Fernandez M, Pirondi S, Antonelli T, Ferraro L, Giardino L, Calzà L
Department of Veterinary Morphophysiology and Animal Production (DIMORFIPA), University of Bologna, Bologna, Italy.
J Neurosci Res. 2005 Oct 1;82(1):115-25. doi: 10.1002/jnr.20608.
The hippocampus is extremely sensitive to microenvironmental signals and toxic events, including massive glutamate release. Despite the extensive literature related to the cascade of molecular events triggered in postsynaptic neurons, the distinction between proapoptotic and survival pathways is still being discussed. In this study, we have investigated the role of c-Fos in glutamate-induced toxicity in primary cultures of hippocampal neurons by using antisense oligonucleotide (ASO) technology. Exposure of cells (5 days in vitro; DIV) to glutamate 0.5 mM for 24 hr caused massive nuclear alteration. An increase in the number of caspase-3-positive cells was also observed 24 hr after glutamate treatment. The expression of c-fos and c-jun immediate-early genes was increased 30 min after glutamate exposure. The study of c-Fos and c-Jun protein expression revealed an increase in the number of cells positive for both antibodies. To investigate whether the expression of c-Fos protein after glutamate treatment was related to cell death activation or cell survival pathways, cells were exposed to 5 microM of c-fos ASO at 4 DIV, 24 hr before glutamate treatment. The presence of the ASO in the medium significantly decreased the number of altered nuclei, and this was associated with a significant reduction in the number of c-Fos-positive cells after glutamate treatment. Exposure of cells to the c-fos ASO under the conditions described above decreased caspase-3 immunostaining induced by glutamate. These results suggest that the synthesis of c-Fos protein after glutamate exposure favors cell death pathway activation in which caspase-3 is also involved.
海马体对微环境信号和毒性事件极为敏感，包括大量谷氨酸释放。尽管有大量文献涉及突触后神经元中触发的分子事件级联反应，但促凋亡途径和存活途径之间的区别仍在讨论之中。在本研究中，我们通过使用反义寡核苷酸（ASO）技术，研究了c-Fos在海马神经元原代培养物中谷氨酸诱导的毒性中的作用。将细胞（体外培养5天；DIV）暴露于0.5 mM谷氨酸中24小时，导致大量核改变。谷氨酸处理24小时后，还观察到caspase-3阳性细胞数量增加。谷氨酸暴露30分钟后，c-fos和c-jun即刻早期基因的表达增加。对c-Fos和c-Jun蛋白表达的研究显示，两种抗体阳性的细胞数量增加。为了研究谷氨酸处理后c-Fos蛋白的表达是否与细胞死亡激活或细胞存活途径有关，在谷氨酸处理前24小时，于4 DIV时将细胞暴露于5 μM的c-fos ASO中。培养基中ASO的存在显著减少了核改变的数量，这与谷氨酸处理后c-Fos阳性细胞数量的显著减少有关。在上述条件下将细胞暴露于c-fos ASO可降低谷氨酸诱导的caspase-3免疫染色。这些结果表明，谷氨酸暴露后c-Fos蛋白的合成有利于细胞死亡途径的激活，其中caspase-3也参与其中。
