Methamphetamine Induces Dopamine D1 Receptor-Dependent Endoplasmic Reticulum Stress-Related Molecular Events in the Rat Striatum

Methamphetamine (METH) is an illicit toxic psychostimulant which is widely abused. Its toxic effects depend on the release of excessive levels of dopamine (DA) that activates striatal DA receptors. Inhibition of DA-mediated neurotransmission by the DA D1 receptor antagonist, SCH23390, protects against METH-induced neuronal apoptosis. The initial purpose of the present study was to investigate, using microarray analyses, the influence of SCH23390 on transcriptional responses in the rat striatum caused by a single METH injection at 2 and 4 hours after drug administration. We identified 545 out of a total of 22,227 genes as METH-responsive. These include genes which are involved in apoptotic pathways, endoplasmic reticulum (ER) stress, and in transcription regulation, among others. Of these, a total of 172 genes showed SCH23390-induced inhibition of METH-mediated changes. Among these SCH23390-responsive genes were several genes that are regulated during ER stress, namely ATF3, HSP27, Hmox1, HSP40, and CHOP/Gadd153. The secondary goal of the study was to investigate the role of DA D1 receptor stimulation on the expression of genes that participate in ER stress-mediated molecular events. We thus used quantitative PCR to confirm changes in the METH-responsive ER genes identified by the microarray analyses. We also measured the expression of these genes and of ATF4, ATF6, BiP/GRP78, and of GADD34 over a more extended time course. SCH23390 attenuated or blocked METH-induced increases in the expression of the majority of these genes. Western blot analysis revealed METH-induced increases in the expression of the antioxidant protein, Hmox1, which lasted for about 24 hours after the METH injection. Additionally, METH caused DA D1 receptor-dependent transit of the Hmox1 regulator protein, Nrf2, from cytosolic into nuclear fractions where the protein exerts its regulatory functions. When taken together, these findings indicate that SCH23390 can provide protection against neuronal apoptosis by inhibiting METH-mediated DA D1 receptor-mediated ER stress in the rat striatum. Our data also suggest that METH-induced toxicity might be a useful model to dissect molecular mechanisms involved in ER stress-dependent events in the rodent brain

METHAMPHETAMINE PRECONDITIONING CAUSES DIFFERENTIAL CHANGES IN STRIATAL TRANSCRIPTIONAL RESPONSES TO LARGE DOSES OF THE DRUG

Methamphetamine (METH) is a toxic drug of abuse, which can cause significant decreases in the levels of monoamines in various brain regions. However, animals treated with progressively increasing doses of METH over several weeks are protected against the toxic effects of the drug. In the present study, we tested the possibility that this pattern of METH injections might be associated with transcriptional changes in the rat striatum, an area of the brain which is known to be very sensitive to METH toxicity and which is protected by METH preconditioning. We found that the presence and absence of preconditioning followed by injection of large doses of METH caused differential expression in different sets of striatal genes. Quantitative PCR confirmed METH-induced changes in some genes of interest. These include small heat shock 27 kD proteins 1 and 2 (HspB1 and HspB2), brain derived neurotrophic factor (BDNF), and heme oxygenase-1 (Hmox-1). Our observations are consistent with previous studies which have reported that ischemic or pharmacological preconditioning can cause reprogramming of gene expression after lethal ischemic insults. These studies add to the growing literature on the effects of preconditioning on the brain transcriptome

Involvement of Dopamine Receptors in Binge Methamphetamine-Induced Activation of Endoplasmic Reticulum and Mitochondrial Stress Pathways

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D1 receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D1 and D2 receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D1 or D2 receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58IPK, in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D2-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors

Methamphetamine Preconditioning Alters Midbrain Transcriptional Responses to Methamphetamine-Induced Injury in the Rat Striatum

Methamphetamine (METH) is an illicit drug which is neurotoxic to the mammalian brain. Numerous studies have revealed significant decreases in dopamine and serotonin levels in the brains of animals exposed to moderate-to-large METH doses given within short intervals of time. In contrast, repeated injections of small nontoxic doses of the drug followed by a challenge with toxic METH doses afford significant protection against monoamine depletion. The present study was undertaken to test the possibility that repeated injections of the drug might be accompanied by transcriptional changes involved in rendering the nigrostriatal dopaminergic system refractory to METH toxicity. Our results confirm that METH preconditioning can provide significant protection against METH-induced striatal dopamine depletion. In addition, the presence and absence of METH preconditioning were associated with substantial differences in the identity of the genes whose expression was affected by a toxic METH challenge. Quantitative PCR confirmed METH-induced changes in genes of interest and identified additional genes that were differentially impacted by the toxic METH challenge in the presence of METH preconditioning. These genes include small heat shock 27 kD 27 protein 2 (HspB2), thyrotropin-releasing hormone (TRH), brain derived neurotrophic factor (BDNF), c-fos, and some encoding antioxidant proteins including CuZn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx)-1, and heme oxygenase-1 (Hmox-1). These observations are consistent, in part, with the transcriptional alterations reported in models of lethal ischemic injuries which are preceded by ischemic or pharmacological preconditioning. Our findings suggest that multiple molecular pathways might work in tandem to protect the nigrostriatal dopaminergic pathway against the deleterious effects of the toxic psychostimulant. Further analysis of the molecular and cellular pathways regulated by these genes should help to provide some insight into the neuroadaptive potentials of the brain when repeatedly exposed to drugs of abuse

Effects of METH on the transcript levels of pro-death genes.

<p>(A) METH caused rapid induction in Chop/Gadd153 mRNA levels. (B) Gadd34 was up-regulated at late time-points. Key to statistics: ** = p<0.01, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the SCH group. ! = p<0.05; !! = p<0.01; !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p

Binge METH injections caused time-dependent increases in the expression of the ER chaperone, BiP/GRP-78, and of the co-chaperone, P58^IPK.

<p>Levels of (A) BiP/GRP-78 and (B) P58^IPK transcripts were rapidly increased at 30 min after METH injections. RT-PCR was performed on total RNA isolated from the striatal tissue. Data were obtained from RNA isolated from six animals per group and determined individually. The levels of mRNA were normalized to clathrin mRNA levels. Values obtained for the treatment groups were compared by analysis of variance (ANOVA) followed by post-hoc analyses when ANOVA revealed significant changes. Key to statistics: * = p<0.05; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ### = p<0.001, in comparison to the SCH group. ! = p<0.05; !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p

METH caused differential effects on ER stress genes.

<p>Binge toxic doses of METH have differential effects on the members of the ATF family of transcription factors (A–F). Key to statistics: * = p<0.05; ** = p<0.01; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the SCH group. ! = p<0.05; !! = p<0.01, in comparison of METH group to the SCH+METH group.</p

The effects of METH and raclopride on the expression of the Bcl-2 family of proteins and cytochrome c.

<p>(A) Representative immunoblots. (B) Pretreatment with raclopride attenuated METH-induced decreases in Bcl-2 protein levels, and (C, D) increases in Bax and Bad expression. In contrast, raclopride was ineffective to block cytochrome c induction (E). Protein expression was normalized to α-Tubulin. Key to statistics: ** = p<0.01; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the Rac group. ! = p<0.05; !! = p<0.01; !!! = p<0.001, in comparison of METH group to the Rac+METH group.</p

Effects of METH injections and SCH23390 treatment on the expression of stress response regulated proteins.

<p>(A) Representative immunoblots showing the effects of METH and SCH23390. (A–E) Pretreatment with SCH23390 blocked the METH-induced changes on ATF3 (B), ATF4 (C), CHOP (D) and caspase-12 (E). Protein expression was normalized to α-Tubulin. Key to statistics: * = p<0.05; *** = p<0.001, in comparison to the Saline group. ## = p<0.01; ### = p<0.001, in comparison to the SCH group. !! = p<0.01; !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p