靶向抑制Dyrk1A对APP外显子7可变剪接和Thr668磷酸化的影响及其分子机制研究

The deposition of amyloid β(Aβ) in brains is a common feature in Alzheimer's disease (AD). Aβ peptide is a cleaved product of amyloid precursor protein (APP), whereas the function of APP holoprotein is not yet established.In adult human brain, APP gene undergoes an alternative splicing regulation, which leads to the generation of six isoforms. Among all these isoforms, APP770, APP751 (APP-KPI) and APP695 are reported to be correlated tightly to the Aβ production. In addition, hyper-phosphorylation of APP Thr668 is related to APP confirmation change leading to abnormal Aβ production. In our preliminary study, we found the kinase activty of Dyrk1A was elevated in AD brains. Dyrk1A is a SR protein kinase and was also reported to phosphorylate at APP Thr668. In this study, we will construct APP mini-gene to mimic APP exon 7 alternative splicing in vivo and detect the changes of splicing pattern when overexpression of Dyrk1A in cell lines. Next, we will establish the stable expression of APP695 in neuronal cell lines such as SH-SY5Y cells or N2a cells, and then detect the APP Thr668 phosphorylation after treating the cells with Dyrk1A inhibitor, EGCG or Harmine. Also, we will compare the changes of Aβ levels by Elisa analysis. Furthermore, we propose to down-regulate Dyrk1A level by feeding Ts65Dn mice with EGCG diet to evaluate its role in modifying Aβ production in mice brains. Finally, we will also detect the behavior change between EGCG feeding group and none feeding group. Our aim for this study is to investigate the regulation of Dyrk1A on alternative splicing of APP gene and phosphorylation on Thr668, and to explore the effect of Dyrk1A inhibitors on Aβ production in Ts65Dn brain.This study will help us to better understand the molecular mechanisms of AD early pathogenesis and provide some new evidence for searching effective intervention strategy.

β淀粉样蛋白（Aβ）聚集形成的老年斑是阿尔茨海默病（AD）的重要病理特征之一，由淀粉样前体蛋白（APP）多次水解形成。人APP基因表达受可变剪接调控，其产物APP-KPI+/KPI-之间的比例与Aβ产生呈正相关。另外APPThr668的磷酸化增加改变APP构象也导致Aβ增多。我们发现AD患者脑内激酶Dyrk1A活性增加，它可磷酸化剪接因子和APP668。本研究在神经细胞及动物水平探讨Dyrk1A促进APP-KPI生成和Thr668磷酸化的分子机制及其对Aβ生成的影响。应用Dyrk1A抑制剂EGCG和Harmine，观察其对神经细胞和TS65Dn模式鼠脑内Aβ变化以及行为学改变。本研究探讨靶向抑制Dyrk1A对AD病理的影响，探索AD早期病变机制，从而为临床治疗AD的新药创制提供分子理论依据。

β淀粉样蛋白（Aβ）聚集形成的老年斑是阿尔茨海默病（AD）的重要病理特征之一，由淀粉样前体蛋白（APP）多次水解形成。人APP基因表达受可变剪接调控，其产物APPKPI+/KPI-之间的比例与Aβ产生呈正相关。我们发现AD患者脑内激酶Dyrk1A活性增加，可通过磷酸化剪接因子从而改变其剪接活性。. 我们从分子、细胞以及整体动物水平研究了靶向抑制Dyrk1A对APP-KPI生成的影响以及对小鼠行为学的改变。主要内容如下：1.研究APP微型基因的构建和在细胞系内表达情况，以及Dyrk1A对APP表达的影响；2.研究剪接因子ASF和Dyrk1A在神经细胞的相互作用；3.研究APP/PS1转基因鼠脑内抑制Dyrk1A活性对APP-KPI、APPThr668和对Aβ生成的影响；4. Dyrk1A抑制剂EGCG和Harmine对神经细胞和Ts65Dn小鼠脑内APP可变剪接、APPThr668磷酸化以及对Aβ产生的影响；5. EGCG对Ts65Dn小鼠行为学的影响。. 研究结果显示：1.APP微型基因可以在细胞系中很好表达出APP各剪接异构体；2.Dyrk1A在细胞系中促进APP-KPI表达，EGCG或Harmine抑制Dyrk1A活性可以降低APP-KPI水平，而对APPThr668磷酸化无明显影响；3.神经细胞中Dyrk1A与剪接因子ASF在细胞核内有共定位，两者有很好的相互作用；4.喂食Dyrk1A抑制剂EGCG降低了APP/PS1小鼠脑内的APP-KPI水平，同时发现Aβ生成量减少；5.EGCG对模式小鼠Ts65Dn的APP-KPI的生成有一定抑制作用，对APPThr668无明显影响；同时对受损的行为学有一定程度的改善。. 通过此研究，我们发现EGCG作为绿茶中茶多酚的主要成分之一，可以一定程度抑制Dyrk1A活性，下调APP-KPI生成，从而减轻Aβ在脑内的生成，对AD早期预防和治疗提供分子理论依据。