Biphasic mechanism of the toxicity induced by 1-methyl-4-phenylpyridinium ion (MPP+) as revealed by dynamic changes in glucose metabolism in rat brain slices

1-Methyl-4-phenylpyridinium (MPP+) is a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease. The classical mechanism of MPP+ toxicity involves its entry into cells through the dopamine transporter (DAT) to inhibit aerobic glucose metabolism, while recent studies suggest that an oxidative mechanism may contribute to the toxicity of MPP+. However, it has not been adequately determined what role these two mechanisms play in the development of neurotoxicity after MPP+ loading in the brain. To clarify this issue, MPP+ was added directly to fresh rat brain slices and the dynamic changes in the cerebral glucose metabolic rate (CMRglc) produced by MPP+ were serially and two-dimensionally measured using the dynamic positron autoradiography technique with [(18)F]2-fluoro-2-deoxy-D-glucose as a tracer. MPP+ dose-dependently increased CMRglc in each of the brain regions examined, reflecting enhanced glycolysis compensating for the decrease in aerobic metabolism. Treatment with DAT inhibitor GBR 12909 significantly attenuated the enhanced glycolysis induced by 10 microM MPP+ in the striatum. Treatment with free radical spin trap alpha-phenyl-N-tert-butylnitrone (PBN) significantly attenuated the enhancement of glycolysis induced by 100 microM MPP+ in all brain regions. These results suggest that the mechanism of the toxicity of MPP+ is biphasic and consists of a DAT-mediated mechanism selective for dopaminergic regions at a lower concentration of MPP+ (10 microM), and an oxidative mechanism that occurs at a higher concentration of MPP+ (100 microM) and is not restricted to dopaminergic regions.     

Rat model of Parkinson's disease: chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+)

Mitochondrial dysfunction is observed in sporadic Parkinson's disease (PD) and may contribute to progressive neurodegeneration. While acute models of mitochondrial dysfunction have been used for many years to investigate PD, chronic models may better replicate the cellular disturbances caused by long-standing mitochondrial derangements and may represent a better model for neurotherapeutic testing. This study sought to develop a chronic model of PD that has the advantages of continuous low level toxin delivery, low mortality, unilateral damage to minimize aphagia and adipsia as well as minimal animal handling to reduce stress-related confounds. Infusion by osmotic minipump of the complex I toxin, 1-methyl-4-phenylpyridinium (MPP+), for 28 days into the left cerebral ventricle in rats caused a selective ipsilateral loss of nigral tyrosine hydroxylase immunoreactive somata (35% loss). In animals that were sacrificed 14 days after the chronic MPP+ administration, there was an even greater loss of nigral tyrosine hydroxylase cells (65% loss). Lewy-body-like structures that stained positive for ubiquitin and alpha-synuclein were found in striatal neurons near the infusion site but were not observed in nigral neurons. At the electron microscope level, however, swollen and abnormal mitochondria were observed in the nigral dopamine neurons, which may represent the early formation of an inclusion body. There were no animal deaths with the chronic treatment regimen that was utilized, and the magnitude of nigrostriatal neuronal loss was relatively consistent among the animals. This model of progressive neurodegeneration of nigrostriatal dopamine neurons may be useful for studying neuroprotective therapeutic agents for PD.     

Iron mediates neuritic tree collapse in mesencephalic neurons treated with 1-methyl-4-phenylpyridinium (MPP+)

Studies in post-mortem tissues of patients with Parkinson’s disease (PD) and in mice treated with 6-hydroxydopamine have shown a decrease in the length of axon and dendrites of striatal neurons. However, the etiology of the morphological changes and their relationship to inhibition of mitochondrial complex I and the cellular levels of iron and glutathione (GSH) have not been described. In this study, we characterized the effect of MPP+, an inhibitor of mitochondria complex I, on the integrity of the neuritic tree of midbrain dopaminergic neurons, and determined the influence of iron and cellular levels of GSH on this degeneration. Sub-maximal concentrations of MPP+ induced a drastic dose-dependent reduction of neurites, without modification of the soma or apparent cell death. Concurrent treatment with MPP+ and non-toxic concentrations of iron accelerated the process of degeneration, whereas neurons grown on a medium low in iron showed enhanced resistance to MPP+ treatment. MPP+-induced neurite shortening depended on the redox state of neurons. Pre-treatment with the general antioxidant N-acetyl cysteine protected neurons from degeneration. Treatment with sub-maximal concentrations of the inhibitor of GSH synthesis buthionine sulfoximine (BSO), in conjunction with iron and MPP+, produced massive cell death, whereas treatment with BSO plus MPP+ under low iron conditions did not damage neurons. These results suggest that under conditions of inhibition of mitochondrial complex I caused by MPP+, the accumulation of iron and the concurrent decrease in GSH results in the loss of the dendritic tree prior to cell death, of dopaminergic neurons in PD.     

Attenuation of 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity by deprenyl in organotypic canine substantia nigra cultures

Summary Systemic administration of MPTP to experimental animals induces neurodegeneration of dopaminergic neurons in the central nervous system. MPTP crosses the blood-brain barrier where it is taken up by astrocytes and converted to MPP⁺ by monamine oxidase-B (MAO-B). Subsequently, MPP⁺ is selectively taken up by dopaminergic neurons upon which it exerts intracellular neurotoxic effects. Systemic administration of the selective MAO-B inhibitor deprenyl prevents the conversion of MPTP to MPP⁺ and by this mechanism is able to protect against MPTP neurotoxicity. Deprenyl has also been reported to exert neuroprotective effects that are independent of its MAO-B inhibitory properties, but since MPP⁺ itself does not cross the blood-brain barrier it is difficult to directly study the MAO-B independentin vivo effects of MPP⁺ itself. One approach is to use organotypic tissue cultures of the canine substantia nigra (CSN) which permit administration of precise concentrations of pharmacological agents directly to mature, well-developed and metabolically active dopaminergic neurons. These neurons as well as other components of the cultures exhibit morphological and biochemical characteristics identical to theirin vivo counterparts. This study was undertaken to evaluate the neuroprotective effects of deprenyl in MPP⁺-treated cultures by measuring changes in the levels of HVA as an indicator of dopamine release and metabolism by dopaminergic neurons and to correlate this indication of dopaminergic function with morphological evidence of survival or loss of dopaminergic neurons in mature CSN cultures. Mature CSN cultures, at 44 days in vitro (DIV), were exposed to either MPP⁺ alone, deprenyl alone or simultaneously to both deprenyl and MPP⁺ or to MPP⁺ following 4 day pretreatment with deprenyl. Exposure to MPP⁺ alone caused significant reduction in HVA levels, evidence of widespread injury and ultimate disappearance of large neurons in the cultures. These effects were attenuated by simultaneous exposure to MPP⁺ and deprenyl and the destructive effects of MPP⁺ appeared to be prevented by pretreatment with deprenyl. Thus the neuroprotective effects of deprenyl on MPP⁺-induced reduction of HVA levels in living cultures appears similar to the effects of deprenyl on dopamine levels and tyrosine hydroxylase activity reported by others in cultures previously exposed to deprenyl and MPP⁺. These studies also confirm that the neuroprotective effects of deprenyl against MPP⁺ in dopaminergic neurons are, at least in part, independent of deprenyl's inhibition of MAO-B.     

A model of chronic neurotoxicity: long-term retention of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) within catecholaminergic neurons

The mechanism by which 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces lesions in the nigrostriatal dopamine system has been extensively studied. MPTP, a lipophilic molecule, enters the brain rapidly where it is converted to the pyridinium metabolite 1-methyl-4-phenylpyridinium (MPP+), by a two-step reaction that requires the enzyme monoamine oxidase. Following this conversion, which occurs primarily in astrocytes, MPP+ is sequestered within monoaminergic neurons by the energy-requiring monoaminergic transporters. Inside the neuron, MPP+ is thought to act as a mitochondrial toxin, slowly sapping the neuron of its energy-producing potential by blocking the action of NADH dehydrogenase. Much attention has been focused on cell death after MPTP administration, but little attention has been paid to the effects of small subtoxic doses of MPTP (i.e., doses that do not produce overt neuropathologic changes), which might occur during environmental exposure to a nigrostriatal toxin. Low doses of MPTP (as little as 1/25th of a toxic dose) produce long-term (greater than 6 weeks) but reversible changes in catecholamine metabolism. These changes are characterized by a decrease in the products of enzymatic oxidative deamination without a concomitant decrease in the amine concentrations (apparent MAO inhibition). Striatal concentrations of MPP+, which is retained in catecholaminergic terminals for similarly long periods, parallel the metabolic changes. Thus, the long-term storage of the MPTP metabolite, MPP+, correlates with altered catecholamine metabolism. The data on the effects of MPTP have been combined into a working model of how MPP+ exerts its effects following subtoxic or toxic doses. The site of this long-term neuronal storage of MPP+ after exposure to subtoxic doses of MPTP is as yet undetermined, but several studies suggest that monoaminergic vesicles may be the primary site, with mitochondria contributing some storage capacity. This vesicular site could represent a potential brain site for the accumulation of toxins during continual or repeated exposure to low levels of MPTP. Induced release from this site might accelerate the toxic interactions with cellular components such as mitochondria.     

Acute effects of 1-methyl-1,4-phenylpyridinium ion (MPP+) on purine metabolism in rat striatum studied in vivo using the microdialysis technique

In order to obtain further insight into the interactions between the purinergic and dopaminergic pathways in the striatum, we studied both metabolisms simultaneously, using a microdialysis technique in 1-methyl-1,4-phenylpyridinium ion (MPP+) unilaterally-denervated conscious rats. In these rats the contralateral side was used as control. The perfusates were collected every 20-25 min using 4 mm dialysis probes, implanted in each striatum, and assayed for dopamine and purine metabolites. After MPP+ administration, all adenosine metabolites - with the exception of uric acid - and dopamine levels were significantly increased in the extracellular medium. However, the time-course change in dopamine level did not correlate with the adenosine and inosine time-courses, suggesting a different mechanism of liberation in response to MPP+ administration.     

Effect of 1-methyl-4-phenylpyridinium (MPP+) on mitochondrial membrane potential in cerebellar neurons: Interaction with the NMDA receptor

Summary The effect of MPP⁺, a dopaminergic neurotoxin, in mitochondrial membrane potential was investigated in dissociated cerebellar granule cells using rhodamine 123 and flow cytometry. MPP⁺ (1 mM) decreased the mitochondrial membrane potential by 30%. Antagonists of the NMDA receptor complex, such as MK-801 (IC₅₀ value of 20.92 ± 0.02 nM), 5,7-dichlorokynurenic acid (IC₅₀ value of 6.46 ± 1.06 M) and D-AP5 (IC₅₀ value of 8.29 ± 0.63 M), inhibited the action of MPP⁺. Neither NBQX, nor riluzole, nor desipramine modified the action of MPP⁺. Dibucaine restored the basal values of mitochondrial membrane potential altered by MPP⁺. Since, in the presence of NMDA, MPP⁺ antagonized the effect of this total agonist, it can be concluded that, in this preparation, MPP⁺ interacts with the NMDA receptor complex as a partial agonist. This interaction could be the result of an allosteric modulation of the NMDA receptor complex by MPP⁺. The decrease of mitochondrial membrane potential induced by MPP⁺ is antagonized by dibucaine, suggesting that this effect is mediated by an activation of phospholipase A₂.     

Metabolic effects of 1-methyl-4-phenylpyridinium (MPP(+)) in primary neuron cultures

Disruption of mitochondrial function has been proposed as an action of 1-methyl-4-phenylpyridinium (MPP(+)) that is responsible for its toxicity. In order to characterize effects of MPP(+) on energy metabolism in primary culture neurons, we monitored levels of several metabolites in cultured rat cerebellar granule cells exposed to MPP(+). The toxin produced a rapid concentration-dependent reduction in intracellular phosphocreatine (PCr), amounting to a 50-80% decrease within 30-60 min at 50 microM, that was maintained through the 1 week exposure interval examined. In contrast, ATP levels remained comparable to those of untreated neurons for approximately 4 days, at that time a 50% reduction in ATP was observed in association with a decrease in cell viability. Acute decreases in PCr were accompanied by increases in creatine such that the total creatine levels were maintained. Lactate levels in the culture medium were significantly increased (from 4.5 to 6.0 mM) within 6 hr after addition of MPP(+), with a concentration dependence similar to that observed for the reduction in PCr. Increased lactate production in the presence of MPP(+) coincided with a more rapid depletion of glucose in the culture medium. MPP(+) induced a rapid and sustained decrease in intracellular pH calculated from the creatine kinase equilibrium, and this acidification is considered primarily responsible for the observed decrease in PCr. These studies provide direct evidence that toxic concentrations of MPP(+) have acute effects on energy metabolism in primary culture neurons, consistent with an increased dependence on glycolysis to meet metabolic demand, but indicate that toxicity is not associated with overt, immediate failure to maintain cellular ATP.     

Ultrastructural alterations induced by 1-methyl-4-phenylpyridinium (MPP+) in canine substantia nigra and rat mesencephalon in vitro

Explants of canine substantia nigra (SN) and rat mesencephalon (MES), grown in organotypic culture, were incubated with 1-methyl-4-phenylpyridinium (MPP+) and examined for ultrastructural changes. Prolonged exposure (3 days) to doses ranging from 0.1 nM to 10 microM MPP+ resulted in total destruction of all constituents (neuronal and glial) of canine SN cultures. No association was noted between MPP+-induced toxicity and age of canine SN cultures. The first ultrastructural change observed in canine SN cultures incubated with 0.1 nM MPP+ was at 3 h. Grossly swollen mitochondria were noted in large nerve cells. Swollen mitochondria were present in all cells of canine SN cultures by 8 h of incubation with MPP+. Only those rat MES cultures with relatively high preincubation levels of homovanillac acid, determined as an index of viable dopaminergic neurons, incubated with MPP+ (10 microM) for up to 8 days exhibited ultrastructural changes, namely, a swelling of mitochondria within the cytoplasm of large nerve cells. These findings suggest that continual exposure to MPP+ in vitro results in a generalized, nonspecific toxicity in those species known to be susceptible to the parent compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in vivo. However, the initial ultrastructural change, i.e., a swelling of mitochondria, may be the same in all species regardless of sensitivity suggesting that the ultimate mechanism underlying MPP+-toxicity relates to mitochondrial function.     

Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells

Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson's disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP+ initiates oxidative stress to cause caspase-3-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP+-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 neuronal cells express key NADPH oxidase subunits gp91phox and p67phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000microM), apocynin (100-1000microM), and diphenylene iodonium (DPI, 3-30microM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300microM MPP+-induced ROS production for over 45min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and SOD also blocked MPP+-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24h significantly suppressed MPP+-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP+-induced increases in caspase-3 enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP+-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP+-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.     

Acute effects of 1-methyl-4-phenylpyridinium ion (MPP+) on dopamine and serotonin metabolism in rat striatum as assayed in vivo by a micro-dialysis technique

Summary The acute effect of 1-methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin derived from 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), was examined by the in vivo micro-dialysis technique. A dialysis cannula was implanted into rat striatum, and the changes in the concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the perfusate every 20 min after administration of MPP⁺ were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED). After MPP⁺ administration the levels of DOPAC, HVA and 5-HIAA were markedly decreased. On the contrary the level of DA was markedly increased and reached a maximum 40 min after beginning of the MPP⁺ administration. By postmortem analysis of the striatal tissue MPP⁺ was proved to cause the inhibition of monoamine oxidase (MAO), especially MAO-B. These results suggest that the acute biochemical changes induced by MPP⁺ in vivo were MAO inhibition and release of DA.     

Inhibition of the neuronal isoform of nitric oxide synthase significantly attenuates 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in vitro

Summary. The possible protection against the toxicity of 1-methyl-4-phenylpyridinium (MPP⁺) afforded by inhibitors of nitric oxide synthase (NOS) and the antagonist of N-methyl-D-aspartate receptor function, MK-801, was studied in a brain-slice superfusion system. Significant decreases in levels of dopamine and its metabolites 3,4-dihyroxyphenylacetic acid (DOPAC) and homovanillic acid were observed following incubation of slices with 25 μM MPP⁺. The activity of intracellular lactate dehydrogenase (LDH), a marker of cell viability, was also significantly decreased. These effects were attenuated by preincubation with I mM 7-nitroindazole (7NI), a selective inhibitor of the neuronal isoform of nitric oxide synthase (NOS). In contrast, the nonspecific NOS inhibitor N^ω-nitro-l-arginine, also at 1 mM, had no effect on levels of dopamine metabolites but did show a small attenuation of the levels of dopamine. 7NI alone caused some increase in levels of dopamine and a decrease in the metabolite DOPAC, which is consistent with it also acting as an inhibitor of monoamine oxidase-B. MK-801 afforded no significant protection of aminergic cells, although changes in LDH activity suggested that there may have been some protection of non-aminergic neurons affected by this, relatively high concentration of MPP⁺. Received December 10, 2001; accepted February 21, 2002     

MALDI Mass Spectrometry Imaging of 1-Methyl-4-phenylpyridinium (MPP+) in Mouse Brain

Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting ~1 % of the population older than 60 years. The administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice is one of the most widely used approach to elucidate the mechanisms of cell death involved in PD. Its toxicity is attributed to its active metabolite 1-methyl-4-phenylpyridinium (MPP⁺). However, the magnitude of the PD-like neurodegeneration induced by MPTP depends on many variables, including the route of administration. Different groups, including us, demonstrated that intranasal (i.n.) administration of MPTP constitutes a new route of toxin delivery to the brain that mimics environmental exposure to neurotoxins. In particular, our previous data showed that mice submitted to acute i.n. MPTP administration displayed a significant decrease of striatal dopamine (DA) and a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. However, little is known about the timing and the anatomical distribution of MPP⁺ after i.n. MPTP administration in mice. In the present study, C57BL/6J mice received one dose of i.n. MPTP (1 mg/nostril) and were sacrificed at two different times after the administration. Using matrix-assisted laser desorption–ionization mass spectrometry imaging, a new technique for the detection of endogenous unlabeled molecules in tissue sections, we showed for the first time the MPP⁺ anatomical distribution in different brain regions. We demonstrated that the toxin first reached almost all the brain areas; however, in a second time MPP⁺ remained highly concentrated in the olfactory bulb, the basal ganglia, the ventral mesencephalon, and the locus coeruleus, regions differently affected in PD.     

1-Methyl-4-phenylpyridinium (MPP+) increases oxidation of cytochrome-b in rat striatal slices

Effects of 1-methyl-4-phenylpyridinium, (the active metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), on reduction/oxidation activity of mitochondrial cytochromes were studied in rat striatal slices using scanning spectrophotometry. The objective was to test the hypothesis that the neurotoxin alters electron transport in the mitochondrial respiratory chain. Incubation of rat striatal slices with MPP+ (1 microM) produced a time-dependent oxidation of Cytochrome-b in a manner consistent with the concept of a block in electron transport in the intramitochondrial respiratory chain between nicotinamide adenine dinucleotide (NAD) and Cytochrome-b. This effect of MPP+ was decreased by co-incubation with a potent dopamine uptake inhibitor (mazindol), or when studied in a tissue with low dopaminergic innervation (hippocampus). The amplitude of Cytochrome-b oxidation was greater than that expected from a selective effect of MPP+ on dopaminergic neurons suggesting that neighboring cells are influenced secondary to the MPP+ effect on dopaminergic terminals.     

热休克蛋白减轻1-甲基-4-苯基-吡啶离子引起的线粒体功能障碍和氧化应激

目的观察热休克蛋白(HSP)对1-甲基-4-苯基-吡啶离子(MPP )引起的线粒体功能障碍和氧化应激的保护作用。方法采用免疫印迹法观察热休克诱导HSP的表达以及转染的HDJ-1基因在细胞内的过表达。通过5,5′,6,6′-四氯-1,1′,3,3′-四乙基苯丙咪唑羰花青碘化物(JC-1)和2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)流式细胞术及荧光显微镜观察MPP 对细胞线粒体膜电势和活性氧族(ROS)的影响以及HSP的保护作用。结果热休克后4h即有Hsp70(7.37±1.17)和HDJ-1(2.32±0.37)增加,并至少持续到72h;同样,转染24h后HDJ-1基因在细胞内过表达(1.26±0·06),并至少持续到72h。MPP 能引起线粒体膜电势降低(60.77±3.68),同时细胞内ROS上升(483.18±16.98)。热休克和HDJ-1基因过表达不仅能维持线粒体膜电势(热休克组68.32±3.42,转HDJ-1组66.13±3.31),而且还能抑制ROS的产生(热休克组449.45±18.80,转HDJ-1组470.56±23.53),其中热休克的作用更强。结论HSP通过保护线粒体功能、减少氧化应激来减轻MPP 毒性,从而发挥保护细胞的作用。     

Calcium overload enhances hydroxyl radical generation by 1-methyl-4 phenylpyridinium ion (MPP+) in rat striatum

We examined whether ouabain-induced Ca(2+) overload increases hydroxyl radical (*OH) generation by 1-methyl-4-phenylpyridinium ion (MPP(+)) in rat striatum. These elevations seem to induce lipid peroxidation of striatum of rats, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. Ouabain enhanced MPP(+)-induced *OH formation trapped as DHBA. Moreover, when iron (II) was administered to MPP(+) then ouabain (100 micro M)-pretreated animals, a marked elevation in the level of DHBA was observed, as compared with the iron (II)-only-treated animals. These results suggests that Ca(2+) overload might enhance *OH generation by MPP(+) in rat striatum.     

1-methyl-4-phenylpyridinium (MPP+) decreases mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) in rat striatum

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP+) into rat striatum. MPP+ (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Deltapsi(m) at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP+ (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP+ (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP+ (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Deltapsi(m) may play a role in MPP+ -induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.     

Differences in the disposition and toxicity of 1-methyl-4-phenylpyridinium in cultured rat and mouse astrocytes

Species difference in the susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was investigated in cultured rat and mouse astrocytes, where 1-methyl-4-phenylpyridinium (MPP⁺), the toxic mediator of MPTP, is formed. Type A monoamine oxidase (MAO) predominated in both rat and mouse astrocytes, while its activity was slightly higher in mouse cells compared to rat cells; MAO-B activity, on the other hand, was significantly lower in mouse astrocytes than in rat astrocytes. Because both types of MAO have been reported to make similar contributions to MPP⁺ production in astrocytes, their total activity was examined and results indicated that there was no significant difference between these two species. In additon, MPP^± caused a dose dependent loss of cell viability as judged by the amount of lactate dehydrogenase released into the incubation medium. The toxicity of MPP^± on astrocytes started to be seen after a 2 day incubation period. Mouse astrocytes were more vulnerable to MPP^± than rat astrocytes. The threshhold values for MPP^± toxicity in mouse and rat cultures were 10 ±M and 70 ±M, respectively. After addition of [³H] MPP^± to the medium, intracellular [³H] MPP^± was found to increase in both cultures. Mouse astrocytes accumulated more MPP^± than rat astrocytes (150 pmol/mg protein vs. 65 pmol/mg protein). When astrocytes were allowed to accumulate [³H] MPP^± and then incubated in fresh medium medium not containing [³H] MPP^±, intracellular levels of [³H] MPP^± in both cells rapidly declined (110 pmol/protein in mouse vs. 40 pmol/mg protein in rat of MPP^± been released). These results indicated that (1) MPP^± could cross the plasma membrane of astrocytes despite of its charged chemical structure, (2) mouse astrocytes had a higher capacity for MPP^± accumulation (approximately 2-fold), as well as release (approximately 2.7-fold), than rat astrocytes, and (3) mouse astrocytes were more vulnerable to MPP^± than rat astrocytes.     

Death by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP) and protection by EGF in GH3 cells

In this study we investigated the uptake and effect of a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP⁺) on a clonal strain, GH3 cells, established from rat anterior pituitary. Although the level was very low compared with that in PC12 cells, a clonal rat pheochromocytoma cell line, there was a detectable amount of tyrosine hydroxylase protein in GH3 cells. The levels of monoamines including dopamine in GH3 cells were also very low compared with those in PC12 cells.

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was incorporated to GH3 cells in a concentration-dependent manner and the uptake was inhibited by nomifensine, an inhibitor of dopamine transporter. Addition of 200 μM MPP⁺ stimulated the leakage of lactate dehydrogenase (LDH) after a lag of 24 h. Pretreatment with 50 ng/ml of epidermal growth factor (EGF), but not nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), protected against MPP⁺-induced cell death. These findings show that: (1) MPP⁺ uptake to GH3 cells was via an effective dopamine transport system and causes delayed cell death, and (2) EGF protects against MPP⁺-induced cell death. A possible role for GH3 cells as dopaminergic neurons is discussed.     

1-Methyl-4-phenylpyridinium (MPP+) increases oxidation of Cytochrome-b in rat striatal slices

Effects of 1-methyl-4-phenylpyridinium, (the active metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), on reduction/oxidation activity of mitochondrial cytochromes were studied in rat striatal slices using scanning spectrophotometry. The objective was to test the hypothesis that the neurotoxin alters electron transport in the mitochondrial respiratory chain. Incubation of rat striatal slices with MPP+ (1 μM) produced a time-dependent oxidation of Cytochrome-b in a manner consistent with the concept of a block in electron transport in the intramitochondrial respiratory chain between nicotinamide adenine dinucleotide (NAD) and Cytochrome-b. This effect of MPP+ was decreased by co-incubation with a potent dopamine uptake inhibitor (mazindol), or when studied in a tissue with low dopaminergic innervation (hippocampus). The amplitude of Cytochrome-b oxidation was greater than that expected from a selective effect of MPP+ on dopaminergic neurons suggesting that neighboring cells are influenced secondary to the MPP+ effect on dopaminergic terminals.