miR-122对APP非淀粉样代谢途径的调节以及miR-122基因多态与阿尔茨海默病易感性关联分析

Alzheimer's disease (AD) is caused by accumulation of Aβ, which is generated in the amyloidogenic pathway by sequential proteolysis of β-amyloid precursor protein (APP) through β- and γ-secretase. However, the vast majority of APP is constitutively processed within the non-amyloidogenic pathway by α-secretase, which prevents formation of Aβ and gives rise to the neurotrophic and neuroprotective cleavage product sAPPα. ADAM10 (a disintegrin and metalloprotease 10) has been identified as the major α-secretase in vivo, and therefore regulation of either expression and/or activity of ADAM10 has been emerged as a valuable strategy for prevention and/or for treatment of Alzheimer's disease.. MicroRNAs (miRNAs) became a tool for interfering with protein expression via impeding translation of single genes or destabilizing their mRNAs. In our previous , ADAM10 was experimentally validated as a target of miR-122 and were repressed by the microRNA. Moreover, miR-122 level is significantly elevated in cerebral spinal fluid of AD. Here we propose a project to further investigate the significance of miR-122 in APP non-amyloidogenic pathway. First, we manipulated levels of miR-122 using mimic to increase, or antagomir to reduce levels. By quantitative analysis of human APP processing products, we will determine whether miR-122 could direct APP processing toward the α-secretase, which results in a reduction in the production of Aβ. Secondly, to evaluate the biological role of miR-122 in AD brain in vivo, we will alter brain miR-122 levels by injecting miR-122 antagomir in a transgenic AD mouse model and then test whether inhibition of miR-122 in the brain could delay plaque formation and alleviate cognitive defects. Finally, we will perform a case-control study to investigate whether a single-nucleotide polymorphism in miR-122 gene affects the amount of mature miR-122, and contributes to the genetic predisposition to AD. The findings of this research will not only provide new insights into regulation of non-amyloidogenic APP processing, but may also provide targets for development of diagnostic biomarkers and therapeutics of AD.

淀粉样前体蛋白(amyloid precursor protein, APP) 在β、γ分泌酶的催化下产生的Aβ是AD发病的关键因素。而在正常生理情况下，大部分APP在α分泌酶的催化下经非淀粉样蛋白代谢途径产生具有神经营养作用的sAPPα（同时抑制Aβ的产生）。ADAM10是神经细胞内最重要的α分泌酶，其表达调控备受注目。前期实验我们发现miR-122可以抑制ADAM10的表达，而且AD患者有miR-122表达升高的现象。以此为切入点，我们准备进一步采用体外细胞转染的方法探讨调控miR-122对APP非淀粉样代谢途径的影响；并在此基础上观察脑内注射miR-122抑制剂对AD模型小鼠脑内老年斑沉积、神经元凋亡、以及学习记忆功能的影响；此外，我们还将观察miR-122基因多态是否会影响体内miR-122的生物合成,继而影响AD易感性。本项目的实施可以为AD的治疗策略提供新的思路和突破点。

本项目的研究目标是通过抑制miRNA的表达，从而增加ADAM10的表达，使APP的代谢向非淀粉样蛋白生成途径倾斜，减少Aβ的产生。该目标已经初步完成。.1．筛选出可能对ADAM10有调控作用的miRNA有四个，分别为：miR-103、miR-107、miR-122、miR-1306；.2．四个miRNA在正常小鼠及AD小鼠海马中均有表达。.3．SH-SY5Y细胞转染miRNA inhibitor 24h后与对照组相比，实验组miR-103、miR-107、miR-1306、miR-122的表达量均下降；转染miR-103 inhibitor、miR-1306 inhibitor 24h后与对照组相比，ADAM10 mRNA的表达量上升；转染miR-107 inhibitor、miR-122 inhibitor 24h后与对照组相比，ADAM10 mRNA的表达量无显著性差异；.4．在双荧光素酶实验结果中，对于miR-103，实验组与对照组相比，荧光素酶活性差异无统计学意义（P>0.05）；对于miR-1306，实验组与对照组相比，荧光素酶活性差异有统计学意义（P<0.05）；.5．SH-SY5Y-APP695细胞转染miR-1306 inhibitor 24h后与对照组相比，ADAM10蛋白、sAPPα蛋白的表达水平上升，sAPPβ蛋白、细胞内Aβ42蛋白的表达水平下降。.我们的结论认为，抑制miR-1306的表达能提高ADAM10的表达水平，促使APP往非淀粉样蛋白生成途径代谢，从而提高sAPPα的表达，减少sAPPβ及Aβ的生成。