Abstract 147: Aberrant Activation of ASK1 Mediates Proinflammatory and Neurotoxic Microglial Responses After Cerebral Ischemia/Reperfusion

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Introduction: Neuronal death and proinflammatory microglial reactions both contribute to ischemic/reperfusion (I/R) brain injury. However, the molecular interplay of these two events is poorly understood.

Hypothesis: Activation of ASK1, a key MAPKKK upstream of JNK and p38 MAPK signaling cascades, mediates neuronal death and microglial neurotoxicity after I/R.

Methods: We created ASK1 kinase dead knock-in mice (ASK1-ki, K716R) with universal loss of ASK1 kinase activity. Brain injury and neurobehavioral deficits were assessed after 1h MCAO and reperfusion. Cell-permeable small peptides mimicking the docking sites on ASK1 for MKK3/6 (TAT-M3i) or MKK4/7 (TAT-M4i) were delivered into primary neurons and microglia, or into mice intranasally to inhibit ASK1-mediated p38 or JNK signaling, respectively.

Results: MCAO induced robust ASK1/JNK/p38 activation in WT but not in ASK1-ki mice. ASK1-ki mice had reduced infarct (by 41.8%, p≤0.01, n=8) and improved sensorimotor and cognitive functions up to 28d vs. WT littermates. In primary cultures, OGD-induced neuronal death was attenuated in ASK1-ki neurons or in WT neurons treated with TAT-M4i. Conditioned medium from post-OGD neurons stimulated microglia toward the cytotoxic M1 phenotype, promoting the release of neurotoxic cytokines and NO. These responses were suppressed in ASK1-ki microglia, or WT microglia treated with TAT-M3i or p38α shRNA. Consistently, ASK1-ki mice had markedly reduced microglial M1 (but not M2) markers and proinflammatory cytokines after MCAO vs. WT. Finally, intranasal delivery of TAT-M4i and TAT-M3i (2, 24, and 48h after MCAO), but not either peptide alone, conferred long-term protection and improved sensorimotor/cognitive performance of WT mice up to 35d.

Conclusions: ASK1 is an essential mediator of prodeath neuron-microglia interactions after I/R. Inhibiting ASK1 signaling is a promising and clinically feasible strategy to achieve sustained protection after stroke.

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