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Involvement of sigma-1 receptor in astrocyte activation induced by methamphetamine via up-regulation of its own expression.

Zhang Y, Lv X, Bai Y, Zhu X, Wu X, Chao J, Duan M, Buch S, Chen L, Yao H - J Neuroinflammation (2015)

Bottom Line: Although it has been documented that methamphetamine induces astrocyte activation, the mechanism(s) underlying this effect remain poorly understood.Subsequently, CREB translocated into nucleus and interacted with the promoter of σ-1R resulting in increased expression of σ-1R with a concomitant increase in expression of GFAP.This effect was inhibited in cells treated with the σ-1R antagonist-BD1047, thereby implicating the role of σ-1R in the activation of astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Medical School of Southeast University, 87 Dingjiaqiao, Nanjing, Jiangsu, 210009, China. 1904334402@qq.com.

ABSTRACT

Background: Although it has been documented that methamphetamine induces astrocyte activation, the mechanism(s) underlying this effect remain poorly understood. We thus sought to examine the molecular mechanisms involved in methamphetamine-mediated activation of astrocytes with a focus on the role of sigma-1 receptor (σ-1R) in this process.

Methods: The expression of σ-1R and glial fibrillary acidic protein (GFAP) was examined by reverse transcription PCR (RT-PCR), real-time PCR, Western blot, and immunofluorescent staining; phosphorylation of cell signaling pathways was detected by Western blot analysis. Immunoprecipitation was used to determine the interaction between σ-1R and p-Src. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of cAMP-response element-binding protein (CREB) with the promoter of σ-1R. The role of σ-1R in astrocyte activation was further validated in σ-1R knockout (KO) mice by Western blot combined with immunofluorescent staining.

Results: Exposure of primary rat astrocytes to methamphetamine increased the expression of σ-1R via the activation of Src, ERK mitogen-activated protein kinase, and downstream CREB pathways. Subsequently, CREB translocated into nucleus and interacted with the promoter of σ-1R resulting in increased expression of σ-1R with a concomitant increase in expression of GFAP. This effect was inhibited in cells treated with the σ-1R antagonist-BD1047, thereby implicating the role of σ-1R in the activation of astrocytes. In vivo relevance of these findings was further corroborated in σ-1R KO mice that were administered methamphetamine. In the methamphetamine administered mice, there was a failure of the drug to induce activation of astrocytes, an effect that was evident in wild-type (WT) mice exposed to methamphetamine.

Conclusions: The study presented herein demonstrates that methamphetamine-mediated activation of astrocytes involved up-regulation of σ-1R through a positive-feedback mechanism. Understanding the regulation of σ-1R expression could provide insights into the development of potential therapeutic strategies for astrocyte activation induced by methamphetamine.

No MeSH data available.


Related in: MedlinePlus

Involvement of σ-1R in methamphetamine-mediated σ-1R expression in primary rat astrocytes. (A) Pretreatment of primary rat astrocytes with the σ-1R antagonist-BD1047 (10 μM) inhibited methamphetamine-mediated increased expression of σ-1R. Representative immunoblots and the densitometric analysis of σ-1R/GAPDH from the three separate experiments are presented. (B) Immunofluorescent staining for σ-1R (green) and GFAP (red) in primary rat astrocytes treated with methamphetamine or σ-1R antagonist-BD1047 (10 μM). Scale bars all indicated 10 μm. (C) Intensity of σ-1R immunofluorescence was quantified in five areas of the slide using the Image J software. IOD-integrated optical density. All the data are presented as mean ± SD of three individual experiments. *P < 0.05 and **P < 0.01 versus control group; #P < 0.05 versus methamphetamine-treated group.
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Fig2: Involvement of σ-1R in methamphetamine-mediated σ-1R expression in primary rat astrocytes. (A) Pretreatment of primary rat astrocytes with the σ-1R antagonist-BD1047 (10 μM) inhibited methamphetamine-mediated increased expression of σ-1R. Representative immunoblots and the densitometric analysis of σ-1R/GAPDH from the three separate experiments are presented. (B) Immunofluorescent staining for σ-1R (green) and GFAP (red) in primary rat astrocytes treated with methamphetamine or σ-1R antagonist-BD1047 (10 μM). Scale bars all indicated 10 μm. (C) Intensity of σ-1R immunofluorescence was quantified in five areas of the slide using the Image J software. IOD-integrated optical density. All the data are presented as mean ± SD of three individual experiments. *P < 0.05 and **P < 0.01 versus control group; #P < 0.05 versus methamphetamine-treated group.

Mentions: Since methamphetamine mediates signaling via binding to σ-1Rs, we next wanted to dissect the detailed mechanism(s) underlying methamphetamine-mediated enhanced expression of σ-1R. Interestingly, as shown in Figure 2A, pretreatment of primary rat astrocytes with the σ-1R antagonist-BD1047 significantly inhibited methamphetamine-mediated up-regulation of σ-1R. In addition to up-regulation of σ-1R expression, treatment of primary rat astrocytes with methamphetamine also resulted in translocation of σ-1R into the plasma membrane as evidenced by the fact that methamphetamine treatment resulted in clustering and polarization of σ-1R within the cell membrane. This effect was significantly inhibited in cells pretreated with BD1047 (Figure 2B,C). Taken together, these findings suggest that σ-1R plays a critical role in methamphetamine-mediated up-regulation of σ-1R.Figure 2


Involvement of sigma-1 receptor in astrocyte activation induced by methamphetamine via up-regulation of its own expression.

Zhang Y, Lv X, Bai Y, Zhu X, Wu X, Chao J, Duan M, Buch S, Chen L, Yao H - J Neuroinflammation (2015)

Involvement of σ-1R in methamphetamine-mediated σ-1R expression in primary rat astrocytes. (A) Pretreatment of primary rat astrocytes with the σ-1R antagonist-BD1047 (10 μM) inhibited methamphetamine-mediated increased expression of σ-1R. Representative immunoblots and the densitometric analysis of σ-1R/GAPDH from the three separate experiments are presented. (B) Immunofluorescent staining for σ-1R (green) and GFAP (red) in primary rat astrocytes treated with methamphetamine or σ-1R antagonist-BD1047 (10 μM). Scale bars all indicated 10 μm. (C) Intensity of σ-1R immunofluorescence was quantified in five areas of the slide using the Image J software. IOD-integrated optical density. All the data are presented as mean ± SD of three individual experiments. *P < 0.05 and **P < 0.01 versus control group; #P < 0.05 versus methamphetamine-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340104&req=5

Fig2: Involvement of σ-1R in methamphetamine-mediated σ-1R expression in primary rat astrocytes. (A) Pretreatment of primary rat astrocytes with the σ-1R antagonist-BD1047 (10 μM) inhibited methamphetamine-mediated increased expression of σ-1R. Representative immunoblots and the densitometric analysis of σ-1R/GAPDH from the three separate experiments are presented. (B) Immunofluorescent staining for σ-1R (green) and GFAP (red) in primary rat astrocytes treated with methamphetamine or σ-1R antagonist-BD1047 (10 μM). Scale bars all indicated 10 μm. (C) Intensity of σ-1R immunofluorescence was quantified in five areas of the slide using the Image J software. IOD-integrated optical density. All the data are presented as mean ± SD of three individual experiments. *P < 0.05 and **P < 0.01 versus control group; #P < 0.05 versus methamphetamine-treated group.
Mentions: Since methamphetamine mediates signaling via binding to σ-1Rs, we next wanted to dissect the detailed mechanism(s) underlying methamphetamine-mediated enhanced expression of σ-1R. Interestingly, as shown in Figure 2A, pretreatment of primary rat astrocytes with the σ-1R antagonist-BD1047 significantly inhibited methamphetamine-mediated up-regulation of σ-1R. In addition to up-regulation of σ-1R expression, treatment of primary rat astrocytes with methamphetamine also resulted in translocation of σ-1R into the plasma membrane as evidenced by the fact that methamphetamine treatment resulted in clustering and polarization of σ-1R within the cell membrane. This effect was significantly inhibited in cells pretreated with BD1047 (Figure 2B,C). Taken together, these findings suggest that σ-1R plays a critical role in methamphetamine-mediated up-regulation of σ-1R.Figure 2

Bottom Line: Although it has been documented that methamphetamine induces astrocyte activation, the mechanism(s) underlying this effect remain poorly understood.Subsequently, CREB translocated into nucleus and interacted with the promoter of σ-1R resulting in increased expression of σ-1R with a concomitant increase in expression of GFAP.This effect was inhibited in cells treated with the σ-1R antagonist-BD1047, thereby implicating the role of σ-1R in the activation of astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Medical School of Southeast University, 87 Dingjiaqiao, Nanjing, Jiangsu, 210009, China. 1904334402@qq.com.

ABSTRACT

Background: Although it has been documented that methamphetamine induces astrocyte activation, the mechanism(s) underlying this effect remain poorly understood. We thus sought to examine the molecular mechanisms involved in methamphetamine-mediated activation of astrocytes with a focus on the role of sigma-1 receptor (σ-1R) in this process.

Methods: The expression of σ-1R and glial fibrillary acidic protein (GFAP) was examined by reverse transcription PCR (RT-PCR), real-time PCR, Western blot, and immunofluorescent staining; phosphorylation of cell signaling pathways was detected by Western blot analysis. Immunoprecipitation was used to determine the interaction between σ-1R and p-Src. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of cAMP-response element-binding protein (CREB) with the promoter of σ-1R. The role of σ-1R in astrocyte activation was further validated in σ-1R knockout (KO) mice by Western blot combined with immunofluorescent staining.

Results: Exposure of primary rat astrocytes to methamphetamine increased the expression of σ-1R via the activation of Src, ERK mitogen-activated protein kinase, and downstream CREB pathways. Subsequently, CREB translocated into nucleus and interacted with the promoter of σ-1R resulting in increased expression of σ-1R with a concomitant increase in expression of GFAP. This effect was inhibited in cells treated with the σ-1R antagonist-BD1047, thereby implicating the role of σ-1R in the activation of astrocytes. In vivo relevance of these findings was further corroborated in σ-1R KO mice that were administered methamphetamine. In the methamphetamine administered mice, there was a failure of the drug to induce activation of astrocytes, an effect that was evident in wild-type (WT) mice exposed to methamphetamine.

Conclusions: The study presented herein demonstrates that methamphetamine-mediated activation of astrocytes involved up-regulation of σ-1R through a positive-feedback mechanism. Understanding the regulation of σ-1R expression could provide insights into the development of potential therapeutic strategies for astrocyte activation induced by methamphetamine.

No MeSH data available.


Related in: MedlinePlus