Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons.
Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons.
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谷氨酸对原代培养皮质神经元溶酶体膜透化的影响
DOI：
10.3892/mmr.2016.4819
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发表时间：
2016-03
影响因子：
3.4
通讯作者：
Yin W
中科院分区：
文献类型：
--
作者：
Yan M;Zhu W;Zheng X;Li Y;Tang L;Lu B;Chen W;Qiu P;Leng T;Lin S;Yan G;Yin W
谷氨酸是中枢神经系统的主要神经递质。谷氨酸介导的兴奋性毒性是脑损伤的主要原因。最近的研究表明，溶酶体膜透化（LMP）参与了非人灵长类动物缺血相关的神经元死亡。本研究旨在探讨谷氨酸对原代培养皮层神经元溶酶体稳定性的影响。谷氨酸处理30分钟诱导溶酶体膜的透化，通过吖啶橙子再分布和细胞质中组织蛋白酶B的免疫荧光进行评估。NMDA受体拮抗剂MK-801和钙螯合剂乙二醇双（2-氨基乙基醚）-N，N，N′，N′-四乙酸抑制谷氨酸兴奋毒性，使溶酶体免于透化。钙蛋白酶和活性氧（ROS）在诱导LMP的作用也进行了研究。谷氨酸处理后的Ca 2+超载诱导钙蛋白酶的激活和ROS的产生，这是神经元死亡的两个主要贡献者。据报道，溶酶体相关膜蛋白2（LAMP 2）和热休克蛋白（HSP）70是促进癌细胞中LMP的两种钙蛋白酶底物;然而，发现钙蛋白酶被谷氨酸激活，但只有LAMP 2随后被降解。此外，用钙蛋白酶抑制剂calpeptin和SJA 6017治疗LMP并没有减轻，这两种药物阻断了钙蛋白酶底物α-胞衬蛋白的切割。结果表明，LMP显着减轻治疗与抗氧化剂N-乙酰-L-半胱氨酸，表明LMP参与早期谷氨酸兴奋性毒性可能部分介导的ROS，而不是钙蛋白酶激活。总之，这些数据揭示了ROS介导的LMP在早期谷氨酸兴奋性毒性中的作用。
Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death in non-human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK-801 and the calcium chelator ethylene glycolbis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal-associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α-fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N-Acetyl-L-cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity.