miR-99a/AC1在唐氏综合征认知功能障碍中的作用及机制

Down syndrome or Down's syndrome （DS）, also known as trisomy 21, is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. DS is the most common chromosome abnormality in humans, occurring in about 1 per 1000 babies born each year. However, the molecular mechanisms of cognitive deficits of DS are still unknown. We found that miR-99a might bind to the 3` untranslated region of adenylyl cyclase 1 （AC1） using bioinformatics softwares. We also found that overexpression of miR-99a decreased the expression of AC1 at the mRNA and protein levels as detected by quantitative RT-PCR and Western Blot in cultured primary neurons.It has been reported that AC1 and its down stream signals play an important role in learning and memory. However, the role of miR-99a in regulating AC1, in synaptic and intrinsic plasticity,and in learning and memory is still unknown and need to be explored. Therefore, we hypothesize that miR-99a plays a vital role in cognitive deficits in DS by regulating AC1. In the present study, we will first explore whether miR-99a regulates AC1 based on our previous research. Then the role of miR-99a in synaptic and intrinsic plasticity, and in learning and memory will be detected. At last, whether miR-99a exerts its function in DS cognitive deficits through regulating AC1 will be determined. Taken together, this projects aims to elucidate the precise role of miR-99a/AC1 in memory and in DS cognitive defictis. These findings will provide novel evidence for the development of new therapeutic strategies to treat DS.

唐氏综合征（Down syndrome，DS）是导致人类先天性认知功能障碍最常见的原因，但其机制尚未完全阐明。本课题组利用生物信息学软件分析发现，人类21号染色体转录的miR-99a可结合在腺苷酸环化酶1（AC1） mRNA的3'端非翻译区。已知AC1及其下游信号通路在认知功能调节中发挥关键作用，且我们的前期研究发现在神经元中过表达miR-99a可显著抑制AC1 mRNA和蛋白表达，然而miR-99a是否通过调控AC1信号通路在DS认知功能障碍中发挥关键作用尚未可知。在前期研究基础上，本项目拟首先阐明miR-99a是否可以调控AC1的表达；然后利用DS小鼠模型明确miR-99a/AC1对神经元内在可塑性和突触可塑性的调控作用；最后探讨miR-99a/AC1在DS认知功能障碍中的作用机制。总之，本项目期望系统揭示miR-99a/AC1在DS发病机理中的作用及其机制，以探索干预DS的新靶标。

唐氏综合征（Down syndrome，DS），亦称为21三体，是导致先天性智力低下最常见的原因，但其认知功能障碍机制尚待阐明。人类21号染色体转录的miR-99a在DS患者海马和皮质中表达显著升高，然而miR-99a在DS 认知功能障碍中的作用及机制尚未可知。本项目主要研究结果如下：（1）在小鼠海马组织过表达mir-99a，可降低ADCY1和PSD-95的表达，但不影响synapsin I的表达；（2）在转轮实验中，海马中过表达mir-99a小鼠与对照组相比，其在转轮上的停留时间无明显差异，提示mir-99a并不影响运动能力；（3）在场景恐惧实验中，训练实验结束 24h后，对照组小鼠的凝滞时间高于miR-99a过表达组小鼠，提示海马中过表达miR-99a导致小鼠学习记忆能力受损；（4）在水迷宫实验中，miR-99a过表达小鼠的reversal learning的能力大大下降，这提示认知的灵活程度—认知刚性（cognitive rigidity）大大下降；（5）对照组小鼠和miR-99a过表达组小鼠进臂总次数，开放臂进入次数，闭臂进入次数，以及开放臂和闭臂停留时间比例等均无差异，这提示miR-99a并不影响小鼠的焦虑行为；（6）为进一步探讨海马中miR-99a对小鼠学习记忆能力影响的机制，我们取对照组和miR-99a过表达小鼠的海马组织进行了RNA-seq测序分析。结果表明，海马中过表达miR-99a导致多种基因mRNA表达变化，且亦导致多种microRNAs表达变化。Q-RT-PCR显示，所验证的基因表达水平与RNA-seq结果相符，提示RNA-seq结果可靠。综上所述，这些研究结果表明海马中过表达的miR-99a，通过调控和影响多种基因的表达，在学习记忆中发挥了重要作用。这些研究结果将深化目前人们对DS发生发展的认识，并为干预DS提供了新的理论和实验依据。