Abstract
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.
Highlights
Global cerebral ischemia is a pathological condition in which brain tissue is subjected to reduced levels of oxygen and glucose due to impairment in blood supply to the entire brain, causing biochemical modifications in the normal functioning of neurons that can lead to injury in specific neuronal subpopulations
There are biochemical alterations that occur in neurons submitted to ischemic insults, which include changes in the expression levels and the molecular composition of proteins related with synaptic transmission, such as the ionotropic glutamate receptors of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and the N-methyl-D-aspartate (NMDAR) types, among other proteins [11,12,13,14] that can be implicated in the mechanisms promoting either cell death or cell survival
In order to characterize the neuronal injury induced by in vitro oxygen and glucose deprivation, primary hippocampal neuronal cultures were subjected to different periods of OGD, followed by 24 h incubation in culture conditioned medium, after which cell viability was evaluated by analysis of the nuclear morphology
Summary
Global cerebral ischemia is a pathological condition in which brain tissue is subjected to reduced levels of oxygen and glucose due to impairment in blood supply to the entire brain, causing biochemical modifications in the normal functioning of neurons that can lead to injury in specific neuronal subpopulations. One of the main features of transient global cerebral ischemia is the delayed death of the pyramidal neurons of the CA1 region of the hippocampus, which occurs hours to days after the insult. This time-window between the end of the transient ischemic insult and the first signs of neuronal demise is believed to be associated with the activation of competing programs of gene expression, in which some will facilitate cell survival, whereas others will contribute to neuronal death [1]. There are biochemical alterations that occur in neurons submitted to ischemic insults, which include changes in the expression levels and the molecular composition of proteins related with synaptic transmission, such as the ionotropic glutamate receptors of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and the N-methyl-D-aspartate (NMDAR) types, among other proteins [11,12,13,14] that can be implicated in the mechanisms promoting either cell death or cell survival
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