ZEB1 links p63 and p73 in a novel neuronal survival pathway rapidly induced in response to cortical ischemia.

BACKGROUND: Acute hypoxic/ischemic insults to the forebrain, often resulting in significant cellular loss of the cortical parenchyma, are a major cause of debilitating injury in the industrialized world. A clearer understanding of the pro-death/pro-survival signaling pathways and their downstream targets is critical to the development of therapeutic interventions to mitigate permanent neurological damage. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that the transcriptional repressor ZEB1, thought to be involved in regulating the timing and spatial boundaries of basic-Helix-Loop-Helix transactivator-mediated neurogenic determination/differentiation programs, functions to link a pro-survival transcriptional cascade rapidly induced in cortical neurons in response to experimentally induced ischemia. Employing histological, tissue culture, and molecular biological read-outs, we show that this novel pro-survival response, initiated through the rapid induction of p63, is mediated ultimately by the transcriptional repression of a pro-apoptotic isoform of p73 by ZEB1. We show further that this phylogenetically conserved pathway is induced as well in the human cortex subjected to episodes of clinically relevant stroke. CONCLUSIONS/SIGNIFICANCE: The data presented here provide the first evidence that ZEB1 induction is part of a protective response by neurons to ischemia. The stroke-induced increase in ZEB1 mRNA and protein levels in cortical neurons is both developmentally and phylogenetically conserved and may therefore be part of a fundamental cellular response to this insult. Beyond the context of stroke, the finding that ZEB1 is regulated by a member of the p53 family has implications for cell survival in other tissue and cellular environments subjected to ischemia, such as the myocardium and, in particular, tumor masses.

Cardiotrophin-1 protects cortical neuronal cells against free radical-induced injuries in vitro.

Cardiotrophin-1 (CT-1) was initially defined as a mediator of cardiomyocyte hypertrophy. Additional studies have showed that CT-1 enhanced survival of differentiated cardiac muscle cells and inhibited cardiac myocyte apoptosis after serum deprivation or cytokine stimulation. Moreover, CT-1 has recently been shown to act as a neuroregulatory cytokine in the peripheral nervous system. However, its effects in the central nervous system have not been determined. In the present study, we evaluated whether CT-1 protects cultured cortical neurons against oxidative injuries caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine (SIN-1). CT-1 reduced neuronal cell death caused by FeSO4 and also attenuated the neurotoxic effect of SIN-1 in a dose-dependent manner. These results indicate that CT-1 is neuroprotective in an in vitro model of cerebral ischemia. This study indicates that further evaluation of CT-1 in acute brain injury should be investigated in vivo.

Erythropoietin after focal cerebral ischemia activates the Janus kinase-signal transducer and activator of transcription signaling pathway and improves brain injury in postnatal day 7 rats.

Erythropoietin (Epo) plays a central role in erythropoiesis but also has neuroprotective properties. Recently, Epo-related neuroprotective studies used a hypoxic-ischemic neonatal model, which is different from focal stroke, a frequent cause of neonatal brain injury. We report on the effects of Epo treatment given after focal stroke and its potential neuroprotective mechanisms in postnatal day 7 rats with focal cerebral ischemia (FCI) achieved by occlusion of the middle cerebral artery. The experimental groups included sham operation, FCI plus vehicle, and FCI plus Epo. In the Epo-treated group, pups received a single intraperitoneal injection of 1000 U/kg 15 min after FCI or three injections of 100, 1000, or 5000 U/kg, starting at 15 min and repeated at 1 and 2 d after FCI. Epo treatment produced significant reductions in the mean infarct area and volume at 1 and 3 d after FCI, demonstrated by 2,3,5-triphenyltetrazolium chloride staining. Terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining showed a markedly reduced number of TUNEL-positive cells in the Epo-treated group when compared with the vehicle control 3 d after FCI (p<0.01). The most effective dose after FCI was 1000 U/kg for 3 d. Immunoanalyses showed that Epo induced a significant increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription-5 expressions at 1 and 3 d and up-regulated Bcl-xL expression by 24 h after FCI but did not affect Epo receptor or NF-kappaB expression. In conclusion, Epo given after FCI in neonatal rats provides significant neuroprotection, mediated possibly by activation of the Janus kinase-signal transducer and activator of transcription-Bcl-xL signaling pathways.

Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain.

Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development.