Methamphetamine Neurotoxicity Research Articles

Involvement of the α1D-adrenergic Receptor in Methamphetamine-Induced Hyperthermia and Neurotoxicity in Rats

Methamphetamine (METH) is a psychostimulant that damages nigrostriatal dopaminergic terminals, primarily by enhancing dopamine and glutamate release. α₁-adrenergic receptor (AR) subtype involved in METH-induced neurotoxicity in rats was investigated using selective α₁-AR antagonists. METH neurotoxicity was evaluated by (1) measuring body temperature; (2) determining tyrosine hydroxylase (TH) immunoreactivity levels; (3) examining levels of dopamine and its metabolites; and (4) assessing glial fibrillary acidic protein (GFAP) and microglial immunoreactivity in the striatum. METH caused a decrease in dopamine and TH levels and induced hyperthermia which is an exacerbating factor of METH neurotoxicity. Concurrently, METH increased GFAP expression and the number of activated microglia. Pretreatment with prazosin, a nonselective α₁-AR antagonist, completely abolished METH-induced decrease in both dopamine and TH and caused a partial reduction in hyperthermia. Prazosin also prevented METH-induced increase in both GFAP expression and the number of activated microglia. In vivo microdialysis analysis revealed that prazosin, however, does not alter the METH-induced dopamine release in the striatum. The neuroprotective effects of prazosin could be mimicked by a selective α(1D) antagonist, BMY 7378, but not by selective α(1A) or α(1B) antagonists. These results suggest that the α(1D)-AR is involved in METH-induced hyperthermia and neurotoxicity in rats.

Further steps in the role of autophagy in methamphetamine toxicity

Methamphetamine (METH) abuse is known to cause a variety of disorders Incluing depression and psychosis. METH induces nigrostriatal damage in animal models and in humans consisting of intracellular alterations in nigral DA cell bodies, degeneration of DA terminals and decreased striatal DA levels. Following METH exposure, the number of nigral cell bodies is quite preserved but autophagy-like vacuoles and cytoplamic accumulation of misfolded proteins are observed. The DA-containing PC12 cell lines represent a simple model of METH toxicity and are commonly used in vitro to understand the pathophysiology of DA neurons. We analyzed at morphological level the effects of plasmid-dependent autophagy modulation on METH toxicity in PC12 cell line. We profited from the high number of authophagic like vacuoles induced by low doses of METH in order to isolate cell fraction in which to study their origin, dynamic structure and molecular composition. We found that authophagy-like vacuoles are positive both for autophagy and proteasome markers. The modulation of autophagy via a p62 containing plasmid protected from METH toxicity, while the inhibition of autophagy machinery worsened METH neurotoxicity. The present data substantiate the protective role of the autophagy machinery in METH-induced DA toxicity where the pro-autophagy protein p62 possesses a key role.

Perseverative behavior in rats with methamphetamine-induced neurotoxicity

Methamphetamine induces monoamine depletions thought to contribute to cognitive and behavioral dysfunctions. Previously, we reported that methamphetamine-induced neurotoxicity is associated with impaired formation of stimulus–response associations. Additionally, subjective observations suggested that behavioral flexibility might be affected. Thus, the present study examined whether methamphetamine neurotoxicity induces perseverative behavior. Rats were pretreated with (±)-methamphetamine (4 × 10 mg/kg, 2-hr intervals) or saline. Three weeks later, rats were trained to press a lever on one side of an operant chamber and then retrieve the reinforcer from a magazine on the opposite side until they reached criterion (>50 reinforcers/30-min). After four consecutive sessions performing the task at criterion, rats were sacrificed and brains removed for monoamine determinations. Methamphetamine-pretreated rats had ∼50% loss of striatal dopamine and prefrontal serotonin. Methamphetamine- and saline-pretreated rats were not different in the number of sessions required to reach criterion or in the total numbers of lever presses and/or head entries made across the four consecutive sessions at criterion-level performance. However, methamphetamine-pretreated rats earned fewer reinforcers, because they made extra lever-presses and head entries when they should have been retrieving the reinforcer or returning to the lever. Latencies for methamphetamine-pretreated rats to switch between the two behaviors also were significantly slower than latencies for controls. Interestingly, the degree of additional lever-presses negatively correlated with serotonin-transporter binding in the prefrontal cortex, even in saline-pretreated controls. These data suggest that methamphetamine-induced partial monoamine toxicity is associated with perseveration and that the degree of perseveration may depend on serotonin innervation of the frontal cortex.

L-Ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1

Methamphetamine (METH) is a drug of abuse which causes neurotoxicity and increased risk of developing neurodegenerative diseases. We previously found that METH induces heme oxygenase (HO)-1 expression in neurons and glial cells, and this offers partial protection against METH toxicity. In this study, we investigated the effects of l-ascorbate (vitamin C, Vit. C) on METH toxicity and HO-1 expression in neuronal/glial cocultures. Cell viability and damage were evaluated by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release, respectively. Neuronal and glial localization of HO-1 were identified by double immunofluorescence staining. Reactive oxygen species (ROS) production was measured using the fluorochrome 2′,7′-dichlorofluorescin diacetate. HO-1 mRNA and protein expression were examined by RT-qPCR and Western blotting, respectively. Results show that Vit. C induced HO-1 mRNA and protein expressions in time- and concentration-dependent manners. Inhibition of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) significantly blocked induction of HO-1 by Vit. C. HO-1 mRNA and protein expressions were significantly elevated by a combination of Vit. C and METH, compared to either Vit. C or METH alone. Pretreatment with Vit. C enhanced METH-induced HO-1 expression and attenuated METH-induced ROS production and neurotoxicity. Pharmacological inhibition of HO activity abolished suppressive effects of Vit. C on METH-induced ROS production and attenuated neurotoxicity. We conclude that induction of HO-1 expression contributes to the attenuation of METH-induced ROS production and neurotoxicity by Vit. C. We suggest that HO-1 induction by Vit. C may serve as a strategy to alleviate METH neurotoxicity.

Peripheral Ammonia as a Mediator of Methamphetamine Neurotoxicity

Ammonia is metabolized by the liver and has established neurological effects. The current study examined the possibility that ammonia contributes to the neurotoxic effects of methamphetamine (METH). The results show that a binge dosing regimen of METH to the rat increased plasma and brain ammonia concentrations that were paralleled by evidence of hepatotoxicity. The role of peripheral ammonia in the neurotoxic effects of METH was further substantiated by the demonstration that the enhancement of peripheral ammonia excretion blocked the increases in brain and plasma ammonia and attenuated the long-term depletions of dopamine and serotonin typically produced by METH. Conversely, the localized perfusion of ammonia in combination with METH, but not METH alone or ammonia alone, into the striatum recapitulated the neuronal damage produced by the systemic administration of METH. Furthermore, this damage produced by the local administration of ammonia and METH was blocked by the GYKI 52466 [4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzamine hydrochloride], an AMPA receptor antagonist. These findings highlight the importance of ammonia derived from the periphery as a small-molecule mediator of METH neurotoxicity and more broadly emphasize the importance of peripheral organ damage as a possible mechanism that mediates the neuropathology produced by drugs of abuse and other neuroactive molecules.

Protective Role for the Disulfide Isomerase PDIA3 in Methamphetamine Neurotoxicity

Methamphetamine abuse continues to be a worldwide problem, damaging the individual user as well as society. Only minimal information exists on molecular changes in the brain that result from methamphetamine administered in patterns typical of human abusers. In order to investigate such changes, we examined the effect of methamphetamine on the transcriptional profile in brains of monkeys. Gene expression profiling of caudate and hippocampus identified protein disulfide isomerase family member A3 (PDIA3) to be significantly up-regulated in the animals treated with methamphetamine as compared to saline treated control monkeys. Methamphetamine treatment of mice also increased striatal PDIA3 expression. Treatment of primary striatal neurons with methamphetamine revealed an up-regulation of PDIA3, showing a direct effect of methamphetamine on neurons to increase PDIA3. In vitro studies using a neuroblastoma cell line demonstrated that PDIA3 expression protects against methamphetamine-induced cell toxicity and methamphetamine-induced intracellular reactive oxygen species production, revealing a neuroprotective role for PDIA3. The current study implicates PDIA3 to be an important cellular neuroprotective mechanism against a toxic drug, and as a potential target for therapeutic investigations.

Parkin protects dopaminergic terminals against methamphetamine neurotoxicity by inducing inclusion formation

Viral vector‐mediated delivery of parkin pervents dopaminergic (DAergic) neurodegeneration in Parkinsonˈs disease models. One of the mechanisms underlying parkin neuroprotection is to promote the formation of á‐synuclein inclusions. It is unclear whether parkin protects against methamphetamine (METH)‐induced degeneration of DAergic terminals. Here we investigated whether parkin overexpression protects against METH neurotoxicity and whether the parkin effect is due to the formation of inclusions. Parkin‐containing adeno‐associated viral 2/6 vector was injected into the left substantia nigra in rats with/without METH treatment. Parkin‐immunoreactivity (IR) in the left substantia nigra and striatum was higher than that of right side. A large portion of parkin‐positive DAergic neurons and terminals expressed tyrosine hydroxylase (TH). Animals overerexpressing parkin showed reductions in METH‐induced decrease in TH‐IR in the striatum. Parkin‐mediated neuroprotection was associated with an increase of punctuated á‐synuclein immunostaining in the striatum. These data suggest that parkin overexpression attenuates METH‐induced damage to striatal DAergic terminals, which may be via increasing á‐synuclein inclusions. It also suggests that future clinical applications to prevent METH neurotoxicity might involve the mediation of parkin function. Support: NIH DA023085

The effects of low-dose methamphetamine pretreatment on endoplasmic reticulum stress and methamphetamine neurotoxicity in the rat midbrain

Methamphetamine (METH) neurotoxicity is involved in METH-related deaths. It has been suggested that the midbrain, together with the striatum, is affected by METH neurotoxicity and the endoplasmic reticulum (ER) stress is induced in the processes of METH neurotoxicity. In this study, we examined the effects of low-dose METH administration for 5d on GRP78 and C/EBP homologous protein (CHOP), both of which are induced under ER stress, and METH neurotoxicity in the rat midbrain. We showed that 1mg/kg of METH induced an increase in GRP78 protein and mRNA expression 1d after the last injection, but had no effect on the levels of CHOP, tyrosine hydroxylase (TH), or GFAP. Secondly, we evaluated the induction of ER stress and the extent of METH neurotoxicity in the midbrain of animals pretreated with METH. In animals pretreated with saline, we observed elevated CHOP levels, together with decreased TH levels and increased GFAP levels, indicative of METH neurotoxicity, after neurotoxic METH administration, while there was no significant change in GRP78 levels. In contrast, low-dose METH (1.0mg/kg) pretreatment increased GRP78 levels and inhibited the induction of CHOP in the midbrain without METH neurotoxicity. These findings of ER stress in animals pretreated with METH were associated with an early increase in SOD1 levels and upregulation of Bcl-2. Therefore, our study suggests that pretreatment with low-dose METH may be protective against METH neurotoxicity in the midbrain, leading to the suppression of oxidative stress and apoptotic mechanisms, in part via ER stress-related pathways. Because chronic human METH abusers administrate low-dose METH repeatedly over an extended period before lethal injection, investigation of the pathophysiology of METH neurotoxicity in animals pretreated with low-dose METH might provide useful information on the pathophysiology of chronic and/or lethal METH use in cases of METH-related deaths.

Rhynchophylline Protects Cultured Rat Neurons against Methamphetamine Cytotoxicity

Rhynchophylline (Rhy) is an active component isolated from species of the genus Uncaria which has been used for the treatment of ailments to the central nervous system in traditional Chinese medicine. Besides acting as a calcium channel blocker, Rhy was also reported to be able to protect against glutamate-induced neuronal death. We thus hypothesize that Rhy may have neuroprotective activity against methamphetamine (MA). The primary neurons were cultured directly from the cerebral cortex of neonatal rats, acting as in vitro model in the present study. The neurotoxicity of MA and the protective effect of Rhy were evaluated by MTT assay. The effects of MA, Rhy or their combination on intracellular free calcium concentration ([Ca2+]i) were determined in individual neocortical neurons by the Fluo-3/AM tracing method. The MTT assay demonstrated that MA has a dose-dependent neurotoxicity in neuronal cultures. The addition of Rhy prior to the exposure to MA prevented neuronal death. Time course studies with the Fluo-3/AM probe showed that Rhy significantly decreased neuronal [Ca2+]i which was elevated by the exposure to MA. Our results suggested that Rhy can protect the neuronal cultures against MA exposure and promptly attenuate intracellular calcium overload triggered by MA challenge. This is the first report demonstrating an inhibitory effect of Rhy against MA impairment in cultured neurons in vitro.

Therapeutic Approaches to Neuroprotective Activity by Complementary and Alternative Medicines

Therapeutic Approaches to Neuroprotective Activity by Complementary and Alternative Medicines

Contrasting Effects of the Neuropeptides Substance P, Somatostatin, and Neuropeptide Y on the Methamphetamine-Induced Production of Striatal Nitric Oxide in Mice.

Several laboratories have shown that methamphetamine (METH) neurotoxicity is associated with increases of nitric oxide (NO) production in striatal tissue and blockade of NO production protects from METH. Because substance P modulates NO production, we tested the hypothesis that intrinsic striatal neuropeptides such as somatostatin and neuropeptide Y (NPY) modulate striatal NO production in the presence of METH. To that end, METH (30 mg/kg, IP) was injected into adult male mice alone or in combination with pharmacological agonists or antagonists of the neurokinin-1 (substance P), somatostatin or NPY receptors and 3-nitrotyrosine (an indirect index of NO production) was assessed utilizing HPLC or a histological method. Pre-treatment with the systemic neurokinin-1 receptor antagonist WIN-51,708 significantly attenuated the METH-induced production of striatal 3-NT measured at two hours post-METH. Conversely, intrastriatal injection of NPY1 or 2 receptor agonists inhibited the METH-induced production of striatal 3-NT. Similarly, intrastriatal infusion of the somatostatin receptor agonist octreotide attenuated the METH-induced striatal production of 3-NT. Taken together, our results suggest the hypothesis that the neuropeptide substance P is pro-damage while the neuropeptides somatostatin and NPY are anti-damage in the presence of METH by targeting the production of NO.

Methamphetamine neurotoxicity decreases phasic, but not tonic, dopaminergic signaling in the rat striatum

Neurotoxic doses of methamphetamine (METH) are known to cause depletions in striatal dopamine (DA) tissue content. However, the effects of METH-induced insults on dopaminergic neurotransmission are not fully understood. Here, we employed fast-scan cyclic voltammetry at a carbon-fiber microelectrode in the anesthetized rat striatum to assess the effects of a neurotoxic regimen of METH on phasic and tonic modes of dopaminergic signaling and underlying mechanisms of DA release and uptake. Extracellular DA was electrically evoked by stimulation of the medial forebrain bundle mimicking tonic and phasic firing patterns for dopaminergic cells and was monitored simultaneously in both the dorsomedial and dorsolateral striatum. Kinetic analysis of evoked recordings determined parameters describing DA release and uptake. Striatal DA tissue content was quantified by high performance liquid chromatography with electrochemical detection. METH-pretreatment (four doses of 7.5 or 10.0 mg/kg s.c.) induced DA depletions of ∼ 40% on average, which are reported in both striatal subregions. METH pre-treatment significantly decreased the amplitude of signals evoked by phasic, but not tonic, stimulation. Parameters for DA release and uptake were also similarly reduced by ∼ 40%, consistent with effects on evoked phasic-like responses and DA tissue content. Taken together, these results suggest that METH-pretreatment selectively diminishes phasic, but not tonic, dopaminergic signaling in the dorsal striatum.

Neuroprotective Effect of Resveratrol Against Methamphetamine-Induced Dopaminergic Apoptotic Cell Death in a Cell Culture Model of Neurotoxicity

A growing body of evidence suggests that oxidative stress-mediated cell death signaling mechanisms may exert neurotoxic effects of methamphetamine (MA)-induced dopaminergic neuronal loss. However, the means by which oxidative stress induced by MA causes neurodegeneration remains unclear. In recent years, resveratrol has garnered considerable attention owing to its antioxidant, anti-inflammatory, anti-aging, and neuroprotective properties. In the present study, we sought to investigate the neuroprotective effects of resveratrol against apoptotic cell death in a mesencephalic dopaminergic neuronal cell culture model of MA neurotoxicity. MA treatment in the N27 dopaminergic neuronal cell model produced a time-dependent activation of the apoptotic cascade involving caspase-3 and DNA fragmentation. We found that the caspase-3 activation preceded DNA fragmentation. Notably, treatment with resveratrol almost completely attenuated MA-induced caspase-3 activity, but only partially reduced apoptotic cell death. We conclude that the neuroprotective effect of resveratrol is at least in part mediated by suppression of caspase-3 dependent cell death pathways. Collectively, our results demonstrate that resveratrol can attenuate MA-induced apoptotic cell death and suggest that resveratrol or its analogs may have therapeutic benefits in mitigating MA-induced dopaminergic neurodegeneration.

Dopamine D2-receptor knockout mice are protected against dopaminergic neurotoxicity induced by methamphetamine or MDMA

Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), amphetamine derivatives widely used as recreational drugs, induce similar neurotoxic effects in mice, including a marked loss of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum. Although the role of dopamine in these neurotoxic effects is well established and pharmacological studies suggest involvement of a dopamine D2-like receptor, the specific dopamine receptor subtype involved has not been determined. In this study, we used dopamine D2 receptor knock-out mice (D2R−/−) to determine whether D2R is involved in METH- and MDMA-induced hyperthermia and neurotoxicity. In wild type animals, both drugs induced marked hyperthermia, decreased striatal dopamine content and TH- and DAT-immunoreactivity and increased striatal GFAP and Mac-1 expression as well as iNOS and interleukin 15 at 1 and 7days after drug exposure. They also caused dopaminergic cell loss in the SNpc. Inactivation of D2R blocked all these effects. Remarkably, D2R inactivation prevented METH-induced loss of dopaminergic neurons in the SNpc. In addition, striatal dopamine overflow, measured by fast scan cyclic voltammetry in the presence of METH, was significantly reduced in D2R−/− mice. Pre-treatment with reserpine indicated that the neuroprotective effect of D2R inactivation cannot be explained solely by its ability to prevent METH-induced hyperthermia: reserpine lowered body temperature in both genotypes, and potentiated METH toxicity in WT, but not D2R−/− mice. Our results demonstrate that the D2R is necessary for METH and MDMA neurotoxicity and that the neuroprotective effect of D2R inactivation is independent of its effect on body temperature.

Methamphetamine oxidatively damages parkin and decreases the activity of 26S proteasome in vivo

Methamphetamine (METH) is toxic to dopaminergic (DAergic) terminals in animals and humans. An early event in METH neurotoxicity is an oxidative stress followed by damage to proteins and lipids. The removal of damaged proteins is accomplished by the ubiquitin-proteasome system (UPS) and the impairment of this system can cause neurodegeneration. Whether dysfunction of the UPS contributes to METH toxicity to DAergic terminals has not been determined. The present investigation examined the effects of METH on functions of parkin and proteasome in rat striatal synaptosomes. METH rapidly modified parkin via conjugation with 4-hydroxy-2-nonenal (4-HNE) to decrease parkin levels and decreased the activity of the 26S proteasome while simultaneously increasing chymotrypsin-like activity and 20S proteasome levels. Prior injections of vitamin E diminished METH-induced changes to parkin and the 26S proteasome as well as long-term decreases in DA and its metabolites' concentrations in striatal tissue. These results suggest that METH causes lipid peroxidation-mediated damage to parkin and the 26S proteasome. As the changes in parkin and 26S occur before the sustained deficits in DAergic markers, an early loss of UPS function may be important in mediating the long-term degeneration of striatal DAergic terminals via toxic accumulation of parkin substrates and damaged proteins.

A2A receptors and methamphetamine toxicity: a role of adenosine as an endogenous neurotoxin

Adenosine A2A are a class of purinergic receptors largely expressed in dopamine (DA)-rich areas of the central nervous system. In particular, they are abundant within basal ganglia, where they modulate the activity of various neurotransmitters, including DA. Despite the lack of knowledge on their fine physiological mechanisms, it is worth to mention that A2A antagonists prevent neuronal death and dyskinesia in Parkinsonism. Moreover the neuroprotective effects observed after blockade of adenosine A2A receptors in several models of neurotoxicity suggests a toxic effect for endogenous adenosine In the light of these evidences, in the present study, by using in vitro models of DA neurons, we investigated: (i) whether A2A antagonists protect DA containing neurons against methamphetamine (METH); (ii) whether activation of A2A receptors produce neurodegeneration. This was done either using A2A agonist receptor NECA or the endogenous compound adenosine; (iii) whether specific cell mechanisms are involved in these phenomena. We found that A2A antagonists protect DA cells against METH neurotoxicity. Moreover, we found that NECA and adenosine both produced a toxic effects. In the light of the key role of autophagy in modulating the survival of DA neurons we found that A2A antagonists increase, while A2A agonists decrease, autophagy. These results suggest that neuroprotection induced by A2A antagonists may be mediated by enhancement of autophagy. As expected we found that pre-treatment with a non-adenosine related inducer of autophagy produced the same protective effects obtained with A2A antagonists. Our data indicate for the first time, that A2A antagonists are protective in DA neurons against METH. Such an effect appear to be mediated by the enhancement of autophagy. On the other hand we found that activation of A2A receptor produces neurotoxicity. Interestingly these effects was reproduced by administering endogenous adenosine. This suggests that adenosine may produce neurodegeneration by inhibiting the autophagy pathway.

Nucleus Accumbens Invulnerability to Methamphetamine Neurotoxicity

Methamphetamine (Meth) is a neurotoxic drug of abuse that damages neurons and nerve endings throughout the central nervous system. Emerging studies of human Meth addicts using both postmortem analyses of brain tissue and noninvasive imaging studies of intact brains have confirmed that Meth causes persistent structural abnormalities. Animal and human studies have also defined a number of significant functional problems and comorbid psychiatric disorders associated with long-term Meth abuse. This review summarizes the salient features of Meth-induced neurotoxicity with a focus on the dopamine (DA) neuronal system. DA nerve endings in the caudate-putamen (CPu) are damaged by Meth in a highly delimited manner. Even within the CPu, damage is remarkably heterogeneous, with ventral and lateral aspects showing the greatest deficits. The nucleus accumbens (NAc) is largely spared the damage that accompanies binge Meth intoxication, but relatively subtle changes in the disposition of DA in its nerve endings can lead to dramatic increases in Meth-induced toxicity in the CPu and overcome the normal resistance of the NAc to damage. In contrast to the CPu, where DA neuronal deficiencies are persistent, alterations in the NAc show a partial recovery. Animal models have been indispensable in studies of the causes and consequences of Meth neurotoxicity and in the development of new therapies. This research has shown that increases in cytoplasmic DA dramatically broaden the neurotoxic profile of Meth to include brain structures not normally targeted for damage. The resistance of the NAc to Meth-induced neurotoxicity and its ability to recover reveal a fundamentally different neuroplasticity by comparison to the CPu. Recruitment of the NAc as a target of Meth neurotoxicity by alterations in DA homeostasis is significant in light of the numerous important roles played by this brain structure.

Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.

Human Prostaglandin H Synthase (hPHS)-1 and hPHS-2 in Amphetamine Analog Bioactivation, DNA Oxidation, and Cytotoxicity

Neurotoxicity of the amphetamine analogs methamphetamine (METH) and 3,4-methylenedioxyamphetamine (MDA) (the active metabolite of ecstasy) may involve their prostaglandin H synthase (PHS)-dependent bioactivation to free radical intermediates that generate reactive oxygen species and oxidatively damage cellular macromolecules. We used Chinese hamster ovary-K1 (CHO-K1) cell lines either untransfected or stably expressing human PHS-1 (hPHS-1) or hPHS-2 to investigate hPHS isozyme-dependent oxidative damage and cytotoxicity. Both METH and MDA (250-1000 μM) caused concentration-independent cytotoxicity in hPHS-1 cells, suggesting maximal bioactivation at the lowest concentration. In hPHS-2 cells, with half the activity of hPHS-1 cells, METH (250-1000 μM) cytotoxicity was less than that for hPHS-1 cells but was concentration dependent and increased by exogenous arachidonic acid (AA), which increased hPHS activity. Whereas 10 μM MDA and METH were not cytotoxic, at 100 μM both analogs caused AA-dependent and concentration-dependent increases in cytotoxicity and DNA oxidation in both hPHS-1/2 cells. The hPHS-2 isozyme appeared to provide more efficacious bioactivation of these amphetamine analogs. Acetylsalicylic acid, an irreversible inhibitor of both hPHS-1 and hPHS-2, blocked cytotoxicity and DNA oxidation in both cell lines and untransfected CHO-K1 cells lacking PHS activity were similarly resistant. Accordingly, isozyme-dependent hPHS-catalyzed bioactivation of METH and MDA can cause oxidative macromolecular damage and cytotoxicity, which may contribute to their neurotoxicity.

The role of endogenous serotonin in methamphetamine‐induced neurotoxicity to dopamine nerve endings of the striatum

Methamphetamine (METH) is a neurotoxic drug of abuse that damages the dopamine (DA) neuronal system in a highly delimited manner. The brain structure most affected by METH is the striatum where long-term DA depletion and microglial activation are maximal. Endogenous DA has been implicated as a critical participant in METH-induced neurotoxicity, most likely as a substrate for non-enzymatic oxidation by METH-generated reactive oxygen species. The striatum is also extensively innervated by serotonin (5HT) nerve endings and this neurochemical system is modified by METH in much the same manner as seen in DA nerve endings (i.e., increased release of 5HT, loss of function in tryptophan hydroxylase and the serotonin transporter, long-term depletion of 5HT stores). 5HT can also be modified by reactive oxygen species to form highly reactive species that damage neurons but its role in METH neurotoxicity has not been assessed. Increases in 5HT levels with 5-hydroxytryptophan do not change METH-induced neurotoxicity to the DA nerve endings as revealed by reductions in DA, tyrosine hydroxylase and dopamine transporter levels. Partial reductions in 5HT with p-chlorophenylalanine are without effect on METH toxicity, despite the fact that p-chlorophenylalanine largely prevents METH-induced hyperthermia. Mice lacking the gene for brain tryptophan hydroxylase 2 are devoid of brain 5HT and respond to METH in the same manner as wild-type controls, despite showing enhanced drug-induced hyperthermia. Taken together, the present results indicate that endogenous 5HT does not appear to play a role in METH-induced damage to DA nerve endings of the striatum.