兴奋性氨基酸转运体2在神经退行性变中的作用

谷氨酸是脑内必需的兴奋性神经递质之一,兴奋性氨基酸转运体（Excitatory amino acid transporterEAAT）2是最主要的谷氨酸转运体,负责脑内90%以上的谷氨酸再摄取,调节突触间隙的谷氨酸浓度。EAAT2功能紊乱导致胞外谷氨酸过量积聚,在多种神经退行性疾病的发病过程中起重要作用,如阿尔茨海默病、亨廷顿舞蹈病、肌萎缩侧索硬化等。对于人EAAT2启动子的研究发现,NF-kB在星形胶质细胞中对EAAT2表达起关键作用。通过筛选1 040种FDA批准的化合物,发现多种β-内酰胺类抗生素如头孢曲松钠等是EAAT2的转录激活剂,可以增加EAAT2的蛋白表达水平,产生神经保护作用。     

Regulation of ethanol-sensitive EAAT2 expression through adenosine A1 receptor in astrocytes

Adenosine-regulated glutamate signaling in astrocytes is implicated in many neurological and neuropsychiatric disorders. In this study, we examined whether adenosine A1 receptor regulates EAAT2 expression in astrocytes using pharmacological agents and siRNAs. We found that adenosine A1 receptor-specific antagonist DPCPX or PSB36 decreased EAAT2 expression in a dose-dependent manner. Consistently, knockdown of A1 receptor in astrocytes decreased EAAT2 mRNA expression while overexpression of A1 receptor upregulated EAAT2 expression and function. Since A1 receptor activation is mainly coupled to inhibitory G-proteins and inhibits the activity of adenylate cyclase, we investigated the effect of forskolin, which activates adenylate cyclase activity, on EAAT2 mRNA levels. Interestingly, we found that forskolin reduced EAAT2 expression in dose- and time-dependent manners. In contrast, adenylate cyclase inhibitor SQ22536 increased EAAT2 expression in dose- and time-dependent manners. In addition, forskolin blocked ethanol-induced EAAT2 upregulation. Taken together, these results suggest that A1 receptor-mediated signaling regulates EAAT2 expression in astrocytes.     

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)₂D₃ formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na⁺-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (I_glu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased I_glu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.