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

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

Methamphetamine induces endoplasmic reticulum stress related gene CHOP/Gadd153/ddit3 in dopaminergic cells

We examined the toxicity of methamphetamine and dopamine in CATH.a cells, which were derived from mouse dopamine-producing neural cells in the central nervous system. Use of the quantitative real-time polymerase chain reaction revealed that transcripts of the endoplasmic reticulum stress related gene (CHOP/Gadd153/ddit3) were considerably induced at 24–48 h after methamphetamine administration (but only under apoptotic conditions), whereas dopamine slightly induced CHOP/Gadd153/ddit3 transcripts at an early stage. We also found that dopamine and methamphetamine weakly induced transcripts for the glucose-regulated protein 78 gene (Grp78/Bip) at the early stage. Analysis by immunofluorescence microscopy demonstrated an increase of　CHOP/Gadd153/ddit3 and Grp78/Bip proteins at 24 h after methamphetamine administration. Treatment of CATH.a cells with methamphetamine caused a re-distribution of dopamine inside the cells, which mimicked the presynaptic activity of neurons with cell bodies located in the ventral tegmental area or the substantia nigra. Thus, we have demonstrated the existence of endoplasmic reticulum stress in a model of presynaptic dopaminergic neurons for the first time. Together with the recent evidence suggesting the importance of presynaptic toxicity, our findings provide new insights into the mechanisms of dopamine toxicity, which might represent one of the most important mechanisms of methamphetamine toxicity and addiction

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

Methamphetamine Causes Differential Alterations in Gene Expression and Patterns of Histone Acetylation/Hypoacetylation in the Rat Nucleus Accumbens

Methamphetamine (METH) addiction is associated with several neuropsychiatric symptoms. Little is known about the effects of METH on gene expression and epigenetic modifications in the rat nucleus accumbens (NAC). Our study investigated the effects of a non-toxic METH injection (20 mg/kg) on gene expression, histone acetylation, and the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2, in that structure. Microarray analyses done at 1, 8, 16 and 24 hrs after the METH injection identified METH-induced changes in the expression of genes previously implicated in the acute and longterm effects of psychostimulants, including immediate early genes and corticotropin-releasing factor (Crf). In contrast, the METH injection caused time-dependent decreases in the expression of other genes including Npas4 and cholecystokinin (Cck). Pathway analyses showed that genes with altered expression participated in behavioral performance, cell-to-cell signaling, and regulation of gene expression. PCR analyses confirmed the changes in the expression of c-fos, fosB, Crf, Cck, and Npas4 transcripts. To determine if the METH injection caused post-translational changes in histone markers, we used western blot analyses and identified METH-mediated decreases in histone H3 acetylated at lysine 9 (H3K9ac) and lysine 18 (H3K18ac) in nuclear sub-fractions. In contrast, the METH injection caused time-dependent increases in acetylated H4K5 and H4K8. The changes in histone acetylation were accompanied by decreased expression of HDAC1 but increased expression of HDAC2 protein levels. The histone acetyltransferase, ATF2, showed significant METH-induced increased in protein expression. These results suggest that METH-induced alterations in global gene expression seen in rat NAC might be related, in part, to METH-induced changes in histone acetylation secondary to changes in HAT and HDAC expression. The causal role that HATs and HDACs might play in METH-induced gene expression needs to be investigated further

Methamphetamine Increases LPS-Mediated Expression of IL-8, TNF-α and IL-1β in Human Macrophages through Common Signaling Pathways

The use of methamphetamine (MA) has increased in recent years, and is a major health concern throughout the world. The use of MA has been associated with an increased risk of acquiring HIV-1, along with an increased probability of the acquisition of various sexually transmitted infections. In order to determine the potential effects of MA exposure in the context of an infectious agent, U937 macrophages were exposed to various combinations of MA and bacterial lipopolysaccharide (LPS). Treatment with MA alone caused significant increases in the levels of TNF-α, while treatment with both MA and LPS resulted in significant increases in TNF-α, IL-1β and the chemokine IL-8. The increases in cytokine or chemokine levels seen when cells were treated with both LPS and MA were generally greater than those increases observed when cells were treated with only LPS. Treatment with chemical inhibitors demonstrated that the signal transduction pathways including NF-kB, MAPK, and PI3-Akt were involved in mediating the increased inflammatory response. As discussed in the paper, these pathways appear to be utilized by both MA and LPS, in the induction of these inflammatory mediators. Since these pathways are involved in the induction of inflammation in response to other pathogens, this suggests that MA-exacerbated inflammation may be a common feature of infectious disease in MA abusers

Rituximab therapy for juvenile-onset systemic lupus erythematosus

Rituximab (RTX), an anti-CD20 monoclonal antibody, has been proposed for use in the therapy of systemic lupus erythematosus (SLE). We present the initial long-term experience of the safety and efficacy of rituximab for treatment of SLE in children. Eighteen patients (mean age 14 ± 3 years) with severe SLE were treated with rituximab after demonstrating resistance or toxicity to conventional regimens. There was a predominance of female (16/18) and ethnic African (13/18) patients. All had lupus nephritis [World Health Organization (WHO) classes 3–5] and systemic manifestations of vasculitis. Clinical disease activity of the SLE was scored with the SLE-disease activity index 2K (SLEDAI-2K). Patients were followed-up for an average of 3.0 ± 1.3 years (range 0.5 to 4.8 years). B-cell depletion occurred within 2 weeks in all patients and persisted for up to 1 year in some. Clinical activity scores, double-stranded DNA (dsDNA) antibodies, renal function and proteinuria [urine protein to creatinine ratio (Upr/cr)] improved in 93% of the patients. Five patients required multiple courses of RTX for relapse, with B-cell repopulation. One died of infectious endocarditis related to severe immunosuppression. In conclusion, our data support the efficacy of rituximab as adjunctive treatment for SLE in children. Although rituximab was well tolerated by the majority of patients, randomized controlled trials are required to establish its long-term safety and efficacy

Mitochondrial Fragmentation Is Involved in Methamphetamine-Induced Cell Death in Rat Hippocampal Neural Progenitor Cells

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca2+) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH

Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.</p> <p><b>Methods</b></p> <p>We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).</p> <p><b>Results</b></p> <p>METH induced microglial cell death in a concentration-dependent manner (EC₅₀ = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.</p> <p><b>Conclusions</b></p> <p>These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.</p