Abstract 22: Apoptosis Signal-Regulating Kinase 1 (ASK1) is a Novel Master Molecular Switch Controlling Microglia/Macrophage Reactions That Impact Short- and Long-Term Stroke Outcomes

Abstract

Introduction: Recent studies suggest that the functional status of microglia/macrophages (Mi/MΦ) profoundly impacts stroke outcome. However, the molecular mechanisms regulating Mi/MΦ reactions after brain ischemia/reperfusion (I/R) are poorly understood. Hypothesis: Activation of ASK1, a key MAPKKK upstream of multiple inflammation-regulating pathways, critically contributes to I/R brain injury via priming Mi/MΦ towards a neurotoxic phenotype. Methods: We generated mice with cell-specific, tamoxifen-induced knockout of ASK1 in neurons (nKO) or Mi/MΦ (mKO). Brain injury, neuroinflammation, and various behavioral deficits were assessed after 1 h MCAO and reperfusion. Results: MCAO induced transient ASK1-MKK4-JNK3 activation (6-24 h) in neurons and persistent ASK1-MKK3-p38/STAT1 activation (3-5 d) in Mi/MΦ in WT, but not in ASK1 nKO or mKO mice, respectively. ASK1 nKO reduced infarct sizes by 28.5% at 2 d after MCAO (p<0.01 vs WT, n=8-10/group), but failed to prevent the release of HMGB1 from necrotic cells and Mi/MΦ activation, or exert any effect on long term (28 d) stroke outcomes. In contrast to neuronal ASK1 KO, ASK1 mKO markedly improved both short and long term sensorimotor/cognitive functions after I/R (p<0.01 vs WT, n=14-16/group). ASK1 mKO attenuated the activation of p38α and STAT1 (immunostaining), inhibited the expression of 12 proinflammatory markers in Mi/MΦ (FACS-sorting followed by qPCR; p<0.05, n=4/group), and mitigated brain inflammation (ELISA array; 19 out of 80 markers, p<0.05, n=5/group) 3 d after I/R. In consistent with the role of p38α and STAT1 as downstream mediators of ASK1 signaling in Mi/MΦ, either p38α or STAT1 mKO alleviated neuroinflammation (3 d) and improved long term (28 d) stroke outcomes (n=12/group), partially mimicking the effects of ASK1 mKO. Conclusions: ASK1-dependent activation of p38α and STAT1 promotes inflammation, I/R brain injury, and maladaptive behavior by priming Mi/MΦ towards a neurotoxic phenotype.