270. Regulation of cell survival related genes in cerebellum following hypoxia preconditioning
H. Xu and F. Sharp
Neurology Department and M.I.N.D Institute, University of California at Davis Medical Center, Sacramento, California, USA
Objectives: Hypoxia Preconditioning (HP) in the brain is a well-known endogenous protective paradigm in which pre-exposure to sub-lethal hypoxia can protect the brain from injury due to lethal hypoxia or ischemia occurring 24 to 72 h later.
1
Though HP has been studied extensively, its mechanisms are still not understood. Since de novo gene transcription and protein expression are required for the hypoxia preconditioning, we have extended previous studies by performing whole genome expression profiling of individual brain regions of adult mice and carefully following the entire time course of hypoxia preconditioning.
Methods: Adult C57BL/6 male mice (8 to 9 weeks old) were exposed to systemic hypoxia (8% O2) for 3 h and allowed to recover in normoxia for 24 h. Animals were sacrificed and the brains were removed and dissected into individual brain regions at multiple time points during the 3 h hypoxia and subsequent 24 h of reoxygenation. Total RNA was purified and gene expression assessed with Affymetrix Mouse Expression 430 2.0 arrays. One-Way ANOVA with false discovery rate correction for multiple comparisons was used to identify differentially regulated genes. The differentially regulated genes were then subjected to a variety of bioinformatics analyses including functional annotation, pathway and promoter sequence analyses.
Result: Among the brain regions examined, cerebellum demonstrated the largest numbers of gene expression changes following hypoxia preconditioning. The greatest numbers of regulated genes in cerebellum were observed immediately after the 3 h of hypoxia and 1 h after reoxygenation. In contrast to the forebrain, where down regulation of gene expression predominated over up-regulation events during this time period, cerebellum exhibited a predominant up-regulation of genes following HP. Compared to hippocampus, substantially more genes involved in cell survival regulation were up-regulated in cerebellum during this time period. Besides hypoxia inducible factor (HIF), various nuclear receptor transcription factors were found to play important roles in regulating the unique gene expression response cerebellum after HP. Most notably, a novel group of genes regulated by transcription factor hepatic nuclear factor 4A (HNF4A) have been identified as being hypoxia responsive genes in cerebellum.
Conclusions: HP induces both time-dependent and region-dependent gene expression responses. The substantial number of unique genes regulated in cerebellum may be critical not only for the survival of the organism during systemic hypoxia, but also for the maturation of neuroprotection effect of HP. Region-specific transcription factors, including HNF4A, likely contribute to region specific genomic responses to hypoxia preconditioning.
489. STAT3 regulates the transcription of the mouse Mn-SOD gene as a neuroprotectant in cerebral ischemic reperfusion
J.E. Jung, G.S. Kim, P. Narasimhan and P.H. Chan
Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA
Objectives: Cerebral ischemic reperfusion increases superoxide anion O2− from mitochondria in the brain.
1
These mitochondrial anions are rapidly removed by manganese superoxide dismutase (Mn-SOD), a primary mitochondrial antioxidant enzyme. Mn-SOD scavenges superoxide radicals and its overexpression provides neuroprotection following cerebral ischemia and reperfusion.
2
Mice with Mn-SOD deficiency exhibit large infarct volume after ischemic reperfusion.
3
However, the mechanism underlying the regulation of Mn-SOD expression and its role in neuroprotection after ischemic reperfusion are still unclear. In this study, we identified the signal transducer and transcriptional activator 3 (STAT3) as a transcriptional factor of the mouse Mn-SOD gene and elucidated the novel mechanism for O2− generation from mitochondria via a decrease in Mn-SOD expression by STAT3 deactivation after transient focal cerebral ischemia (tFCI).
Methods: We employed in vivo tFCI by way of 45 mins of transient middle cerebral artery occlusion, and in models of 2.5 h of oxygen glucose deprivation/24 h of reoxygenation in vitro. We cloned and constructed 1779 bp length of mouse Mn-SOD promoter containing STAT3 putative binding motifs in 5′ flank of mouse Mn-SOD gene. This clone was ligated with pGLU vector and then used in a luciferase assay for analysis of transcription activity of Mn-SOD promoter.
Results: We found that Mn-SOD expression is rapidly down-regulated (50% reduction, P<0.05) by reperfusion in the cerebral ischemic brain. Interestingly, we also found that STAT3 is usually recruited into the putative STAT3 binding sites of the mouse Mn-SOD promoter and up-regulates the transcription of the mouse Mn-SOD gene in the normal mouse brain. It was revealed that STAT3 is highly phosphorylated at Y705 in the normal mouse brain. However, at early post-reperfusion periods after tFCI, STAT3 is significantly dephosphorylated (60 to 70% reduction, P<0.05) and the recruitment of STAT3 into the promoter of Mn-SOD is completely diminished. In addition, the transcriptional activity of the mouse Mn-SOD gene was significantly reduced (70% reduction, P<0.05) in primary cortical neurons whose STAT3 activity is disrupted by STAT3-specific siRNA. Moreover, we found that STAT3 deactivated by reperfusion induces the accumulation of O2− (50 to 60% increase, P<0.05) from mitochondria through a reduction in the Mn-SOD expression caused by the loss of STAT3 activity under reperfusion as well as neuronal cell death (50 to 70%, P<0.05). We also found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death using SOD2 (−/+) heterozygous knockout mice.
Conclusion: Our study demonstrates that STAT3 is a novel transcriptional factor of the mouse Mn-SOD gene and is a pivotal mediator in the generation of reactive oxygen species, which trigger neuronal cell death from mitochondria after cerebral ischemic injury.
This work was supported by National Institutes of Health grants P50 NS014543, RO1 NS025372, RO1 NS036147, and RO1 NS038653.
874. Effects of glucose availability on HIF1alpha function in SH-SY5Y cells subjected to hypoxia-reoxygenation
T. Santalucia1, A. Serra-Perez1, E. Berra2 and A. Planas1
1Brain Ischemia and Neurodegeneration, IIBB-CSIC/IDIBAPS, Barcelona; 2Cell Biology & Stem Cells Unit, CICbioGUNE, Derio, Spain
Objectives: Hypoxia-induced Factor (HIF) regulates the cellular response against decreasing oxygen concentrations by activating genes involved in ATP production by glycolysis, among other mechanisms. HIF is a transcription factor that consists of an oxygen-regulated subunit (HIFalpha) and a constitutively expressed one (HIFbeta). HIFalpha expression is low under normoxia owing to proteasomal degradation. This is due to oxygen-dependent hydroxylation of certain proline residues in HIFalpha catalysed by prolylhydroxylases (PHD). Hypoxia inhibits degradation causing accumulation of HIFalpha.
1
Despite the regulation of HIFalpha expression and its transcriptional activity under hypoxia have been the subject of intense study, the putative contribution of glucose has not been examined so thoroughly.
Methods: SH-SY5Y (SH) cells were incubated in anoxic conditions for 15 h and either in the presence or absence of glucose. Cells were then allowed to reoxygenate and incubated for variable periods prior to preparing cell extracts.
2
These were analysed for the expression of HIF-1alpha and some of its target genes by Western blotting or ELISA. HIF transcriptional activity was measured by transient transfection with a luciferase reporter vector. Functionality of the proteasome was monitored by using SH clones engineered to express a ubiquitin-EGFP fusion protein that is constitutively targeted for proteasomal degradation.
3
Results: The expression of HIF-1 target genes (GLUT-1, HKII and VEGF) and the transcriptional activity of HIF after a period of 15 h of anoxia were higher in the presence of glucose in the culture medium. These results were paralleled by differences in the expression of HIF-1alpha. HIF-1alpha was immediately degraded at reoxygenation following incubation of the cells in anoxia and in the presence of glucose. Surprisingly, the HIF-1alpha expressed under conditions of oxygen and glucose deprivation (OGD) remained stable after reoxygenation of the cells. The analysis of the proteasome activity under both conditions revealed no overt differences. However, we observed a specific change in the apparent molecular weight of HIF-1alpha at the end of the 15 h OGD incubation. Whether this is related to the stabilisation is currently under study, together with the possible contribution of metabolites intervening in the PHD reaction.
4
Conclusions: The presence of glucose during anoxia enhances the induction of HIF-1alpha as well as of HIF-1 responsive genes, while OGD conditions are less favourable for the activation of HIF-1 in SH cells. Moreover, our data suggest the existence of some mechanism under OGD conditions that either affects the degradation of HIF-1alpha or renders it resistant to degradation triggered by oxygen.
Acknowledgements: ASP has a predoctoral fellowship from AGAUR (Generalitat de Catalunya). TS is a “Ramón y Cajal” scientist (Spanish Ministry of Science and Innovation, MICINN). Project funded by grants from MICINN (SAF2005–05793-C02–02 and SAF2008–04515-C02–02).
886. Calcium-dependent transcription in cerebral ischemia: role of astrocytes
M. Sobrado1, B.G. Ramírez2, M. Arbonés3, D. Fernández-López1, I. Lizasoaín1, M.A. Moro1 and E. Cano2
1Complutense University of Madrid; 2National Centre of Cardiovascular Research (CNIC), Madrid; 3Center for Genomic Regulation (CRG-UPF), Barcelona, Spain
Background: Increase in intracellular calcium concentration [Ca2+]i is a common feature of many neurodegenerative, ischemic and ageing-related processes that likely involve astrocytes. Work from our laboratory, demonstrated the existence of an inducible calcium/calcineurin/Nuclear Activated Factor of T cells (NFAT) signalling pathway in primary cultured astrocytes. Moreover, the increase in [Ca2+]i activates NFAT-dependent mRNA and protein expression of targets such as cyclooxygenase 2 and Rcan 1.4 in this cell type [1]. Here, we have studied the induction of the calcineurin/NFAT signalling pathway in response to ischemia in astrocytes, using “in vivo” and “in vitro” experimental ischemia models in rodents. We show here that there is a specific up-regulation of total Rcan 1.4 protein in brain cortex after ischemia. We have focused in the induction of Rcan protein in astrocytes around the infarcted area.
Methods and results: Western blot and quantitative RT-PCR showed that oxygen and glucose deprivation (OGD) activates calcium/calcineurin/NFAT pathway in astrocytes cultures. Protein and mRNA time course shows that RCAN 1.4 increases at early times after OGD. We have found that Rcan 1.4 overexpression in astrocytes, using adenoviral vector, inhibits the expression of inflammatory markers such as Cyclooxigenase. The occlusion of middle cerebral artery for 1 h produced an increased expression of RCAN 1.4 as soon as 1 h after ischemia and it was maximal 24 h after reperfusion. At the moment, we are carrying out a more thorough analysis of the inflammation process after brain ischemia, and the role of Rcan1.4 overexpression.
Conclusions: These results demonstrate the activation of calcium/calcineurin/NFAT pathway and the up-regulation of RCAN 1.4 after experimental cerebral ischemia. Moreover, our results suggest that the study of the calcium-dependent signalling pathway in general and the Ca/CN/NFAT pathway in particular in the ischemic context, together with its role in the regulation of genes-induced might be essential for a better understanding of neuroprotection and recovery of damaged tissue.
*These authors contributed equally to this work.
