Fernandes Joana, Vieira Marta, Carreto Laura, Santos Manuel A S, Duarte Carlos B, Carvalho Ana Luísa, Santos Armanda E
CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal.
PLoS One. 2014 Jun 24;9(6):e99958. doi: 10.1371/journal.pone.0099958. eCollection 2014.
Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia.
短暂性全脑缺血会引起脑细胞转录组的深刻变化，这部分与影响缺血反应的基因的诱导或抑制有关。然而，海马神经元对全脑缺血选择性易损性的机制仍有待阐明。为了确定缺血性损伤引起的分子变化，我们将海马原代培养物进行氧糖剥夺（OGD），这是一种全脑缺血的体外模型，会导致具有兴奋性毒性成分的延迟性神经元死亡。为了研究经历OGD的海马神经元转录组的变化，在OGD后的早期（7小时）和延迟期（24小时）提取总RNA，并用于全基因组RNA微阵列分析。我们观察到，OGD后7小时基因普遍受到抑制，而在24小时则普遍出现基因表达的诱导。与转录和RNA生物合成等功能相关的基因在OGD后的两个孵育期都受到高度调控，证实对缺血的反应是一个动态且协调的过程。我们的分析表明，OGD后，突触蛋白的基因，如编码PICK1、GRIP1、TARPγ3、钙黏蛋白-2/3、SAPAP2和SNAP-25的基因被下调。此外，OGD降低了GluA1 AMPA受体亚基以及NMDA受体的GluN2A和GluN2B亚基的mRNA和蛋白表达水平，但增加了GluN3A亚基的mRNA表达，从而改变了海马神经元中离子型谷氨酸受体的组成。总之，我们的结果呈现了体外缺血在海马神经元中引发的表达谱，并表明OGD激活了一个转录程序，导致编码突触蛋白的基因表达下调，提示缺血后突触蛋白质组可能会发生变化。
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