下调miR-138表达对阿尔茨海默病的保护作用及机制研究

Amyloid-β (Aβ) deposition is an early pathological hallmark of Alzheimer's disease (AD). MiR-138, a brain-enriched microRNA, negatively regulates dendritic spine morphogenesis, and is significantly increased in brains of AD patients. Our previous work found that miR-138 was elevated in the hippocampus of 12-month-old AD transgenic mice (Tg2576 mice) as well as cell models. Forced overexpression of miR-138 promoted tau phosphorylation by targeting retinoic acid receptor α (RARA). In addition, miR-138 also remarkably increased Aβ production, but the underling mechanism was not clear. This application is based on our previous work, and proposes to investigate the effect of miR-138 on Aβ production and the underlying molecular mechanisms in both 4-month-old Tg2576 mice and N2a/APP cell line, which stably expresses human amyloid precursor protein (APP). Furthermore, ELISA, immunohistochemistry, Golgi staining, and Morris Water Maze will be used to study the protective effects of strategies targeting miR-138 or its target gene on Aβ production and deposition, learning and memory defect in 12-month Tg2576 mice by injecting miR-138 antagomir or its target gene overexpressed via Adeno-associated virus(AAV) into the hippocampus of 12-month-old Tg2576 mice. The results of this project may reveal new strategies for prevention and treatment of AD.

细胞外的淀粉样蛋白β（Aβ）沉积是阿尔茨海默病(AD)的早期病理改变。MiR-138是一种脑内富集，能负向调节突触形态发生的microRNA，并在AD病人脑内表达显著升高。我们发现miR-138在12月龄AD转基因小鼠以及细胞模型中表达升高，并通过抑制维甲酸受体α表达促进tau蛋白磷酸化。此外，miR-138也能显著增加Aβ水平，但其具体机制尚不明确。本项目基于前期研究，拟通过在4月龄Tg2576转基因小鼠以及过表达淀粉样蛋白前体蛋白（APP）的N2a/APP细胞中研究miR-138对Aβ生成的影响及其分子机制。最后在12月龄Tg2576小鼠海马中注射miR-138抑制剂或其靶基因的过表达腺病毒，运用ELISA，免疫组化，高尔基染色，水迷宫等技术研究下调miR-138或上调其靶基因表达对12月龄Tg2576小鼠AD样Aβ沉积和学习记忆障碍的保护作用，该项目研究结果有望为AD防治提供新靶点。

突触功能障碍和APP异常剪切是阿尔茨海默病(AD)的早期病理改变。近年来，包括microRNA（miRNA）和环状RNA（circRNAs）在内的非编码RNA被广泛发现参与了AD的发病过程。miR-138是一种脑内富集，能负向调节突触形态发生的miRNA，并在AD病人脑内表达显著升高。我们发现与对照小鼠相比，APP/PS1小鼠海马中miR-138的表达呈现年龄依赖性升高。在2月龄APP/PS1小鼠海马中，通过注射miR-138激动剂上调miR-138表达，发现上调miR-138能够抑制ADAM10表达，促进Aβ产生，诱导突触和学习/记忆缺陷。在4月龄APP/PS1小鼠海马中，通过注射miR-138抑制剂下调miR-138表达，发现抑制miR-138表达能够改善4月龄APP/PS1小鼠上述AD样病变。体外实验发现，过表达Sirt1（miR-138的靶标）改善了miR-138诱导的ADAM10抑制和Aβ升高。通过软件预测，我们发现circRNA HDAC9（circHDAC9）含有miR-138结合位点。在Aβ寡聚体处理的N2a细胞和4月龄APP/PS1小鼠海马中，circHDAC9的表达与miR-138呈负相关。FISH发现circHDAC9与miR-138在N2a细胞质中共定位。体外实验发现，circHDAC9作为miR-138海绵，降低miR-138表达，逆转miR-138诱导的Sirt1抑制和Aβ的过量产生。此外，circHDAC9在轻度认知障碍（MCI）和AD患者中表达均显著低于对照人群。因此，这些结果表明circHDAC9/miR-138/Sirt1信号通路可能AD中突触功能障碍和APP异常剪切的重要调节因素，为AD患者提供了新的治疗靶点。