ZINC PROMOTES FOCAL CEREBRAL ISCHEMIA-REPERFUSION INJURY THROUGH ACTIVATING CDK5 BY TYR15 PHOSPHORYLATION

Abstract

Aberrant CDK5 activation plays a key role in mediating ischemic stroke injury, while the underlying mechanism for CDK5 activation remains incompletely clarified. Hippocampal CA1 neurons are highly sensitive to ischemic injury, it was discovered that a large number of ionic zinc accumulated in that area after ischemia-reperfusion. Whether zinc is involved in CDK5 activation in ischemia-reperfusion injury and the regulation pathway has not been elucidated. MCAO rat model was established, with or without intraperitoneal injection of zinc chelator CQ or intraventricular injection of tyrosine kinase SFK inhibitor PP2 before embolization. After 6 hours of reperfusion, rat were sacrificed and brains were separated. TTC staining was performed to detect the area of brain infarction. The protein levels of total CDK5, phosphorylated CDK5 at Y15, p25 and p35 were detected by immunoblotting. The activity of CDK5 was detected by using Histone H1 as substrate. At the cellular level, N2a cells were incubated with ZnSO4 for 3 hours, with or without pretreatment with CQ or PP2. The protein test wan same as before. GFP-tagged wild-type or Y15F-mutated CDK5 were overexpressed in N2a cells and then cells were incubated with ZnSO4. After the treatment, exogenously expressed CDK5 was pull-down by using anti-GFP antibody and CDK5 activities were measured. In the rat MCAO model, CDK5 was activated by ischemia-reperfusion injury, and the level of Y15 phosphorylated CDK5 was increased remarkably. CQ or PP2 pretreatment significantly decreased the brain infarction area, reversed neuron loss, CDK5 activation and CDK5-Y15 phosphorylation, but not p35 cleavage to p25. At the cellular level, ZnSO4 treatment resulted in CDK5 activation, and CDK5-Y15 phosphorylation, which could also be reversed by CQ or PP2. The unphosphorylated form of CDK5 – Y15F mutant could not be phosphorylated and activated by zinc treatment. Activation of CDK5 by zinc was not dependent on p35 cleaved into p25 pathway. During stroke, cellular released zinc activates the tyrosine protein kinase, which further leads to Y15 phosphorylation and activation of CDK5, thus mediates neuronal damage. The CDK5 activation does not depend on p35 cleavage. These results revealed a novel mechanism of abnormal activation of CDK5 during ischemia-reperfusion injury.