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

NeuroToxicology 28 (2007) 672–678

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 Nobuyuki Maruoka a,b, Tetsuhito Murata a,*, Naoto Omata a, Yasuhiro Takashima a, Yasuhisa Fujibayashi b, Yuji Wada a b

a Department of Neuropsychiatry, University of Fukui, Fukui 910-1193, Japan Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193, Japan

Received 6 August 2006; accepted 9 February 2007 Available online 23 February 2007

Abstract 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 [18F]2-fluoro-2-deoxy-D-glucose as a tracer. MPP+ dosedependently 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 mM MPP+ in the striatum. Treatment with free radical spin trap a-phenyl-N-tert-butylnitrone (PBN) significantly attenuated the enhancement of glycolysis induced by 100 mM 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 mM), and an oxidative mechanism that occurs at a higher concentration of MPP+ (100 mM) and is not restricted to dopaminergic regions. # 2007 Elsevier Inc. All rights reserved. Keywords: 1-Methyl-4-phenylpyridinium; Neurotoxicity; GBR 12909; a-Phenyl-N-tert-butylnitrone; [18F]2-fluoro-2-deoxy-D-glucose; Brain slice

1. Introduction 1-Methyl-4-phenylpyridinium (MPP+), the pyridinium metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a well-known neurotoxin which causes a clinical syndrome similar to Parkinson’s disease (PD) in humans and in non-human primates and is used as a suitable drug in various experimental models to study new therapeutic strategies in PD (Adams and Odunze, 1991; Przedborski and Jackson-Lewis, 1998). MPTP, when administered systemically, is metabolized in the brain by astrocyte monoamine oxidase-B to MPP+. The MPP+ is then taken up specifically by dopaminergic neurons, where it exerts an

* Corresponding author. Tel.: +81 776 61 8363; fax: +81 776 61 8136. E-mail address: [email protected] (T. Murata). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.02.008

intraneuronal toxic effect resulting in degeneration of the neurons (Langston et al., 1984; Markey et al., 1984). Therefore, MPP+ is thought to be the ultimate mediator of the toxic effects of MPTP (Langston et al., 1984; Markey et al., 1984). Although the neurotoxic action of MPP+ is under discussion, it is thought that the mechanism of toxicity of this compound may involve uptake by the dopamine transporter (DAT) (Javitch et al., 1985), subsequent accumulation into the mitochondria (Ramsay and Singer, 1986) and inhibition of NADH: ubiquinone oxidoreductase in complex I of the electron transport chain (Przedborski and Jackson-Lewis, 1998; Tipton and Singer, 1993). The consequent depletion of the energy supply has been shown to be related to the cytotoxicity of MPP+ in various cell cultures (Chalmers-Redman et al., 1999; Storch et al., 2000; Tipton and Singer, 1993). Supportive evidence indicating a role for mitochondrial involvement in PD is the fact

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that exposure to rotenone, pesticides and other mitochondrial toxicants can lead to similar pathologies (Betarbet et al., 2000). Other factors, such as generation of reactive-oxygen species, may contribute to the toxicity of MPP+ (Akaneya et al., 1995; Cassarino et al., 1999; Cleeter et al., 1992; Fall and Bennett, 1999; Fallon et al., 1997; Hasegawa et al., 1990; Ramsay and Singer, 1992; Schulz et al., 1995). The involvement of oxidative stress in idiopathic Parkinson’s disease has been extensively reviewed (Berg et al., 2001; Jenner, 1998; Schuman and Madison, 1994). On the other hand, several studies indicated that MPP+ did not produce oxygen free radicals and antioxidants did not attenuate MPP+ neurotoxicity (Fonck and Baudry, 2003; Lee et al., 2000; Sanchez-Ramos et al., 1988). Therefore, the involvement of the oxidative mechanism in the neurotoxicity of MPP+ is less clear. Furthermore, it has not been adequately determined in what way these two mechanisms (i.e. the DAT-mediated and the oxidative mechanisms) contribute to the development of neurotoxicity after MPP+ loading in the brain. There is significant evidence supporting the hypothesis that MPP+ toxicity corresponds to parallel changes in glycolytic activity. For example, in neuronal cells and astrocytes, MPP+ toxicity occurs in parallel to reductions in phosphocreatine and ATP (Marini and Nowak, 2000; Storch et al., 2000; Wu et al., 1992), with a simultaneous increase in lactic acid and glucose utilization, and the subsequent depletion of extracellular glucose (Fall and Bennett, 1999; Marini and Nowak, 2000; Mazzio and Soliman, 2003; Wu et al., 1992). Moreover, preserving the cellular ATP concentration through sustaining cellular glucose metabolism is a preeminent factor in deterring MPP+-induced toxicity (Chalmers-Redman et al., 1999; Storch et al., 2000), whereas a glucose-deficient environment exacerbates MPP+-induced toxicity in vitro (Wu et al., 1992). These data indicate a paramount role of the impairment of glucose metabolism in the development of MPP+-mediated toxicity in the brain. However, since MPP+ itself does not cross the blood–brain barrier, most previous studies have been conducted on cell preparations (Marini and Nowak, 2000; Mazzio and Soliman, 2003; Wu et al., 1992), and it is difficult to directly map the effects of MPP+ itself on cerebral glucose metabolism. As an imaging technique in living brain slices, we have developed the ‘‘dynamic positron autoradiography technique’’ (dPAT), which utilizes positron emitter-labeled ligands as probes and a radioluminography plate as the detector (Murata et al., 1999; Omata et al., 2000). Serial twodimensional images of radioactivity in the slices can be constructed quantitatively with a short exposure time while the brain tissue is still alive in the incubation solution because of the high specific radioactivity of the radiotracers, high energy of positrons, and high sensitivity of the radioluminography plate. dPAT is especially useful for examining the effect of MPP+ itself on cerebral glucose metabolism because MPP+ and/or other agents can be applied directly to brain slices at precisely controlled concentrations for specific time intervals. In the present study, MPP+ was added directly (rather than being generated from MPTP) to fresh rat brain slices and the dynamic changes in the cerebral glucose metabolic rate

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(CMRglc) produced by MPP+ were serially and twodimensionally measured using dPAT with [18F]2-fluoro-2deoxy-D-glucose ([18F]FDG) as a tracer. In addition, to clarify how the classical mechanism of toxicity of MPP+, i.e. entry through the DAT (Javitch et al., 1985) and blockade of complex I of the respiratory chain (Przedborski and Jackson-Lewis, 1998; Tipton and Singer, 1993), and the oxidative mechanism are involved in the promotion of the neurotoxicity, the effects of the addition of GBR 12909 as a specific DAT inhibitor and aphenyl-N-tert-butylnitrone (PBN) as a free radical scavenger on the MPP+-induced changes in glucose metabolism were quantitatively evaluated. 2. Materials and methods 2.1. Materials MPP+ iodide, GBR 12909 and PBN were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were from Nacalai Tesque Inc. (Kyoto, Japan). 2.2. Dynamic positron autoradiography technique All animal procedures were approved by the Animal Care and Use Committee of the University of Fukui in accordance with the Guidelines for Animal Experiments, University of Fukui. Male Wistar rats (250–300 g) were decapitated under diethyl ether anesthesia and their brains were removed. Sagittal brain slices (300 mm in thickness) were prepared with a microslicer (DTK-2000, Dosaka EM, Kyoto, Japan), and incubated as previously described (Murata et al., 1999; Omata et al., 2000). The system is shown schematically in Fig. 1. The outer chamber was filled with Krebs-Ringer solution and the inner chamber was immersed in it. The bottom of the inner

Fig. 1. Schematic side view of the apparatus for incubation and the radioactivity signal detected on the radioluminography plate. (a) brain slice (300 mm thick); (b) nylon sheet (300 mm thick) as a slice phantom for measuring the background noise; (c) 300-mm-thick bathing solution layer; (d) radioluminography plate (replaced every 20 min); (e) stainless steel ring; (f) outer chamber; (g) inner chamber; (h) hole on the side wall of the inner chamber; (i) nylon net (80 mm thick); (j) polyvinylidene chloride film (10 mm thick); (k) Krebs-Ringer solution containing [18F]FDG [36 8C, pH 7.3–7.4, and bubbled with 95% O2/ 5% CO2 gas (oxygen partial pressure 650–700 mmHg)]; (l) polytetrafluoroethylene catheter for bubbling. A (*), B (**) and C (***) were defined as the radioactivity signal [photostimulated luminescence (PSL)/mm2] on the radioluminography plate detected beneath the brain region of interest and the bathing medium solution, respectively. Images were obtained in a dark environment at 36 8C.

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chamber was made of a nylon net, and the bottom of the outer chamber was a 10-mm-thick polyvinylidene chloride film that could be penetrated by the beta and gamma rays of 18F. The prepared slices were placed in the inner chamber and covered with a 300-mm-thick stainless steel ring whose upper side was covered by a nylon net. The incubation volume was 80 ml. During the incubation, the Krebs-Ringer solution was bubbled with a mixture of 95% O2 and 5% CO2. 18F was produced by 18 O (p,n) 18F nuclear reactions, and [18F]FDG was produced by the method of Hamacher et al. (1986) using an automated [18F]FDG synthesis system (NKK Co. Ltd., Tokyo, Japan). The specific radioactivity of [18F]FDG was 1–2 Ci/mmol at the end of the synthesis, and the total concentration (labeled plus unlabeled) used in the experiment was 0.51–1.12 mg/ml (2.8– 6.2 mM). After 1 h of pre-incubation, the slices were incubated in Krebs-Ringer solution containing [18F]FDG diluted to 150 kBq/ml. The exposed radioluminography plates (BAS-MP 2040S, Fuji Photo Film Co., Tokyo, Japan) were scanned using a BAS-1500 (Fuji Photo Film Co.). The pixel size was 100 mm. The regions of the brain slices were identified by referring to a brain map of the rat (Paxinos and Watson, 1998). A three-compartment model using the Gjedde–Patlak graphical method was applied to the image data for determination of the net influx constant of [18F]FDG (=K, proportional to the CMRglc) (Murata et al., 1999; Omata et al., 2000). The Gjedde–Patlak graphical method (Gjedde, 1981; Patlak et al., 1983) is based on the following equation under the equilibrium conditions (Huang et al., 1980; Sokoloff et al., 1977): Ci ðtÞ ¼K Cp ðtÞ

Z 0

t

Cp ðtÞdt þV Cp ðtÞ

where Ci*(t), representing the sum of Ce*(t) ([18F]FDG in the tissue) plus Cm*(t) ([18F]FDG-6-PO4 in the tissue), is the total brain tissue radioactivity, Cp*(t) ([18F]FDG in the bathing medium) is the input function, and V is related to the effective distribution volume of the tracer [18F]FDG. Therefore, K is estimated from the slope of the linear portion of the graph, Rt Ci*(t)/Cp*(t) (vertical Y-axis) versus 0 Cp ðtÞdt=Cp ðtÞ

(horizontal X-axis). The rate of delivery of the tracer to the brain slices is determined by the diffusion rate because there is a specified concentration of [18F]FDG in the medium without interference by blood-borne factors (the product of cerebral perfusion rate and extraction fraction, as in the living brain). Therefore, the slope does not have the units of the net influx constant defined by Gjedde–Patlak (K, ml/g per min) (Gjedde, 1981; Patlak et al., 1983) but is a fractional rate constant for phosphorylation of [18F]FDG (k3 , min1) (Murata et al., 1999; Omata et al., 2000). Linear regression analysis was used to calculate the slopes (¼ k3 ) from the Gjedde–Patlak plot under each equilibrium state. The slices were then incubated with MPP+ (10 or 100 mM), and the effects of the agent on the slopes (¼ k3 ) were evaluated. As the doses of MPP+, 10 and 100 mM were selected, because MPP+ reached concentrations of 10–100 mM in the brains of mice and monkeys exposed to MPTP (Irwin and Langston, 1985; Irwin et al., 1987). The concentrations used are in the same range as those used in previous studies which investigated MPP+-induced changes in glucose metabolism in cell preparations (Marini and Nowak, 2000; Mazzio and Soliman, 2003; Wu et al., 1992). MPP+ was dissolved in distilled water. In addition, GBR 12909 (10 mM) or PBN (1 mM) was administered at 140 min (i.e. 30 min prior to MPP+ administration), and its effect on the slope (¼ k3 ) was evaluated. Both GBR 12909 and PBN were dissolved in dimethyl sulfoxide (DMSO). The final concentration of the vehicle (DMSO) in the incubation medium was 1%. DMSO at this concentration had no effect on [18F]FDG uptake. 2.3. Statistical analysis The presented values are shown as the means  S.D. The Mann–Whitney U-test was used to evaluate the significance of differences. P < 0.05 was considered statistically significant. 3. Results We quantitatively evaluated serial changes in the regional CMRglc when MPP+ (10 or 100 mM) was loaded in the bathing

Fig. 2. Time-resolved pseudocolor images of [18F]FDG uptake in sagittally sectioned rat brain slices. Time zero is when [18F]FDG was added to the bathing medium containing brain slices. Two typical slices under the control conditions (a) and before and after the loading of 10 mM MPP+ with its diagram (b) for two representative time periods (100–120 and 500–520 min) are shown. The filled circles in the diagram represent the five brain regions examined in the present study (frontal cortex, caudate putamen, thalamus, hippocampus, and cerebellum). For decay correction, the color-coding was based on the relative uptake ratio (see text for further explanation).

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solution at 170 min. Fig. 2 shows time-resolved images of [18F]FDG uptake of two typical slices under the control condition (a) and loading of 10 mM MPP+ (b) for two representative time periods, 100–120 and 500–520 min. Patlak plots at 20-min intervals in the striatum when 10 or 100 mM MPP+ was loaded are indicated in Fig. 3. The graphs indicate that the slope (¼ k3 ), reflecting the fractional rate constant for phosphorylation of [18F]FDG, was increased by both 10 and 100 mM MPP+ as compared to that of the unloaded control. Similar results were obtained in each of the five brain regions examined (Tables 1 and 2). These data indicate that MPP+ increased the k3 values in a dose-dependent manner (Fig. 3; Tables 1 and 2). We investigated the influence exerted by the administration of GBR 12909 (10 mM) or PBN (1 mM) on the slope (¼ k3 ) after MPP+ loading. In the striatum, treatment with GBR 12909 significantly attenuated the increase in k3 induced by 10 mM MPP+ loading [Fig. 3(a); Table 1]. A similar effect was not obtained in the other four brain regions examined (Table 1). On the other hand, treatment with GBR 12909 had no significant effect on the increase in k3 induced by 100 mM MPP+ loading in any of the five brain regions examined [Fig. 3(b); Table 2]. Treatment with PBN significantly attenuated the increase in k3 induced by 100 mM MPP+ loading in each of the five brain regions examined [Fig. 3(b); Table 2]. On the other hand, treatment with PBN had no significant effect on the increase in k3 induced by 10 mM MPP+ loading in any of the five brain regions examined [Fig. 3(a); Table 1]. Neither GBR 12909 nor PBN alone had an effect on [18F]FDG uptake (data not shown). 4. Discussion Our data indicate that 10 and 100 mM MPP+ increased the slope of the graph (¼ k3 ), reflecting the fractional rate constant for phosphorylation of [18F]FDG, by around 2- to 4-fold and 5to 15-fold, respectively, as compared to that of the unloaded control. This was thought to reflect enhanced glycolysis compensating for the decrease in aerobic metabolism when oxidative phosphorylation was inhibited at the respiratory chain level by MPP+. This possibility is plausible because mitochondrial inhibition is associated with an increase of glucose consumption through anaerobic glycolysis in several types of cultured cells, including neurons (Pauwels et al., 1985),

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Fig. 3. Effects of 10 mM MPP+ administration (a) and 100 mM MPP+ administration (b) on the Gjedde–Patlak plots of [18F]FDG uptake in the striatum of rat brain slices. MPP+ was administered alone or co-administered with either dopamine transporter (DAT) inhibitor GBR 12909 or free radical spin trap aphenyl-N-tert-butylnitrone (PBN). Ordinate: [Ci*(t)/Cp*(t)] expressed in terms of the radioactivityRsignal ratio (=(A  B)/(C  B) in Fig. 1) vs. Abscissa: time t in min indicating [ 0 Cp ðtÞ dt=Cp ðtÞ] (see text for further explanation). Time zero is when [18F]FDG was added to the bathing medium containing brain slices. The point at which each agent was applied is indicated by an arrow. Values are mean  S.D. obtained in six slices (S.D. is shown only for the uppermost and lowermost lines).

and because in our previous studies using dPAT, increases in the slope of the graph (¼ k3 ) were induced by the inhibition of aerobic glucose metabolism by hypoxia (Murata et al., 1999; Omata et al., 2000) and mitochondrial toxicants such as antimycin-A and 2,4-dinitrophenol (Murata et al., 1999). Our results are consistent with previous studies showing an increased rate of glucose use after MPP+ administration in murine neuroblastoma cells (Mazzio and Soliman, 2003), rat

Table 1 Effect of 10 mM MPP+ administration on the fractional rate constant of [18F]FDG Brain region

Control

10 mM MPP+

10 mM MPP+ and GBR 12909

10 mM MPP+ and PBN

Frontal cortex Striatum Thalamus Hippocampus Cerebellum

4.31  0.83 3.97  1.25 2.24  0.83 1.82  0.31 2.45  0.69

10.32  1.11* 14.30  1.15* 6.87  1.90* 8.35  0.65* 4.64  0.44*

13.22  0.39* 10.04  0.51*,# 4.44  1.02* 7.98  0.73* 7.28  0.97*

12.66  1.50* 13.05  1.66* 5.51  2.03* 9.20  0.83* 5.68  0.61*

The k3 (1000), indicating the fractional rate constant of [18F]FDG, was obtained from the slope of the regression equation (y = ax + b) fitted to Gjedde–Patlak plots using the linear regression analysis. y = Ci*(t)/Cp*(t) in terms of the radioactivity signal ratio on the imaging plate; x: time (min) after the start of incubation; a: slope of the line; b: intercept. All values are means  S.D. obtained from six slices. * P < 0.05; significantly different from the control. # P < 0.05; significantly different from 10 mM MPP+ group.

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Table 2 Effect of 100 mM MPP+ administration on the fractional rate constant of [18F]FDG Brain region

Control

100 mM MPP+

100 mM MPP+ and GBR 12909

100 mM MPP+ and PBN

Frontal cortex Striatum Thalamus Hippocampus Cerebellum

5.17  0.68 3.77  0.74 1.91  0.37 1.71  0.29 2.43  0.48

25.54  3.48* 33.66  3.30* 26.29  5.38* 27.58  5.06* 25.33  3.08*

27.87  4.56* 31.99  4.35* 29.47  6.17* 26.90  5.23* 23.60  2.43*

16.58  2.20*,# 21.52  2.48*,# 14.44  4.22*,# 17.05  4.00*,# 12.28  2.19*,#

The k3 (1000), indicating the fractional rate constant of [18F]FDG, was obtained from the slope of the regression equation (y = ax + b) fitted to Gjedde–Patlak plots using the linear regression analysis. y = Ci*(t)/Cp*(t) in terms of the radioactivity signal ratio on the imaging plate; x: time (min) after the start of incubation; a: slope of the line; b: intercept. All values are means  S.D. obtained from six slices. * P < 0.05; significantly different from the control. # P < 0.05; significantly different from 100 mM MPP+ group.

cerebellar granule cells (Marini and Nowak, 2000) and mouse astrocytes (Wu et al., 1992). Depletion of the energy supply has been shown to be related to the neurotoxicity of MPP+ (Chan et al., 1993; Fonck and Baudry, 2003; Scotcher et al., 1990; Wu et al., 1992). Energy depletion by MPP+ can be prevented in several models by stimulating ATP production by increasing glucose consumption in anaerobic glycolysis (ChalmersRedman et al., 1999; Storch et al., 2000). In our study, the toxicity of MPP+ (at both 10 and 100 mM) was not restricted to dopaminergic regions such as the striatum; rather, MPP+ induced significant overall toxicity in the brain, as shown by enhanced glycolysis. These results were consistent with previous studies which showed that MPP+ was no longer selective for dopaminergic neurons but affected all cells when high concentrations of MPP+ (above 10 mM) were administered to both rat brain slices (O’Byrne and Tipton, 2002) and rat mesencephalic cell cultures (Callier et al., 2002; SanchezRamos et al., 1988). Impairment of glucose metabolism lessens the regional selectivity of MPTP, making the loss of ATP evident in areas of the brain (e.g. the cerebellum) that are normally spared by MPTP administration (Chan et al., 1993). Therefore, a possible mechanism of the reduced regional selectivity of the MPP+ toxicity could be that, while administration of relatively low concentrations of MPP+ may cause mitochondrial inhibition only in neuronal cells able to accumulate relatively high levels of MPP+ via the dopaminergic uptake system (Javitch et al., 1985), in the case of relatively high concentrations of MPP+, the threshold concentration for MPP+ action may be lowered by excessive ATP depletion, and inhibition of mitochondrial respiration may become evident even in cells that do not actively accumulate MPP+ (Scotcher et al., 1990). In our study, the protective effect of GBR 12909 against the toxicity of 10 mM MPP+ was partial and observed only in the striatum, while GBR 12909 was not protective against glycolytic enhancement induced by 100 mM MPP+. A possible explanation for these findings is that while a protective effect was observed only in the striatum since GBR 12909 binds preferentially to the striatum (Lundkvist et al., 1997), mechanisms that involve uptake systems other than DAT may diminish the protective effect of GBR 12909 in the striatum. For example, cationic amino acid transporter has been reported to mediate such an alternative way of MPP+ entrance

into cerebellar granule cells (Gonzalez-Polo et al., 2001). Moreover, a higher dose of MPP+ (100 mM) could overcome the actions of GBR 12909 in preventing the enhanced glycolysis elicited by MPP+. A previous report showed that mazindol, a dopamine uptake blocker, is effective in blocking the action of low-dose (7.5 mg/kg) MPTP, but ineffective against high-dose (30 mg/kg) MPTP (Hess, 1990). Our study showed the protective action of PBN against the enhanced glycolysis induced by 100 mM MPP+ in all brain regions examined, which suggested that the oxidative mechanism was likely to be involved in the 100 mM MPP+induced toxicity. This may also account for the reduced regional selectivity of 100 mM MPP+-induced toxicity; at this concentration, MPP+ may induce oxidative stress which is not restricted to dopaminergic regions. Free radicals themselves are capable of causing respiratory chain defects involving complex I (Hartley et al., 1993; Hillered and Ernster, 1983). The interaction of MPP+ with complex I induces free radical generation, which in turn leads to further inhibition of complex I activity (Cleeter et al., 1992). Therefore in our study, it is possible that inhibition of complex I induced by 100 mM MPP+ could initiate free radical production, leading to further complex I inhibition and free radical generation. This sequence of events could further enhance glycolysis compensating for the inhibition of mitochondrial respiration. PBN might be protective in lowering glycolytic enhancement induced by 100 mM MPP+ since it could inhibit the free radical-mediated mitochondrial inhibition. In our study, however, PBN did not protect against glycolytic enhancement induced by 10 mM MPP+. Although the reason for the lack of protection against the MPP+ toxicity at the concentration of 10 mM MPP+ is not clear, our data indicate that the toxicity of 10 mM MPP+ is still considerably dependent on the classical mechanism of toxicity of MPP+, involving entry through the DAT (Javitch et al., 1985), and blockade of complex I of the respiratory chain (Przedborski and Jackson-Lewis, 1998; Tipton and Singer, 1993) or is related to unknown mechanisms which are distinct from both the classical and the oxidative mechanism. This may also account for why the enhancement of CMRglc induced by 10 mM MPP+ was smaller than that of 100 mM MPP+; in contrast to 100 mM MPP+, 10 mM MPP+ could not induce a secondary inhibition of complex I which is mediated via free radical release. Nevertheless, it appears that inhibitory effects

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of MPP+ and oxygen radicals on complex I activity are synergistic. Nitric oxide potentiates the MPP+-induced inhibition of complex I in rat brain mitochondria (Cleeter et al., 2001). MPP+ sensitizes cells to oxidative stress (Lee et al., 2000). Therefore, it is safe to say that although oxygen free radicals may not be a primary cause of MPP+-induced neurotoxicity, oxidative stress may enhance the vulnerability of cells to MPP+. Further studies are needed to explore the complexity of mechanisms underlying the MPP+ toxicity and the pathogenesis of PD as well as other neurodegenerative disorders. Our experimental system has the potential to be very useful for further studies on the mechanisms of MPP+ toxicity and on the role of glucose metabolism in the action of MPP+ as well as other neurotoxicants. This system may also be particularly valuable given the increasing interest in the relationship between energy impairment and human neurodegenerative disorders (Beal, 1992; Di Monte, 1991; Wallace, 1992). In conclusion, we investigated in what way the two mechanisms (i.e., the DAT-mediated and the oxidative mechanisms) contribute to the development of neurotoxicity after MPP+ loading in the brain, and our findings suggested that the mechanism of the toxicity of MPP+ is biphasic, consisting of a DAT-mediated mechanism selective for dopaminergic regions at a lower concentration of MPP+ (10 mM), and an oxidative mechanism that occurs at a higher concentration of MPP+ (100 mM) and is not restricted to dopaminergic regions. Acknowledgements This work was supported in part by the 21st Century COE ‘‘Biomedical Imaging Technology Integration Program’’ from the Japan Society for the Promotion of Science (JSPS). References Adams JD Jr, Odunze IN. Biochemical mechanisms of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine toxicity. Could oxidative stress be involved in the brain? Biochem Pharmacol 1991;41:1099–105. Akaneya Y, Takahashi M, Hatanaka H. Involvement of free radicals in MPP+ neurotoxicity against rat dopaminergic neurons in culture. Neurosci Lett 1995;23:53–6. Beal MF. Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann Neurol 1992;31:119–30. Berg D, Gerlach M, Youdim MB, Double KL, Zecca L, Riederer P, et al. Brain iron pathways and their relevance to Parkinson’s disease. J Neurochem 2001;79:225–36. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci 2000;3:1301–6. Callier S, Le Saux M, Lhiaubet AM, Di Paolo T, Rostene W, Pelaprat D. Evaluation of the protective effect of oestradiol against toxicity induced by 6-hydroxydopamine and 1-methyl-4-phenylpyridinium ion (MPP+) towards dopaminergic mesencephalic neurones in primary culture. J Neurochem 2002;80:307–16. Cassarino DS, Parks JK, Parker WD Jr, Bennett JP Jr. The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism. Biochim Biophys Acta 1999;1453:49–62.

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