cAMP反应元件结合蛋白介导磷酸化ERK1/2在癫痫苔藓纤维出芽中的作用及机制探讨
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摘要
目的:
观察细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase 1/2,ERK1/2)信号转导通路抑制剂(U0126)对体外培养海马神经元癫痫样放电后磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response-element binding protein, pCREB)、生长相关蛋白(growth associated prorein,GAP-43)和突触体素(synaptophysin, SYP)表达变化的影响,探讨ERK/CREB转导通路在癫痫苔藓纤维出芽(mossy fiber sprouting, MFS)中的作用。
方法:
采用24 h内新生Wistar大鼠,海马神经元用含10%胎牛血清的Neurobasal培养液培养于37℃、5%CO2细胞培养箱。将细胞分为:1.正常对照组(control):正常培养液培养至第9d时,换用正常细胞外液处理3h,运用膜片钳测定细胞活性;2.模型组(model):将海马神经元培养至第9d时,将培养液换成无镁细胞外液处理3h,去标本运用膜片钳测定细胞放电情况,确定细胞癫痫样放电模型建立成功;3.U0126组:将海马神经元培养至第9d时,将培养液换成无镁细胞外液,同时加入10μm/l U0126,3 h后换成正常培养液。在模型组和U0126组中,分别取6个时间点(无镁细胞外液处理3h后0min,30min,2h,6h,12h和24h)的标本进行相应实验。采用免疫荧光双重标记各组中每个时间点的pERK1/2和pCREB的表达,并在激光共聚焦扫描显微镜下拍照。运用Western-blot检测不同时间点各组细胞GAP-43和SYP的表达水平。
结果:
1.运用EPC-10系统进行全细胞模式记录海马神经元癫痫样放电:正常对照组中,神经元培养至第9d时,换成正常细胞外液处理3h,显示神经元在多数时间内处于静息状态,偶尔出现动作电位;模型组中,神经元培养至第9d时,换成无镁细胞外液处理3h,神经元阵发性出现连续的稳定的l0～35mV动作电位,放电频率5～l7Hz,经无镁处理3h,换成正常维持培养基24h后,90%以上的神经元仍呈癫痫样放电。
2.免疫荧光双重标记pERK1/2和pCREB的表达:在正常对照组中,可以观察到pCREB在神经元胞核内有轻度表达,pERK1/2主要在神经元胞浆内表达;模型组中,0min时,可以观察到pCREB在神经元胞核内表达,pERK1/2在神经元胞核与胞浆内皆有表达,30min时,两者的表达达到了高峰,2h时开始减弱,6h,12h和24h仍然有所表达,强度与2h时接近;U0126组中,各时间点的pERK1/2表达完全被抑制,pCREB的荧光强度绝对值与模型组中相应时间点的相比较明显减弱(均p <0.01),并且各时间点的表达强度相近。
3.Western-blot检测GAP-43和SYP的表达水平:GAP-43和SYP的表达趋势一样。在正常对照组中,发现GAP-43和SYP都有轻度表达;在模型组中,GAP-43和SYP在各个时间点的都有表达,30min时,表达达到高峰,与pERK1/2的表达趋势相似;在U0126组中,六个时间点的GAP-43和SYP表达强度与模型组中相应的时间点比较减弱明显(均p <0.01),同时,该组中各时间点的表达强度相近。
结论:
1.通过Neurobasal培养液加胎牛血清所培养的海马神经元活性好,经无镁细胞外液处理3h后,所获得的神经元癫痫样放电模型是一种持续的稳定癫痫细胞模型,为今后进一步研究癫痫的发病机制提供了条件。
2.海马神经元癫痫样放电后ERK/CREB信号转导通路明显被激活,同时反应突触可塑性的蛋白GAP-43和SYP的表达明显增强,此外,ERK1/2的特异性抑制剂U0126在阻断ERK/CREB信号转导通路的同时,明显减弱了GAP-43和SYP的表达, GAP-43和SYP是癫痫MFS的两大标记物,
3.本研究在细胞水平上探讨了癫痫MFS和ERK/CREB信号转导通路之间的关系。
Objective:
To observe the effects of the inhibitor of extracellular signal-regulated protein kinase 1/2 on the expression of phosphorylated cAMP response-element binding protein, growth associated prorein and synaptophysin after epileptiform discharge of hippocampal neurons, and investigate the effects of ERK/CREB on epilepsy mossy fiber sprouting
Methods:
Neonatal Wistar(<24 hours) rats were adopted. Hippocampal neurons were cultured in Neurobasal medium supplemented with 10% fetal bovine serum, and placed into a 37oC, 5% CO2 cell culture incubator. The neurons were devided into:1.Normal control group: On day 9 after beginning the incubation, we replaced the maintenance medium with normal extracellular fluid for three hours, then recorded the discharge using whole-cell patch clamp techniques. 2.Model group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid for three hours, then recorded the epileptiform activity using whole-cell patch clamp techniques. 3.U0126 group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid and 10μm/l U0126 for three hours. We evaluated two groups, model and U0126, and six time points, 0 minutes, 30 minutes, 2 hours, 6 hours, 12 hours, and 24 hours following epileptiform discharge. We investigated changes in pERK1/2 and pCREB expression by double-label immunofluorescence, for each sample, photos were collected using a laser scanning confocal microscope. Using western blotting, we observed changes in expression of GAP-43 and synaptophysin.
Results:
1.Hippocampal neurons epileptiform discharge was recorded by EPC-10 system in whole-cell patch clamp. Action potential could be recorded in neurons, who had been treated with normal extracellular fluid for 3 hours. Epileptiform discharges of neurons recordings from cultured neurons that were exposed to magnesium-free extracellular fluid for 3 hours manifested larger, longer duration synaptic potentials and multiple action potentials. These evolved into continuous tonic high-frequency burst discharges that resolved into recurrent epileptiform discharges after return to normal medium.
2.Following immunofluorescence double labeling, differentially expressed products were detected. In the normal control group, pCREB were observed in the nucleus, and pERK1/2 were detected in the cytoplasm predominantly. pERK1/2 could be observed at 0 min, and the expression peaked at 30 min. The expression pattern of pCREB was the same as pERK1/2 in the model group. In the U0126 group, pERK1/2 expression was inhibited completely. Moreover, pCREB expression also was partially inhibited, and the intensity of its expression was similar at each time point.
3. We measured the levels of SYP and GAP-43 by western blotting.In normal neurons, both SYP and GAP-43 were observed. In model group, both proteins were detected at the first time point, and changes in expression followed the same pattern as pERK1/2. On the other hand, in U0126 group, neurons expression of both proteins were reduced across all six time points.
Conclusions:
1.Neurons cultured in Neurobasal medium with fetal bovine serum was fit to be treated with magnesium-free extracellular fluid for three hours. The neuronal epileptiform discharge model was stabile for next study.
2.ERK/CREB signal transduction pathway is functionally regulated by phosphorylation, which was activated after neuronal epileptiform discharge, and accompanied by increased levels of GAP-43 and SYP, which were considered to be markers of MFS. Moreover, bouth the activation of the signal transduction pathway was inhibited, and the expression of the markers were decreased by U0126.
3.In cellular level, these findings indicate that MFS after epileptic seizure are related to activation of the ERK/CREB signal transduction pathway.
观察细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase 1/2,ERK1/2)信号转导通路抑制剂(U0126)对体外培养海马神经元癫痫样放电后磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response-element binding protein, pCREB)、生长相关蛋白(growth associated prorein,GAP-43)和突触体素(synaptophysin, SYP)表达变化的影响,探讨ERK/CREB转导通路在癫痫苔藓纤维出芽(mossy fiber sprouting, MFS)中的作用。
方法:
采用24 h内新生Wistar大鼠,海马神经元用含10%胎牛血清的Neurobasal培养液培养于37℃、5%CO2细胞培养箱。将细胞分为:1.正常对照组(control):正常培养液培养至第9d时,换用正常细胞外液处理3h,运用膜片钳测定细胞活性;2.模型组(model):将海马神经元培养至第9d时,将培养液换成无镁细胞外液处理3h,去标本运用膜片钳测定细胞放电情况,确定细胞癫痫样放电模型建立成功;3.U0126组:将海马神经元培养至第9d时,将培养液换成无镁细胞外液,同时加入10μm/l U0126,3 h后换成正常培养液。在模型组和U0126组中,分别取6个时间点(无镁细胞外液处理3h后0min,30min,2h,6h,12h和24h)的标本进行相应实验。采用免疫荧光双重标记各组中每个时间点的pERK1/2和pCREB的表达,并在激光共聚焦扫描显微镜下拍照。运用Western-blot检测不同时间点各组细胞GAP-43和SYP的表达水平。
结果:
1.运用EPC-10系统进行全细胞模式记录海马神经元癫痫样放电:正常对照组中,神经元培养至第9d时,换成正常细胞外液处理3h,显示神经元在多数时间内处于静息状态,偶尔出现动作电位;模型组中,神经元培养至第9d时,换成无镁细胞外液处理3h,神经元阵发性出现连续的稳定的l0～35mV动作电位,放电频率5～l7Hz,经无镁处理3h,换成正常维持培养基24h后,90%以上的神经元仍呈癫痫样放电。
2.免疫荧光双重标记pERK1/2和pCREB的表达:在正常对照组中,可以观察到pCREB在神经元胞核内有轻度表达,pERK1/2主要在神经元胞浆内表达;模型组中,0min时,可以观察到pCREB在神经元胞核内表达,pERK1/2在神经元胞核与胞浆内皆有表达,30min时,两者的表达达到了高峰,2h时开始减弱,6h,12h和24h仍然有所表达,强度与2h时接近;U0126组中,各时间点的pERK1/2表达完全被抑制,pCREB的荧光强度绝对值与模型组中相应时间点的相比较明显减弱(均p <0.01),并且各时间点的表达强度相近。
3.Western-blot检测GAP-43和SYP的表达水平:GAP-43和SYP的表达趋势一样。在正常对照组中,发现GAP-43和SYP都有轻度表达;在模型组中,GAP-43和SYP在各个时间点的都有表达,30min时,表达达到高峰,与pERK1/2的表达趋势相似;在U0126组中,六个时间点的GAP-43和SYP表达强度与模型组中相应的时间点比较减弱明显(均p <0.01),同时,该组中各时间点的表达强度相近。
结论:
1.通过Neurobasal培养液加胎牛血清所培养的海马神经元活性好,经无镁细胞外液处理3h后,所获得的神经元癫痫样放电模型是一种持续的稳定癫痫细胞模型,为今后进一步研究癫痫的发病机制提供了条件。
2.海马神经元癫痫样放电后ERK/CREB信号转导通路明显被激活,同时反应突触可塑性的蛋白GAP-43和SYP的表达明显增强,此外,ERK1/2的特异性抑制剂U0126在阻断ERK/CREB信号转导通路的同时,明显减弱了GAP-43和SYP的表达, GAP-43和SYP是癫痫MFS的两大标记物,
3.本研究在细胞水平上探讨了癫痫MFS和ERK/CREB信号转导通路之间的关系。
Objective:
To observe the effects of the inhibitor of extracellular signal-regulated protein kinase 1/2 on the expression of phosphorylated cAMP response-element binding protein, growth associated prorein and synaptophysin after epileptiform discharge of hippocampal neurons, and investigate the effects of ERK/CREB on epilepsy mossy fiber sprouting
Methods:
Neonatal Wistar(<24 hours) rats were adopted. Hippocampal neurons were cultured in Neurobasal medium supplemented with 10% fetal bovine serum, and placed into a 37oC, 5% CO2 cell culture incubator. The neurons were devided into:1.Normal control group: On day 9 after beginning the incubation, we replaced the maintenance medium with normal extracellular fluid for three hours, then recorded the discharge using whole-cell patch clamp techniques. 2.Model group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid for three hours, then recorded the epileptiform activity using whole-cell patch clamp techniques. 3.U0126 group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid and 10μm/l U0126 for three hours. We evaluated two groups, model and U0126, and six time points, 0 minutes, 30 minutes, 2 hours, 6 hours, 12 hours, and 24 hours following epileptiform discharge. We investigated changes in pERK1/2 and pCREB expression by double-label immunofluorescence, for each sample, photos were collected using a laser scanning confocal microscope. Using western blotting, we observed changes in expression of GAP-43 and synaptophysin.
Results:
1.Hippocampal neurons epileptiform discharge was recorded by EPC-10 system in whole-cell patch clamp. Action potential could be recorded in neurons, who had been treated with normal extracellular fluid for 3 hours. Epileptiform discharges of neurons recordings from cultured neurons that were exposed to magnesium-free extracellular fluid for 3 hours manifested larger, longer duration synaptic potentials and multiple action potentials. These evolved into continuous tonic high-frequency burst discharges that resolved into recurrent epileptiform discharges after return to normal medium.
2.Following immunofluorescence double labeling, differentially expressed products were detected. In the normal control group, pCREB were observed in the nucleus, and pERK1/2 were detected in the cytoplasm predominantly. pERK1/2 could be observed at 0 min, and the expression peaked at 30 min. The expression pattern of pCREB was the same as pERK1/2 in the model group. In the U0126 group, pERK1/2 expression was inhibited completely. Moreover, pCREB expression also was partially inhibited, and the intensity of its expression was similar at each time point.
3. We measured the levels of SYP and GAP-43 by western blotting.In normal neurons, both SYP and GAP-43 were observed. In model group, both proteins were detected at the first time point, and changes in expression followed the same pattern as pERK1/2. On the other hand, in U0126 group, neurons expression of both proteins were reduced across all six time points.
Conclusions:
1.Neurons cultured in Neurobasal medium with fetal bovine serum was fit to be treated with magnesium-free extracellular fluid for three hours. The neuronal epileptiform discharge model was stabile for next study.
2.ERK/CREB signal transduction pathway is functionally regulated by phosphorylation, which was activated after neuronal epileptiform discharge, and accompanied by increased levels of GAP-43 and SYP, which were considered to be markers of MFS. Moreover, bouth the activation of the signal transduction pathway was inhibited, and the expression of the markers were decreased by U0126.
3.In cellular level, these findings indicate that MFS after epileptic seizure are related to activation of the ERK/CREB signal transduction pathway.
引文
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[10]Heloisa A,Simone k,Luiz E.Plastic changes and disease-modifying effects of scopolamine in the pilocarpine model of epilepsy in rats[J]. Epilepsia, 2005,46(suppl.5):118-124.
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[12]E. Lynd-balta, W. H. Pilcher,A. Joseph. Ampa receptor alterations procede mossy fiber sprouting in young children with temporal lobe epilepsy[J]. Neuroscience, 2004,126 : 105-114.
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[15]Koyama R, Yamada MK, Fujisawa S, et al.Brain-derived neurotrophic factor induces hyperexcitable reentrant circuits in the dentate gyrus[J].J Neurosci,2004,24(33):7215-7224.
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[17]Muddanna S, Bharathi H, Ashok K. Fetal hippocampal CA3 cell grafts enriched with FGF-2 and BDNF exhibit robust long-term survival and integration and suppress aberrant mossy fiber sprouting in the injured middle-aged hippocampus[J]. Neurobiology of Disease, 2006, 21 :276-290.
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