L-glutamate released from activated microglia downregulates astrocytic L-glutamate transporter expression in neuroinflammation: the 'collusion' hypothesis for increased extracellular L-glutamate concentration in neuroinflammation.
L-glutamate released from activated microglia downregulates astrocytic L-glutamate transporter expression in neuroinflammation: the 'collusion' hypothesis for increased extracellular L-glutamate concentration in neuroinflammation.
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DOI：
10.1186/1742-2094-9-275
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发表时间：
2012-12-23
影响因子：
9.3
通讯作者：
Sato K
中科院分区：
文献类型：
--
作者：
Takaki J;Fujimori K;Miura M;Suzuki T;Sekino Y;Sato K
在中枢神经系统中，星形细胞l -谷氨酸（L-Glu）转运体维持细胞外l -谷氨酸低于神经毒性水平，但其功能因神经炎症而受损。小胶质细胞因炎症而激活；然而，激活的小胶质细胞与L-Glu转运蛋白受损之间的相关性尚不清楚。我们使用由星形胶质细胞、小胶质细胞和神经元组成的混合培养。为了量化L-Glu转运蛋白的功能，我们测量了L-Glu加入培养基（起始浓度为100 μM） 30分钟后的细胞外L-Glu。我们确定了脂多糖（LPS）处理的最佳条件，以建立无细胞死亡的炎症模型。我们检测了L-Glu转运蛋白的主要亚型以及这些转运蛋白在炎症模型中表达水平的变化。然后，我们研究了激活的小胶质细胞在L-Glu转运蛋白表达变化中的作用及其在炎症模型中的潜在机制。由于LPS （10 ng/mL, 72 h）使L-Glu残留量显著增加，但不影响细胞活力，因此我们采用这种条件建立无细胞死亡的炎症模型。GLAST是主要的L-Glu转运蛋白亚型，其表达在炎症模型中下降。由于它们释放L-Glu，激活的小胶质细胞被证明是L-Glu摄取显著减少所必需的。连续应用L-Glu可显著降低星形胶质细胞L-Glu的摄取和GLAST的表达。半通道抑制剂卡贝诺洛酮（CBX）抑制活化小胶质细胞释放L-Glu，改善炎症模型中GLAST表达的下降。此外，星形细胞内L-Glu自身升高引起GLAST下调。我们的研究结果表明，激活的小胶质细胞通过自身释放L-Glu触发细胞外L-Glu的升高，星形胶质细胞内L-Glu水平升高导致星形胶质细胞内L-Glu转运蛋白下调，导致细胞外L-Glu进一步升高。我们的数据提出了新的假设，即激活的小胶质细胞与星形胶质细胞勾结，导致神经炎症早期细胞外L-Glu升高。
In the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown. We used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 μM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model. Because LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST. Our findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.
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