Figuera-Losada Mariana, Thomas Ajit G, Stathis Marigo, Stockwell Brent R, Rojas Camilo, Slusher Barbara S
Johns Hopkins Drug Discovery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.
Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.
Biochem Biophys Rep. 2017 Jan 17;9:266-272. doi: 10.1016/j.bbrep.2016.12.009. eCollection 2017 Mar.
The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System x cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC values in agreement with previous studies using established cell lines.
中枢神经系统中的炎症反应涉及活化的小胶质细胞。在正常情况下，它们通过吞噬作用清除受损神经元。另一方面，神经退行性疾病被认为涉及小胶质细胞的慢性活化，导致过量谷氨酸、促炎细胞因子和活性氧的释放，从而导致神经元死亡。系统x胱氨酸/谷氨酸反向转运体（SXC）是一种在中枢神经系统的小胶质细胞和星形胶质细胞中发现的不依赖钠的异二聚体转运体，它将胱氨酸导入细胞并输出谷氨酸。已证明SXC在包括多发性硬化症、肌萎缩侧索硬化症、神经艾滋病、帕金森病和阿尔茨海默病在内的神经退行性疾病中上调。因此，SXC抑制剂可用于治疗以神经炎症和谷氨酸兴奋性毒性为特征的疾病。我们报告了一种基于原代小胶质细胞的检测方法的优化，用于筛选SXC抑制剂。使用脂多糖（LPS）激活大鼠原代小胶质细胞，并分别通过荧光和放射性监测谷氨酸释放和胱氨酸摄取。LPS诱导的谷氨酸释放随着细胞密度、孵育时间和LPS浓度的增加而增加。以96孔板形式优化了筛选SXC抑制剂的条件，随后用于评估SXC抑制剂。已知的SXC抑制剂柳氮磺胺吡啶、S-4CPG和埃拉斯汀可阻断谷氨酸释放和胱氨酸摄取，而S-4CPG的无活性对映体R-4CPG则不能抑制谷氨酸释放或胱氨酸转运。此外，使用原代小胶质细胞评估了几种埃拉斯汀类似物，发现其EC值与先前使用已建立细胞系的研究结果一致。
