Elsevier

Experimental Neurology

Volume 237, Issue 1, September 2012, Pages 238-245
Experimental Neurology

Protection from cerebral ischemia by inhibition of TGFβ-activated kinase

https://doi.org/10.1016/j.expneurol.2012.05.019Get rights and content

Abstract

Objective

Transforming growth factor-β-activated kinase (TAK1) is a member of the mitogen-activated protein kinase family that plays important roles in apoptosis and inflammatory signaling, both of which are critical components of stroke pathology. TAK1 has recently been identified as a major upstream kinase that phosphorylates and activates adenosine monophosphate-activated protein kinase (AMPK), a major mediator of neuronal injury after experimental cerebral ischemia. We studied the functional role of TAK1 and its mechanistic link with AMPK after stroke.

Methods

Male mice were subjected to transient middle cerebral artery occlusion (MCAO). The TAK1 inhibitor 5Z-7-oxozeaenol was injected either intracerebroventricularly or intraperitoneally at various doses and infarct size and functional outcome after long term survival was assessed. Mice with deletion of the AMPK α2 isoform were utilized to assess the contribution of downstream AMPK signaling to stroke outcomes. Levels of pTAK1, pAMPK, and other TAK1 targets including the pro-apoptotic molecule c-Jun-N-terminal kinase (JNK)/c-Jun and the pro-inflammatory protein cyclooxygenase-2 were also examined.

Results

TAK1 is critical in stroke pathology. Delayed treatment with a TAK1 inhibitor reduced infarct size and improved behavioral outcome even when given several hours after stroke onset. This protective effect may be independent of AMPK activation but was associated with a reduction in JNK and c-Jun signaling.

Conclusions

Enhanced TAK1 signaling, via activation of JNK, contributes to cell death in ischemic stroke. TAK1 inhibition is a novel therapeutic approach for stroke as it is neuroprotective with systemic administration, has a delayed therapeutic window, and demonstrates sustained neuroprotective effects.

Highlights

► Stroke injury propagation involves complex molecular processes. ► Inhibiting transforming growth factor-β-activated kinase 1 (TAK1) reduced injury. ► TAK1 is a critical molecule in deciding neuronal fate in stroke.

Introduction

Transforming-growth-factor-beta-activated kinase-1 (TAK1), also known as mitogen-activated protein kinase kinase kinase-7 (MAPKKK7), is a mitogen-activated protein kinase that can be activated by TGF-β, tumor necrosis factor-alpha (TNF-α) and other cytokines including interleukin-1 (IL-1) (Ninomiya-Tsuji et al., 1999, Sakurai et al., 2003, Yamaguchi et al., 1995). Activation of TAK1 requires association with TAK1-binding protein 1 (TAB1) and phosphorylation at multiple sites including threonine-184 and threonine-187 (Sakurai et al., 2000, Shibuya et al., 1996). The function of TAK1 has been studied in a number of cell lines with conflicting results. Several studies have demonstrated an anti-apoptotic response to TAK1 activation (Omori et al., 2008, Thiefes et al., 2005), however others have suggested that TAK1 is pro-apoptotic via activation of downstream JNK signaling (Sorrentino et al., 2008). TAK1 is also emerging as a possible contributing factor to pro-inflammatory signaling through induction of the transcription factor AP-1 with subsequent expression of inflammatory genes including COX-2 (Kumar et al., 2009, Ninomiya-Tsuji et al., 2003, Zhang et al., 2010). Inflammation and apoptosis both contribute to ischemic neuronal cell death (Sims and Muyderman, 2010, Tuttolomondo et al., 2009), and due to the delayed activation of these pathways after injury, may represent an attractive target for the development of neuroprotective agents.

TAK1 has been recently identified as one of the three upstream kinases that phosphorylates and activates adenosine monophosphate-activated protein kinase (AMPK) (Momcilovic et al., 2006), a key metabolic enzyme and sensor of cellular metabolic state (Li and McCullough, 2010). AMPK's activity is primarily regulated by the cellular AMP:ATP ratio (Li and McCullough, 2010) but AMPK can also be activated by other cellular stressors including ischemia and hypoxia. AMPK is activated by phosphorylation at threonine 172 via an upstream kinase triggering a cascade of events that reduces the activity of anabolic enzymes and increases catabolic pathways, thereby maintaining ATP levels. Although activation of AMPK appears to be a protective adaptive response to stress in peripheral tissues, in the brain, ischemia-induced AMPK activation exacerbates injury by enhancing metabolic failure and inducing lactic acidosis (Li and McCullough, 2010, Li et al., 2007, Li et al., 2010a, Li et al., 2010b). Deletion of the catalytic isoform of AMPK responsible for phosphorylation and activation of AMPK is neuroprotective, as is pharmacological inhibition of AMPK. We hypothesized that inhibition of TAK1, due to its function as an upstream activator of AMPK (Li and McCullough, 2010), would lead to a reduction in stroke-induced AMPK phosphorylation and subsequent neuroprotection. TAK1 was recently shown to be activated in rodents after neonatal hypoxic-ischemic injury (Nijboer et al., 2009), in a rodent model of middle cerebral artery occlusion (MCAO), and in vitro after oxygen glucose deprivation (Neubert et al., 2011) but the signaling pathways induced by TAK and actions on AMPK signaling are unknown. In the present study, the function of TAK1 was studied in a rodent model of ischemic stroke using the highly specific small molecule TAK1 inhibitor 5Z-7-oxozeaenol (Ninomiya-Tsuji et al., 2003, Sicard et al., 2009) and putative downstream targets of TAK1 were investigated.

Section snippets

Stroke model

C57BL/6 mice were purchased from Charles River Laboratories (Willimantic, Connecticut, USA). Experiments were performed according to NIH guidelines for the care and use of animals in research and under protocols approved by the University of Connecticut Health Center Animal Care and Use Committee. Prior to experiments, animals were randomized into vehicle and drug groups. All treatments were performed by a blinded investigator. Focal transient cerebral ischemia was induced in male mice (20 to 25

Intracerbroventricular application of 5Z-7-oxozeaenol reduced stroke injury

ICV administration of 0.32 μg of 5Z-7-oxozeaenol resulted in no significant differences in infarct volume (Cortex: 53.66 ± 5.72% for vehicle versus 39.01 ± 7.15% for drug, p = 0.151; Striatum, 48.89 ± 7.98% for vehicle versus 33.41 ± 7.01 for drug, p = 0.142; in whole-hemisphere, 44.58 ± 5.67% for vehicle versus 32.58 ± 5.93%, p = 0.149, n = 7 vehicle, 9 drug). However, injection of a higher dose (1.6 μg) significantly reduced cortical (Fig. 1a) (59.04 ± 4.98% for vehicle versus 35.00 ± 7.10% for drug, p = 0.014) and

Discussion

TAK1 has recently been implicated as a contributor to cell death in several in vitro and in vivo models (Sicard et al., 2009, Tang et al., 2008). This study reveals several novel and important findings related to the role of TAK1 signaling in cerebral ischemia. First, levels of pTAK were significantly lower in the ischemic brain compared to sham. Second, direct intracerebroventricular delivery of 5Z-7-oxozeaenol, a TAK1 inhibitor, reduced the severity of experimental stroke as measured by both

Conclusion

TAK1 inhibition is neuroprotective after induced cerebral ischemia and the mechanism may be independent of AMPK signaling. We demonstrated that 5Z-7-oxozeaenol has the potential to reduce the severity of ischemic stroke through its effects on TAK1 and downstream signaling via JNK. This work has uncovered a new, yet unexplored signaling pathway involved in ischemic cell death.

Acknowledgment

This work was supported by National Institutes of Health grants R01 NS050505 and NS055215 (to L.D.M.) and American Heart Association grant 09SDG2261435 (to J.L.). Disclosure/Conflict of Interest: B.J. White: nothing to declare; S. Tarabishy: nothing to declare; V.R. Venna: nothing to declare; B. Manwani: nothing to declare; S. Benashki: nothing to declare; L.D. McCullough: nothing to declare; J. Li: nothing to declare.

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