Dataset Information

Role of C/EBP-? in Methamphetamine-Mediated Microglial Apoptosis.

ABSTRACT: Methamphetamine (MA) is a widely abused psychoactive drug that primarily damages the nervous system. However, the involvement of MA in the survival of microglia remains poorly understood. CCAAT-enhancer binding protein (C/EBP-?) is a transcription factor and an important regulator of cell apoptosis. Lipocalin2 (lcn2) is a known apoptosis inducer and is involved in many cell death processes. We hypothesized that C/EBP-? is involved in MA-induced lcn2-mediated microglial apoptosis. To test this hypothesis, we measured the protein expression of C/EBP-? after MA treatment and evaluated the effects of silencing C/EBP-? or lcn2 on MA-induced apoptosis in BV-2 cells and the mouse striatum after intrastriatal MA injection. MA exposure increased the expression of C/EBP-? and stimulated the lcn2-mediated modulation of apoptosis. Moreover, silencing the C/EBP-?-dependent lcn2 upregulation reversed the MA-induced microglial apoptosis. The in vivo relevance of these findings was confirmed in mouse models, which demonstrated that the microinjection of anti-C/EBP-? into the striatum ameliorated the MA-induced decrease survival of microglia. These findings provide a new insight regarding the specific contributions of C/EBP-?-lcn2 to microglial survival in the context of MA abuse.

SUBMITTER: Chen X

PROVIDER: S-EPMC6712175 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Role of C/EBP-β in Methamphetamine-Mediated Microglial Apoptosis.

Chen Xuebing X Lu Jiancong J Zhao Xu X Chen Chuanxiang C Qiao Dongfang D Wang Huijun H Yue Xia X

Frontiers in cellular neuroscience 20190821

Methamphetamine (MA) is a widely abused psychoactive drug that primarily damages the nervous system. However, the involvement of MA in the survival of microglia remains poorly understood. CCAAT-enhancer binding protein (C/EBP-β) is a transcription factor and an important regulator of cell apoptosis. Lipocalin2 (lcn2) is a known apoptosis inducer and is involved in many cell death processes. We hypothesized that C/EBP-β is involved in MA-induced lcn2-mediated microglial apoptosis. To test this hy ...[more]

PMID: 31496936

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Project description:HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30-60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton-Weiss-Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS.

Project description:BackgroundNeuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer's disease (AD) pathology. Amyloid beta (A?) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether A? activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors.MethodsThe presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric A?1-42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain.ResultsAcute applications of human amylin or A?1-42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNF? and IL-1?, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNF?, and IL-1?. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls.ConclusionMicroglial amylin receptors mediate A?-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Dataset Information

Role of C/EBP-? in Methamphetamine-Mediated Microglial Apoptosis.

Publications

Role of C/EBP-β in Methamphetamine-Mediated Microglial Apoptosis.

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