Environmental Estrogen-Like Chemicals and Hydroxyl Radicals Induced by MPTP in the Striatum: A Review

Oxygen free radical formation has been implicated in lesions caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP⁺) by type B monoamine oxidase (MAO) in the brain, the etiology of this disease remains obscure. This review focuses on the role of an environmental neurotoxin chemically related to MPP⁺-induced free radical generation in the pathogenesis of Parkinson's disease. Environmental-like chemicals, such as para-nonylphenol or bisphenol A, significantly stimulated hydroxyl radical (OH) formation in the striatum. Allopurinol, a xanthine oxidase inhibitor, prevents para-nonylphenol and MPP⁺-induced OH generation. Tamoxifen, a synthetic nonsteroidal antiestrogen, suppressed the OH generation via dopamine efflux induced by MPP⁺. These results confirm that free radical production might make a major contribution at certain stages in the progression of the injury. Such findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson's disease and traumatic brain injuries.     

Nitroindazole compounds inhibit the oxidative activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to neurotoxic pyridinium cations by human monoamine oxidase (MAO)

Monoamine oxidase (MAO) B is a mitochondrial enzyme selectively involved in the oxidative activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to toxic pyridinium cations producing Parkinsonism in animal models. Various synthesized 5-nitroindazoles, 6-nitroindazole and the neuroprotectant 7-nitroindazole were examined as inhibitors of MAO and as antioxidants and radical scavengers. The oxidation of MPTP by human MAO-B and mitochondria was assessed by HPLC. Simple nitroindazoles inhibited MPTP oxidation to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) in a competitive and reversible manner. 5-Nitroindazole (IC₅₀=0.99 µM, K_i=0.102 µM) and 6-nitroindazole (IC₅₀=2.5 µM) were better inhibitors of human MAO-B than 7-nitroindazole (IC₅₀=27.8 µM). 6-Nitroindazole also inhibited MAO-A. Nitroindazole isomers were good hydroxyl radical (OH^?) scavengers, with 5-nitro-, 6-nitro- and 7-nitroindazole showing similar activity (k ~10¹⁰ M^?1 s^?1). Neuroprotective actions of nitroindazoles (7-nitroindazole) could be linked to their MAO-inhibitory and antiradical properties besides inhibition on nitric oxide synthase (NOS). 5-Nitro- and 6-nitroindazole, previously reported as weak NOS inhibitors, were better inhibitors of human MAO-B and more active against MPTP neurotoxin oxidation (lower MPDP⁺ and MPP⁺ levels) than 7-nitroindazole and acted as good radical scavengers and could be potential neuroprotective agents in addition to MAO-B inhibitors.     

2-Deoxyglucose Enhances 1-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine-Induced ATP Loss in the Mouse Brain

Abstract: The effects of 2-deoxyglucose (2-DG), an inhibitor of the uptake and use of glucose, on ATP loss caused by the neurotoxicant 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) were determined in the mouse brain. 2-DG alone had no effect on brain ATP levels, but when administered 30 min before MPTP exposure, 2-DG significantly enhanced MPTP-induced ATP reduction. This was reflected as an increase in ATP loss in the striatum (from 15 to 27%) as well as a significant decrease in ATP in the cerebellar cortex, an area of the brain that was not affected after exposure to MPTP alone. In mice pretreated with 2-DG, striatal ATP levels remained significantly decreased for >8 h after MPTP administration. In contrast, ATP levels in the cerebellar cortex returned to normal values within 4 h from MPTP exposure. Mazindol, a catecholamine uptake blocker, completely protected against MPTP-induced loss of striatal ATP in the absence of 2-DG, but it only partially prevented striatal ATP decrease after administration of both 2-DG and MPTP; mazindol was also ineffective in protecting against ATP loss caused by 2-DG and MPTP in the cerebellar cortex. 2-DG/MPTP-induced ATP loss appeared to be associated with the presence of the 1 -methyl-4-phenylpyridinium (MPP⁺) metabolite because (1) the pattern of ATP recovery in the striatum and cerebellar cortex appeared to reflect the pattern of MPP⁺clearance from these areas of the brain (i.e., significant MPP⁺ levels persisted longer in the striatum than in the cerebellar cortex), and (2) ATP decrease was completely prevented by blocking the conversion of MPTP to MPP⁺with the monoamine oxidase B inhibitor deprenyl. Data indicate that impairment of glucose metabolism dramatically enhances the effects of MPTP/MPP⁺ on cerebral energy supplies, making these effects relatively nonselective for dopaminergic neurons of the nigrostriatal pathway.     

Involvement of Hepatic Aldehyde Oxidase in Conversion of 1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+) to 1-Methyl-4-phenyl-5,6-dihydro-2-pyridone

To obtain direct evidence of the involvement of aldehyde oxidase (AO), a cytosolic molybdoflavoenzyme, in the metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we investigated thein vitrometabolism of MPTP and the two-electron-oxidized 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP⁺) by using mouse liver enzyme preparations. Incubation of MPTP with mitochondrial fraction gave exclusively 1-methyl-4-phenylpyridinium (MPP⁺); this reaction was inhibited by deprenyl, a monoamine oxidase (MAO)-B inhibitor, and KCN. When the mitochondrial fraction was combined with the cytosolic fraction, MPP⁺formation was markedly decreased, while a large amount of 1-methyl-4-phenyl-5,6-dihydro-2-pyridone (MPTP lactam) was newly formed. Incubation of MPDP⁺with the cytosolic fraction led to rapid formation of MPTP lactam with concomitant disappearance of the substrate. The cytosol-dependent formation of MPTP lactam was inhibited by known AO inhibitors, such as menadione, norharman, and KCN. The activity of cytosol in MPTP lactam formation was completely duplicated by purified mouse liver AO. These results indicate that AO catalyzes the metabolic conversion of MPDP⁺, produced from MPTP by MAO-B, to MPTP lactam. This metabolic pathway might be an important detoxification route, averting the formation of toxic MPP⁺.     

Striatal and Urinary DOPAC/DA Ratio May Indicate a Long-Lasting DA Release Enhancement by MPP+ and MPTP

The DOPAC/DA ratio in mouse striatum, in striatal synaptosomes, and in rat urine after MPP⁺ and MPTP neurotoxin administrations to the animals was followed temporally. The neurotoxins were given intraperitoneally and, in some experiments, to enhance the sensitivity, the animals were subsequently reserpinized before either sacrifice or 24 hour urine collection. MPP⁺ treatment, followed by saline, weakly lowered mouse striatal DOPAC/DA ratio up to 6 hours; in reserpinized animals, however, the neurotoxin reduced striatal ratio potently and for longer periods. Similarly, MPP⁺ reduced rat (saline treated) urinary DOPAC level and DOPAC/DA ratio in the short term (1.0 hr) while the neurotoxin effects could still be detected following longer periods up to 27 days in reserpinized animals. A single MPTP treatment (90 min.), followed by preparation of striatal synaptosomal fraction and its incubation (37°C) with or without reserpine, also led to a reduced DOPAC/DA ratio. Although mainly the pooled peripheral effect is directly indicated by urinary DOPAC/DA ratio, MPP⁺ may reduce DA oxidation in the CNS and may similarly affect the amine oxidation in the peripheral tissues. The CNS and peripheral effects differ, however, in respect to dose-sensitivity and time course. The similarities between the CNS and peripheral effects suggest that a blunted rise of urinary DOPAC/DA ratio after reserpine challenge could be utilized as a peripheral marker of MPP⁺ action in the CNS, a marker that is not currently available.     

Studies on the Characterization of the Inhibitory Mechanism of 4'-Alkylated 1-Methyl-4-Phenylpyridinium and Phenylpyridine Analogues in Mitochondria and Electron Transport Particles

Abstract: 1-Methyl-4-phenylpyridinium (MPP⁺), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′-alkylated analogues of MPP⁺. These derivatives had IC₅₀ values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC₅₀ values of corresponding 4′-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ～600-fold, versus only a 36-fold, increase in the IC₅₀ of MPP⁺ versus 4′-pentyl-MPP⁺, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′-decyl-MPP⁺ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol. In addition, the lipophilic anion TPB^? was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.     

Uptake of N-methyl-4-phenylpyridinium ion (MPP+) into PC12h pheochromocytoma cells

The uptake and accumulation of N-methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin produced by oxidation of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), into PC12h pheochromocytoma cells were examined. Concentration gradients of MPP⁺ were established at its low concentrations of 10 to 100 nM. Uptake of MPP⁺ into PC12h cells was mediated by saturable, carrier mediated transport systems with two different kinetic properties; a high-affinity and low-capacity system and a low-affinity and high-capacity system. The apparent K_m values of these two systems were obtained to be 254.4 ± 96.5 nM and 23.1 ± 6.9 μM, respectively, and the maximal uptake velocity was obtained to be 8.47 ± 1.72 and 28.6 ± 5.2 pmol/min/mg protein, respectively. The uptake by a high-affinity system was mediated by a carrier system common to dopamine and noradrenalin and MPTP itself proved to be taken up by this system, which was further confirmed by the inhibition of the MPP⁺ uptake by nomifensine and mazindol. The uptake was inhibited by metabolic inhibitors, such as carbonyl cyanide m-chlorophenyl hydrazone, sodium cyanide and 2,4-dinitrophenol, and the uptake was inhibited by ouabain and nigercin. By subcellular fractionation, MPP⁺ taken up was found to be localized mainly in cytosol fraction, but a definite amount of MPP⁺ was found also in mitochondrial fraction.     

AMP-activated protein kinase is activated in Parkinson’s disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP⁺) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP⁺ in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP⁺-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP⁺ in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.     

2′,3′-Dideoxycytidine Protects Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

DNA polymerase-β (DNA pol-β) plays a crucial role in the pathogenesis of Parkinson’s disease (PD). The aim of this study was to investigate the neuroprotective effects of a DNA polymerase-β inhibitor 2′,3′-dideoxycytidine (DDC) in PD models. In the in vitro studies, primary cultured neurons were challenged with 1-methyl-4-phenylpyridinium ion (MPP⁺). The expression of DNA pol-β was assessed using western blot. The neuroprotective effect of DNA pol-β knockdown and DNA pol-β inhibitor DDC was determined using cell viability assay and caspase-3 activity assay. We found that MPP⁺ induced neuronal death and the activation of caspase-3 in a dose-dependent manner. The expression of DNA pol-β increased after the neurons were exposed to MPP⁺. DNA pol-β siRNA or DNA pol-β inhibitor DDC attenuated neuronal death induced by MPP⁺. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, MPTP treatment triggered behavioral deficits and nigrostriatal lesions. Pretreatment with DDC attenuated MPTP-induced behavioral deficits, dopaminergic neuronal death and striatal dopamine depletion in the MPTP mouse model. These results indicate that DNA pol-β inhibitors may present a novel promising therapeutic option for the neuroprotective treatment of PD.

     

Neuroprotective Effect of Acute and Chronic Administration of Copper (II) Sulfate against MPP+ Neurotoxicity in Mice

Neurodegenerative effects of MPP⁺, the main metabolite of MPTP include dopamine (DA) depletion and enhanced lipid peroxidation (LPO) in mice striata, both associated to free radicals overproduction. Since copper is related to several antioxidant enzymes, we tested its neuroprotective effect against MPP⁺-induced neurotoxicity (20 g/3 l). CuSO₄ pretreatment was administrated by either acute (2.5 mg/kg, i.p) or chronic (350 or 700 mg/l doses through drinking water, for 30 days) schemes. Acute administration blocked MPP⁺-induced striatal LPO only when administered 16 or 24 hours before MPP⁺, and prevented the DA-depleting effect only at 24 hours. Chronic CuSO₄ prevented the LPO increase, and blocked the DA depletion only at the higher dose used (700 mg/l). Neuroprotective effect of CuSO₄ was dependent on the dose and the time of pretreatment, which suggest that this lag could be related with mechanisms of activation or synthesis of copper-dependent proteins responsible of cellular defense against MPP⁺.     

Inhibition of mitochondrial respiration by 1,2,3,4-tetrahydroisoquinoline-like endogenous alkaloids in mouse brain

Since the discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism, it has been postulated that (a) MPTP-like toxin(s) such as 1,2,3,4-tetrahydroisoquinoline (TIQ) may induce Parkinson's disease. As the neuronal degeneration in MPTP-induced parkinsonism is thought to be caused by the inhibition of the mitochondrial respiration by 1-methyl-4-phenylpyridinium ion (MPP⁺), we studied the effects of TIQ-like alkaloids including dopaminederived ones on the mitochondrial respiration using mouse brains. TIQ, tetrahydropapaveroline (THP), and tetrahydropapaverine (THPV) produced significant inhibition of the state 3 and 4 respiration and respiratory control ratio supported by glutamate + malate, the activity of Complex 1 and the ATP synthesis. Among those compounds, THPV was most potent. Toxic properties of these compounds on mitochondria were quite similar to that of MPP⁺. Our results support the hypothesis that (a) MPTP- or MPP⁺-like substance(s) may be responsible for the nigral degeneration in Parkinson's disease.Abbreviations used MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine - MPP⁺ 1-methyl-4-phenylpyridinium ion - ATP adenosine triphosphate - ADP Adenosine diphosphate - TCL tricarboxylic acid - TIQ cycle: 1,2,3,4-Tetrahydroisoquinoline - THPV Tetrahydropapaverine - THP Tetrahydropaveroline     

Phosphatidylinositol Transfer Protein Expression Altered by Aging and Parkinson Disease

1. Phosphatidylinositol transfer proteins (PI-TP) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids from endoplasmic reticulum to the other membranes and indirectly for lipid mediated signaling. Till now little is known about PI-TPs in brain aging and neurodegeneration. The aim of this study was to investigate expression of PI-TP in the brain during aging and in animal's model of Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, in vitro, effect of 1-methyl-4-phenyl-pyridine cation (MPP⁺) on PI-TP, tyrosine hydroxylase (TH) protein level, and viability of cells was investigated.2. Wistar rats 4, 24, and 36 months old and C57/BL mice and rat pheochromocytoma (PC12) cell line were used for the studies. Mice C57/BL received three injections of MPTP in saline at 2 h intervals in a total dose of 40 mg/kg and then after 3, 7, and 14 days they were used for the investigation. PC12 cells were treated with increasing concentration (50–300 μM) of MPP⁺ for 24 h at 37°C. The level of PI-TP_{α and β} and TH were determined using Western Blot analysis.3. Our data indicated that PI-TP_{α and β} level decreased in brain of 36 months old rat by 20% comparing to the control value (4 months old). In animal's model of PD, PI-TP_{α and β} level was significantly lower by 85, 69, 64% in striatum at 3, 7, and 14 days after MPTP injection, respectively, compared to the control value. MPP⁺ decreased PI-TP_{α and β}, TH expression, and viability of PC12 cells in a dose-dependent manner. H₂O₂, menadione, and NO donor significantly decreased the PI-TP level and viability of PC12 cells.4. Our results indicate the lower protein expression of PI-TP_{α and β} in aged brain and in PD and suggest that oxidative stress may be responsible for the alteration of PI-TP.     

TRPC1 inhibits apoptotic cell degeneration induced by dopaminergic neurotoxin MPTP/MPP

Disturbances in Ca²⁺ homeostasis have been implicated in a variety of neuropathological conditions including Parkinson's disease (PD). However, the importance of store-operated Ca²⁺ entry (SOCE) channels in PD remains to be investigated. In the present study, we have scrutinized the significance of TRPC1 in 1-methyl-4-phenyl-1,2,3,6-tetrahyrdro-pyridine (MPTP)-induced PD using C57BL/6 animal model and PC12 cell culture model. Both sub-acute and sub-chronic treatments of MPTP significantly reduced TRPC1, and tyrosine hydroxylase levels, but not TRPC3, along with increased neuronal death. Furthermore, MPTP induces mitochondrial dysfunction, which was associated with reduced mitochondrial membrane potential, decreased level of Bcl₂, Bcl-xl, and an altered Bcl-xl/Bax ratio thereby initiating apoptosis. Importantly, TRPC1 overexpression in PC12 cells showed significant protection against MPP⁺ induced neuronal apoptosis, which was attributed to the restoration of cytosolic Ca²⁺ and preventing loss of mitochondrial membrane potential. Silencing of TRPC1 or addition of TRPC1 channel blockers decreased mitochondrial membrane potential, whereas activation of TRPC1 restored mitochondrial membrane potential in cells overexpressing TRPC1. TRPC1 overexpression also inhibited Bax translocation to the mitochondria and thereby prevented cytochrome c release and mitochondrial-mediated apoptosis. Overall, these results provide compelling evidence for the role of TRPC1 in either onset/progression of PD and restoration of TRPC1 levels could limit neuronal degeneration in MPTP mediated PD.     

Evaluation of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium cations by monoamine oxidase (MAO) enzymes and its use to search for new MAO inhibitors and protective agents

Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of amines and neurotransmitters and inhibitors of MAO are useful as neuroprotectants. This work evaluates the human MAO-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, to the directly-acting neurotoxic metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) measured by High-Performance Liquid Chromatography (HPLC), and this approach is subsequently used as a new method for screening of MAO inhibitors and protective agents. Oxidation of MPTP by human MAO-B was more efficient than by MAO-A. R-Deprenyl, a known neuroprotectant, norharman (β-carboline), 5-nitroindazole and menadione (vitamin K3) inhibited MAO-B and reduced the formation of toxic pyridinium cations. Clorgyline and the β-carbolines, harman and norharman, inhibited the oxidation of MPTP by MAO-A. Cigarette smoke, as well as the naturally occurring β-carbolines (norharman and harman) isolated from smoke and coffee inhibited the oxidation of MPTP by MAO-B and/or MAO-A, suggesting protective effects against MPTP. The results show the suitability of the approach used to search for new MAO inhibitors with eventual neuroprotective activity.     

Effect of pyruvate on rat heart thiol status during ischemia and hypoxia followed by reperfusion

The effect of the neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP⁺) on acetylcholinesterase (AchE) activity was investigated. The MPP⁺ was found to inactivate the enzyme in a dose dependent manner. The kinetic parameter, K_m for the substrate (acetylthiocholine), was found to be 0.216 mM and K_i for MPP⁺ for the inactivation of AChE was found to be 0.197 mM. It was found that MPP⁺ is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate inactivation of AChE to be a linear mixed-type inhibition. The inactivation of AChE by MPP⁺ was partially recovered by either dilution or gel exclusion chromatography. These data suggest that once MPP⁺ enters the basal ganglia of the brain, it can inactivate the AChE and thereby increase the acetylcholine level in the basal ganglia, leading to potential cell dysfunction. It appears likely that the nigrostriatal toxicity by MPP⁺ leading to Parkinson's disease-like syndrome may, in part, be mediated via the AChE inactivation.     

Neuroprotective effects of ginkgetin against neuroinjury in Parkinson's disease model induced by MPTP via chelating iron

Abstract

Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP⁺) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP⁺-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.     

Involvement of tubulin in MPP+ neurotoxicity on NGF-differentiated PC12 cells.

In vivo, the neurotoxin MPTP is oxidated to MPP⁺, which is toxic to dopaminergic neurons. In this paper, we have used MPP⁺ as a tool to evoke neurotoxicity in the PC12 cell line and investigate the intracellular events that are involved. A cytotoxicity test, performed on undifferentiated and NGF-differentiated PC12 cells, showed that MPP⁺ is much more toxic on differentiated cells and indicated the suitable range of concentrations for studying the starting events evoked by the neurotoxin. By indirect immunofluorescence we have shown that the localisation of α - and β -tubulin in NGF-differentiated cells was modified by a 24 h treatment with 15 μmol/l MPP⁺. A biochemical analysis was performed on cell extracts and the results showed that MPP⁺ treatment induced an increase in α -tubulin levels and a decrease in β -tubulin levels. These results suggest the involvement of the two microtubule proteins in MPP⁺ neurotoxicity on NGF-differentiated PC12 cells.     

In vitro oxidation of MPTP by primate neural tissue: A potential model of MPTP neurotoxicity

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a parkinsonian syndrome in humans and primates. We have previously found that metabolism of MPTP to a quaternary species is necessary for the expression of its neurotoxic effects. We now report that the metabolism of MPTP occurs in primate brain tissue $\text{in vitro}$, and present a model of MPTP neurotoxicity which incorporates our findings to date.Since the toxicity of MPTP is metabolism dependent, we propose that the $\text{in vitro}$ metabolism of MPTP by brain tissue should provide a useful model for studying selected aspects of MPTP neurotoxicity.     

Relative stability of α-melanotropin and related analogues to rat brain homogenates

α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle⁴, D-Phe⁷]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–10-NH₂, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–11-NH₂ is totally resistant to inactivation by rat brain homogenate. Both [Nle⁴, D-Phe⁷]-fragments are resistant to degradation by rat serum, but [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH_4–10-NH₂ and Ac-[Nle⁴]-α-MSH_4–11-NH₂ are rapidly inactivated under both conditions. The cyclic melanotropin, [ ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ ]-α-MSH_4–10-NH₂ analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ ]-α-MSH_4–10-NH₂ is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Substrate-Dependent Inhibition of the Human Organic Cation Transporter OCT2: A Comparison of Metformin with Experimental Substrates

The importance of the organic cation transporter OCT2 in the renal excretion of cationic drugs raises the possibility of drug-drug interactions (DDIs) in which an inhibitor (perpetrator) drug decreases OCT2-dependent renal clearance of a victim (substrate) drug. In fact, there are clinically significant interactions for drugs that are known substrates of OCT2 such as metformin. To identify drugs as inhibitors for OCT2, individual drugs or entire drug libraries have been investigated in vitro by using experimental probe substrates such as 1-methyl-4-phenylpyridinium (MPP⁺) or 4–4-dimethylaminostyryl-N-methylpyridinium (ASP⁺). It has been questioned whether the inhibition data obtained with an experimental probe substrate such as MPP⁺ or ASP⁺ might be used to predict the inhibition against other, clinical relevant substrates such as metformin. Here we compared the OCT2 inhibition profile data for the substrates metformin, MPP⁺ and ASP⁺. We used human embryonic kidney (HEK 293) cells stably overexpressing human OCT2 as the test system to screen 125 frequently prescribed drugs as inhibitors of OCT2-mediated metformin and MPP⁺ uptake. Data on inhibition of OCT2-mediated ASP⁺ uptake were obtained from previous literature. A moderate correlation between the inhibition of OCT2-mediated MPP⁺, ASP⁺, and metformin uptake was observed (pairwise r _s between 0.27 and 0.48, all P < 0.05). Of note, the correlation in the inhibition profile between structurally similar substrates such as MPP⁺ and ASP⁺ (Tanimoto similarity T = 0.28) was even lower (r _s = 0.27) than the correlation between structurally distinct substrates, such as ASP⁺ and metformin (T = 0.01; r _s = 0.48) or MPP⁺ and metformin (T = 0.01; r _s = 0.40). We identified selective as well as universal OCT2 inhibitors, which inhibited transport by more than 50% of one substrate only or of all substrates, respectively. Our data suggest that the predictive value for drug-drug interactions using experimental substrates rather than the specific victim drug is limited.