Department of Neurology, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21287, USA.
Glia. 2010 Feb;58(3):277-86. doi: 10.1002/glia.20922.
Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.
星形胶质细胞谷氨酸转运体 EAAT2/GLT1 可防止中枢神经系统中谷氨酸引起的兴奋性毒性。EAAT2/GLT1 的表达受神经元的动态调节。肌萎缩侧索硬化症 (ALS) 的发病机制涉及星形胶质细胞功能障碍，包括 EAAT2/GLT1 的大量丧失。基因启动子的 DNA 甲基化是调节基因表达的最重要表观遗传机制之一。DNA 甲基化在不同条件下，特别是在 ALS 中对星形胶质细胞 EAAT2/GLT1 表达的调节作用尚未得到探索。在这项研究中，我们建立了一种使用基于 GFP 的荧光激活细胞分选方法从 BAC GLT1 eGFP 小鼠体内和体外选择性分离纯星形胶质细胞群体的程序。从该程序中分离的星形胶质细胞是 GFAP+和 GLT1+，并对神经元刺激作出反应，从而能够对这些星形胶质细胞中的 GLT1 启动子进行直接甲基化分析。为了研究 DNA 甲基化在生理和病理 EAAT2/GLT1 表达中的作用，通过 bisulfite 测序法分析了体外和体内模型或死后 ALS 运动皮层中星形胶质细胞中 EAAT2/GLT1 启动子的甲基化状态。GLT1 启动子上选择性 CpG 位点的 DNA 去甲基化与神经元刺激后星形胶质细胞中 GLT1 mRNA 水平的升高高度相关；然而，在来自人类肌萎缩侧索硬化症患者死后运动皮层的 EAAT2 启动子的 CpG 位点上发现低水平的甲基化。总之，GLT1 启动子上选择性 CpG 位点的高甲基化参与抑制 GLT1 启动子的激活，但这种调节在 ALS 患者星形胶质细胞 EAAT2 表达功能障碍中不起作用。
