The role of phospholipid methylation in 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced neurotoxicity in PC12 cells

Excessive methylation has been proposed to be involved in the pathogenesis of Parkinson's disease (PD), via mechanisms that involve phospholipid methylation. Meanwhile, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to stimulate phospholipid methylation via the oxidized metabolite, 1-methyl-4-phenyl-pyridinium (MPP+), in the rat brain and liver tissues. In the present study, we investigated the effect of MPP+ on phosphatidylethanolamine N-methyltransferases (PENMT) and the potential role of this pathway in MPP(+)-induced neurotoxicity using PC12 cells. The results obtained indicate that MPP+ stimulated phosphatidylethanolamine (PTE) methylation to phosphatidylcholine (PTC) and correspondingly increased the formation of lysophosphatidylcholine (lyso-PTC). Moreover, the addition of S-adenosylmethionine (SAM) to the cell culture medium increases MPP(+)-induced cytotoxicity. The incubation of 1mM MPP+ and various concentrations of SAM (0-4 mM) decreased the viability of PC12 cells from 80% with MPP+ alone to 38% viability with 4 mM SAM for 4 days incubation. The data also revealed that the addition of S-adenosylhomocysteine (SAH), a methylation inhibitor, offered significant protection against MPP(+)-induced cytotoxicity, indicating that methylation plays a role in MPP(+)-induced cytotoxicity. Interestingly, lyso-PTC showed similar actions to MPP+ in causing many cytotoxic changes with at least 10 times higher potency. Lyso-PTC induced dopamine release and inhibited dopamine uptake in PC12 cells. Lyso-PTC also caused the inhibition of mitochondrial potential and increased the formation of reactive oxygen species in PC12 cells. These results indicate that phospholipid methylation pathway might be involved in MPP+ neurotoxicity and lyso-PTC might play a role in MPP(+)-induced neurotoxicity.     

(-)-Epigallocatechin gallate regulates dopamine transporter internalization via protein kinase C-dependent pathway

Dopamine transporter (DAT) provides not only an integral component of dopaminergic neurotransmission but also a molecular gateway for the accumulation of some neurotoxins such as 1-methyl-4-phenylpyridinium (MPP(+)), a metabolite of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Previous study reported that the neuroprotective effects of green tea polyphenols against MPP(+)-induced neurotoxicity were related to its inhibitory effect on MPP(+) uptake via DAT in dopaminergic cells. To extend the study, we investigated (-)-epigallocatechin gallate (EGCG), a monomer of green tea polyphenols, on DAT internalization in DAT-overexpressed PC12 cells. We found that EGCG (1-100 microM) can induce a dose-dependent inhibition of dopamine uptake in DAT-PC12 cells. In parallel, treatment of EGCG decreased membrane-bound DAT by 15% to 60%. Furthermore, protein kinase C (PKC) inhibitor GF109203X at 2 microM can markedly diminish the inhibitory effects of EGCG on dopamine uptake and reverse the EGCG-induced internalization of DAT. In addition, semiquantitative RT-PCR analysis indicated that EGCG did not affect DAT mRNA expression in the PC12 cells. These data suggest that EGCG exerts its inhibitory effect on DAT by modulating DAT internalization, in which PKC activation may be involved.     

蛇床子素对MPP~＋损伤PC12细胞的保护作用研究

目的观察蛇床子素（osthole）对1-甲基-4-苯基吡啶离子（MPP＋）诱导PC12细胞损伤的神经保护作用。方法将MPP＋加入培养的PC12细胞中,建立多巴胺能神经元损伤模型,加入不同浓度的蛇床子素预处理细胞（0.01、0.05、0.1mmol/L）。处理24h后用噻唑蓝（MTT）比色法检测细胞活性;以乳酸脱氢酶（LDH）活性测定反映细胞的损伤程度;采用Westernblot法检测Bax、Bcl-2蛋白的表达,分析Bax/Bcl-2比值变化,以及检测细胞色素C的改变。结果蛇床子素可以明显减少MPP＋诱导的PC12细胞活性的降低,LDH的释放,Bax/Bcl-2比值的增高以及细胞色素C的释放（P〈0.05）。结论蛇床子素对MPP＋诱导的PC12细胞损伤具有保护作用。     

14-3-3蛋白过表达减轻1-甲基-4-苯基吡啶离子对PC12细胞的毒性

目的 研究14—3—3蛋白过表达对1-甲基-4苯基吡啶离子（MPP^＋诱导的PC12细胞死亡的影响作用及其可能的机制。方法 构建pcDNA3．1（＋）-14—3—3真核表达质粒,用脂质体2000转染PCI2细胞;Westernn blot技术检测PC12细胞中14—3—3蛋白、Bcl-2蛋白,和BAD蛋白的表达;然后分别用MTT法、酶标仪及流式细胞仪检测PC12细胞的活力、caspase的活性及PC12细胞的凋亡率。结果 （1）将pcDNA3．1（＋）-14—3—3质粒转染PCI2细胞3周后,14—3—3蛋白的表达显著增加;（2）MPP^＋诱导PC12细胞存活率的下降是剂量依赖性的,当MPP^＋的浓度达100μmol／L时,PC12细胞的存活率丧失约50％;（3）caspase的活性随着MPP^＋浓度的增加而增高,当MPP^＋浓度到达100μmol／L时caspase的活性也到达最大值,而当MPP^＋浓度超过100μmol／L时,caspase的活性急剧下降;（4）用100μmol／L的MPP^＋处理PC12细胞24h后,PC12细胞的凋亡率为26．5％,14—3—3蛋白的过表达使PC12细胞的凋亡率下降到8．6％;（5）用100μmol／LMPP^＋处理PC12细胞后,Bcl-2蛋白的表达趋于下调而BAD蛋白的表达上调,14—3-3蛋白的过表达能显著的增加Bcl-2蛋白的表达而使BAD蛋白的表达下调。结论 14—3—3蛋白过表达通过上调Bcl-2蛋白的表达并下调BAD蛋白的表达,减少了MPP^＋诱导的PC12细胞的凋亡,从而发挥对PC12细胞的保护作用。这些结果可能为PD的治疗提供新的药物靶点。     

Modulation of 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells by K<Subscript>ATP</Subscript> channel block

The present study investigated the effect of 5-hydroxydecanoate, a selective mitochondrial K(ATP) channel blocker, on the cytotoxicity of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 5-Hydroxydecanoate and glibenclamide (a cell surface and mitochondrial K(ATP) channel inhibitor) reduced the MPP(+)-induced cell death and GSH depletion and showed a maximal inhibitory effect at 5 and 10 microM, respectively. Addition of 5-hydroxydecanoate attenuated the MPP(+)-induced nuclear damage, changes in the mitochondrial membrane permeability and increase in the reactive oxygen species formation in PC12 cells. The results show that 5-hydroxydecanote may prevent the MPP(+)-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the cytochrome c release and caspase-3 activation. This effect appears to be accomplished by the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. The blockade of mitochondrial K(ATP) channels seems to prevent the MPP(+)-induced neuronal cell damage.     

The antioxidant ebselen prevents neurotoxicity and clinical symptoms in a primate model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), via its major metabolite 1-methyl-4-phenylpyridinium (MPP(+)), produces in primates including humans clinical, biochemical, and neuropathological changes similar to those which occur in idiopathic Parkinson's disease. Ebselen is an antioxidant drug with glutathione peroxidase-like activity and a proven neuroprotective action in stroke patients. Here we show that Ebselen, when administered before, during, and after MPTP injections, prevents both neuronal loss and clinical symptoms in a primate MPTP model of Parkinson's disease. Ebselen also prevents peroxide radical overproduction induced by serum withdrawal in cultured PC12 cells and hydroxyl radical generation induced by the mitochondrial toxin, MPP(+), in vivo in rat brain. Moreover, Ebselen inhibits MPP(+)-induced toxicity in PC12 cells, without interacting with the dopamine uptake system. Our results demonstrate that compounds which prevent mitochondrial dysfunction and free radical production may be useful as preventive treatment of Parkinson's disease.     

Toxic effects of MPP and MPTP in PC12 cells independent of reactive oxygen species formation

MPTP is a toxin presumed to damage dopamine-secreting neurons by an oxygen free radical-mediated mechanism. Two steps in MPTP metabolism are the primary candidates for oxygen free radical generation: (a) MPTP oxidation to MPP(+) by a monoamine oxidase and (b) NADH dehydrogenase inhibition by MPP(+). In order to test the idea that MPTP toxicity is mediated by oxygen free radicals, we assessed lipid peroxidation and the effects of antioxidants in dopaminergic PC12 cells treated with MPTP or MPP(+). For comparison purposes, we also examined the effects of the pro-oxidant tert-butyl-hydroperoxide (TBHP) and of the dopaminergic toxin 6-hydroxydopamine (6-OHDA) in PC12 cells. MPTP and MPP(+), unlike TBHP, failed to induce lipid peroxidation in PC12 cells after a 4-h exposure. All toxins tested (MPTP, MPP(+), TBHP and 6-OHDA) caused a dose-dependent decrease in [(3)H]dopamine ((3)H-DA) uptake in PC12 cultures. The hydroperoxide scavengers glutathione and N-acetyl-cysteine and the superoxide and peroxide scavenger EUK-134 protected PC12 cells from TBHP- and 6-OHDA-induced decrease in (3)H-DA uptake. However, no protection by these antioxidants at various concentrations and time regimens was observed against MPTP- or MPP(+)-induced decreases in (3)H-DA uptake in PC12 cells. In addition, incubation of PC12 cells with the energy-rich substrate, NADH, attenuated MPP(+)-induced decrease in (3)H-DA uptake. These results suggest that MPTP-induced toxicity in dopaminergic PC12 cell cultures, does not involve oxygen free radical production, but rather may be caused by impairment in energy metabolism.     

Subtoxic concentration of manganese synergistically potentiates 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells

Endogenous or exogenous substances that are toxic to dopaminergic cells have been proposed as possible cause of idiopathic Parkinson's disease (PD). 1-Methyl-4-phenylpyridinium (MPP(+)) and manganese are dopaminergic neurotoxins causing a parkinsonism-like syndrome. Here, we studied the possible synergistic reaction between these two neurotoxins using rat PC12 pheochromocytoma cells. MPP(+) induced a delayed neurotoxicity in PC12 cells. Although low concentration of manganese did not cause cell damage, it markedly enhanced MPP(+)-induced neurotoxicity with characteristics of apoptosis, such as DNA laddering and activation of caspase-3. To understand the mechanism of enhancement of subtoxic concentration of manganese on MPP(+)-induced neurotoxicity, we investigated the reactive oxygen species (ROS) generation using a molecular probe, 2',7'-dichlorofluorescein diacetate. Although subtoxic concentration of manganese alone did not induce ROS increase, it significantly enhanced the ROS generation induced by MPP(+). We also determined the intracellular MPP(+) content. A time- and concentration-dependent increase of MPP(+) levels was found in PC12 cells treated with MPP(+). The accumulation of MPP(+) by PC12 cells was not affected by manganese. Taken together, these studies suggest that co-treatment with MPP(+) and manganese may induce synergistic neurotoxicity in PC12 cells and that subtoxic concentration of manganese may potentiate the effect of MPP(+) by an ROS-dependent pathway.     

Econazole attenuates cytotoxicity of 1-methyl-4-phenylpyridinium by suppressing mitochondrial membrane permeability transition

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The effect of econazole against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells was assessed in relation to the mitochondrial membrane permeability changes. Treatment of PC12 cells with MPP(+) resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Econazole (0.25-2.5 microM) inhibited the cytotoxicity of MPP(+) or rotenone. The addition of econazole (0.5 microM) significantly attenuated the MPP(+)-induced mitochondrial damage, elevation of intracellular Ca(2+) level and cell death. However, because of the cytotoxicity, econazole at 5 microM did not attenuate the toxicity of MPP(+). The results show that econazole at the low concentrations may reduce the MPP(+)-induced viability loss in PC12 cells by suppressing the mitochondrial permeability transition, leading to activation of caspase-3 and the elevation of intracellular Ca(2+) levels, which are associated with the increased formation of ROS and depletion of GSH.     

Transient receptor potential vanilloid subtype 1 contributes to mesencephalic dopaminergic neuronal survival by inhibiting microglia-originated oxidative stress

The present study examined whether capsaicin (CAP), an agonist of transient receptor potential vanilloid subtype 1 (TRPV1) can prevent 1-methyl-4-phenylpyridinium (MPP(+))-induced dopaminergic (DA) neuronal death in the substantia nigra (SN). Unilateral injection of MPP(+) into the median forebrain bundle of rat brain resulted in a significant loss of nigral DA neurons, assessed by tyrosine hydroxylase (TH) immunostaining. In parallel, activation of microglia, visualized by OX-42 and OX-6 immunostaining were also observed in the SN, where degeneration of nigral neurons was found. By contrast, MPP(+) neurotoxicity was partially inhibited by co-treatment with MPP(+) and CAP. Interestingly, CAP significantly decreased not only immunoreactivity of OX-42 and OX-6 but also production of microglia-derived reactive oxygen species (ROS) in the SN of MPP(+)-treated rats. In experiments designed to further verify effectiveness of CAP against microglia-derived neurotoxicity, CAP inhibited ROS production and blocked MPP(+)-induced death of DA neurons in co-cultures of mesencephalic neurons and microglia, but not in microglia-free, neuron-enriched mesencephalic cultures. This beneficial effect was reversed by capsazepine, an antagonist of TRPV1, expressed in microglia, indicating TRPV1 involvement. Our data demonstrate for the first time that CAP may inhibit microglial activation-mediated oxidative stress via TRPV1, suggesting that CAP and its analogs may have therapeutic value by inhibiting microglial activation and/or ROS generation that occurs in Parkinson's disease.     

Dopamine-releasing action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP) in the neostriatum of the rat as demonstrated in vivo by the push-pull perfusion technique: dependence on sodium but not calcium ions

This study examined the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+) on the levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in push-pull perfusates of the striatum in chloral hydrate-anaesthetized rats. In control animals the levels of DA and DOPAC remained stable for at least 6 h and responded rapidly to a depolarizing stimulus of 25 mM K+. This K+-induced DA release was Ca2+-dependent since no stimulation was observed when the striatal sites were perfused with high K+ in a Ca2+-free medium containing 2 mM EGTA thus verifying that the striatal sites were functionally active. MPTP (0.025 and 0.05 microgram/microliter) stimulated DA release and inhibited DOPAC output in a dose-related manner. MPP+ (0.01, 0.025 and 0.05 microgram/microliter) produced a more robust dose-dependent increase in DA levels in the perfusates; however, the level of suppression of DOPAC was similar to that in response to MPTP. The effect of MPP+ on DA release was attenuated by 10(-6) M benztropine, the DA re-uptake blocker and completely inhibited by 10 micrograms/kg i.p. benztropine and 10(-4) M ouabain, the Na+, K+-ATPase (Na pump) inhibitor. However, although these substances prevented the MPP+-induced release of DA, the levels of DOPAC in the perfusates did not recover and remained completely suppressed suggesting that MPP+ may inhibit extraneuronal rather than intraneuronal monoamine oxidase (MAO). Perfusion of the striatal sites with a Ca2+-free medium containing 2 mM EGTA did not prevent the MPP+-induced DA release indicating that MPP+ does not release DA from the striatal DA terminals by the Ca2+-dependent process of exocytosis. The responses of DA and DOPAC to 25 mM K+ were markedly suppressed in animals treated with MPTP and MPP+, these effects being most severe with the highest dose of MPP+. Moreover, this suppression of the K+-induced responses persisted in animals perfused with MPP+ in the presence of benztropine or ouabain, thus suggesting that MPP+ may have potent deleterious membrane effects. These studies have provided the first direct in vivo demonstration of the action of MPTP and MPP+ and the neuropharmacological basis of this action on DA metabolism in the rat striatum. The results show that the elevated levels of DA in the striatal perfusates are due to a direct action of MPTP and MPP+ on the nigrostriatal DA terminals and cannot be fully accounted for solely by their inhibition of MAO activity and/or inhibition of DA re-uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imidaprilat, an angiotensin-converting enzyme inhibitor exerts neuroprotective effect via decreasing dopamine efflux and hydroxyl radical generation induced by bisphenol A and MPP+ in rat striatum

The present study examined the ability of antioxidant effects of angiotensin-converting enzyme (ACE) inhibitor, imidaprilat, on the synergistic effect of bisphenol A and 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical (*OH) formation and dopamine (DA) efflux in extracellular fluid of rat striatum. Bisphenol A clearly enhanced OH formation and DA efflux induced by MPP(+). When imidaprilat was infused in bisphenol A and MPP(+)-treated rats, DA efflux and OH formation significantly decreased, as compared with that in the bisphenol A and MPP(+) treated control. These results suggest that ACE inhibitors may protect against the synergistic effect of bisphenol A and MPP(+)-induced OH formation via suppressing DA efflux in the rat striatum.     

MPP-induced increases in extracellular potassium ion activity in rat striatal slices suggest that consequences of MPP neurotoxicity are spread beyond dopaminergic terminals

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces symptoms similar to idiopathic Parkinson's disease in primates. A metabolite of MPTP, MPP+ (1-methyl-4-phenylpyridinium), is actively accumulated by dopaminergic (DA) terminals and selectively destroys nigrostriatal DA neurons. The mechanism of this effect remains unknown but reports that MPP+ inhibits electron transport in isolated mitochondria and increases oxidation of cytochrome b in striatal slices suggest that depression of ATP production is involved. To relate metabolic effects of MPP+ with tissue electrophysiology, extracellular potassium ion activity [K+]o was measured by microelectrodes simultaneous to optical monitoring of reduction/oxidation (redox) activity of cytochrome b during superfusion of MPP+ onto rat striatal and hippocampal slices. MPP+ increased oxidation of cytochrome b and increased [K+]o in slices of striatum. These increases were greater than expected from a selective effect of MPP+ on DA terminals which likely comprise no more than 3% of the total striatal mass. These effects of MPP+ were slowed by a dopamine uptake inhibitor (mazindol) and did not occur in hippocampal slices. These findings indicate that MPP+ influences ion transport as well as metabolic activity and that these actions require the presence of functioning DA terminals. However, the large amplitudes of the MPP+-induced changes suggest that consequences of MPP+-neurotoxicity are not ultimately confined to DA terminals. Two hypothesis are proposed: that energy failure in DA terminals results in leakage of neurotoxic substances or metabolites altering membrane conductance properties of adjacent cells and thereby placing additional demand upon ion transport pumps and mitochondrial oxidative phosphorylation; or that there is secondary uptake of MPP+ leading to mitochondrial inhibition in cells neighboring DA terminals.     

Role of CYP2E1 in the mouse model of MPTP toxicity

It has been shown that diethyldithiocarbamate (DDC) potentiates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice as a result of increased levels of 1-methyl-4-phenylpyridinium ion (MPP(+)) in the striatum. Brain CYP2E1 inhibition by DDC in C57Bl mice was responsible for increased toxicity and striatal MPP(+) accumulation. However, CYP2E1-null mice did not show any enhanced sensitivity to MPTP or any MPP(+) accumulation. This unexpected finding suggested that the CYP2E1-null mice compensate with other isozymes as already described for acetaminophen-induced liver damage. MPP(+) intoxication of mesencephalic cell cultures from CYP2E1-null mice indicated a reduced sensitivity of dopaminergic (DA) neurons from knockout animals. Surprisingly, MPP(+) cell distribution under these conditions indicated that the toxin accumulates more intracellularly in knockout cultures, suggesting further that CYP2E1 has a role in MPP(+) storage and efflux.     

Effect of serotonergic, corticostriatal and kainic acid lesions on the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6- tetrahydropyridine (MPTP) in mice

In mice, prior destruction of striatal 5-hydroxytryptamine (5-HT) neurons by intrastriatal or intraventricular injections of 5,7-dihydroxytryptamine did not abolish or attenuate DA depletions produced in striatum by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). This suggests that although they contain monoamine oxidase type B, the oxidative conversion of MPTP to 1-methyl-4-phenyl-pyridinium ion (MPP+) does not take place in 5-HT neurons. Likewise, decortication and kainic acid lesions did not prevent or enhance striatal MPTP-induced DA decrements suggesting that corticostriatal projections and striatal neurons are not involved in the mechanisms of MPTP neurotoxicity.     

H2O2预处理通过ERK1/2和p38 MAPK信号通路上调14-3-3蛋白在PC12

OBJECTIVE: To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP(+)) and to explore the potential mechanisms. METHODS: The viability and apoptosis of PC12 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6'-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phosphorylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2). RESULTS: The cell viability decreased and the number of apoptotic cells increased dramatically in MPP(+) group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP(+)-treated PC12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC12 cells induced by HPP. CONCLUSION: HPP protects PC12 cells against MPP(+) toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.     

Effects on dopamine metabolism of MPTP and MPP+ infused through a push-pull cannula into the caudate nucleus of awake adult male rats

Our previous data demonstrated that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) exerted potent inhibition on endogenous 3,4-dihydroxyphenylacetic acid (DOPAC) output and potent stimulation on endogenous dopamine (DA) release from the rat corpus striatum superfused in vitro. In this report, using a push-pull perfusion technique, we examined in vivo the acute effects of MPTP and MPP+ on DA metabolism in the rat caudate nucleus (CN). MPTP or MPP+ in modified Krebs-Ringer phosphate buffer at concentrations of 10(-6), 10(-5) and 10(-4) M was administered directly into the CN for 15 min, each 90 min apart. Thirty minutes after the infusion of 10(-6) M MPP+, DOPAC output was reduced to a significantly lower value and subsequent infusions of high concentrations of MPP+ further decreased DOPAC output. Homovanillic acid (HVA) output was also decreased by MPP+ infusions, however, at higher concentrations. In respect to DA release, 1 of 10, 4 of 10 and 7 of 10 animals responded with significant increases to 10(-6), 10(-5) and 10(-4) M MPP+, respectively. On the other hand, MPTP was effective in reducing DOPAC output only at 10(-4) M and ineffective in altering DA and HVA output at all doses tested. In addition, neither drugs had a significant effect on 5-hydroxyindoleacetic acid. Accompanying the dramatic changes in DA metabolism caused by MPP+, two uncommon behavioral syndromes were also observed; tremor-body twist and body shaking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-steroidal anti-inflammatory drug sodium salicylate,but not diclofenac or celecoxib,protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats

We evaluated the hydroxyl radical (*OH) scavenging action of nonsteroidal anti-inflammatory drugs (NSAIDs), sodium salicylate (SA), diclofenac and celecoxib in Fenton's reaction and their neuroprotective effects in 1-methyl-4-phenylpyridinium (MPP(+))-induced striatal dopamine (DA) depletion in rats. Salicylate hydroxylation procedure employing HPLC-electrochemistry was used to assay formation of *OH in Fenton's reaction in test tubes. While SA dose- and time-dependently hydroxylated itself and inactivated *OH, celecoxib (up to 10 mM) showed no effect on *OH formation and diclofenac caused a reduction in *OH generation only at high doses (100 microM-10 mM). Administration of the non-selective cyclooxygenase (COX) inhibitor, SA (50, 100 mg/kg, i.p.) significantly attenuated striatal DA depletion caused by intrastriatal infusion of MPP(+) (100 nmol in 4 microl). Treatment with another nonselective, reversible COX inhibitor, diclofenac (5, 10 mg/kg) did not protect against MPP(+)-induced DA depletion. The selective COX-2 inhibitor, celecoxib (2.5-50 mg/kg) treatment exacerbated MPP(+)-induced decrease in DA. Failure of celecoxib or diclofenac to render protection in animals against MPP(+)-induced DA depletion indicates absence of prostaglandin involvement in MPP(+) action. These results also suggest that the neuroprotective ability of SA is independent of prostaglandin mediation. A relationship between inactivation of *OH by SA and its ability to protect DA depletion in the striatum caused by MPP(+) indicates a direct involvement of *OH in the action of this neurotoxin. The present study establishes potent neuroprotective activity of SA and suggests the use of aspirin in adjuvant therapy in Parkinson's disease.     

Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells

A large body of experimental evidence supports a role for oxidative stress as a mediator of nerve cell death in Parkinson's disease. To better understand the cellular insult of oxidative stress on dopaminergic neurons, we studied the cytotoxic effect of the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite, 1-methyl-4-phenyl pyridium (MPP(+)), on several parameters of cell distress using neuronal PC12 cells. We also measured the level of protein expression for the dopamine transporter and the estrogen receptors alpha and beta. Since estrogens have been reported to prevent neuronal degeneration caused by increased oxidative burden, we investigated the ability of 17beta-estradiol, the stereoisomer 17alpha-estradiol, and several phytoestrogens to rescue neuronal PC12 cells submitted to MPP(+)-induced cytotoxicity. Our results consistently show a protective effect of 17alpha-estradiol, 17beta-estradiol and certain phytoestrogens such as quercetin and resveratrol, in neuronal PC12 cells treated with MPP(+). In our cellular paradigm, phytoestrogens coumestrol, genistein, and kaempferol did not revert MPP(+)-induced cellular death. By Western blot, we demonstrated that administration of MPP(+) alone decrease dopamine transporter expression, while treatments with MPP(+) together with 17alpha-estradiol, 17beta-estradiol, quercetin, or resveratrol could restore dopamine transporter protein expression to control levels. Moreover, the same treatments did not modulate alpha estrogen receptor or beta estrogen receptor expression. By these studies, we aim to provide more evidence for the involvement of phytoestrogens in the process of neuroprotection and to test our hypothesis that some of these compounds may act as neuroprotective molecules and have a lesser hormonal effect than estrogens.     

MPP+-induced pathophysiology demonstrates advantages of neurotoxicology studies in brain slices

Since MPTP and its metabolite MPP+ produce nigrostriatal lesions and symptoms similar to Parkinson's disease, recent studies have aimed toward defining their selectivity and neurotoxic mechanisms. In mitochondria in vitro, MPP+ blocked electron transport and decreased oxygen consumption. However, these effects were not selective to striatal mitochondria or even to mitochondria from brain, they required concentrations of MPP+ much greater than those found in vivo, and physiological actions could not be related to intramitochondrial changes. Lower doses of MPP+ did produce highly selective degeneration of dopaminergic (DA) neurons in cell cultures. We report here that MPP+ provoked large (80%) oxidations of cytochrome b and large K+o increments (approximately 30 mM) in rat striatal slices. These effects were slowed by mazindol, which inhibits DA uptake, and were markedly attenuated in rat hippocampal slices which have little DA input. Since DA terminals comprise only 2-4% of the striatal mass, the large MPP+-induced changes suggest that while MPP+ neurotoxicity in brain requires the presence of functioning DA terminals, effects are not confined to these terminals. Such studies illustrate the complexity of MPP+ neurotoxicity and demonstrate the importance of investigations in models such as brain slices with an extracellular space and intracellular relationships as in intact brain.