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Evaluation of Microglia/Macrophage Cells from Rat Striatum and Prefrontal Cortex Reveals Differential Expression of Inflammatory-Related mRNA after Methamphetamine.

ABSTRACT: RNA sequencing (RNAseq) can be a powerful tool in the identification of transcriptional changes after drug treatment. RNAseq was utilized to determine expression changes in Fluorescence-activated cell sorted (FACS) CD11b/c+ cells from the striatum (STR) and prefrontal cortex (PFC) of male Sprague-Dawley rats after a methamphetamine (METH) binge dosing regimen. Resident microglia and infiltrating macrophages were collected 2 h or 3 days after drug administration. Gene expression changes indicated there was an increase toward an overall pro-inflammatory state, or M1 polarization, along with what appears to be a subset of cells that differentiated toward the anti-inflammatory M2 polarization. In general, there were significantly more mRNA expression changes in the STR than the PFC and more at 2 h post-binge METH than at 3 days post-binge METH. Additionally, Ingenuity® Pathway Analysis along with details of RNA expression changes revealed cyclo-oxygenase 2 (COX2)-driven prostaglandin (PG) E2 synthesis, glutamine uptake, and the Nuclear factor erythroid2-related factor 2 (NRF2) canonical pathway in microglia were associated with the binge administration regimen of METH.

SUBMITTER: Kays JS

PROVIDER: S-EPMC6955783 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Evaluation of Microglia/Macrophage Cells from Rat Striatum and Prefrontal Cortex Reveals Differential Expression of Inflammatory-Related mRNA after Methamphetamine.

Kays Joanne S JS Yamamoto Bryan K BK

Brain sciences 20191125 12

RNA sequencing (RNAseq) can be a powerful tool in the identification of transcriptional changes after drug treatment. RNAseq was utilized to determine expression changes in Fluorescence-activated cell sorted (FACS) CD11b/c+ cells from the striatum (STR) and prefrontal cortex (PFC) of male Sprague-Dawley rats after a methamphetamine (METH) binge dosing regimen. Resident microglia and infiltrating macrophages were collected 2 h or 3 days after drug administration. Gene expression changes indicated ...[more]

PMID: 31775383

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Project description:BackgroundMethamphetamine (MA), is an extremely addictive stimulant that adversely affects the central nervous system. Accumulating evidence indicates that molecular mechanisms such as oxidative stress, apoptosis, and autophagy are involved in the toxicity of MA. Considering experimental animal studies exhibiting MA-induced neurotoxicity, the relevance of these findings needs to be evidently elucidated in human MA users. It is generally assumed that multiple chemical substances released in the brain following MA-induced metabolic activation are primary factors underlying damage of neural cells. Hence, this study aimed to investigate the role of autophagy and apoptosis as well as oxidative stress in the brain of postmortem MA-induced toxicity.Materials and methodsIn this study, we determine the gene expression of autophagy and apoptosis, including BECN1, MAP1ALC3, CASP8, TP53, and BAX genes in ten healthy controls and ten chronic users of MA postmortem dorsolateral prefrontal cortex (DLPFC) by real-time polymerase chain reaction. Also, we applied immunohistochemistry in formalin-fixed and paraffin-embedded human brain samples to analyze brain-derived neurotrophic factor (BDNF). Also, spectrophotometry was performed to measure glutathione (GSH) content.ResultsThe expression level of apoptotic and autophagic genes (BECN1, MAP1ALC3, CASP8, TP53, and BAX) were significantly elevated, while GSH content and BDNF showed substantial reductions in DLPFC of chronic MA users. Discussion: Our data showed that MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. Also, MA induces multiple functional and structural perturbations in the brain, determining its toxicity and possibly contributing to neurotoxicity.DiscussionOur study showed BDNF-positive cells as well as GSH amount, displayed significant declines in DLPFC of MA user. MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. However, further investigations are needed to throw light on the cellular and molecular mechanisms that act in the various regions of the addicted brain, especially in DLPFC.

Project description: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.

Project description:Methamphetamine (METH) is a psychostimulant that can cause long-lasting neurodegenerative effects in humans and animals. These toxic effects appear to occur, in part, via activation of dopamine (DA) D1 receptors. This paper assessed the possibility that the DA D1 receptor antagonist, SCH23390, might inhibit METH-induced changes in the expression of several members of immediate early genes (IEGs) which are known to control more delayed expression of other genes. We found that injections of METH (4x10 mg/kg, given at 2 h intervals) caused significant increases in c-fos and fra-2 expression which lasted from 30 min to 4 h. Pre-treatment with SCH23390, given 30 min before each METH injection, completely blocked METH-induced expression of c-fos, but only partially inhibited fra-2 mRNA expression. These results were confirmed by Western blot analysis which showed METH-induced changes in c-Fos protein expression that were blocked by pretreatment with SCH23390. There were also delayed METH-induced DA D1 receptor-dependent effects on fosB mRNA expression. Even though fra-1 expression was not affected by pretreatment with METH alone, the repeated injections of SCH23390 caused substantial decreases in fra-1 mRNA expression in both the presence and absence of METH. The repeated injections of METH caused no changes in the mRNAs for c-jun, junB or junD. However, there were significant increases in the phosphorylation of c-Jun protein (ser63). Phosphorylation of c-Jun occurred in a delayed fashion (16 and 24 h after the last METH injections) and was attenuated by SCH23390 pretreatment. Interestingly, SCH23390 given alone caused significant decreases in phospho-c-Jun at all time-points. The METH injections also caused delayed induction in the expression of members of the Egr family of transcription factors in a DA D1 receptor-dependent fashion. Repeated injections of SCH23390 caused substantial suppression of basal striatal egr-1 and egr-2 mRNA expression but not of that of egr-3. Both crem and arc mRNA levels were induced by METH in a SCH23390-sensitive fashion. Moreover, multiple injections of SCH23390 given alone caused marked inhibition of basal arc expression. These results show that multiple injections of METH can differentially affect the expression of several IEGs, some of which occurred in a DA D1 receptor dependent fashion. The SCH23390-mediated suppression of basal fra-1, egr-1, and egr-2 mRNA levels suggests that their basal expression in the striatum might be dependent on tonic stimulation of the DA D1 receptor.

Dataset Information

Evaluation of Microglia/Macrophage Cells from Rat Striatum and Prefrontal Cortex Reveals Differential Expression of Inflammatory-Related mRNA after Methamphetamine.

Publications

Evaluation of Microglia/Macrophage Cells from Rat Striatum and Prefrontal Cortex Reveals Differential Expression of Inflammatory-Related mRNA after Methamphetamine.

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