We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.

译文

我们研究了自噬 (一种细胞大分子和细胞器的受控溶酶体降解) 在体外营养剥夺期间谷氨酸兴奋性毒性中的作用。在低葡萄糖血清/无氨基酸细胞培养基中孵育与谷氨酸协同作用,增加AMP/ATP比,并在SH-SY5Y人神经母细胞瘤细胞中引起兴奋性毒性坏死。谷氨酸抑制饥饿触发的自噬,这通过减少细胞内酸化,降低LC3标点和LC3-I转化为自噬体相关LC3-II,降低自噬beclin-1和ATG5的表达,选择性自噬靶NBR1的增加以及自噬囊泡数量的减少来证实。在PC12大鼠嗜铬细胞瘤细胞中观察到类似的结果。NMDA拮抗剂美金刚逆转了饥饿的SH-SY5Y细胞中谷氨酸介导的兴奋性毒性和自噬抑制,并被NMDA激动剂D-天冬氨酸和ibotenate模仿。谷氨酸减少了饥饿触发的能量传感器AMP激活的蛋白激酶 (AMPK) 的磷酸化,而不影响自噬的主要负调节剂雷帕霉素复合物1的哺乳动物靶标的活性。这与自噬转录激活剂 (FOXO3,ATF4) 和参与自噬起始 (ULK1,ATG13,FIP200) 、自噬体成核/延伸 (ATG14,beclin-1,ATG5) 和自噬货物递送至自噬体 (SQSTM1) 的分子的mRNA水平降低有关。组成型活性AMPK的过表达减轻了谷氨酸介导的自噬转录抑制。通过AMPK过表达或二甲双胍进行的遗传或药理学AMPK激活,以及通过TFEB过表达或氯化锂进行的遗传或药理学自噬诱导,降低了营养剥夺的SH-SY5Y细胞对谷氨酸兴奋性毒性的敏感性。这些数据表明,AMPK依赖性细胞保护性自噬的转录抑制参与了体外营养剥夺过程中谷氨酸介导的兴奋性毒性。

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