Abstract 197: Post-stroke Inhibition of TGF-β-activated Kinase by 5Z-7-oxozeaenol Improves Histological and Behavioral Outcomes in a Mouse Model of Ischemic Stroke

Abstract

Background: Transforming growth factor-β activated kinase 1 (TAK1) activates several intracellular signaling cascades, affecting cell proliferation, inflammation, and apoptosis. It is also a putative kinase for adenosine monophosphate protein kinase (AMPK). 5Z-7-oxozeaenol is a selective inhibitor of TAK1. We and others have previously reported that this drug reduced infarct volume and levels of activated TAK1when administered prior to ischemia in a mouse model of ischemic stroke. We investigated the possible clinical utility of the drug by administering it after ischemia and measuring infarct volumes and behavioral outcomes at 7 days. We also explored the relationship between TAK1 and AMPK by examining outcomes of TAK1 inhibition in AMPK knockout (KO) mice. Methods: Stroke was induced by 90 minutes of reversible middle cerebral artery occlusion (MCAO) in male mice. In one cohort, wild-type (WT) and AMPK KO mice were treated with drug or vehicle via intraperitoneal (IP) injection 2 hours after stroke onset and stroke severity was measured by tissue staining at 24 hours. In a second cohort, WT and AMPK KO mice were treated with drug or vehicle 3.5 hours after stroke onset and stroke severity was measured using the novel-object recognition test (NORT) and by tissue staining at 7 days. Proteins were analyzed by western blot in a third cohort of mice that were sacrificed 4 hours after onset of ischemia. Results: IP injection of 5.0 mg/kg of drug 2 hours after onset of MCAO resulted in a significant reduction in whole-hemisphere infarct volume after 24 hours of reperfusion (49.18±4.18% vehicle vs.17.81±5.86% drug, p=0.001, n=7 vehicle, 8 drug) and after 7 days of reperfusion (57.16±2.38% vehicle vs. 38.35±8.97% drug, p=0.049, n=8 vehicle, 8 drug), and significantly increased the percentage of time that the mice spent exploring a novel object (59.85 ± 3.62% drug vs. 33.15 ± 5.04% vehicle, p=0.001, n=7 vehicle, 7 drug). In AMPK KO mice, there was also a reduction in whole-hemisphere infarct volume. The drug reduced levels of the TAK1 target JNK as well as the JNK target c-Jun. Conclusions: Post-stroke inhibition of TAK1 was neuroprotective and this effect was sustained for 7 days. It was also associated with improvement in the NORT, suggesting preservation of memory function. Together, these findings suggest that 5Z-7-oxozeaenol may have clinical utility for patients suffering ischemic stroke. 5Z-7-oxozeaenol reduced levels of the TAK1 target JNK and its target, c-Jun, suggesting that the benefits of inhibition are due to decreased levels of the transcription factor complex AP-1, of which c-Jun is a major component. The sustained protective effect in AMPK KO mice suggests that AMPK is not important to the mechanism of neuroprotection following TAK1 inhibition.