谷氨酰胺酶在TNF-α诱导神经元损伤和凋亡中的作用及其定位调控机制探讨

The mechanisms of the pathogenesis of HAND (HIV-1-associated neurocognitive disorders) are still unknown to us. Recent studies have indicated that proinflammatory cytokines are very critical for the generation and process of HAND. The concentration of glutamate is critical for HAND and glutaminase is the main enzyme responsible for glutamate production in mammalian cells. The expression level of type I glutamines（GLS 1）is much higher than type II glutaminase（GLS2）in the central nervous system. Our previous studies indicated that tumor necrosis factor-α (TNF-α), a proinflammatory cytokine that are typically elevated in HAND states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following TNF-α treatment.We found that TNF-α significantly upregulated the GLS1 expression and specifically increase GLS1 levels in cytosol and in the extracellular fluid.The up-regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV-1 encephalitis.We also confirmed that neurons express the most GLS1 among all the cell types in the CNS. So we hypothesize that GLS1 plays a very important role in TNF-α-induced neuronal death and apoptosis. To verify this hypothesis, we will overexpress or knockdown GLS1 in neurons and then investigate the relationship between GLS1 and neuronal death and apoptosis. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for HAND.

HIV-1相关的认知功能障碍（HAND）的具体发病机制尚不完全清楚。现已证实：细胞因子与HAND的发生及发展密切相关；HAND中一个重要的犯罪分子是谷氨酸；谷氨酰胺酶（GLS）是催化生成谷氨酸的关键酶；CNS中的GLS1(I型)远多于GLS2。我们证实了TNF-α调控GLS1的表达和定位、显著提高胞内外的谷氨酸浓度及诱导神经元损伤，并最终导致细胞凋亡。HIV脑炎动物模型结果显示，注射HIV-1感染的MØ组的GLS1较PBS组显著增加，且GLS1与β-Ⅲ-tubulin存在共定位。体外实验也提示神经元的GLS1表达最丰富。据此假设TNF-α影响GLS1的定位在细胞凋亡中起关键作用。我们拟采用过表达或抑制GLS验证GLS与TNF-α诱导神经元凋亡的关系，并探讨活性氧、线粒体通透性变化是否参与该凋亡信号传导通路。本研究将深化对细胞因子诱导细胞凋亡的分子机制认识，还为阐明HAND的发病机制提供依据

细胞外囊泡（Extracellular vesicles，EVs）是从细胞表面释放的一种双层膜结构的囊泡，它包含蛋白质、脂质和核酸，在细胞与细胞交流中发挥重要作用。在神经炎症中，细胞外囊泡释放量增加，但是其释放机制尚不清楚。谷氨酰胺酶（Glutaminase）是定位于线粒体的一种代谢酶，可以将谷氨酰胺分解为谷氨酸。在我们前期的研究中，发现肿瘤坏死因子（tumor necrosis factor-α，TNF-α）可以促进神经元谷氨酰胺酶表达。但是，在神经炎症环境下，小鼠星形胶质细胞中谷氨酰胺酶的表达及作用尚不清楚。我们假设TNF-α促进小鼠星形胶质细胞谷氨酰胺酶表达，进而促进星形胶质细胞细胞外囊泡的释放。.本课题通过电子显微镜、纳米粒子追踪技术和免疫印迹的方法发现TNF-α处理星形胶质细胞引起细胞外囊泡释放量增加。此外，TNF-α显著增加星形胶质细胞谷氨酰胺酶表达及谷氨酸的产生，这表明TNF-α增强了谷氨酰胺酶活性。此外，通过使用氨酰胺酶的抑制剂预处理星形胶质细胞，降低TNF-α引起的细胞中活性氧（reactive oxygen species，ROS）水平，表明谷氨酰胺酶参与神经炎症中星形胶质细胞的氧化应激。由TNF-α引起的细胞外囊泡释放增加可以被谷氨酰胺酶抑制剂、抗氧化剂阻断，类似结果在敲除谷氨酰胺酶的星形胶质细胞中也出现。这表明，炎性环境下，谷氨酰胺酶在星形胶质细胞细胞外囊泡释放过程中发挥了重要作用。综合上述结果，神经炎症中，TNF-α通过谷氨酰胺酶促进星形胶质细胞细胞外囊泡释放。.这些结果证明，在神经炎症中，TNF-α通过谷氨酰胺酶促进星形胶质细胞细胞外囊泡释放。.项目资助发表SCI收录论文1篇，待发表论文2篇。已协助培养硕士生3名，其中1名已经取得硕士学位。项目投入经费23万元，支出20.67万元，各项支出基本与预算相符。结余经费2.329万元，剩余经费计划用于本项目研究后续支出。