The regulation of astrocytic glutamate transporters by intracellular calcium signaling

• Main Authors: Yu-peng Liu, 劉于鵬
• Other Authors: Shun-Fen Tzeng
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/71368932648132976202

博士 === 國立成功大學 === 生命科學系碩博士班 === 97 === During the CNS injury, the accumulation of extracellular glutamate induces neuronal excitotoxicity, leading to secondary tissue damage. Astrocytes are responsible for the clearance of extracellular glutamate primarily through glial specific glutamate transporters (GLT-1) and Na+-dependent glutamate/aspartate transporters (GLAST). In first part of study, by using an in vitro model of cadmium-induced cellular stress, we found that the activity of glutamate uptake by astrocytes was inhibited through the downregulation of GLAST expression. The blockage of cadmium-induced rise of intracellular Ca2+ levels by pre-exposure to Ca2+ chelators can restore GLAST transcription and then increase astrocytes to uptake extracellular glutamate, suggesting that Ca2+-dependent signaling exerts a negative control in GLAST expression. This suggestion was supported by the findings showing the reduction of GLAST mRNA in astrocytes after treatment with Ca2+-ionophore A23187. In addition, the promoter activity assay by transfection of astrocytes with human GLAST promoter showed that a Ca2+ signaling inhibited the expression of GLAST mRNA at the transcriptional levels, which was possibly due to an increase in the binding activity of Ca2+-sensitive activator protein-1 (AP-1) and cAMP response element binding protein (CREB) to their specific elements of the GLAST promoter. Persistent release of high ATP from damaged neurons and activated glia also occurs after the occurrence of traumatic CNS injury, which results in progressive cell death through the effect of its ionotropic P2X and metabotropic P2Y receptors. The continuous study was performed to examine the effect of ATP on GLAST expression in astrocytes, since ATP can evoke [Ca2+]i in glia through the activation of ionotropic P2X. Our in vivo findings indicated that GLAST mRNA levels were reduced at 24 h post traumatic injury to the rat spinal cord tissue. Furthermore, exposure of cultured astrocytes to ATP and its P2X7R agonist, BzATP, for 24 h caused a significant reduction in astrocytic GLAST mRNA levels and their glutamate uptake activity. This inhibition was abolished by the blockade of the P2X7R by an irreversible P2X7R blocker, oxATP, and by P2X7R gene knockdown using the approach of lentivirus-short hairpin RNA (shRNA). In addition, the reduction in the expression levels of GLAST mRNA was mediated by PI3K-IP3R-CaMKII cascades triggered by a P2X7R-mediated Ca2+ influx. Interestingly, the GLAST promoter and RNA decay assays indicated that the P2X7R-triggered signaling induced the post-transcriptional regulation of GLAST expression through a Ca2+-dependent PI3K cascade. These findings indicate that prolonged activation of P2X7R by sustained releases of ATP in the injured CNS may reduce GLAST mRNA stability via Ca2+-dependent signaling, suggesting that the blockage of P2X7R may recover astrocytic GLAST function and prevent neurons from glutamate-induced excitotoxicity. Together, the stress-induced Ca2+ influx may trigger different signaling pathways, depending on the source of stress. In addition, the GLAST gene expression can be regulated via transcriptional and post-transcriptional mechanisms, involving the binding of transcription factors and the mRNA stability respectively, by the robust increase of intracellular Ca2+ levels.