective effects of ropinirole, a dopamine agonist

Recent information suggests that free radicals are closely involved in the pathogenesis and/or progression of Parkinsons disease (PD). High-dose levodopa therapy has been suggested to increase oxidative stress, thereby accelerating the progression of PD. Based on this viewpoint, free radical scavenging, antioxidant and neuroprotective agents which may prevent the progression of PD have recently attracted considerable attention. For example, ergot derivative dopamine (DA) agonists have been reported to scavenge free radicals in vitro and show a neuroprotective effect in vivo. Non-ergot DA agonists have also recently been used in the treatment of PD despite the lack of substantial evidence for any free radical scavenging activity or antioxidant activity. The present study was conducted to assess the in vitro free radical scavenging and antioxidant activities of ropinirole, a non-ergot DA agonist, as well as its glutathione (GSH), catalase and superoxide dismutase (SOD) activating effects and neuroprotective effect in vivo. Ropinirole scavenges free radicals and suppresses lipid peroxidation in vitro, but these activities are very weak, suggesting that the antioxidant effect of ropinirole observed in vitro may be a minor component of its neuroprotective effect in vivo. Administration of ropinirole for 7 days increased GSH, catalase and SOD activities in the striatum and protected striatal dopaminergic neurons against 6-hydroxydopamine (6-OHDA) in mice. Pre-treatment with sulpiride prevented ropinirole from enhancing striatal GSH, catalase and SOD activities and abolished the protection of dopaminergic neurons against 6-OHDA. Our findings indicate that activation of GSH, catalase and SOD mediated via DA D2 receptors may be the principal mechanism of neuroprotection by ropinirole.

Neuroprotective effects of non-steroidal anti-inflammatory drugs by direct scavenging of nitric oxide radicals

Recently, it has been reported that inflammatory processes are associated with the pathophysiology of Alzheimers disease and that treatment of non‐steroidal anti‐inflammatory drugs reduce the risk for Alzheimers disease. In the present study, we examined nitric oxide radical quenching activity of non‐steroidal anti‐inflammatory drugs and steroidal drugs using our established direct in vitro nitric oxide radical detecting system by electron spin resonance spectrometry. The non‐steroidal anti‐inflammatory drugs, aspirin, mefenamic acid, indomethacin and ketoprofen directly and dose‐dependently scavenged generated nitric oxide radicals. In experiments of nitric oxide radical donor, NOC18‐induced neuronal damage, these four non‐steroidal drugs significantly prevented the NOC18‐induced reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of inducible nitric oxide synthase‐like immunoreactivity. However, ibuprofen, naproxen or steroidal drugs, which had less or no scavenging effects in vitro, showed almost no protective effects against NOC18‐induced cell toxicity. These results suggest that the protective effects of the former four non‐steroidal anti‐inflammatory drugs against apoptosis might be mainly due to their direct nitric oxide radical scavenging activities in neuronal cells. These direct NO· quenching activities represent novel effects of non‐steroidal anti‐inflammatory drugs. Our findings identified novel pharmacological mechanisms of these drugs to exert not only their anti‐inflammatory, analgesic, antipyretic activities but also neuroprotective activities against neurodegeneration.

Glial cells protect neurons against oxidative stress via transcriptional up-regulation of the glutathione synthesis.

Abstract: We examined the effects of oxidative stress on rat cultured mesencephalic neurons and glial cells. Glial cells were more resistant to 6‐hydroxydopamine (6‐OHDA) and H2O2 toxicity than neurons. In glial cells, incubation with 6‐OHDA and H2O2 induced a significant increase in the expression of γ‐glutamylcysteine synthetase (the rate‐limiting enzyme in glutathione synthesis) mRNA, which correlated well with increased TPA‐response element (TRE)‐binding activity. Furthermore, a subsequent elevation in cellular total glutathione content was also observed. In neurons, both agents decreased TRE‐binding activity, and these cells failed to up‐regulate the glutathione synthesis. We also examined the mechanisms of the neuroprotective effects of glial cells using a glia conditioned medium. Neurons maintained in glia conditioned medium up‐regulated the level of TRE‐binding activity, γ‐glutamylcysteine synthetase mRNA expression, and total glutathione content in response to 6‐OHDA or H2O2, and became more resistant to both agents than cells maintained in a normal medium. Neurons maintained in normal medium failed to up‐regulate the glutathione synthesis. Our results suggest that transcriptional up‐regulation of glutathione synthesis in glial cell appears to mediate brain glial cell resistance against oxidative stress, and that glial cells protect neurons via transcriptional up‐regulation of the antioxidant system.

Apoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive quinone generation in neuroblastoma cells.

Neurotoxic properties of L-dopa and dopamine (DA)-related compounds were assessed in human neuroblastoma SH-SY5Y cells with reference to their structural relationship. L-Dopa and its metabolites containing two free hydroxyl residues on their benzene ring showed toxicity in the cell, which was prevented by superoxide dismutase (SOD) and reduced glutathione (GSH), but not by catalase. Furthermore, a synthetic derivative of DA, 3-hydroxy-4-methoxyphenethylamine (HMPE) containing methoxy residue at position 4 in the benzene ring, exerted partial cytotoxicity, which was not prevented by SOD, GSH or catalase. However, the metabolites containing methoxy residue at position 3 failed to show a toxic effect in the SH-SY5Y cells. Moreover, DA induced apoptotic cell death, which was observed by nuclear and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and measurement of caspase-3 activity; this compound up-regulated apoptotic factor p53 while down-regulating anti-apoptotic factor Bcl-2. In the cell-free in vitro electron spin resonance (ESR) spectrometry, DA possessing two hydroxyl groups showed generation of DA-semiquinone radicals, which were markedly prevented by addition of SOD or GSH but not by catalase. On the other hand, methylation of one of the hydroxyl residues on the benzene ring of DA converted DA to an unoxidizable compound (3-MT or HMPE), and caused it to lose the property to produce semiquinone radicals. It has been previously reported that SOD acting as a superoxide:semiquinone oxidoreductase prevents quinone formation, and that reduced GSH through forming a complex with DA-quinone prevents quinone binding to the thiol group of the intact protein. Therefore, the present results suggest that DA and its metabolites containing two hydroxyl residues exert cytotoxicity mainly due to generation of highly reactive quinones.

Methamphetamine-induced dopaminergic neurotoxicity is regulated by quinone formation-related molecules

Recently, the neurotoxicity of dopamine (DA) quinone formation by auto‐oxidation of DA has focused on dopaminergic neuron‐specific oxidative stress. In the present study, we examined DA quinone formation in methamphetamine (METH)‐induced dopaminergic neuronal cell death using METH‐treated dopaminergic cultured CATH.a cells and METH‐injected mouse brain. In CATH.a cells, METH treatment dose‐dependently increased the levels of quinoprotein (protein‐bound quinone) and the expression of quinone reductase in parallel with neurotoxicity. A similar increase in quinoprotein levels was seen in the striatum of METH (4 mg/kg X4, i.p., 2 h interval)‐injected BALB/c mice, coinciding with reduction of DA transporters. Furthermore, pretreatment of CATH.a cells with quinone reductase inducer, butylated hydroxyanisole, significantly and dose‐dependently blocked METH‐induced elevation of quinoprotein, and ameliorated METH‐induced cell death. We also showed the protective effect of tyrosinase, which rapidly oxidizes DA and DA quinone to form stable melanin, against METH‐induced dopaminergic neurotoxicity in vitro and in vivo using tyrosinase null mice. Our results indicate that DA quinone formation plays an important role, as a dopaminergic neuron‐specific neurotoxic factor, in METH‐induced neurotoxicity, which is regulated by quinone formation‐related molecules.

Parkin attenuates manganese-induced dopaminergic cell death.

Manganese as environmental factor is considered to cause parkinsonism and induce endoplasmic reticulum stress‐mediated dopaminergic cell death. We examined the effects of manganese on parkin, identified as the gene responsible for familial Parkinsons disease, and the role of parkin in manganese‐induced neuronal cell death. Manganese dose‐dependently induced cell death of dopaminergic SH‐SY5Y and CATH.a cells and cholinergic Neuro‐2a cells, and that the former two cell types were more sensitive to manganese toxicity than Neuro‐2a cells. Moreover, manganese increased the expression of endoplasmic reticulum stress‐associated genes, including parkin, in SH‐SY5Y cells and CATH.a cells, but not in Neuro‐2a cells. Treatment with manganese resulted in accumulation of parkin protein in SH‐SY5Y cells and its redistribution to the perinuclear region, especially aggregated Golgi complex, while in Neuro‐2a cells neither expression nor redistribution of parkin was noted. Manganese showed no changes in proteasome activities in either cell. Transient transfection of parkin gene inhibited manganese‐ or manganese plus dopamine‐induced cell death of SH‐SY5Y cells, but not of Neuro‐2a cells. Our results suggest that the attenuating effects of parkin against manganese‐ or manganese plus dopamine‐induced cell death are dopaminergic cell‐specific compensatory reactions associated with its accumulation and redistribution to perinuclear regions but not with proteasome system.

Direct evidence for expression of dopamine receptors in astrocytes from basal ganglia

Expression of dopamine receptors (DA-Rs) in astrocytes was examined in vitro and in vivo using primary cultured astrocytes and brain slices from rat basal ganglia. Astrocytes from basal ganglia expressed DA D1-, D3-, D4- and D5-receptors and D4-mediated signal transduction in response to DA, suggesting possible involvement of astrocytes in the pharmacological action of atypical antipsychotic drugs and in DA response in some neurological diseases.

Neuroprotective effects of zonisamide target astrocyte.

Recent double‐blind, controlled trials in Japan showed that the antiepileptic agent zonisamide (ZNS) improves the cardinal symptoms of Parkinsons disease. Glutathione (GSH) exerts antioxidative activity through quenching reactive oxygen species and dopamine quinone. GSH depletion within dopaminergic neurons impairs mitochondrial complex I activity, followed by age‐dependent nigrostriatal neurodegeneration. This study examined changes in GSH and GSH synthesis‐related molecules, and the neuroprotective effects of ZNS on dopaminergic neurodegeneration using 6‐hydroxydopamine–injected hemiparkinsonian mice brain and cultured neurons or astrocytes.

Methamphetamine-induced neurotoxicity in mouse brain is attenuated by ketoprofen, a non-steroidal anti-inflammatory drug

We examined effects of non-steroidal anti-inflammatory drugs (NSAIDs) on methamphetamine (METH)-induced neurotoxicity. Marked reduction of dopamine transporter-positive signals and accumulation of microglial cells in the striatum after METH injections (4 mg/kg x4, i.p. with 2 h-interval) were significantly and dose-dependently attenuated by four injections of ketoprofen (2 or 5 mg/kg x4, s.c.) given 30 min prior to each METH injection, but not by either a low or high dose of aspirin. The present results suggest that the protective effects of ketoprofen against METH-induced neurotoxicity and microgliosis might be based on its inhibitory activity on inflammatory response or on microglia activation, but not on its cyclooxygenase-inhibiting property. This provides a possible new strategy against METH-induced neurotoxicity using commonly used NSAIDs.