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Coenzyme Q10 and nicotinamide and a free radical spin trap protect against MPTP neurotoxicity
EXPERIMENTAL
NEUROLOGY
132,
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BRIEF COMMUNICATION Coenzyme Q10and Nicotinamide and a Free Radical Spin Trap Protect against MPTP Neurotoxicity J~~RGB. SCHULZ, D. Ross HENSHAW, RUSSELL T. MATTHEWS, Neurochemistry
AND M. FLINT
BEAL’
Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
interruption of oxidative phosphorylation which results in decreased levels of ATP (6). This may then lead to neuronal depolarization and secondary excitotoxicity mediated by activation of voltage-dependent NMDA receptors (2). A consequence of activation of these receptors is an influx of calcium which is accumulated by mitochondria. Levels of calcium which are achieved in the mitochondria following activation of NMDA receptors markedly enhance free radical generation (8). If this scenario is indeed accurate then agents which improve mitochondrial energetics ought to be neuroprotective against MPTP toxicity. Similarly, agents which are free radical scavengers should have neuroprotective effects. We recently found that both coenzyme QiO and nicotinamide can protect against neurotoxicity and ATP depletions produced by the mitochondrial toxin malonate in uivo (4). Furthermore the combination of the two agents exerts more potent neuroprotective effects than either agent administered alone. We also found that several free radical spin trap compounds can exert neuroprotective effects against both excitotoxicity and mitochondrial toxins in uiuo (25). In the present study Academic Preee. Inc. we therefore examined whether coenzyme Qlo and nicotinamide or a free radical spin trapping compound could exert neuroprotective effects against MPTP neurotoxic1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine MPTP) ity in mice. is a neurotoxin which produces clinical, biochemical, Male Swiss-Webster mice (30-35 g, Taconic Farms, and neuropathologic changes in both human and nonhuGermantown, NY) were treated with either normal man primates, which are analogous to those observed in saline or MPTP hydrochloride (Research Biochemicals, idiopathic Parkinson’s disease (PD). The neurotoxic Natick, MA). MPTP was administered in 0.1 ml water, effects of MPTP are thought to be mediated by l-methylpH adjusted to 7.4, at a dose of 10 mg/kg ip at 2-h 4-phenylpridinium (MPP+ 1, which is a metabolite formed intervals for either four or six doses or 20 mg/kg ip at by the action of monoamine oxidase B (28). MPP+ is 2-h intervals for 4 doses. Coenzyme QiO tablets (Vitaline selectively accumulated by the high afhnity dopamine Formulas, Ashland, OR) were crushed and added to uptake system and subsequently taken up into mitochonground rat chow (Agway Prolab 3200, Syracuse, NY). ’ dria of dopaminergic neurons. It then disrupts oxidative Animals in each group received either normal rat chow phosphorylation by inhibiting complex I of the mitochonor oral coenzyme QIO at 400 mg/kg in 15 g of rodent drial electron transport chain (20, 28). This leads to chow for 10 days prior to MPTP administration. The animals were checked daily to be certain they consumed 1 To whom correspondenceshould be addressed.Fax: (617) 724- the full dose of coenzyme Qro. The mice then remained on coenzyme Q10 for 2 more days, before being switched 1480. 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) produces Parhinsonism in both experimental animals and in man. MPTP is metabolized to 1-methyl-4phenylpridinium, an inhibitor of mitochondrial complex I. MPTP administration produces ATP depletions in UIUO,which may lead to secondary excitotoxicity and free radical generation. If this is the case then agents which improve mitochondrial function or free radical scavengers should attenuate MPTP neurotoxicity. In the present experiments three regimens of MPTP administration produced varying degrees of striatal dopamine depletion. A combination of coenzyme QIO and nicotinamide protected against both mild and moderate depletion of dopamine. In the MPTP regimen which produced mild dopamine depletion nicotinamide or the free radical spin trapiV-tert-butyl-a-(2zulfophenyl)nitrone were also effective. There was no protection with a MPTP regimen which produced severe dopamine depletion. These results show that agents which improve mitochondrial energy production (coenzyme QIO and nicotinamide) and free radical scavengers can attenuate mild to moderate MPl’P neurotoxicity. Q1~135
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to normal rat chow. Nicotinamide and S-PBN were obtained from Aldrich (Milwaukee, WI). Mice received doses of either 200 mglkg nicotinamide or 100 mg/kg S-PBN ip 0.5 h before every MPTP injection and every 8 h for 48 h after the first MPTP injection. Eleven or twelve animals were used in each group. Animals were sacrificed at 1 week. The striata were rapidly dissected and placed in chilled 0.1 M perchloric acid. Tissue was subsequently sonicated and aliquots taken for protein measurements using a fluorometric assay. Dopamine, 3,4dihydroxyphenylacetic acid (DOPAC) and homovanillic acid HVA) were measured by high performance liquid chromatography with 16-electrode electrochemical detection (5). Concentrations of dopamine and metabolites are expressed as nglmg protein, mean + SEM. The ability of S-PBN to inhibit the dopamine transporter was tested with an in vitro assay for 13Hldopamine uptake into striatal mouse synaptosomes (11). In our system dopamine uptake was saturable with half-maximal uptake at 60 n&f and a V,, value of 2.2 nmollg of tissuelmin. For the inhibition studies with S-PBN we used 0.13 fl 13H]dopamine as substrate and 10 l&f mazindol to block the specific binding. The statistical significance of differences was determined by one-way analysis of variance (ANOVA) followed by Fisher protected least significant difference (PLSD) post-hoc test to compare group means. Treatment with four doses of MPTP at a dose of 20 mg/kg ip resulted in severe dopamine depletions of approximately 80% (Fig. 11. Under these circumstances treatment with coenzyme QiO significantly worsened both dopamine and HVA depletions. None of the other treatments had any significant effects on MPTP neuro.toxicity under this regimen. We therefore examined
120
DOPAC
HVA
FIG. 1. Effects of treatment with coenzyme Q,o, nicotinamide, and S-PBN on dopamine depletions induced by 4 x 20 mg/kg MPTP at 7 days. ###P < 0.001 as compared tb controls using ANOVA followed by Fisher PLSD post-hoc test. *P < 0.05 as compared to MPTPtreated mice. Error bars indicate SEM.
t l
160 140 cl20 5 2100 0. F 60 \ F 60 40 20 0 DOPAC
HVA
FIG. 2. Effects of treatment with coenzyme Qlo, nicotinamide, and S-PBN on dopamine depletions induced by 6 x 10 mg/kg MPTP at 7 days. ##P < 0.01, ###P < 0.001 as compared to controls using ANOVA followed by Fisher PLSD post-hoc test. *P < 0.05, **P < 0.01, ***P i 0.001 as compared to MPTP-treated mice. Error bars indicate SEM.
whether two milder regimens of MPTP-induced dopamine depletion might be attenuated by treatment with either coenzyme Qio, nicotinamide, or S-PBN. Treatment with six doses of 10 mg/kg MPTP at 2-h intervals produced a 44% depletion in dopamine levels while DOPAC and HVA levels were depleted by 32 and 39%, respectively, at 1 week (Fig. 2). Following this treatment regimen there is a mild attenuation of MPTP-induced dopamine depletions with either coenzyme Qio or nicotinamide alone; however, these results were not significant. The combination of nicotinamide with coenzyme Qlo completely protected against the dopamine depletion. Similarly there was significant protection against both DOPAC and HVA depletions. The depletion of HVA induced by this regimen of MPTP was also blocked by either coenzyme Qio or nicotinamide treatment alone. We then examined an even milder regimen of MPTPinduced neurotoxicity. Treatment with four doses of 10 mg/kg MPTP at 2-h intervals produced a significant 30% depletion of dopamine levels, while DOPAC and HVA levels were depleted by 19 and 9%, respectively, at 1 week (Fig. 3). Treatment with coenzyme Qio alone produced a mild attenuation of the dopamine depletion which was not significant. Significant neuroprotective effects, however, were obtained with treatment with nicotinamide alone, or the combination of coenzyme Qio with nicotinamide. Treatment with the free radical spin trap S-PBN was also effective. Both nicotinamide and S-PBN prevented DOPAC depletions. Nicotinamide was effective in preventing HVA depletions. The other agents mildly attenuated HVA depletions but the results were not significant. In vitro S-PBN at 1, 10, and 100 a did not inhibit MAO-B activity. The K; of deprenyl was 0.3 CJM with
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HVA
FIG. 3. Effects of treatment with coenzyme Qlo, nicotinamide, and S-PBN on dopamine depletions induced by 4 x 10 mg/kgMPTP at 7 days. #P < 0.05, ##P < 0.01 as compared to controls using ANOVA followed by Fisher PLSD post-hoc test. **P < 0.01, ***P < 0.001 as compared to MPTP-treated mice. Error bars indicate SEM.
complete inhibition at 1.2 a. S-PBN at 1, 10, and 100 fl did not inhibit the [3H]dopamine uptake into synaptosomes in vitro, whereas the Kj of mazindol was 0.05 fl with complete inhibition at 1 fl. In the present experiments we examined whether several agents which are putative enhancers ofmitochondrial energy metabolism could produce neuroprotective effects against MPTP-induced dopaminergic neurotoxicity in uiuo. MPTP produces depletions of ATP in ho, and its metabolite MPP+ also produces depletions of ATP and increases in lactate concentrations when administered intrastriatally in uiuo (6, 27). We hypothesized that this energy depletion may result in secondary excitotoxic cell death which is accompanied by oxidative stress (2). Studies of the neuroprotective effects of excitatory amino acid antagonists against MPTP and MPP’ neurotoxicity in rodents and mice have been controversial; however, two studies in primates showed neuroprotective effects (12, 32). If energy impairment plays a role in MPTP-mediated neurotoxicity in uiuo, then agents which improve mitochondrial energetics should be neuroprotective. Coenzyme Qlo is an essential component of the electron transport chain where it serves as an electron donor and acceptor. It has been proposed that it could bridge a defect in the electron transport chain (19). It also improves membrane fluidity and acts as an antioxidant (10). Administration of coenzyme Qlo results in increased activity of the mitochondrial electron transfer system both in vitro and in uiuo (15, 22). It protects against ischemia-reperfusion injury in the heart (15). It also increases ATP production in cultured cardiac myocytes (16). Coenzyme Qlo protects against glutamate neurotoxicity in cultured cerebellar neurons (9). It
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produces clinical improvement in some but not all patients suffering with mitochondrial myopathies or encephalomyopathies (1, 14, 26). Nicotinamide is an essential precursor of NADH, which is a substrate for both complex I of the electron transport chain as well as a number of dehydrogenase enzymes involved in the citric acid cycle. It shows some efficacy in the treatment of patients with mitochondrial encephalopathies (17). Nicotinamide can also block the enzyme poly (ADPribose) polymerase, which is associated with cell death activated by hydrogen peroxide and nitric oxide (23,311. We recently found that treatment with either coenzyme Qlo or nicotinamide alone could significantly attenuate striatal lesions produced by the mitochondrial toxin malonate (4). Furthermore, we showed that the combination of the two agents was more effective than either agent alone. Both coenzyme Qlo and nicotinamide block ATP depletions and lactate increases in uiuo (4). In the present experiments we examined whether either coenzyme Qlo alone, nicotinamide alone, or the combination of the two agents could protect against MPTPinduced neurotoxicity in uiuo. The combination of coenzyme Qlo with nicotinamide significantly protected against mild and moderate deplet,ions of dopamine produced by MPTP; however, it was ineffective with more severe depletions. With mild dopaminergic neurotoxicity, nicotinamide produced neuroprotective effects by itself. Coenzyme Qlo alone showed a slight neuroprotective effect; however, this result was not significant. We also examined whether the free radical spin trapping agent S-PBN could also attenuate lesions produced by either mild, moderate, or severe regimens of MPTP-induced dopamine depletion. S-PBN had no significant neuroprotective effects in regimens of either severe or moderate dopamine depletion; however, it significantly protected against mild MPTP-induced neurotoxicity. These results are consistent with prior studies, suggesting that free radical generation is involved in MPTP neurotoxicity. MPP+ and MPTP increase the generation of free radicals (71, and MPTP-induced neurotoxicity was significantly attenuated in transgenic mice, which overexpress the enzyme copper/zinc superoxide dismutase (18). The present results provide further evidence implicating both impaired energetics and free radical generation in the pathogenesis of MPTPinduced neurotoxicity. They, however, suggest that under conditions of more severe dopamine depletion other mechanisms of neurotoxicity may occur. These mechanisms may involve activation of non-NMDA receptors followed by influx of calcium and activation of proteases and endonucleases. Our prior work showed that intrastriatal malonate neurotoxicity which produces moderate ATP depletion is blocked by NMDA antagonists (3), whereas 3-nitropropionic acid, which produces a more severe energy depletion, requires a non-NMDA antago-
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nist to show protection (29). Similar observations were initially made in vitro by Zeevalk and Nicklas (30). The present results have implications for the treatment of neurodegenerative diseases, in particular, PD. A number of studies have shown that there is a deficiency of complex I of the electron transport chain in the substantia nigra, platelets, and muscle tissue of patients with Parkinson’s disease (24). It is, therefore, possible that the mechanism of cell death may be similar to that which occur5 following administration of MPTP in uiuo, which also results in a complex I defect. In PD the decrease in complex I activity in the substantia nigra is relatively mild in the range of 30-40% (13, 211. The degree of energy depletion and oxidative stress in PD are therefore more likely to be mimicked by the less severe regimens of MPTP administration utilized in the present experiments. Under these circumstances treatment with both coenzyme Q10 in combination with nicotinamide, or S-PBN showed neuroprotective effects. These results provide further evidence that MPTP may produce neurotoxic effects by a mechanism involving energy depletion and free radical generation (2). Furthermore, they suggest that therapeutic strategies to improve mitochondrial energy metabolism or to scavenge free radicals might prove useful in PD. ACKNOWLEDGMENTS The eecretariaI aesi&e.nce of Sharon Melanaon is greatfully acknowledged. This work wee supported by NIH Grants NS 16367 and NS 31579. J.B.S. ia supported by Fellowship of the Deutache Forachungsgemeinschaft (Schu 932/l-2). .
REFERENCES
1. ABE, K., H. FUJIMURA, Y. NISHIKAWA, et al. 1991. Marked reduction in CSF lactate and pyruvate levels after CoQ therapy in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes. Acta Neurol. &and. 03: 356 359. 2. BEAL, M. F. 1992. Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann. Neural. 31: 119-130. 3. BEAL, M. F., E. BROUILLET, B. JENKINS, et al. 1993. Agedependent striatal excitotoxic lesions produced by the endogenoue mitochondriaI inhibitor malonate. J. Neurochem. 61: 1147-1150. 4. BP&, M. F., D. R. HENSHAW,B. G. JENKINS, et al. 1994. Coenzyme Qio and nicotinamide blockstriatal lesions produced by mitochondrialt.~xinmalonate.Ann.Neurol.38:882-888. 5. BEAL, M. F., W. R. MATBON, K. J. SWAIZTZ,et al. 1990. Kynurenine pathway measurements in Huntin@on’s disease striatum: evidence for reduced formation of kynurenic acid. J. Neurochem. 55: 1327-1339. 6. CHAN, P., L. E. DELANNEY, I. IRWIN, et al. 1991. Rapid ATP loss caueed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mouee brain. J. Neumchem. 51: 348-351. 7. CHRTEH, C. C., G. KRISHNA, P. TUISI, et al. 1992. Intracranial microdialysia of saIicyIic acid ‘to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: Effecta of MPP+. Free Radicals Biol. Med. 13: 581-583.
mitochon8. DYKENS, J. A. 1994. Isolated cerebral and cerebek dria produce free radicals when exposed to elevated Ca*+ and Na+: Implications for neurodegeneration. J. Neurochem. 63: 584-591. 9. FAVIT, A., F. NICOLETTI, U. SCAPAGNINI,et al. 1992. Ubiquinone protects cultured neurons against spontaneous and excitotoxininduced degeneration. J. Cereb. Blood Flow Metab. 12: 638-645. 10. FREI, B., M. C. KIM, AND B. N. AMES. 1990. Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations. Pmt. Natl. Acad. Sci. USA 81: 4879-4883. 11. JAVITCH, J. A., R. J. D’I&ATO, S. M. STIUTTMA~TER,et al. 1985. Parkinsonism-inducing neurotoxin, N-methyl-Cphenyl-1,2,3,6tetrahydropyridine: uptake of the metabolite N-methyl-4phenylpyridine by dopamine neurons explains selective toxicity. Pmc Nat1 Acod Sci USA 82: 2173-2177. 12. LANGE, K. W., P.-A. LQSCHMANN, E. SOFIC, et al. 1993. The competitive NMDA antagonist CPP protects substantia nigra neurons from MPTP-induced degeneration in primates. NaunynSchmiedebergs Arch. Pharmacol. 348: 586-592. 13. MANN, V. M., J. M. COOPER,D. KRIGE, et al. 1992. Brain, skeletal muscle and platelet homogenate mitochondrial function in Parkinson’s disease. Brain 115: 333-342. 14. MATHEWS, P. M., B. FORD, R. J. DAND~D, et al. 1993. Coenzyme Qio with multiple vitamins is generally ineffective in treatment of mitochondrial disease. Neurology 43: 884-890. 15. OHHARA, H., H. KANAIDE, R. YOSHIMURA,et al. 1981. A protective effect of coenzyme Qia on ischemia and reperfusion of the isolated perfused rat heart. J. Mol. Cell. Cardiol. 13: 65-74. 16. OKAMOTO, T., K. KOBAYASHI, T. TAKAHASHI, et al. 1993. Biochemical roles of coenzyme Q on cultured ventricular myocytes: Effects of coenxyme Q on mitochondrial ATP production. 8th Znternational Coensyme Q Symposium. 17. PENN, A. M. W., J. W. K. LEE, P. THUILLIER, et al. 1992. MELAS syndrome with mitochondrial tRNALeuuua mutation: Correlation of clinical state, nerve conduction, and muscle 31P magnetic resonance spectroscopy during treatment with nicotinamide and riboflavin. Neurology 42: 2147-2152. 18. PRZEDBORSKI, S., V. KOSTIC, V. JACKSON-LEWIS, et al. 1992. Transgenic mice with increased Cu/Zn-superoxide dismutaee activity are resistant to N-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced neurotoxicity. J. Neurosci. 12: 1658-1667. 19. PRZYREMBEL, H. 1987. Therapy of mitochondrial disorders. J. Znher. Me&b. Dis. 10: 129-146. 20. RAMSAY, R. R., M. J. KRUEGER, S. K. YOUNGSTER,et al. 1991. Interaction of 1-methyl-4-phenylpyridinium ion (MPP+ ) and its analogs with the rotenonelpiericidin binding site of NADH dehydrogenase. J. Neurochem. 56: 1184-1190. 21. SCHAPIRA, A. H. V., J. M. COOPER, D. DEXTER, et al. 1990. Mitochondrial complex I deficience in Parkinson’s disease. J. Neumchem. 54: 823-827. 22. SCHNEIDER,H., J. J. LEMASTER, AND C. R. HACKEMBROEK. 1982. Lateral diffusion of ubiquinone during electron transfer in phospholipidand ubquinone-enriched mitochondriai membrane. J. Biol. Chem. 267: 10789-10793. 23. SCHRAUFSTATTER,I. U., P. A. HYSLOP, D. B. HINSHAW,et al. 1986. Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase. Proc. Natl. Acad. Sci. USA 83: 4908-4912. 24. SCHULZ,J. B., AND M. F. BEAL. 1994. Mitochondriel dysfunction in movement disorders. Curr. Opin. Neurol. 7: 333-339. 25. SCHULZ,J. B., D. R. HENSHAW,D. SIWEK, et al. 1994. Involvement of free radicals in excitotoxicity in vivo. J. Neumchem., in press. 26. SHOFFNER, J. M., M. T. LOTT, A. S. VOLJA~~C, et al. 1989. Spontaneous Kearns/Sayre/chronic external ophthalmoplegia
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27.
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plus syndrome associated with a mitochondrial DNA deletion: A slip-replication model and metabolic therapy. Proc. Natl. Acad. Sci. USA 86: 7952-7956. STOREY, E., B. T. HYMAN, B. JENKINS, et al. 1992. 1-Methyl-4phenylpyridinium produces excitotoxic lesions in rat striatum as a result of impairment of oxidative metabolism. J. Neurochem. 58: 1975-1978. TIPTON, K. F., AND T. P. SINGER. 1993. Advances in our understanding of the mechanisms of the neurotoxicity of MPTP and related compounds. J. Neurochem. 61: 1191-1206. TURSKI, L., AND H. IKONOMIDOU.1994. StriataI toxicity of 3-nitro-
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propionic acid prevented by the AMPA antagonist NBQX. SOC. Neurosci. Abst. 20: 1677. 30. ZEEVALK, G. D., AND W. J. Nrc~w\s. 1990. Chemically induced hypoglycemia and anoxia: relationship to glutamate receptormediated toxicity in retina. J. Pharm. Exp. 2%~. 253: 12851292. 31. ZHANG,J., U. L. DAWSON,T. M. DAWSON,et al. 1994. Nitric oxide activation of poly(ADP-ribose) synthetaae in neurotoxicity. Science 83: 687-689. 32 ZUDDAS,A., G. OBERTO, F. VAGLINI, et al. 1992. MK-801 prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Parkinsonism in primates. J. Neurochem. 59: 733-739.
NEUROLOGY
132,
2’i%233
(19%)
BRIEF COMMUNICATION Coenzyme Q10and Nicotinamide and a Free Radical Spin Trap Protect against MPTP Neurotoxicity J~~RGB. SCHULZ, D. Ross HENSHAW, RUSSELL T. MATTHEWS, Neurochemistry
AND M. FLINT
BEAL’
Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
interruption of oxidative phosphorylation which results in decreased levels of ATP (6). This may then lead to neuronal depolarization and secondary excitotoxicity mediated by activation of voltage-dependent NMDA receptors (2). A consequence of activation of these receptors is an influx of calcium which is accumulated by mitochondria. Levels of calcium which are achieved in the mitochondria following activation of NMDA receptors markedly enhance free radical generation (8). If this scenario is indeed accurate then agents which improve mitochondrial energetics ought to be neuroprotective against MPTP toxicity. Similarly, agents which are free radical scavengers should have neuroprotective effects. We recently found that both coenzyme QiO and nicotinamide can protect against neurotoxicity and ATP depletions produced by the mitochondrial toxin malonate in uivo (4). Furthermore the combination of the two agents exerts more potent neuroprotective effects than either agent administered alone. We also found that several free radical spin trap compounds can exert neuroprotective effects against both excitotoxicity and mitochondrial toxins in uiuo (25). In the present study Academic Preee. Inc. we therefore examined whether coenzyme Qlo and nicotinamide or a free radical spin trapping compound could exert neuroprotective effects against MPTP neurotoxic1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine MPTP) ity in mice. is a neurotoxin which produces clinical, biochemical, Male Swiss-Webster mice (30-35 g, Taconic Farms, and neuropathologic changes in both human and nonhuGermantown, NY) were treated with either normal man primates, which are analogous to those observed in saline or MPTP hydrochloride (Research Biochemicals, idiopathic Parkinson’s disease (PD). The neurotoxic Natick, MA). MPTP was administered in 0.1 ml water, effects of MPTP are thought to be mediated by l-methylpH adjusted to 7.4, at a dose of 10 mg/kg ip at 2-h 4-phenylpridinium (MPP+ 1, which is a metabolite formed intervals for either four or six doses or 20 mg/kg ip at by the action of monoamine oxidase B (28). MPP+ is 2-h intervals for 4 doses. Coenzyme QiO tablets (Vitaline selectively accumulated by the high afhnity dopamine Formulas, Ashland, OR) were crushed and added to uptake system and subsequently taken up into mitochonground rat chow (Agway Prolab 3200, Syracuse, NY). ’ dria of dopaminergic neurons. It then disrupts oxidative Animals in each group received either normal rat chow phosphorylation by inhibiting complex I of the mitochonor oral coenzyme QIO at 400 mg/kg in 15 g of rodent drial electron transport chain (20, 28). This leads to chow for 10 days prior to MPTP administration. The animals were checked daily to be certain they consumed 1 To whom correspondenceshould be addressed.Fax: (617) 724- the full dose of coenzyme Qro. The mice then remained on coenzyme Q10 for 2 more days, before being switched 1480. 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) produces Parhinsonism in both experimental animals and in man. MPTP is metabolized to 1-methyl-4phenylpridinium, an inhibitor of mitochondrial complex I. MPTP administration produces ATP depletions in UIUO,which may lead to secondary excitotoxicity and free radical generation. If this is the case then agents which improve mitochondrial function or free radical scavengers should attenuate MPTP neurotoxicity. In the present experiments three regimens of MPTP administration produced varying degrees of striatal dopamine depletion. A combination of coenzyme QIO and nicotinamide protected against both mild and moderate depletion of dopamine. In the MPTP regimen which produced mild dopamine depletion nicotinamide or the free radical spin trapiV-tert-butyl-a-(2zulfophenyl)nitrone were also effective. There was no protection with a MPTP regimen which produced severe dopamine depletion. These results show that agents which improve mitochondrial energy production (coenzyme QIO and nicotinamide) and free radical scavengers can attenuate mild to moderate MPl’P neurotoxicity. Q1~135
279
0014-4999/95$9.00
Copyright01995byAcademicPrem,Inc. All rightsof reproductionin anyform reserved.
280
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to normal rat chow. Nicotinamide and S-PBN were obtained from Aldrich (Milwaukee, WI). Mice received doses of either 200 mglkg nicotinamide or 100 mg/kg S-PBN ip 0.5 h before every MPTP injection and every 8 h for 48 h after the first MPTP injection. Eleven or twelve animals were used in each group. Animals were sacrificed at 1 week. The striata were rapidly dissected and placed in chilled 0.1 M perchloric acid. Tissue was subsequently sonicated and aliquots taken for protein measurements using a fluorometric assay. Dopamine, 3,4dihydroxyphenylacetic acid (DOPAC) and homovanillic acid HVA) were measured by high performance liquid chromatography with 16-electrode electrochemical detection (5). Concentrations of dopamine and metabolites are expressed as nglmg protein, mean + SEM. The ability of S-PBN to inhibit the dopamine transporter was tested with an in vitro assay for 13Hldopamine uptake into striatal mouse synaptosomes (11). In our system dopamine uptake was saturable with half-maximal uptake at 60 n&f and a V,, value of 2.2 nmollg of tissuelmin. For the inhibition studies with S-PBN we used 0.13 fl 13H]dopamine as substrate and 10 l&f mazindol to block the specific binding. The statistical significance of differences was determined by one-way analysis of variance (ANOVA) followed by Fisher protected least significant difference (PLSD) post-hoc test to compare group means. Treatment with four doses of MPTP at a dose of 20 mg/kg ip resulted in severe dopamine depletions of approximately 80% (Fig. 11. Under these circumstances treatment with coenzyme QiO significantly worsened both dopamine and HVA depletions. None of the other treatments had any significant effects on MPTP neuro.toxicity under this regimen. We therefore examined
120
DOPAC
HVA
FIG. 1. Effects of treatment with coenzyme Q,o, nicotinamide, and S-PBN on dopamine depletions induced by 4 x 20 mg/kg MPTP at 7 days. ###P < 0.001 as compared tb controls using ANOVA followed by Fisher PLSD post-hoc test. *P < 0.05 as compared to MPTPtreated mice. Error bars indicate SEM.
t l
160 140 cl20 5 2100 0. F 60 \ F 60 40 20 0 DOPAC
HVA
FIG. 2. Effects of treatment with coenzyme Qlo, nicotinamide, and S-PBN on dopamine depletions induced by 6 x 10 mg/kg MPTP at 7 days. ##P < 0.01, ###P < 0.001 as compared to controls using ANOVA followed by Fisher PLSD post-hoc test. *P < 0.05, **P < 0.01, ***P i 0.001 as compared to MPTP-treated mice. Error bars indicate SEM.
whether two milder regimens of MPTP-induced dopamine depletion might be attenuated by treatment with either coenzyme Qio, nicotinamide, or S-PBN. Treatment with six doses of 10 mg/kg MPTP at 2-h intervals produced a 44% depletion in dopamine levels while DOPAC and HVA levels were depleted by 32 and 39%, respectively, at 1 week (Fig. 2). Following this treatment regimen there is a mild attenuation of MPTP-induced dopamine depletions with either coenzyme Qio or nicotinamide alone; however, these results were not significant. The combination of nicotinamide with coenzyme Qlo completely protected against the dopamine depletion. Similarly there was significant protection against both DOPAC and HVA depletions. The depletion of HVA induced by this regimen of MPTP was also blocked by either coenzyme Qio or nicotinamide treatment alone. We then examined an even milder regimen of MPTPinduced neurotoxicity. Treatment with four doses of 10 mg/kg MPTP at 2-h intervals produced a significant 30% depletion of dopamine levels, while DOPAC and HVA levels were depleted by 19 and 9%, respectively, at 1 week (Fig. 3). Treatment with coenzyme Qio alone produced a mild attenuation of the dopamine depletion which was not significant. Significant neuroprotective effects, however, were obtained with treatment with nicotinamide alone, or the combination of coenzyme Qio with nicotinamide. Treatment with the free radical spin trap S-PBN was also effective. Both nicotinamide and S-PBN prevented DOPAC depletions. Nicotinamide was effective in preventing HVA depletions. The other agents mildly attenuated HVA depletions but the results were not significant. In vitro S-PBN at 1, 10, and 100 a did not inhibit MAO-B activity. The K; of deprenyl was 0.3 CJM with
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5100 5 z 60 E ' 60 e 40 20 0
Dopamine
DOPAC
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FIG. 3. Effects of treatment with coenzyme Qlo, nicotinamide, and S-PBN on dopamine depletions induced by 4 x 10 mg/kgMPTP at 7 days. #P < 0.05, ##P < 0.01 as compared to controls using ANOVA followed by Fisher PLSD post-hoc test. **P < 0.01, ***P < 0.001 as compared to MPTP-treated mice. Error bars indicate SEM.
complete inhibition at 1.2 a. S-PBN at 1, 10, and 100 fl did not inhibit the [3H]dopamine uptake into synaptosomes in vitro, whereas the Kj of mazindol was 0.05 fl with complete inhibition at 1 fl. In the present experiments we examined whether several agents which are putative enhancers ofmitochondrial energy metabolism could produce neuroprotective effects against MPTP-induced dopaminergic neurotoxicity in uiuo. MPTP produces depletions of ATP in ho, and its metabolite MPP+ also produces depletions of ATP and increases in lactate concentrations when administered intrastriatally in uiuo (6, 27). We hypothesized that this energy depletion may result in secondary excitotoxic cell death which is accompanied by oxidative stress (2). Studies of the neuroprotective effects of excitatory amino acid antagonists against MPTP and MPP’ neurotoxicity in rodents and mice have been controversial; however, two studies in primates showed neuroprotective effects (12, 32). If energy impairment plays a role in MPTP-mediated neurotoxicity in uiuo, then agents which improve mitochondrial energetics should be neuroprotective. Coenzyme Qlo is an essential component of the electron transport chain where it serves as an electron donor and acceptor. It has been proposed that it could bridge a defect in the electron transport chain (19). It also improves membrane fluidity and acts as an antioxidant (10). Administration of coenzyme Qlo results in increased activity of the mitochondrial electron transfer system both in vitro and in uiuo (15, 22). It protects against ischemia-reperfusion injury in the heart (15). It also increases ATP production in cultured cardiac myocytes (16). Coenzyme Qlo protects against glutamate neurotoxicity in cultured cerebellar neurons (9). It
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produces clinical improvement in some but not all patients suffering with mitochondrial myopathies or encephalomyopathies (1, 14, 26). Nicotinamide is an essential precursor of NADH, which is a substrate for both complex I of the electron transport chain as well as a number of dehydrogenase enzymes involved in the citric acid cycle. It shows some efficacy in the treatment of patients with mitochondrial encephalopathies (17). Nicotinamide can also block the enzyme poly (ADPribose) polymerase, which is associated with cell death activated by hydrogen peroxide and nitric oxide (23,311. We recently found that treatment with either coenzyme Qlo or nicotinamide alone could significantly attenuate striatal lesions produced by the mitochondrial toxin malonate (4). Furthermore, we showed that the combination of the two agents was more effective than either agent alone. Both coenzyme Qlo and nicotinamide block ATP depletions and lactate increases in uiuo (4). In the present experiments we examined whether either coenzyme Qlo alone, nicotinamide alone, or the combination of the two agents could protect against MPTPinduced neurotoxicity in uiuo. The combination of coenzyme Qlo with nicotinamide significantly protected against mild and moderate deplet,ions of dopamine produced by MPTP; however, it was ineffective with more severe depletions. With mild dopaminergic neurotoxicity, nicotinamide produced neuroprotective effects by itself. Coenzyme Qlo alone showed a slight neuroprotective effect; however, this result was not significant. We also examined whether the free radical spin trapping agent S-PBN could also attenuate lesions produced by either mild, moderate, or severe regimens of MPTP-induced dopamine depletion. S-PBN had no significant neuroprotective effects in regimens of either severe or moderate dopamine depletion; however, it significantly protected against mild MPTP-induced neurotoxicity. These results are consistent with prior studies, suggesting that free radical generation is involved in MPTP neurotoxicity. MPP+ and MPTP increase the generation of free radicals (71, and MPTP-induced neurotoxicity was significantly attenuated in transgenic mice, which overexpress the enzyme copper/zinc superoxide dismutase (18). The present results provide further evidence implicating both impaired energetics and free radical generation in the pathogenesis of MPTPinduced neurotoxicity. They, however, suggest that under conditions of more severe dopamine depletion other mechanisms of neurotoxicity may occur. These mechanisms may involve activation of non-NMDA receptors followed by influx of calcium and activation of proteases and endonucleases. Our prior work showed that intrastriatal malonate neurotoxicity which produces moderate ATP depletion is blocked by NMDA antagonists (3), whereas 3-nitropropionic acid, which produces a more severe energy depletion, requires a non-NMDA antago-
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nist to show protection (29). Similar observations were initially made in vitro by Zeevalk and Nicklas (30). The present results have implications for the treatment of neurodegenerative diseases, in particular, PD. A number of studies have shown that there is a deficiency of complex I of the electron transport chain in the substantia nigra, platelets, and muscle tissue of patients with Parkinson’s disease (24). It is, therefore, possible that the mechanism of cell death may be similar to that which occur5 following administration of MPTP in uiuo, which also results in a complex I defect. In PD the decrease in complex I activity in the substantia nigra is relatively mild in the range of 30-40% (13, 211. The degree of energy depletion and oxidative stress in PD are therefore more likely to be mimicked by the less severe regimens of MPTP administration utilized in the present experiments. Under these circumstances treatment with both coenzyme Q10 in combination with nicotinamide, or S-PBN showed neuroprotective effects. These results provide further evidence that MPTP may produce neurotoxic effects by a mechanism involving energy depletion and free radical generation (2). Furthermore, they suggest that therapeutic strategies to improve mitochondrial energy metabolism or to scavenge free radicals might prove useful in PD. ACKNOWLEDGMENTS The eecretariaI aesi&e.nce of Sharon Melanaon is greatfully acknowledged. This work wee supported by NIH Grants NS 16367 and NS 31579. J.B.S. ia supported by Fellowship of the Deutache Forachungsgemeinschaft (Schu 932/l-2). .
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