Abstract DP370: The Inhibition of ROCK2 Mediates the Neuroprotection via BRSK1 Signaling Pathway in Ischemic Stroke

Background and Purpose: Brain specific kinases 1 and 2 (BRSK1/2, also named SAD-B/A kinases) are serine-threonine kinases and are specifically expressed in mammalian forebrain and are implicated in various processes including cell polarity and cell cycle regulation. ROCKs (ROCK1 and ROCK2) regulate cell polarity, cell cycle, and cell migration predominantly through enhancing actomyosin contraction and focal adhesions. Increased ROCK activity is found after ischemic stroke, and inhibition of ROCK2 in the brain is an important therapeutic target for ischemic stroke. To determine the interplay between BRSKs and ROCKs in ischemic stroke, we investigated the functions of ROCK2, AMPKalpha, BRSK1, and BRSK2 after experimental ischemic stroke. We hypothesize that the inhibition of ROCK2 mediates BRSK1 signaling pathway and plays an important role in mitigating ischemic stroke damage. Methods: Using ROCK1 and ROCK2 siRNA in mouse hippocampal neuronal cell line (HT-22 cells), we investigated the roles ROCK1/ROCK2 and BRSK1/BRSK2 in brain ischemia. To determine the role of BRSK1/2 in transient focal cerebral ischemia, isoflurane-anesthetized WT (C57Bl/6), ROCK1 +/- and ROCK2 +/- mice (20-22g) underwent 2 h focal cerebral ischemia (Middle Cerebral Artery Occlusion; MCAO) induced with an 8-0 nylon monofilament coated with a silicone resin/hardener mixture introduced into the left internal carotid artery up to the anterior cerebral artery. We measured Sirt1, AMPKalpha, BRSK1, BRSK2, and GAPDH protein levels by western blotting analysis in both in vitro and in vivo experiments. Results: After the ischemic stroke, expression levels of Sirt1, AMPKalpha, BRSK1, and BRSK2 decreased. The Inhibition of ROCK by fasudil (10 mg/kg, i.p.) and KD025 (100 mg/kg, p.o.) significantly reduced infarction volume and increased AMPKalpha, BRSK1, and BRSK2 expression levels in the brain after ischemic stroke compared to those of WT mice. ROCK2 deficient mice showed increased BRSK1 expression, decreased infarction volume and improved neurological deficit score. Conclusions: These findings indicate that ROCK2 deficiency decreased infarction volume in vivo via BRSK1 signaling pathways. These results suggest that inhibition of ROCK2 and activation of BRSK1 may have therapeutic benefits in ischemic stroke.