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NEUROPHARMACOLOGY
Department of Neurology, Mollie and Jerome Levine Neuroscience Division, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey
Received for publication
July 14, 2006
Accepted
November 6, 2006.
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Abstract |
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-(p-fluorophenyl)-1
H,5H-tropane-2-carboxylate1,5-naphthalene (Win 35,428)] attenuated malonate-induced DA overflow in vivo and protected mice against subsequent damage to DA nerve terminals. Consistent with these findings, the DAT inhibitors prevented malonate-induced damage to DA neurons in mesencephalic cultures and also protected against the loss of GABA neurons in this system. The DAT inhibitors did not modify malonate-induced formation of reactive oxygen species or lactate production, indicating that the DAT inhibitors neither exert antioxidant effects nor interfere with the actions of malonate. Taken together, these findings provide direct evidence that mitochondrial impairment and metabolic stress cause striatal DA efflux via the DAT and suggest that disruptions in DA homeostasis resulting from energy impairment may contribute to the pathogenesis of neurodegenerative diseases.
; Rollema et al., 1986; Globus et al., 1988; Slivka et al., 1988; Beal et al., 1993; Ferger et al., 1999; Moy et al., 2000).
Despite the multiple neuronal populations that are affected after these insults, DA nerve terminals are consistently more susceptible to damage (Weinberger et al., 1983; Marey-Semper et al., 1993, 1995; Zeevalk et al., 1995, 1997). The mechanisms involved in the neuronal damage after these insults remain unresolved, but the greater vulnerability of DA neurons has highlighted the neurotransmitter DA itself as a potential mediator of toxicity.
The enzymatic and spontaneous oxidation of DA generates reactive oxygen species (ROS) as well as the electron-deficient DA quinone, which can target nucleophilic sulfhydryl groups in proteins, lipids, and DNA (reviewed in Stokes et al., 1999). Because of the cytotoxic nature of the DA molecule, it is thought that DA acts as an endogenous neurotoxin and contributes to the pathology of neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and ischemia-induced damage in the striatum (Slivka et al., 1988; Hastings et al., 1996; Maragos et al., 1998, 2004; Reynolds et al., 1998; Jakel and Maragos, 2000; Xia et al., 2001; Bozzi and Borrelli, 2006). Thus, the regulation of dopaminergic neurotransmission may be critical in neuronal damage.
The physiological role of the plasmalemmal DA transporter (DAT) is to maintain DA homeostasis in the central nervous system by terminating the synaptic actions of DA via high-affinity uptake into the presynaptic nerve terminals. However, under pathological conditions, increases in extracellular DA can be mediated by reversal of the DAT, also known as carrier-mediated release (Raiteri et al., 1979; Santos et al., 1996; Buyukuysal and Mete, 1999). Reverse transport of DA from the cytosol into the extracellular space can be blocked by uptake inhibitors (Levi and Raiteri, 1993).
The present studies were performed to determine whether reverse transport of DA plays a role in DA efflux under conditions of a metabolic stress. Mitochondrial bioenergetic defects have been implicated in the pathology of a number of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (Beal, 2005; Mattson and Magnus, 2006). Malonate is a reversible inhibitor of succinate dehydrogenase, which is a component of the tricarboxylic acid cycle and complex II of the mitochondrial electron transport chain. The infusion of malonate into the striatum of animals imposes a metabolic stress on several neuronal populations (Beal et al., 1993; Greene et al., 1993) and results in the loss of striatal DA and GABA content as well as the retrograde loss of nigral DA cell bodies (Beal et al., 1993; Sonsalla et al., 1997; Zeevalk et al., 1997; Moy et al., 2000). Moreover, the perfusion of malonate via in vivo microdialysis causes a substantial increase in striatal DA efflux (Ferger et al., 1999; Moy et al., 2000; Xia et al., 2001).
In this study, we hypothesized that carrier-mediated DA release is a component of damage under conditions of energy impairment. To test this hypothesis, we examined the effect of DAT inhibition on DA overflow and damage to DA nerve terminals resulting from intrastriatal malonate infusions in mice. In addition, we evaluated whether DAT is involved in malonate-induced damage to DA and GABA populations in vitro in mesencephalic cultures. Finally, we further characterized the protective effects of DAT inhibition in vitro. Our data strongly support a role for DA and DAT in the damage resulting from malonate treatment in animals and cell cultures.
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Materials and Methods |
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-(p-fluorophenyl)-1H,5H-tropane-2-carboxylate1,5-naphthalene), and 5(6)-carboxyfluorescein diacetate (CFDA) were obtained from Sigma-Aldrich (St. Louis, MO). GBR 13098 [1-(2-(bis(4-fluophenyl methoxy) ethyl)-4-(3-(4-fluorophenyl)-propyl)piperazine) dimethane sulfonate] was a gift from Gist-Brocades (Delft, The Netherlands). Mazindol was acquired through Novartis (East Hanover, NJ) and was dissolved with a small volume of 0.1 N HCl that was diluted to a final concentration of 0.001 N HCl. 2',7'-Dichlorofluorescein diacetate (DCF) was purchased from Molecular Probes (Eugene, OR). Animals. All in vivo experiments were conducted in male Swiss-Webster mice (30–40 g) from Taconic Farms (Germantown, NY), and neuronal cultures for in vitro experiments were obtained from rat fetuses from timed-pregnant rats from Charles River Laboratories, Inc. (Wilmington, MA) in accordance with the Principles of Laboratory Animal Care (National Institutes of Health publication 85-23, revised 1985). All procedures were approved by the local animal care committee. Mice were group-housed (four to six per cage), and pregnant rats were housed individually at 20 to 22°C on a 12-h light/dark cycle with food and water available ad libitum.
Cannula Implantation. Cannulas were stereotaxically implanted 3 to 5 days before treatment as described previously (Giovanni et al., 1994; Moy et al., 2000). Briefly, mice were anesthetized with a combination of pentobarbital (60 mg/kg) and xylazine (5.8 mg/kg). A guide cannula was positioned over the medial aspect of the striatum (anterior 0.6, lateral 2.0, vertical –1.8 from bregma suture) and was cemented in place with Tak-Pak glue (Loctite Corp., Newington, CT). For neurotoxicity studies, this guide cannula was made from a 21-gauge stainless steel needle. For microdialysis experiments, guide cannulas were purchased from Bioanalytical Systems (West Lafayette, IN) to fit their microdialysis probes.
In Vivo Microdialysis and Intrastriatal Injections. Microdialysis and neurotoxicity studies were performed 3 to 5 days after cannula implantation. For microdialysis studies, the extracellular contents of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in freely moving, awake mice as described previously (Giovanni et al., 1994; Moy et al., 2000). The microdialysis probe was implanted 
12 h before testing. The cerebrospinal fluid perfusion fluid was composed of 1.75 mM CaCl2, 147 mM NaCl, and 4 mM KCl in HPLC-grade water, with pH adjusted to 7.0 to 7.4 with NaOH, and filtered through a 0.2-µm filter. The flow rate was 1.0 µl/min. Samples were collected every 15 min. Drug treatment was initiated as described in the legends to the figures only after a stable baseline was achieved (i.e., three values ±15%). For neurotoxicity studies, mice were treated as described in the legends to the figures, and malonate (4 µmol in 2 µ1, pH 7.4–7.6) or saline was infused into the left striatum using a 10-µl glass syringe as described previously (Albers et al., 1996; Moy et al., 2000). All infusions were made over 1 min, and the needle was left in place for 1 min before being slowly withdrawn. Animals were sacrificed 4 to 7 days after treatment, and the striatum was removed, rapidly frozen, and stored at –80°C for HPLC analyses. In experiments in which striatal GABA content determination was necessary, mice received a tail vein injection of 3-mercaptopropionic acid, an inhibitor of glutamate decarboxylase, 2 min before euthanization.
Drug Treatment. The DAT inhibitors, cocaine, mazindol, Win 35,428, and GBR 13098, are pharmacological agents that have been shown to effectively block DAT. These drugs and the doses selected were based on their effectiveness to block DAT activity (Mayer et al., 1986).
Measurement of DA and Metabolites in Dialysate Samples. Dialysate samples were injected into a HPLC system equipped with a 3-µm, C18 Luna ODS column (Phenomenex, Torrance, CA) and an electrochemical detector (Antec Leyden BV, Zoeterwoude, The Netherlands) as described previously (Moy et al., 2000).
Measurement of DA and Metabolites in Tissue Samples. DA and its metabolites were measured by HPLC-electrochemical detection as described previously (Sonsalla et al., 1991; Moy et al., 2000). Briefly, the tissue was homogenized in 0.2 M perchloric acid (10 mg/100 µl). The homogenate was centrifuged at 15,000g for 5 to 10 min, and an aliquot of the supernatant was injected into an high-performance liquid chromatograph equipped with a 3-µm, C18 Luna ODS column and an electrochemical detector (Bioanalytical Systems, Indianapolis, IN).
Enzyme-Linked Immunosorbent Assay TH Assay. TH, the rate-limiting enzyme for DA synthesis, was measured by an enzyme-linked immunosorbent assay method routinely used in our laboratory (Alfinito et al., 2003). Briefly, 96-well microtiter plates were incubated overnight at 4°C with monoclonal anti-TH (1:500; Calbiochem, San Diego, CA) in 8 mM phosphate-buffered saline, pH 7.4. Wells were washed and blocked for 1 h using nonfat dry milk in phosphate-buffered saline. Striatal tissue homogenates were incubated for 1 h at room temperature, the wells were washed, and polyclonal anti-TH (1:500; Calbiochem) and polyclonal anti-rabbit horseradish peroxidase (1:3000; GE Healthcare, Piscataway, NJ) were added and incubated for 1 h. After washing, wells were incubated with Amplex Red (Molecular Probes) horseradish peroxidase substrate according to the protocol of the manufacturer. Reaction product was measured fluorometrically within the linear range of detection (excitation/emission ratio of 530:580 nm).
Mesencephalic Cultures and Treatment. As described previously (Zeevalk et al., 1995; Moy et al., 2000), mesencephalon from embryonic day 15 Sprague-Dawley rats were dissected and pooled in ice-cold Ca2+-Mg2+-free HEPES-buffered saline (HBS), mechanically dissociated, and centrifuged at 1000g for 10 min. The cells were resuspended in DMEM and supplemented with 5 mM glucose, 2 mM glutamine, and 2.2 g/liter bicarbonate, plus 10% fetal bovine serum and 10% horse serum. The dissociated cells were counted in a hemocytometer and plated at 2.5 x 105 cells/cm2, into 24-well trays, onto a substrate previously coated with polyornithine. The cultures were incubated at 37°C in a 95% air, 5% CO2 atmosphere with 100% relative humidity. After 48 h, the culture medium was replaced with l ml of fresh DMEM supplemented with 10% fetal bovine serum and 10% horse serum. On day 8 in vitro, half of the medium was removed before toxin treatment (conditioned medium), supplemented with 5.5 mM glucose, and saved for refeeding of the cultures after toxin treatment. DAT inhibitors were added to some wells 1 h before addition of malonate. On day 9 in vitro, the toxin-containing medium was removed, and the cells were fed with 0.5 ml of conditioned media. High-affinity DA and GABA uptakes were assessed after a 48-h recovery period.
Striatal Cultures and Treatment. Striata from embryonic day 17 Sprague-Dawley rats were dissected and pooled in ice-cold Ca2+-Mg2+-free HBS, mechanically dissociated and centrifuged at 1000g for 10 min as described -previously (Moy et al., 2000). The cells were resuspended in glutamine-free DMEM, supplemented with 10% fetal bovine serum. The dissociated cells were counted in a hemocytometer and plated at 1.0 x 105 cells/cm2, into 24-well trays, onto a substrate previously coated with polyornithine. The cultures were incubated at 37°C in a 95%–5% air-CO2 atmosphere with 100% relative humidity. After 48 h, the culture medium was replaced with l ml of fresh DMEM supplemented with 10% fetal bovine serum. On day 6 in vitro, conditioned medium was removed before treatment with DAT inhibitors in some wells 1 h before the addition of malonate. On day 7 in vitro, the toxin-containing medium was removed, and the cells were fed with 0.5 ml of CM. Neuronal viability was assayed 48 h later.
DA and GABA High-Affinity Uptake. Toxicity in the mesencephalic cultures was assayed by measurement of the high-affinity uptake of [3H]DA (final concentration 20 nM) and [14C]GABA (final concentration 5 µM) as described previously (Zeevalk et al., 1995). Uptake was carried out for 15 min at 37°C. The radioactivity in the tissue was extracted with 1 ml of 95% ethanol and radioactive DA and GABA were quantified by scintillation counting of the extract. Energy-dependent uptake was calculated by subtracting the amount of radioactivity in samples incubated at 4°C (nonspecific radioactivity).
DCF Assay for Free Radical Formation. Free radical formation was monitored in mesencephalic cultures by measuring the generation of DCF (Moy et al., 2000; Zeevalk et al., 2000). In the presence of ROS, the nonfluorescent precursor of DCF is oxidized to DCF, a fluorescent product. At 8 days in vitro, some wells were pretreated with DAT inhibitors for 1 h. All wells were then switched from DMEM serum-containing medium to bicarbonate-buffered Krebs-Ringer (KRB) to which DCF in dimethyl sulfoxide was added (at a final concentration of 5 µM DCF in 1% dimethyl sulfoxide). Cultures were loaded with DCF for 30 min at 37°C and washed three times with KRB. Malonate and DAT inhibitors were added immediately as described in the legends to the figures before DCF fluorescence was monitored at 485-nm excitation and 530-nm emission in a Cytofluor 4000 (Applied Biosystems, Framingham, MA) at 37°C. Readings were taken every 2.5 min for a total of 30 min, and results were expressed as arbitrary fluorescent units.
CFDA Assay for Neuronal Viability. Neuronal survival in striatal cultures was determined using an assay based upon the vital dye CFDA (Petroski and Geller, 1994). Cultures were washed once with HBS (122 mM NaCl, 3.3 mM KCl, 1.2 mM CaCl2, 0.4 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, and 25 mM HEPES at pH 7.3) and incubated with 10 µM CFDA in HBS for 15 min at 37°C. The dye readily enters cells and is converted to the fluorescent carboxyfluorescein (CF) anion by intracellular esterases. The cells were then rinsed and incubated with HBS for 30 min, during which astrocytes pump the dye out of the cells whereas neurons retain the CF for hours. The HBS was aspirated, and the cells were lysed with 0.1% Triton X-100. The fluorescence was measured using a multiwell plate reader at 485 nm excitation and 530 nm emission in a Cytofluor 4000 (Perceptive Biosystems) at 37°C. Fluorescent readings were taken in parallel with external CF standards, and results were expressed as arbitrary fluorescent units.
Measurement of Lactate. Lactate measurement in culture has been described previously (Zeevalk et al., 1995; Moy et al., 2000). At 8 days in vitro, the medium in mesencephalic cultures was switched to KRB, and DAT inhibitors (as indicated in the legends to the figures) were added to the wells for 30 min before the addition of malonate. Lactate in the medium after malonate exposure was determined spectrophotometrically by following the oxidation of NADH by pyruvate in the presence of lactate dehydrogenase.
Statistical Analysis. All results were analyzed by a one-way analysis of variance with Tukey's multiple comparison test (GraphPad Prism, San Diego, CA). A probability value of 0.05 or less was considered statistically significant.
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250-fold that peaked at 30 min after infusion and returned to basal levels within 90 min (Fig. 1A). Intraperitoneal administration of the DAT inhibitors cocaine, mazindol, Win 35,428, or GBR 13098 attenuated the malonate-induced DA efflux by 50 to 75% (Fig. 1A). These inhibitors did not affect the malonate-induced efflux pattern of the DA metabolite, DOPAC (Fig. 1B). Administration of the DAT inhibitors in the absence of a striatal malonate infusion elevated DA efflux 5-fold above baseline (Fig. 1C and inset). This elevation in striatal DA overflow was accompanied by a decrease in striatal DOPAC efflux to 20 to 60% of control values (Fig. 1D). The administration of DAT inhibitors has been previously reported to elicit similar changes in striatal DA overflow (Nakachi et al., 1995; He and Shippenberg, 2000).
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DAT Inhibition Protects against Malonate-Induced Damage to DA and GABA Neurons in Vitro in Mesencephalic Cultures. Malonate-induced toxicity to both DA and GABA neurons in the mesencephalic culture system has been extensively characterized, and we presently used this system to assess the effect of DAT inhibition on malonate-induced damage to these two neuronal populations. Exposure of cultures to malonate produced a greater reduction in high-affinity [3H]DA uptake (loss of 60%) (Fig. 3) than [14C]GABA uptake (loss of 25%) (Fig. 4), consistent with our previous findings (Zeevalk et al., 1995). Coincubation of the cultures with malonate and the DAT inhibitors produced a dose-dependent attenuation of the malonate-induced loss of both high-affinity DA and GABA uptake with relative potencies of GBR13098 > mazindol > Win 35,428 > cocaine (Figs. 3 and 4). Exposure of the cultures to mazindol or GBR 13098 alone resulted in some loss of [3H]DA uptake, but not [14C]GABA uptake, at the higher concentrations (Fig. 3, B and D). The reasons for this decrease are unclear, but similar findings have also been observed with high concentrations of other DAT inhibitors (Bennett et al., 1993).
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DAT Inhibition Does Not Interfere with Malonate-induced Free Radical Generation or Lactate Production. Mitochondrial inhibition leads to a greater reliance on anaerobic glycolysis with consequent increases in lactate production and free radical generation. In the present studies, the effects of DAT inhibition on malonate-induced free radical production and lactate production were evaluated in cultures. Under control conditions, free radical production as measured by DCF fluorescence increased slightly over time and was not affected by exposure to cocaine or mazindol alone (Fig. 7), indicating some free radical formation under basal conditions that is unaffected by DAT inhibition. Malonate exposure produced an increase in fluorescence that was approximately 2-fold above basal levels by 30 min (Fig. 7). Concurrent exposure of the cultures to the DAT inhibitors (cocaine or mazindol) and malonate did not affect the increase in DCF fluorescence (Fig. 7). Similarly, neither cocaine nor mazindol prevented the increase in free radical production produced by menadione, an established free radical generator (data not shown). To determine whether the DAT inhibitors modified malonate-induced lactate production, cultures were exposed to malonate and assayed for lactate levels in the medium and for ATP levels in cell lysates. Mesencephalic cultures treated with malonate plus cocaine or mazindol showed no effect of the DAT inhibitors on the ability of malonate to increase lactate formation and decrease ATP levels (Fig. 8, A and B, respectively). In addition, the DAT inhibitors alone did not affect lactate formation (data not shown) or ATP levels (Fig. 8B).
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25% loss of GABA neurons as determined by the neuronal viability CFDA fluorescent assay. Pretreatment with either cocaine or mazindol at concentrations that protected GABA neurons in mesencephalic cultures did not prevent the loss in GABA neurons in striatal cultures (Fig. 9, A and B).
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; Slivka et al., 1988; O'Dell et al., 1991; Ferger et al., 1999; Moy et al., 2000; Xia et al., 2001). The significance of this release to neuronal damage is underscored by findings that striatal DA depletion prevents or attenuates damage produced by these insults (Weinberger et al., 1985; Globus et al., 1987; Maragos et al., 1998; Ferger et al., 1999; Moy et al., 2000; Xia et al., 2001). Thus, understanding the mechanism of DA release in neurotoxic paradigms can provide insight into the underlying mechanisms involved in neurodegenerative processes. We presently report that DA release induced by the mitochondrial inhibitor malonate is predominately via DAT reversal. This was demonstrated by attenuation of DA release with any one of several DAT inhibitors that have diverse structures (cocaine, mazindol, GBR 13098, and Win 35,428). DAT inhibitors also provided protection against malonate-induced neuronal damage. Several lines of converging evidence from our studies indicate that DAT activity and/or DAT-mediated DA release is critically involved in neuronal damage produced by metabolic stress. First, protection against malonate toxicity was observed with all four DAT inhibitors tested and at doses that attenuated DA release. Second, the compounds protected both in vivo in mouse striatum and in vitro in mesencephalic cultures but failed to provide protection in striatal cultures that are devoid of DA innervation and DAT protein. Third, malonate-induced ROS and lactate production were not modified by DAT inhibitors, indicating that the compounds did not act as antioxidants nor interfere with the ability of malonate to cause a metabolic stress.
Although our data indicate protection against malonate-induced damage by DAT inhibition, it is currently unclear how they protect. We propose that DAT blockade protects against compromises in energy stores by different, but not mutually exclusive, mechanisms: 1) decreased carrier-mediated DA release, 2) decreased energy utilization by DA neurons, and 3) decreased formation and uptake of extracellular DA oxidative products.
In the first scenario, DAT inhibitors may protect against metabolic stresses by reducing carrier-mediated DA efflux. Several conditions can elicit carrier-mediated release of neurotransmitters including a decrease in the Na+ gradient and an increase in the cytoplasmic concentration of a neurotransmitter (Levi and Raiteri, 1993). Metabolic inhibition leads to collapses in electrochemical gradients and membrane potentials (McLaughlin et al., 1998b), conditions that would favor DA efflux by DAT reversal. Another possible effect of metabolic inhibition is disruption of vesicular DA storage that would increase cytosolic DA levels and thus contribute to DA efflux. It is known that disruption of the vesicular pH gradient (i.e., by amphetamines) releases vesicular DA into the cytosol (Sulzer et al., 2005). Moreover, the brain vesicular monoamine transporter, driven by a proton electrochemical gradient generated by the vacuolar ATPase, uses ATP to translocate H+ ions into the vesicle. Thus, metabolic impairment and reduced ATP levels could have a negative impact on vesicular DA storage. The effects of metabolic stress on vesicular function and how DAT inhibitors might modify such actions are presently unknown. We suggest that reduced use of energy stores during DAT blockade provides sufficient ATP to maintain vesicular DA storage. Studies are currently underway to examine the effects of malonate and DAT inhibitors on vesicle function.
A second possible mechanism of protection by DAT inhibition is a decrease in energy needs of DA neurons, allowing for more economical use of the limited energy stores for other crucial cellular processes. DA transport, driven by the Na+ electrochemical gradient across the cell membrane, is indirectly ATP-dependent because Na+/K+-ATPase is required to maintain the Na+ gradient (Harris and Baldessarini, 1973; Nelson, 1998; Masson et al., 1999). The Na+/K+-ATPase uses 30 to 60% of total neuronal ATP and under anoxic conditions, ATP demand by this ATPase can increase to 75% (Cousin et al., 1995; Hochachka et al., 1996). Thus, under conditions of compromised ATP production, as occurs with intrastriatal malonate infusions (Schulz et al., 1995), blocking DAT activity would substantially reduce ATP usage by the neurons. Therefore, our present observations that DAT inhibition prevents malonate-induced damage to DA nerve terminals may be attributable, at least in part, to a decreased metabolic burden and conserved energy stores resulting from blockade of DAT activity.
Thirdly, DAT inhibition may also protect by blocking the generation and uptake of detrimental products of extracellular DA oxidation. Our previous findings that DA depletion before a metabolic insult protects against malonate-induced damage and our present findings demonstrating that DAT inhibitors both reduce malonate-induced extracellular DA and provide neuroprotection suggest that large amounts of extracellular DA or the process by which DA is released from the nerve terminals (via DAT as discussed above) are responsible for the neuronal damage to the DA nerve terminals. A potential consequence of decreased extracellular DA is a reduction in the formation of extracellular ROS and reactive DA quinones. Intrastriatal DA injections are associated with quinone modification of cellular nucleophiles and formation of cysteinyl-catechol conjugates, species indicative of protein alteration (Hastings et al., 1996). The detection of 5-S-cysteinyl-DA adducts provides evidence for the in vivo oxidation of DA and its subsequent reaction with sulfhydryl groups of cellular nucleophilic constituents (Fornstedt et al., 1990). Moreover, exogenously applied DA can be toxic in vitro and in vivo (Graham, 1978; Michel and Hefti, 1990; Filloux and Townsend, 1993; McLaughlin et al., 1998a), and results in free and protein-bound cysteinyl DA formation (Hastings et al., 1996). One of the proteins targeted by the quinones is DAT, and site-directed mutagenesis studies have identified specific residues on DAT as potential targets for the DA quinone (Whitehead et al., 2001). Moreover, cocaine prevents the oxidation of cysteine residues on DAT, presumably by altering the accessibility of the sulfhydryl groups for modification (Ferrer and Javitch, 1998). Although our data support a role for extracellular DA oxidation and quinone formation in toxicity, direct evidence in our model is lacking. Future studies are needed to address the role of quinones, their intracellular or extracellular formation and the proteins targeted in the malonate model.
DA release after metabolic impairment is implicated in damage not only to DA neurons but also to striatal neurons (Reynolds et al., 1998; Ferger et al., 1999; Maragos et al., 1999; Moy et al., 2000). Unfortunately, we were unable to assess the effect of the DAT inhibitors on malonate-induced damage to striatal GABA neurons in vivo as there was no damage to these neurons in the present studies although we have seen malonate-induced damage to GABA neurons in vivo in other experiments, particularly in rat studies (Sonsalla et al., 1997; Zeevalk et al., 1997). However, in primary mesencephalic cultures, used extensively to evaluate mitochondrial inhibition and metabolic stress on neuronal viability, GABA neurons are routinely affected, although to a lesser degree than DA neurons (Marey-Semper et al., 1993, 1995; Zeevalk et al., 1995; McLaughlin et al., 1998a; Moy et al., 2000). Our findings that DAT inhibition attenuates malonate-induced damage to GABA neurons in mesencephalic cultures but not in striatal cultures that lack dopaminergic input, support the premise that extracellular DA has downstream detrimental effects. Also supportive of the contributory role of DA in damage to GABA neurons is the fact that higher concentrations of malonate are needed to produce a similar degree of damage to GABA neurons in striatal cultures than in mesencephalic cultures. Together, these data suggest that the DAT inhibitors may protect GABA neurons in mesencephalic culture by attenuating malonate-induced DA release. This protection could be afforded via reductions in extracellular DA-derived ROS and/or via reduced activation of DA receptor-mediated processes (Xia et al., 2001; Brouillet et al., 2005; Charvin et al., 2005; Bozzi and Borrelli, 2006). If, as proposed, extracellular DA oxidation products contribute extensively to neurodegeneration, then the question may be raised as to why GABA and DA neurons are not equally damaged. Although we do not have an answer, it is tempting to speculate that it may be because the DA oxidation products are formed in closer proximity to the DA nerve terminals and thus DA nerve terminal proteins may be more subject to damage than GABA membrane proteins.
In summary, we report that striatal DA efflux resulting from metabolic inhibition by intrastriatal malonate infusions is mediated in part by reversal of the DAT in the plasma membrane. Pharmacological inhibition of the DAT attenuates the malonate-induced DA efflux and protects against the resultant toxicity to DA nerve terminals. In vitro studies clearly support this finding and also demonstrate that DAT inhibition is protective against malonate-induced damage to GABA neurons. Taken together, our data strongly suggest that mitochondrial dysfunction can affect DA homeostasis, which in turn may contribute to the pathogenesis of neurodegenerative disorders. Specifically, DAT inhibitors may have potential therapeutic applications in the treatment of Parkinson's disease. Decreased striatal DAT activity could be beneficial by 1) prolonging the effects of synaptic DA in neurotransmission and 2) reducing the energy requirements in DA nerve terminals, which would be particularly relevant in Parkinson's disease patients with mitochondrial defects.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DA, dopamine; ROS, reactive oxygen species; DAT, plasma membrane dopamine transporter; WIN 35,428, methyl(–)-3-(p-fluorophenyl)-1H,5H-tropane-2-carboxylate1,5-naphthalene; CFDA, carboxyfluorescein diacetate; GBR 13098, 1-(2-(bis(4-fluophenyl methoxy) ethyl)-4-(3-(4-fluorophenyl)-propyl)piperazine) dimethane sulfonate; DCF, 2',7'-dichlorofluorescein diacetate; DOPAC, dihydroxyphenylacetic acid; HPLC, high-performance liquid chromatography; TH, tyrosine hydroxylase; HBS, HEPES-buffered saline; DMEM, Dulbecco's modified Eagle's medium; CM, conditioned medium; KRB, bicarbonate-buffered Krebs-Ringer; CF, carboxyfluorescein.
1 Current affiliation: MIT Picower Institute, Cambridge, Massachusetts. 
Address correspondence to: Dr. Patricia K. Sonsalla, UMDNJ-Robert Wood Johnson Medical School, Department of Neurology, 675 Hoes Lane, Piscataway, NJ 08854. E-mail: sonsalla{at}umdnj.edu
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