Hypothermia blocks β-catenin degradation after focal ischemia in rats

Abstract

Dephosphorylated and activated glycogen synthase kinase (GSK) 3β hyperphosphorylates β-catenin, leading to its ubiquitin-proteosome-mediated degradation. β-catenin-knockdown increases while β-catenin overexpression prevents neuronal death in vitro; in addition, protein levels of β-catenin are reduced in the brain of Alzheimer's patients. However, whether β-catenin degradation is involved in stroke-induced brain injury is unknown. Here we studied activities of GSK 3β and β-catenin, and the protective effect of moderate hypothermia (30 °C) on these activities after focal ischemia in rats. The results of Western blot showed that GSK 3β was dephosphorylated at 5 and 24 h after stroke in the normothermic (37 °C) brain; hypothermia augmented GSK 3β dephosphorylation. Because hypothermia reduces infarction, these results contradict with previous studies showing that GSK 3β dephosphorylation worsens neuronal death. Nevertheless, hypothermia blocked degradation of total GSK 3β protein. Corresponding to GSK 3β activity in normothermic rats, β-catenin phosphorylation transiently increased at 5 h in both the ischemic penumbra and core, and the total protein level of β-catenin degraded after normothermic stroke. Hypothermia did not inhibit β-catenin phosphorylation, but it blocked β-catenin degradation in the ischemic penumbra. In conclusion, moderate hypothermia can stabilize β-catenin, which may contribute to the protective effect of moderate hypothermia.

Introduction

After stroke, abnormal activities of glycogen synthase kinase 3β (GSK 3β) and β-catenin, two molecules that are downstream of Akt and Wnt survival pathways, are implicated in ischemic injury. In the Akt pathway, activated-Akt phosphorylates and inactivates GSK 3β, thereby stabilizing β-catenin. Activated GSK 3β, on the other hand, phosphorylates β-catenin, resulting its proteosomal degradation (Zhao et al., 2006). Stabilized β-catenin translocates into the nuclei, activating gene expression that supports cell survival (Nelson and Nusse, 2004, Rasola et al., 2007). β-catenin has been shown to decrease in brains of patients with Alzheimer's disease (Fuentealba et al., 2004). In addition, β-catenin-knockdown results in apoptosis, whereas β-catenin overexpression prevents neuronal death in vitro (Li et al., 2007). Thus, we hypothesize that β-catenin degradation is also complicated in brain injury after stroke.

Mild (33–36 °C) or moderate (28–32 °C) hypothermia is one of the strongest neuroprotectants identified to date that protect against cerebral ischemia (Busto et al., 1987, Colbourne et al., 2000, Zhao et al., 2005a), but the exact mechanisms for its protective effects are not fully understood (Zhao et al., 2007). We have previously reported that intra-ischemic moderate hypothermia (30 °C) reduces infarct size in focal ischemia by regulating the Akt survival pathways (Zhao et al., 2005). Moderate hypothermia attenuates reductions in Akt activity after stroke (Zhao et al., 2005a) but does not attenuate reduction in GSK 3β phosphorylation, suggesting that it may not inhibit GSK 3β activity (Zhao et al., 2005a). Since GSK 3β activity is known to exacerbate ischemic injury, and its inhibition reduces infarction (Cimarosti et al., 2005, Kelly et al., 2004, Martinez et al., 2002), it is puzzling that hypothermia can inhibit ischemic injury without blocking GSK 3β activity. To address this paradox, we further examined the protective effects of moderate hypothermia on β-catenin phosphorylation and total protein level of β-catenin (molecules downstream of GSK 3β), in the same focal ischemia model with rats we used before for studying the Akt/GSK 3β pathway.