Mechanisms Of Methamphetamine Toxicity

As discussed above, increases in the extracellular concentrations of both DA and glutamate within the striatum appear to contribute to MA-induced damage to dopaminergic nerve terminals. Many of the manipulations used to identify the role of DA in mediating MA-induced damage also modify MA-induced changes in the extracellular concentrations of glutamate. These findings suggest that the release of both DA and glutamate are obligatory in the MA toxicity cascade (9,10,33,34). For example, D2 receptor blockade decreases glutamate release in the striatum without altering DA overflow, whereas administration of a DA uptake inhibitor decreases the ability of MA to release DA without affecting the striatal increase in glutamate efflux (10). Although these treatments differentially affect the release of DA and glutamate, both are neuroprotective and demonstrate the importance of these neurotransmitters as comediators of MA toxicity.

There are several ways that the actions of DA and glutamate may synergize to mediate the toxicity of MA. High-dose MA treatment has been found to induce the endogenous formation of oxidizing compounds in brain regions susceptible to toxicity (61,62), implicating oxidative stress as an underlying cause of terminal damage. In support of this finding, DA exacerbates glutamate-induced cell death in vitro via an oxidative mechanism (63). Dopaminergic lesions of the nigrostriatal pathway in vivo decreases the excitotoxic effect of intrastriatal infusion of excitatory amino acids (64,65), further implicating interactions between DA and glutamate in MA toxicity. In addition, efflux of both glutamate and DA can lead to the formation of reactive oxygen species and a shift in mitochondrial membrane potential to compromise mitochondrial function and produce metabolic stress and subsequent cell death (66-69).

Overall, there is substantial support for the hypothesis that increased DA and glutamate efflux leads to excitotoxic, oxidative, and metabolic stress and that substrates that attenuate the consequences of such stressors (glutamate receptor antagonists, antioxidants, free-radical scavengers, or substrates for the electron-transport chain) are neuroprotective. Evidence for the ability of DA and glutamate to induce excitotoxic, oxidative, and metabolic stress, as well as evidence for their involvement in MA toxicity, are discussed below (Fig. 5).

Fig. 5. Glutamate and dopamine contribute to MA toxicity by influencing several factors, including excitotoxic stress, oxidative stres, and metabolic stress.

3.1. Excitotoxicity

Increased extracellular glutamate concentrations and overstimulation of ionotropic glutamate receptors leads to a cascade of events that culminate in excitotoxic cell death (for review, see refs, 70 and 71). Initially, stimulation of a-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptors increases intracellular Na+, resulting in depolarization and removal of the voltage-gated Mg+ block from the NMDA receptor channel (72). Further glutamate stimulation at the NMDA receptor increases intracellular Ca2+ and a subsequent sequestration of Ca2+ within the mitochondira via activation of a Ca2+-ATPase. Because the oxidation of pyruvate drives both Ca2+ sequestration and ATP synthesis, an increase in intracellular Ca2+ can shift the balance between these two processes and interrupt ATP synthesis. This eventually leads to the depletion of energy stores, collapse of the mitochondrial membrane potential, and a consequent rise in intracellular Ca2+ levels as Ca2+ is released from mitochondrial stores.

The NMDA receptor activation and elevated levels of intracellular Ca2+ that result from increased extracellular glutamate concentrations can activate a number of enzymes, including calpain, endonu-cleases, phospholipase A2, xanthine oxidase, nitric oxide synthase, and arachidonate (73,74). Each of these enzymes can elicit a sequence of destructive events that lead to the formation of intracellular reactive oxygen species and eventual cell death (27,70). In addition, the free-radical species that are generated further enhance glutamate release, inhibit glutamate reuptake (75-77), and thus promote a feed-forward cycle to augment glutamate-mediated damage.

A consequence of an increase in intracellular Ca2+ is the activation of a Ca2+-dependent protease, calpain. Calpain activation is mediated by excitatory amino acid release and results in the proteolysis of axonal spectrin, a major component of the cytoskeleton (78,79). Activation of calpain is a primary mechanism that contributes to several types of neurodegenerative condition, including glutamate-induced neurotoxicity associated with traumatic brain injury, ischemia, and hyperthermia (80-83). Glutamate-mediated activation of calpain also catalyzes the conversion of xanthine dehydrogenase to xanthine oxidase. Xanthine oxidase, in turn, promotes the catabolism of xanthine and hypoxanthine to uric acid, yielding oxygen free radicals in the process (84). We have recently shown that MA treatment increases the concentration of uric acid in the striatum, providing evidence that glutamate-mediated excitotoxic stress accompanies MA administration (85).

In summary, excitotoxic mechanisms may underlie, in part, the damage to dopaminergic nerve terminals following high-dose MA administration. MA-induced neurotoxicity specifically involves the activation of several glutamate-mediated enzymes, including calpain, xanthine oxidase, and nitric oxide synthase (see Section 3.2). Activation of these enzymes, and other glutamate- and Ca2+- mediated systems, could result in the formation of reactive oxygen species. Together with free radicals that may be formed as a result of increased DA release, these neurotoxic oxygen species can actively participate in cell death.

3.2. Oxidative Stress

There is indirect and direct evidence that MA produces oxidative stress. Oxidative stress is defined as the cytotoxic consequences of reactive oxygen species (e.g., O2-, OH) generated as byproducts of oxidative metabolism. Evidence that indirectly supports the contention that MA leads to oxidative stress is that MA administration results in the production of hydroxyl radicals (OH) in the striatum (85-87). Conversely, antioxidants (e.g.,ascorbic acid) and the spin-trap agent, phenyl-i-butylnitrone, prevent the striatal toxicity produced by MA (85,88,89). Overexpression of the human Cu/Zn-superox-ide dismutase gene, which degrades O2-, also confers protection against the DA-depleting effects of MA (90). In addition, because it is thought that the immediate early gene c-fos plays a protective role in the brain by activating a variety of antioxidant enzyme systems (91,92) or by increasing the levels of trophic factors in the brain (for review, see ref. 93), the induction of c-fos following MA administration (94-96) and the exacerbation of toxicity in c-fos knockout mice (97) further support for the role of oxidative processes in MA-induced damage.

More direct evidence of free-radical-mediated damage by MA would indicate the presence of oxidized proteins (protein nitration), lipids (lipid peroxidation), and DNA (nucleotide oxidation) (98). In fact, all three types of cellular damage occur after MA administration. DA-dependent intracellular oxidataion following exposure to MA produces degeneration of neurite outgrowth in DA neuron cultures (99) and induces apoptosis in intrinsic nondopaminergic neurons in the striatum and frontal cortex of mice in vivo, as determined by TUNEL staining for DNA fragmentation (97). Additionally, MA treatment increases lipid peroxidation in the striatum as evidenced by an increase in malonyldialdehyde production (85,100). Conversely, inhibition of lipid peroxidation attenuates the toxicity produced by MA (101). Furthermore, MA treatment results in protein nitration as evidenced by the formation of 3-nitrotyrosine from peroxnitrite production (102,103).

The mechanistic underpinnings of MA-induced oxidative stress may involve dopamine and glutamate. The increase in cytosolic and extracellular DA produced by MA may induce cytotoxicity via the generation of free-radical species and quinones. DA is enzymatically metabolized to form H2O2 that is then nonenzymatically catalyzed by iron to form OH (29). In addition, DA autoxidation produces cytotoxic quinones, which attack thiol-containing proteins and result in the formation of 5-cysteinyl adducts of DA (104). Consistent with these in vitro findings, intrastriatal injection of high concentrations of DA results in neurotoxicity and in the in vivo formation of protein-bound cysteinyl adducts of DA, both of which are prevented by the coadministration of antioxidants (105). Similar to the effects of MA administration in vivo, free radicals and DA quinones rapidly decrease DA transporter function and inactivate tyrosine hydroxylase in vitro (106-108). Therefore, the massive increase in the extracellular concentrations of DA, such as that produced by MA, could result in the production of hydroxyl free radicals, oxidative stress, and eventual damage to DA terminals.

A compromise in endogenous antioxidant mechanisms (e.g., glutathione) by MA may also contribute to oxidative stress. MA decreases glutathione peroxidase activity (109). Although total glutathione content in the striatum is reduced in the long run after MA (110), we have shown that both reduced glutathione and oxidized glutathione are acutely increased in the striatum following a neurotoxic regimen of MA (111), it is possible that MA-induced oxidative stress results in the rapid recruitment of the endogenous glutathione antioxidant system followed by a lasting decrease associated with neurotoxicity and long-term dopaminergic damage.

Glutamate and glutamate receptor activation also can cause neuronal death through these oxidative mechanisms (112). Several lines of evidence indicate that glutamate exposure and subsequent nitric oxide production lead to a depletion of endogenous antioxidant and energy stores and an accumulation of intracellular peroxides leading to oxidative stress and cell death—a phenomenon known as oxidative glutamate toxicity (113). Glutamate-mediated activation of NMDA receptors, neuronal NOS, and the production of excess nitric oxide (114) can produce neurotoxicity (115,116). Nitric oxide reacts with O2- to form the oxidant, peroxynitrite (ONOO) (112,117). Peroxynitrite and its decomposition product nitrite may contribute to toxicity via oxidation of DA and protein modification (118). Conversely, inhibition of nitric oxide synthesis by administration of the neuronal NOS inhibitor 7-nitroin-dazole, in vivo, protects against DA damage caused by MPTP administration (119,120) and attenuates excitotoxicity following intrastriatal administration of NMDA (116). Moreover, inhibition of neuronal NOS also protects against MA-induced toxicity both in vitro and in vivo (121-124) presumably resulting from the attenuation of hydroxyl radical formation and the consequent decrease in formation of 8-hydroxy-2-deoxyguanosine as well as 3-nitrotyrosine (116,120).

In general, a substantial amount of evidence supports the hypothesis that MA administration leads to the endogenous formation of reactive oxygen species through both dopaminergic and glutamatergic mechanisms and that these reactive compounds mediate toxicity to dopaminergic nerve terminals. However, intimately related to the glutamate-dependent dependent production of oxidative stress and its role in MA toxicity are the effects of glutamate on cellular bioenergetics and the production of metabolic stress.

3.3. Metabolic Stress

Mitochondrial dysfunction, metabolic stress, and disruption of bioenergetic systems that result from high concentrations of extracellular glutamate also contribute to MA-induced neurotoxicity. Alterations in brain energy utilization by low doses of amphetamine and related analogs were reported originally in the 1970s. The results of these early experiments show that low doses of amphetamine and MA rapidly increase metabolism in the cerebral cortex or the whole brain as measured by lactate formation and changes in high-energy substrates such as ATP and phosphocreatine (125). More recent studies have demonstrated that amphetamine and MA increase local cerebral glucose utilization in multiple brain regions within 45 min of drug administration (126,127). In contrast, high-dose treatment with MA decreases cerebral glucose metabolism for weeks to months following drug administration, suggesting that initial increases in energy utilization are followed by lasting impairments in metabolism (128).

Methamphetamine and amphetamine alter energy utilization in a brain-region-specific manner, in that acute increases in glucose utilization appear to be greatest in those brain regions most susceptible to the toxic effects of MA. Our laboratory has demonstrated that MA increases the extracellular concentrations of lactate in the striatum but not in the prefrontal cortex, the latter area being relatively resistant to the long-term DA-depleting effects of MA (129). MA also rapidly and transiently decreases complex IV (cytochrome-c oxidase) activity and ATP concentrations in the striatum but not the hippocampus, a region resistant to the DA-depleting effects of MA (130,131). Because brain-region-dependent changes in metabolism appear to be correlated with depletions of DA, the selective effect of MA-induced energy consumption and subsequent energy depletion may be related to MA-induced glutamate release, oxidative stress, and the long-term depletions of DA.

Stimulant-induced increases in the extracellular concentrations of monoamines may contribute to mitochondrial inhibition. Elevated extracellular DA may compromise mitochondrial function via autoxidation to form quinones and/or the enzymatic degradation of DA to form H2O2 and the generation of hydroxyl radicals (132,133). This hypothesis is especially interesting given the finding that decreased cytochrome-c oxidase activity is restricted to DA-rich brain regions (striatum, nucleus accumbens, and substantia nigra) (130). Reactive oxygen species and DA-derived quinones are known to directly inhibit mitochondrial enzymes associated with energy production (66,134,135). Although DA-mediated inhibition of energy production has not been demonstrated to occur in vivo, in vitro incubation of rat brain mitochondria with DA or DA-derived quinones decreases state 3 (ATP-synthesis coupled) and increases state 4 respiration (67). These studies indicate that reactive DA byproducts may increase proton leakage across the mitochondrial membrane and inhibit the production of energy stores.

Several additional mechanisms could underlie the compromise in metabolic function that follows MA administration. Psychostimulants may increase neuronal energy utilization through the sustained sodium-dependent reversal of monoamine transporters, hyperlocomotion, and the production of hyper-thermia (136-138). The majority of ATP in the neuropil is devoted to the maintenance of ion (e.g., Na+) gradients and the restoration of the membrane potential following depolarization (139-142). Therefore, sustained activation of the ATP-dependent Na+/K+-ATPase following prolonged neuro-transmitter release may lead indirectly to the depletion of substrates for the electron-transport chain. Such a decrease in available precursors may slow or halt the production of ATP through a decline in complex IV activity.

Depletion of striatal ATP stores could significantly contribute to elevated glutamate levels and further potentiate damage following MA administration (for review, see ref. 27). For example, a loss of Na+/K+ ATPase activity could lead to depolarization and release of neuronal glutamate from vesicular stores. In addition, energy failure could contribute to excess extracellular glutamate levels by disrupting or reversing the ATP-dependent glutamate transporter. The conversion of glutamate to glutamine in glia is also ATP dependent. Thus, depletion of energy stores could increase intraglial concentrations of glutamate. Increased intracellular glutamate concentrations could disrupt the concentration-dependent uptake of glutamate into glia, resulting in the accumulation of extracellular glutamate. Thus, in addition to activation of the corticostriatal pathway, MA administration could lead indirectly to elevated extracellular glutamate concentrations by disrupting bioenergetic systems and depleting energy (ATP) stores.

As discussed previously, increased extracellular glutamate concentrations after MA and subsequent NMDA receptor activation may lead to metabolic inhibition via classic excitotoxic mechanisms. Direct inhibition of mitochondrial function induces NMDA receptor-mediated excitotoxic damage that has similarities with damage resulting from MA administration. Almeida et al. (143) reported that neurons exposed to glutamate in vitro had decreased glutathione and ATP content, increased lactate dehy-drogenase activity, decreased mitochondrial enzyme activity (succinate cytochrome-c reductase and cytochrome-c oxidase), and decreased oxygen consumption. Interestingly, increases in the extracellular concentrations of lactate, decreases in ATP content, and inhibition of cytochrome-c oxidase have all been found to occur in vivo following MA administration (129-131). Similarly, local striatal perfusion of mitochondrial inhibitors acutely increases the extracellular concentration of DA and glutamate, depletes ATP, and produces an accumulation of lactate (25,144-146). The long-term effects of mal-onate infusions include damage to striatal DA and, to a lesser extent, 5-HT terminals and a potentiated depletion of DA produced by both systemic and central administration of MA (25,147). Furthermore, removal of excitatory corticostriatal afferents or administration of glutamate receptor antagonists attenuates striatal damage induced by either MA or the metabolic inhibitors malonate and 3-nitropro-prionic acid (148,149). In addition, MA toxicity appears to be dependent on an increase in the release of nitric oxide, via glutamate activation of NMDA receptors, and the subsequent activation of the NOS pathway (150-152). The subsequent production of nitric oxide can lead to the formation of reactive oxygen species (peroxynitrite) and to mitochondrial dysfunction by directly inhibiting complex IV of the electron-transport chain, cytochrome-c oxidase (151,153). Thus, metabolic stress appears to be an important mediator in the excitotoxicity following direct inhibition of mitochondrial enzymes by mal-onate or 3-nitroproprionic, or indirectly following MA administration.

Several studies have demonstrated that manipulation of energy availability, via metabolic inhibition or support of bioenergetic systems, can alter the lasting effects of MA administration. Chan et al. (131) reported that inhibition of metabolism by pretreatment with 2-deoxyglucose exacerbates both MA-induced ATP loss and long-term reduction of striatal DA content (but see ref. 155). Similarly, the local inhibition of complex II via intrastriatal perfusion with malonate synergizes with the local administration of MA to enhance DA toxicity compared to the perfusion of either drug alone (25). Conversely, pretreatment with nicotinamide attenuates both the acute decrease in striatal ATP and the lasting DA depletions following amphetamine administration (155). In addition, the local intrastriatal perfusion of substrates for the electron-transport chain (ubiquinone or nicotinamide) for several hours following MA administration attenuates the long-term loss of DA content (129). Taken together, these data indicate that metabolic deficits and a depletion of energy stores is critical to the loss of monoamine nerve terminals following amphetamine and that the restoration or supplementation of energy production can attenuate the toxicity to MA.

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