Abstract WP320: Neuronal ROCK2 Plays Important Role in Ischemic Stroke: GSK-3beta and Tau Signaling Pathways

Introduction: The role of neuronal cell specific ROCK2 in ischemic stroke in vivo has been poorly investigated. We investigated the pharmacological effect of selective ROCK2 inhibition by selective ROCK2 inhibitor, KD025 and the genetic effect using neuronal specific ROCK2 knockout (ROCK2Eno2-/-) mice. Methods: Isoflurane-anesthetized littermate control (LC) and ROCK2Eno2-/- mice (20-22 g) underwent ischemia (2h middle cerebral artery occlusion (MCAO)/reperfusion (22h after 2h MCAO) injury model. An 8.0 nylon monofilament coated with a silicone was introduced into the left internal carotid artery up to the anterior cerebral artery. To assess the mechanism of ROCK2 deficiency in neuronal cells, we seeded hippocampal neuronal (HT22) cells in 6-well plates and transfected ROCK2 siRNA. Phosphorylated glycogen synthase kinase 3 beta (pGSK-3β) and Tau proteins which were involved in neuroprotection were measured by western blotting from brain lysates and neuronal cells. Infarction volume was measured by TTC staining and evaluation of behavioral was assessed by neurological deficit score (NDS) at 22h after MCAO. Results: In WT mice, ROCK2 inhibition by KD025 (100 mg/kg, p.o.) decreased infarct volumes and improved the NDS in ischemic stroke. ROCK activity (p-MYPT1 to MYPT1 ratio) of the brain lysates by KD025 was decreased dose-dependently in ischemic mice brain. In genetic modified mice, infarction volume (64.6 ± 10.4 vs. LC (102.3 ± 12.4), n=5) of ROCK2Eno2-/- mice was significantly decreased by 37% and the NDSs (1.8 ± 0.4 vs. LC (2.8 ± 0.5), n=5) were improved by 36% compared to the LC groups. Both pGSK-3β and Tau in HT22 cells with ROCK2 siRNA transfection and ROCK2Eno2-/- mice brain was significantly decreased compared to each respective controls. Conclusion: These findings indicate that neuronal ROCK2 plays important role in ischemic stroke via GSK-3β and Tau signaling pathways and inhibition of neuronal specific ROCK2 may have therapeutic benefits in ischemic stroke.