糖尿病大鼠脑细胞周期依赖性蛋白激酶5和蛋白激酶A参与调节Tau蛋白磷酸化

细胞周期依赖性蛋白激酶5(cyclin-dependent kinase5,Cdk-5)及蛋白激酶A(protein kinaseA,PKA)是调节Tau蛋白磷酸化的重要激酶,其对糖尿病(diabetes mellitus,DM)大鼠脑内Tau蛋白磷酸化的作用如何,目前尚不明确.为探讨胰岛素缺乏的DM大鼠海马Cdk-5及PKA对Tau蛋白磷酸化的作用,用链脲佐菌素(streptozotocin,STZ)建立DM大鼠模型,Fura-2负载及荧光测定细胞内游离Ca2 浓度,免疫沉淀法测定Cdk-5活性,放射性配体结合实验检测PKA的活性,蛋白质印迹检测Tau蛋白磷酸化的水平.结果提示:在DM大鼠海马神经元,Ca2 浓度升高,Cdk-5及PKA活性升高,Tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点的磷酸化增强.Cdk-5的特异性抑制剂roscovitine可降低DM大鼠Cdk-5活性,但不能降低PKA活性,使Tau蛋白在Ser198/Ser199/Ser202位点磷酸化水平降低,但不降低Ser396/Ser404位点的磷酸化,roscovitine处理正常大鼠后,上述酶的活性及Tau蛋白的磷酸化无明显变化.首次从整体水平上证实DM大鼠海马Cdk-5及PKA活性升高,协同促进Tau蛋白在Ser198/Ser199/Ser202位点和Ser396/Ser404位点的磷酸化,神经元内游离Ca2 浓度升高可能起重要作用.

Regulation of Tau Phosphorylation by Cdk-5 and PKA in Diabetic Rat Brain

Cyclin-dependent kinase 5(Cdk-5) and protein kinase A(PKA) are the important kinases in the regulation of Tau phosphorylation. However, it is unclear weather they participate in the regulation of tau phosphorylation in diabetic rat brain. The roles of Cdk-5 and PKA in the regulation of abnormal tau hyperphosphorylation in the hippocampus of diabetic rat model have been investigated. The diabetic rat model was induced by intra peritoneal injection with 55 mg/kg streptozotocin. Intracellular free calcium concentration was detected by loading Fura-2 and fluorescent technique in Control, Con Ros, DM, and DM Ros groups, and the activities of Cdk-5 and PKA were measured by immunoprecipitation and by liquid scintillation for incorporated radioactivity respectively, furthermore, Western blotting was used to examine the level of tau phosphorylation by using phosphorylation-dependent and site-specific tau antibodies. The results show that, compared to normal control, the concentration of intracellular calcium in the diabetic rat hippocampus was elevated to 165.92 nmol/L(P < 0.05). Cdk-5 activity was increased by 29% of control, and an increase of PKA activity by 30% of control was also found, both Cdk-5 activity and PKA activity were increased highly compared to normal control(P < 0.01, P < 0.01), furthermore, the immunoreactivity of Tau-1(detection of Ser 198/Ser 199/ Ser202, non-phosphorylated site) was decreased(P < 0.01), the immunoreactivity of PHF-1 sites (detection of ser396/ser404, phosphorylated site) was increased(P < 0.05) in these diabetic rats. After treated the DM rats with roscovitine, a specific Cdk-5 inhibitor, Cdk-5 activity was inhibited to 105% vs control, it was not obviously increased compared to control (P >0.05), but PKA activity remained 127% vs control, it was high compared to control(P < 0.01). Moreover, the hyperphosphorylation of tau at Ser198/Ser199/Ser202 epitopes was reversed (P < 0.01), but the level of phosphorylation of tau at Ser396/Ser404 epitopes was remained highly after roscovitine was administered to DM rats. However, after treated the control rats with roscovitine, Cdk-5 activity was about 104% vs control, and PKA activity was 105% vs control, neither Cdk-5 activity nor PKA activity was higher compared to control(P > 0.05), and the level of tau phosphorylation at Ser198/Ser199/Ser202 and Ser396/Ser404 sites was not increased compared to control too(P > 0.05). These results firstly suggest that the increase of Cdk-5 activity and PKA activity may cause the hyperphosphorylation of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 epitopes in diabetic rat hippocampus, and the intracellular calcium may play a role in this process, however, it is needed to be elucidated in great details.