Rapid ATP Loss Caused by Methamphetamine in the Mouse. Striatum: Relationship Between Energy Impairment and Dopaminergic Neurotoxicity. Piu Chan ...
Journal qf Ncurochemislry Raven Press. Ltd., New York 0 1994 InternationalSociety for Neurochemistry
Rapid Communication
Rapid ATP Loss Caused by Methamphetamine in the Mouse Striatum: Relationship Between Energy Impairment and Dopaminergic Neurotoxicity Piu Chan, Donato A. Di Monte, Jin-Jun Luo, Louis E. DeLanney, Ian Irwin, and J. William Langston The Parkinson’s Institute, Sunnyvale, California, U.S.A.
Abstract: To study the relationship between energy impairment and the effects of &methamphetamine (METH) on dopaminergic neurons, ATP and dopamine levels were measured in the brain of C57BL/6 mice treated with either a single or four injections of METH (10 mg/kg, i.p.) at 2-h intervals. Neither striatal ATP nor dopamine concentrations changed after a single injection of METH, but both were significantly decreased 1.5 h after the multiple-dose regimen. The effects of METH on ATP levels appear to be selective for the striatum, as ATP concentrations were not affected in the cerebellar cortex and hippocampus after either a single or multiple injections of METH. In a second set of experiments, an intraperitoneal injection of 2-deoxyglucose (2-DG; 1 g/kg). an inhibitor of glucose uptake and utilization, was given 30 min before the third and fourth injections of METH. 2-DG significantly potentiated METH-induced striatal ATP loss at 1.5 h and dopamine depletions at 1.5 h and 1 week. These results indicate that a toxic regimen of METH selectively causes striatal energy impairment and raise the possibility that perturbations of energy metabolismplay a role in METH-induceddopaminergic neurotoxicity. Key Words: Methamphetamine-ATP-2-Deoxyglucose -Dopamine-Parkinsonism-Energy metabolism-Striaturn. J. Neurochem. 62,2484-2487 (1994).
d-Methamphetamine (METH) and amphetamine have been shown to cause a long-lasting decrease in striatal levels of dopamine, tyrosine hydroxylase activity, and dopamine uptake binding sites in rodents as well as in nonhuman primates (Kogan et al., 1976; Wagner et al., 1980; Woolverton et al., 1989). The selective damage to the dopaminergic neurons has been further demonstrated in the striatum by using catecholamine fluorescence and silver staining techniques (Ellison et al., 1978; Ricaurte et al., 1984). Although studies have suggested that endogenous dopamine, excitatory amino acids, and oxidative stress are involved in METH toxicity (Gibb and Kogan, 1972; Sonsalla et al., 1989; ODell et al., 199I), the precise sequence ofevents that leads to the injury of nigrostriatal dopaminergic neurons is still unknown. Recently, there has been considerable interest in the possibility that perturbations of energy metabolism play a role in the nigrostriatal injury caused by several neurotoxicants. 1 -Methyl-4-phenyl- I ,2,3,6-tetrahydropyridine (MPTP) causes a selective damage to the nigrostriatal dopamine sys-
tem and parkinsonism in humans and nonhuman primates (Burnset al., 1983; Langstonetal., 1983). MPTPisaninhibitor of mitochondria1 respiration (Nicklas et al., 1985) and has been found to decrease striatal levels of ATP (Chan et al., 199 1). Similarly, 3-nitropropionic acid and malonic acid, which block the mitochondria1 respiratory chain and deplete striatal ATP, induce neuronal degeneration in the striatum (Beal et al., 1993; Brouillet et al., 1993). In addition, experimental evidence suggests the possible involvement of excitotoxic effects in the action of these compounds (Turski et al., 1991; Beal et al., 1993; Brouillet et al., 1993; Chan et al., 1993). It has been suggested that METH also causes excitotoxic effects in the nigrostriatal dopamine system (Sonsalla et al., 1989). However, whether or not METH neurotoxicity involves changes in energy metabolism remains to be determined. In the present study, therefore, a series of experiments was conducted to investigate the effects of METH on mouse brain ATP and the relationship between METH-induced changes in striatal dopamine and ATP levels.
MATERIALS AND METHODS Male C57BL/6 mice 8-10 weeks old (Simonsen Laboratory, San Jose, CA, U.S.A.) were used for all experiments, which were performed in strict accordance with the NIH Guidefor the Care and Use oflaboratory Animals and were approved by the Institutional Animal Care and Use Committee. Animals were injected intraperitoneally with either a single dose of METH (10 mg/kg) or four injections of METH (10 mg/kg) at 2-h intervals because recent work has shown that the four-dose regimen (multiple-dose regimen) of METH induces a long-lasting selective damage to the
Resubmitted manuscript received March 3, 1994; accepted March 4, 1994. Address correspondence and reprint requests to Dr. P. Chan at The Parkinson’s Institute, 1170 Morse Avenue, Sunnyvale, CA 94089, U.S.A. Abbreviations used: 2-DG, 2-deoxyglucose; METH. d-methamphetamine; MPTP, 1-1nethyl-4-phenyl- 1.2,3,6-tetrahydropyridine.
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STRIATAL ATP LOSS BY METHAMPHETAMINE
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TABLE 1. Effecctsof METH on ATP levels in different areas of the mouse brain ATP (nmol/mg of protein) Single dose
Four doses
Area
Control
METH
Control
METH
Striatum Cerebellum Hippocampus
25.57 f 3.19 19.50 k 1.71 2 1.32 f 3.03
24.10 f 3.99 19.15 f 4.15 22.02 4.53
26.78 f 3.16 19.34 f 2.15 2 1.72 f 2.67
22.14 f 2.23" 20.32 f 1.54 22.20 f 1.83
*
ATP levels were measured in the striatum, cerebellar cortex, and hippocampus ofC57BL/6 mice (n = 10 per group) treated with either saline or a single dose or four doses of METH (10 mg/kg, i.p.) at 2-h intervals and killed by microwave irradiation of the brain 1.5 h after the last injection. Data are mean f SEM values. Statistically different ( p < 0.01) from the corresponding control value.
striatal dopaminergic system, whereas a single dose does not (Wagner et al., 1980; Sonsalla and Heikkila, 1986). Controls were injected with saline instead. For the 2-deoxyglucose (2-DG) experiments, animals were divided into four groups (n = 10 per group). Group I received four injections of saline at 2-h intervals. Additional injections of saline were given 30 min before the third and fourth injection. Animals in group I1 were given four injections of saline at 2-h intervals, and additional injections of 2-DG ( 1 g/kg) were given 30 min before the third and fourth injection. In groups 111 and IV, the treatments were identical to group I and 11, respectively, except the four injections of saline given at 2-h intervals were replaced by four doses of METH. Animals were killed by microwave irradiation of the brain (Chan et al., 199I ) at either 1.5 h or 1 week following the last METH injection. ATP was quantified in the striatum, cerebellar cortex, and hippocampus by chemiluminescence using the enzyme-substrate system luciferin-luciferase (Lemasters and Hackenbrock, 1979). Striatal dopamine concentrations were determined by HPLC as previously reported (Finnegan et al., 1988), and proteins were analyzed by the method of Lowry et al. (1 95 I). METH was obtained from the National Institute of Drug Abuse. Other chemicals were purchased from Sigma (St. Louis, MO, U.S.A.). Statistical analysis was performed by the Newman-Keuls post hoc test after a two-way ANOVA.
RESULTS Effects of METH on ATP levels The single-dose regimen of METH had no significant effect on ATP levels in the striatum, cerebellar cortex, and hippocampus (Table 1). In contrast, the multiple-dose regimen produced a statistically significant decrease in striatal ATP concentrations measured I .5 h after the last injection (Table 1). ATP levels returned to near control values by 24 h (data not shown). No decrease in ATP content was found after the multiple-dose regimen in the cerebellar cortex and hippocampus at either time point (Table 1).
Effects of METH on striatal dopamine levels Consistent with previous reports indicating that only a large dose (50-60 mg/kg) or multiple injections of moderate doses of METH induce long-lasting dopamine deficits in the striatum (Wagner et al., 1980; Sonsalla and Heikkila, 1986), a single dose of METH did not cause dopamine depletion,
but the multiple-dose regimen caused an -50% reduction in striatal dopamine concentrations at both 1.5 h and 1 week (Table 2). In addition, no changes were found in serotonin levels in either the striatum or the hippocampus (data not shown), consistent with previous reports that METH induces selective damage to the dopaminergic system in mice (Sonsalla and Heikkila, 1986).
Effects of 2-DG on METH-induced decrease in striatal levels of dopamine and ATP The second set of experiments was designed to evaluate further the relationship between METH-induced striatal ATP loss and dopamine depletion. If energy impairment plays a role in METH neurotoxicity, it is likely that a compromise of glucose metabolism would increase the susceptibility of dopaminergic neurons to METH-induced dopamine depletion. To test this hypothesis, animals were given 2-DG, an inhibitor of glucose uptake and utilization, before the third and fourth injections of METH. As shown in Fig. 1. the METH-induced striatal dopamine depletion was greatly enhanced at 1.5 h (42% reduction in the METH alone group vs. 75% reduction in the 2-DG/METH group as compared with control value). Moreover, this enhancement of METH-induced dopamine depletions was also observed in animals killed 1 week later (Fig. 1). 2-DG also exacerbated the effects of METH on striatal ATP (Fig. I). An -30% loss of ATP as compared with the saline control was observed in animals pretreated with 2DG, whereas only a 20% loss of striatal ATP was found in the METH alone group. The effects of METH on striatal ATP in the presence of 2-DG appeared to remain selective because there were no changes in cerebellar ATP levels (data not shown).
DISCUSSION The effects of METH on brain energy metabolism were first studied in the early 1960s and 1970s, but results were conflicting. Although lactate concentrations were found to be increased in the brain of rodents exposed to low or moderate dose of METH, cerebral ATP levels were reported to be unchanged, decreased, or increased (Lewis and Van Petten, 1962; Nahorski and Rogers, 1973; Sylvia et al., 1977). Because most of these studies focused on the relationship between energy metabolism and psychomotor stimulation, changes in levels of high-energy phosphate compounds were measured in either the cerebral cortex or the whole brain J. Neurochrm., Vol. 62. Nu. 6 , 1994
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P. CHAN ET A L . TABLE 2. Ejfec~sofMETH on dopumine lmels in striaturn of the mouse brain
Dopamine (ng/mg of protein) 1.5 h
1 week
Dose
Control
METH
Control
METH
Single dose Four doses
135.76 k 4.67 151.43 f 19.41
132.72 f 3.3 I 73.28 f 19.13"
130.95 f 7.52 145.14f 8.21
128.30 f 1 1.66 75.98 f 8.37"
" Statistically different ( p < 0.001) from the corresponding control values.
after a single injection of METH. More recent work, however, has shown that only the multiple-dose regimen of METH causes damage to the striatal dopaminergic system (Wagner et al., 1980; Sonsalla and Heikkila. 1986). Thus, the use of a single injection of low or moderate dose of METH and/or the area of brain chosen in the earlier studies may account for the negative results on ATP levels. Our results provide the first description of effects of a neurotoxic regimen of METH on striatal ATP levels. Doses of METH that depleted striatal dopamine also caused a significant and rapid decrease in striatal ATP concentrations. The ATP-depleting effects of METH appear to be selective because they were observed only in the striatum but not in the cerebellar cortex and hippocampus. Another novel finding of this study is that METH-induced ATP loss paralleled dopamine neurotoxicity in that (a) both ATP and dopamine
FIG. 1. Effects of 2-DG on METH-induceddecrease in levels of both striatal ATP and dopamine. Animals (n = 10 per group) were divided into four groups (for details, see Materials and Methods): (a) saline alone (solid column); (b) saline 2-DG (striped column); (c) four doses of METH saline (shaded column); and (d) four doses of METH 2-DG (open column). Striatal ATP and dopamine concentrations were measured 1.5 h and/or 1 week after the last injection of METH. Data are mean -+ SEM (bars) values expressed as percentages of the corresponding control values (ATP, 26.22 f 1.76 nmol/mg of protein; dopamine. 164.43 f 5.18 ng/mg of protein). 'Statistically different ( p < 0.01) from the saline alone control; +statistically different ( p < 0.01) from the METH alone group.
+
+
J . Neuroclirin.. L;d. 62. N o . 6. I494
+
levels were affected only by the multiple-dose regimen of METH, (b) ATP and dopamine levels decreased selectively in the striatum. and (c) 2-DG, an inhibitor ofglucose metabolism, significantly potentiated both METH-induced striatal dopamine depletion and ATP loss. These results, together with an earlier report showing an association between the METH-induced early increase in the regional cerebral glucose consumption and long-lasting dopaminergic neurotoxicity (Pontieri et al., 1990), suggest a correlation between METH-induced perturbations of energy metabolism and dopaminergic neurotoxicity. The mechanism or mechanisms by which METH may cause energy impairment in the nigrostriatal system remain to be identified. One possibility is that METH directly inhibits the mitochondria1 respiratory chain, thereby reducing cellular energy production. Alternatively, the ATP depletion observed in our study could be a consequence of "metabolic stress" caused by METH on dopaminergic neurons; increased energy consumption may be the result of dopamine release and/or activation of glutamate receptors or other energy-demanding processes. Such metabolic stress might ultimately contribute to cell injury or neuronal de-' generation. Our data indicate that an early transient decline of ATP levels may trigger a process or processes that ultimately lead to long-lasting neurotoxic effects on the dopaminergic system. This is supported by the fact that 2-DG not only potentiated METH-induced ATP loss at 1.5 h but also enhanced dopamine depletion at I week. Sonsalla et al. (1989) have shown that antagonists of excitatory amino acid receptors protected against the nigrostriatal dopaminergic damage induced by METH. It is possible, therefore, that perturbations of energy metabolism and excitotoxic effects might link the processes leading to the final dopaminergic toxicity. This is consistent with the hypothesis that energy impairment could secondarily lead to slow excitotoxic neuronal death by increasing the sensitivity of excitatory amino acid receptor activation (for review, see Beal, 1992). Acknowledgment: This work was supported by The Parkinson's Institute. The technical assistance of Wendy Bogart and Marina Fridlib and the help of David Rosner in the preparation of the manuscript are greatly appreciated.
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J. Neurochem.. Vol. 62, No. 6, 1994
