Pathway and gene ontology based analysis of gene expression in a rat model of cerebral ischemic tolerance

Department of Anesthesiology, University of California, San Diego, USA.
Brain Research (Impact Factor: 2.84). 11/2007; 1177(1):103-23. DOI: 10.1016/j.brainres.2007.07.047
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Ischemic tolerance is a phenomenon whereby a sublethal ischemic insult [ischemic preconditioning (IPC)] provides robust protection against subsequent lethal ischemia. Activation of N-methyl-D-aspartate (NMDA) receptors and subsequent new gene transcription are required for tolerance. We utilized the NMDA antagonist, MK801, prior to the IPC stimulus to separate candidate genes from epiphenomenona. Rats were divided into four groups: vehicle/IPC (preconditioned), MK801/IPC (attenuated preconditioning), vehicle/sham (non-preconditioned), and MK801/sham (non-preconditioned). Hippocampi (5/group/time point) were harvested immediately after ischemia as well as 1, 4, and 24 h post-ischemia to profile gene expression patterns using microarray analyses. Extracted mRNAs were pooled and subsequently hybridized to Affymetrix arrays. In addition, groups of rats were sacrificed for Western blot analysis and histological studies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO) analyses were used to identify functionally related groups of genes whose modulation was statistically significant, while hierarchical cluster analysis was used to visualize the fold expression within these groups. Significantly modulated pathways included: MAP kinase signaling pathway, Toll receptor pathway, TGF-beta signaling pathways, and pathways associated with ribosome function and oxidative phosphorylation. Our data suggest that the tolerant brain responds to subsequent ischemic stress by partially downregulating inflammatory and upregulating protein synthesis and energy metabolism pathways.

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    • "Moreover, preconditioning has been shown to facilitate the recovery of protein synthesis following lethal ischemia in preconditioned gerbils [37]. Recently, pathway analysis in a micro-array study of ischemic preconditioning demonstrated an up-regulation of ribosomal pathways following a severe ischemic insult [4]. This seems similar to our findings, however we demonstrate that the up-regulation in ribosomal pathways is already present after the preconditioning stimulus alone, so before the deleterious insult has even occurred, possibly preparing the brain for disruption of ribosomal structure during a subsequent asphyctic insult. "
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    ABSTRACT: Fetal asphyctic (FA) preconditioning is effective in attenuating brain damage incurred by a subsequent perinatal asphyctic insult. Unraveling mechanisms of this endogenous neuroprotection, activated by FA preconditioning, is an important step towards new clinical strategies for asphyctic neonates. Genomic reprogramming is thought to be, at least in part, responsible for the protective effect of preconditioning. Therefore we investigated whole genome differential gene expression in the preconditioned rat brain. FA preconditioning was induced on embryonic day 17 by reversibly clamping uterine circulation. Male control and FA offspring were sacrificed 96 h after FA preconditioning. Whole genome transcription was investigated with Affymetrix Gene1.0ST chip. Data were analyzed with the Bioconductor Limma package, which showed 53 down-regulated and 35 up-regulated transcripts in the FA-group. We validated these findings with RT-qPCR for adh1, edn1, leptin, rdh2, and smad6. Moreover, we investigated differences in gene expression across different brain regions. In addition, we performed Gene Set Enrichment Analysis (GSEA) which revealed 19 significantly down-regulated gene sets, mainly involved in neurotransmission and ion transport. 10 Gene sets were significantly up-regulated, these are mainly involved in nucleosomal structure and transcription, including genes such as mecp2. Here we identify for the first time differential gene expression after asphyctic preconditioning in fetal brain tissue, with the majority of differentially expressed transcripts being down-regulated. The observed down-regulation of cellular processes such as neurotransmission and ion transport could represent a restriction in energy turnover which could prevent energy failure and subsequent neuronal damage in an asphyctic event. Up-regulated transcripts seem to exert their function mainly within the cell nucleus, and subsequent Gene Set Enrichment Analysis suggests that epigenetic mechanisms play an important role in preconditioning induced neuroprotection.
    BMC Neuroscience 06/2013; 14(1):61. DOI:10.1186/1471-2202-14-61 · 2.67 Impact Factor
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    • "The KEGG pathways are compiled from multiple literature sources, and they integrate individual components into a unified pathway. Therefore, the KEGG pathway database was used to further characterize the enrichment of specific pathway components into functionally regulated gene groups [17]. Seventeen KEGG pathways were significantly enriched in genes associated with serum starvation (P<0.01) "
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    ABSTRACT: Background Intervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model. Methodology/Principal Findings Rat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21Cip1 and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with ≥1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27Kip1 and p53 protein expression was upregulated in serum-starved cells. p21Cip1 expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA. Conclusions/Significance Nutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.
    PLoS ONE 03/2013; 8(3):e58806. DOI:10.1371/journal.pone.0058806 · 3.23 Impact Factor
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    • "In brief, results of the present study show that ischemic PC induces a temporal change in the cerebral miRNA profile and many of the altered miRNAs target many mRNAs that are part of the biological pathways that control cellular signaling. Although, the molecular mechanisms of the PC-induced neuroprotection are not yet completely understood, we and others showed that PC leads to extensive changes in the mRNA and protein expression in rodent brain (Dhodda et al. 2004, Stenzel-Poore et al. 2007, Feng et al. 2007). Many proteins reported previously to be altered after PC like heme oxygenases, metallothioneins, heat shock proteins, hypoxia-inducible factor-1, immediate early genes, growth factors, nitric oxide, ion channels, toll-like receptors and kinases were proposed as putative mediators of ischemic tolerance (Zeynalov et al. 2009, Tang et al. 2006, Dhodda et al. 2004, Trendelenburg et al. 2002, Truettner et al. 2002, Pradillo et al. 2009, Gonzalez-Zulueta et al. 2000, Bernaudin et al. 2002). "
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    ABSTRACT: J. Neurochem. (2010) 113, 1685–1691. Cerebral gene expression is known to be significantly influenced by a sublethal ischemic event (pre-conditioning; PC) that induces tolerance to future damaging ischemic events. Small non-coding RNAs known as microRNAs (miRNAs) were recently shown to control the mRNA translation. We currently profiled cerebral miRNAs in the cerebral cortex of rats subjected to PC. The miRNAome reacted quickly and by 6 h following PC, levels of 51 miRNAs were altered (26 up- and 25 down-regulated; > 1.5-fold change). Twenty of these stayed at the altered level even at 3 days after PC. At least nine miRNAs showed > 5-fold change at one or more time points between 6 h to 3 days after PC compared with sham. Bioinformatics analysis showed 2007 common targets of the miRNAs that were up-regulated and 459 common targets of the miRNAs that were down-regulated after PC. Pathways analysis showed that MAP-kinase and Mammalian target of rapamycin (mTOR) signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the up-regulated miRNAs, and Wnt and GnRH signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the down-regulated miRNAs after PC. We hypothesize that alterations in miRNAs and their down-stream mRNAs of signaling pathways might play a role in the induction of ischemic tolerance.
    Journal of Neurochemistry 06/2010; 113(6):1685-91. DOI:10.1111/j.1471-4159.2010.06735.x · 4.28 Impact Factor
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