腺相关病毒介导低表达RCAN1对AD模型小鼠治疗作用的研究

The pathogenesis of Alzheimer's disease (AD) is complex. Until now, the clinical treatments for AD show little effect while the development of new drugs for AD shows no progress. Nevertheless, gene therapy is becoming more and more promising, in which the adeno-associated virus (AAV) is the most widely used vector due to its high expression, security and stability in the nervous system. Studies found that regulator of calcineurin 1 (RCAN1) might be a new target of gene therapy for AD because of its significant correlation to the pathological features of AD. We found that downregulation of RCAN1 in 2EB2 cells could reduce the production of Aβ, make SH-SY5Y cells more tolerant to oxidative stress and relieve apoptosis, but whether it has the same effect in animal models is not clear. Therefore, this study is going to prove whether low expression of RCAN1 has a protection on neurons from AD mice in vitro and an improvement on the pathological damages and cognitive impairments of AD mice in vivo in early and later course of AD by RCAN1 RNAi AAV, and also to explore the mechanism throughout. This study is aimed to further clarify the regulation of RCAN1 in the pathogenesis of AD, to provide a theoretical evidence and practical basis for the treatment of AD by downregulation of RCAN1, and to open a new window for ameliorating the symptoms of AD in its pathogenesis.

阿尔茨海默氏病（AD）的发病机制复杂，缺乏有效的临床治疗对策，近年来针对AD的新药开发也屡遭失败，而基因治疗正展现出良好的应用前景，其中腺相关病毒（AAV）因其在神经系统安全稳定高表达的特性正成为最广泛使用的载体。钙调磷酸酶调节因子1（RCAN1）和AD的病理特征有显著相关性，有潜力成为AD基因治疗的新靶点。我们发现，在细胞中低表达RCAN1可以减少Aβ的产生，使其耐受应激，凋亡减轻，但是否在实验动物中同样有治疗效果尚不明确。因此，本项目拟以RCAN1 RNA干扰的AAV为媒介，观察低表达RCAN1对体外培育的AD模型小鼠神经元是否有保护作用，进而在体内实验中探索发病早期及中晚期低表达RCAN1对其病理改变和认知功能的改善作用，并研究其机制。本研究旨在进一步阐明RCAN1对AD发病的调控作用，为低表达RCAN1治疗AD的临床应用提供理论依据和实践基础，为从发病机制上治疗AD提供一条新思路。

阿尔茨海默氏病（AD）的基因治疗正展现出良好的应用前景，其中腺相关病毒（AAV）因其在神经系统安全稳定高表达的特性正成为最广泛使用的载体。钙调磷酸酶调节因子1（RCAN1）和AD的病理特征有显著相关性，有潜力成为AD基因治疗的新靶点。在本研究中，我们构建了低表达RCAN1的AAV9-RCAN1 shRNA-GFP，观察到其可以减少AD小鼠来源的神经元分泌产生Aβ42，且可以保护细胞耐受氧化应激环境。在体内实验中，于胚胎期AD小鼠侧脑室注射的AAV在脑内表达良好，其促使RCAN1表达降低后可以显著改善3月龄及7月龄时的行为学评价指标，分子病理检测也证实脑内老年斑沉积减少，神经元凋亡减轻。然而，本研究中胚胎期侧脑室注射的AAV在10月龄小鼠脑内表达偏低，具有四环素调控系统的AAV9-TR3G-RCAN1 shRNA-GFP未能通过小鼠含多西环素饮水在其脑内开放表达，因此即时降低RCAN1的表达是否同样对AD小鼠产生治疗作用，仍有待进一步深入探讨。本研究首次在实验动物体内证实了低表达RCAN1对AD症状及病理的改善作用，进一步阐明了RCAN1在AD发病机制中的关键调控作用，为低表达RCAN1治疗AD的临床应用提供理论依据和实践基础，为从发病机制上治疗AD提供一条新思路。