冈田酸诱导大鼠海马神经元Tau蛋白过度磷酸化

OKADAIC ACID INDUCES HYPERPHOSPHORYLATION OF Tau PROTEIN IN THE HIPPOCAMPAL NEURONS OF THE RAT

目的研究蛋白磷酸酯酶抑制剂冈田酸(OA)对海马神经元微管相关蛋白(Tau)磷酸化的影响,建立Tau过度磷酸化的大鼠模型。方法实验随机分为正常组、二甲基亚砜(DMSO)对照组、OA模型组。模型组又分为OA12h、24h、48h和2周组。OA模型组大鼠海马CA1区背侧定向注射1.5μl溶于10%DMSO的OA,DMSO对照组注射1.5μl10%DMSO溶液。通过Bielschowski染色、免疫组织化学染色、蛋白免疫印迹分别观察海马神经元形态的改变和磷酸化Tau的表达水平;检测蛋白磷酸酶2A(PP2A)的活性,了解其动态变化与Tau磷酸化的关系。结果OA模型各组与正常组和DMSO对照组比较,Bielschowski染色示海马神经元胞体和突起着色较深,欠均匀,部分神经元轴丘处浓染成斑块状,但各模型组均未见到老年斑和神经元纤维缠结样改变;免疫组织化学染色示模型组海马神经元Thr231和Ser199202磷酸化Tau蛋白表达增加,与DMSO对照组相比具有显著意义(P<0.05);蛋白免疫印迹提示OA可引起Tau蛋白Thr231、Ser396和Ser199/202位点发生磷酸化,且不同位点磷酸化的稳定性不同,注射OA48h后PP2A的活性明显降低,其变化与Tau蛋白Thr231和Ser396位点的磷酸化改变相一致。结论海马CA1区背侧单次注射OA可诱导建立神经元Tau蛋白过度磷酸化的大鼠模型。

Objective To explore the effect of Okadaic acid(OA),a protein phosphatase inhibitor,on Tau hyperphosphorylation and neuronal microtubule system in the rat hippocampus. Methods OA in a 1.5?μl volume was injected into unilateral dorsal part of rat hippocampal CA1 region in OA Model groups at 12h,24h,48h and 2 weeks before sample collection,respectively.DMSO treatment alone was defined as vehicle control group.The time-course-depend pathological changes and Tau protein hyperphosphorylation were observed by Bielschowsky staining,immunocytochemistry(AT8,Tau-pThr 231)and Western-blot with Tau antibodies(Tau-5,AT8,Tau-pThr 231 and Tau-pSer 396).The activity of PP2A was also assayed. Results 1.After injection with OA,the results of Bielschowsky staining showed that the axons of hippocampal neurons became anachromasis,and the thickened and other pathological changes of neurofibrils in hippocampus were observed,however,the neurofibrils were arranged in an order in the normal and vehicle groups.2.The immunochemistry staining of brain slice showed that the numbers of Tau-AT8 and Tau-pThr 231 immuno-positive stained cells in OA groups were markedly increased(P<0.05)comparing to those in normal and vehicle control groups.For Western-blot detect,hyperphosphorylation of Tau at the sites of Ser 199/202,Thr 231 and Ser 396 was increased comparing to vehicle control groups.Phosphorylation of Ser 199/202(AT8)reached peak at 12h after injection,then declined at 24h,48h and 2 weeks later.Phosphorylation of Thr 231 and Ser 396 increased gradually,reached peak at 48h,and became weak at 2 weeks.The total Tau(Tau5)increased when Tau was much more phosphorylated at the sites of Thr 231 and Ser 396.3. Activity of PP2A decreased significantly after OA injection(P<0.01),it came to the bottom at 48h,and recovered at 2 weeks later.Conclusion OA could induce Tau hyperphosphorylation in the hippocampal neurons of the rat brain.