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首页> 外文期刊>Experimental Eye Research >AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells
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AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells

机译：AMPA受体脱敏是纯化的视网膜神经节细胞中AMPA受体介导的兴奋性毒性的决定因素。

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摘要

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the cc-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retina] ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4-7 Sprague Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for cc-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking lba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 liM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 mu M) did not depolarize the RGC mitochondria] membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of cc-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 mu M) or the combination of s-AMPA and cyclothiazide (50 mu M) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival. (C) 2015 Published by Elsevier Ltd.
机译：已经假设离子型谷氨酸受体（iGLuR）通过介导兴奋性毒性死亡在神经元发病机理中发挥作用。以前关于视网膜iGluR的研究集中在两类受体上：NMDA和非NMDA受体，包括cc-氨基-3-羟基-5-甲基-4-异恶唑丙酸酯受体（AMPAR）和海藻酸酯受体。在这项研究中，我们检查了受体脱敏对AMPAR激活对原代视网膜神经节细胞（RGC）的特异性兴奋毒性作用的作用。从出生后第4-7天的Sprague Dawley大鼠中分离纯化的大鼠RGC。钙成像用于鉴定AMPAR的功能和s-AMPA激动剂的选择性。 s-AMPA处理后进行磷酸化的CREB和ERK1 / 2表达。 s-AMPA兴奋性毒性通过JC-1线粒体膜去极化测定，胱天蛋白酶3/7荧光素酶活性测定，cc-fodrin的免疫印迹分析和Live（钙调素AM）/ Dead（乙锭均二聚体1）测定来确定。本研究使用纯度为98％，缺少lba1和GFAP表达的RGC培养物。分离的产前RGCs表达钙可渗透的AMPAR，而s-AMPA（100 liM）处理培养的RGCs可显着增加CREB的磷酸化，但不能增强ERK1 / 2的磷酸化。使用s-AMPA（100μM）在纯化的RGC中长时间（6小时）激活AMPAR不会使RGC线粒体膜电位去极化。此外，与存在和缺乏营养因子（BDNF和CNTF）相比，用s-AMPA处理培养的RGC不会增加caspase 3/7活性或cc-fodrin（神经细胞凋亡标记物）的裂解。到未经处理的对照。最后，与未处理的对照RGC相比，在s-AMPA处理后观察到RGC的细胞存活率显着增加。然而，用海藻酸（100μM）或s-AMPA和环噻嗪（50μM）的组合处理可防止AMPAR脱敏，大大降低了细胞存活率。 RGC中AMPAR的激活似乎并未激活凋亡的信号级联反应，这提示RGC在体外对AMPA兴奋性毒性不敏感。相反，通过差异激动剂激活防止AMPAR脱敏引起AMPAR介导的兴奋性毒性。 AMPAR在增加CREB磷酸化中的激活取决于钙的存在，这可能有助于解释这种在增加RGC存活率方面的作用。 （C）2015由Elsevier Ltd.出版

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