RNA氧化在散发性阿尔茨海默病老年斑形成中的分子机制研究

Alzheimer’s Disease is the main type of senile dementia, and it is also a significant public health problem in our aging society. Although the amyloid hypothesis for AD has been widely accepted, it can hardly explain the pathogenesis of sporadic AD(SAD）, which accounts for over 90% AD and has rare gene mutations. For the first time, our previous research has showed that RNA oxidative modification in eukaryotic cells would cause RNA and protein variation. Based on this mechanism, we have not only proved that the increase of RNA oxidation could lead to increased kinds and amounts of the Aβ fragment in CHO cells, but also demonstrated increased RNA oxidation could result in the formation of senile plaques in the mouse brain, eventually leading to cognitive impairment in SAM mice. According to these results, we hypothesize that the increase in RNA-DNA Difference of APP gene may be the initial factor inducing increased Aβ secretion and accumulated senile plaques, which are important pathological features of SAD. Therefore, we intend to find out the relationship between increased RNA oxidation and increased DNA-RNA Difference of APP gene，as well as the formation of increased kinds and amounts of the Aβ fragment in CHO-APP-iMTH1 cells. we will further confirm the hypothesis that increased DNA-RNA Difference of APP gene caused by RNA oxidation may induce increased kinds and amounts of the Aβ fragment , the formation of senile plaques and cognitive impairment in SAM mouse model. This study will provide scientific and reasonable evidence to reveal the pathogenesis of SAD roundly.

阿尔茨海默病（AD）是老年痴呆的主要类型，AD 的90%以上为基因突变率极低的散发性阿尔茨海默病（SAD），目前的Aβ级联学说难以解释SAD中Aβ的产生机制。我们的前期研究显示真核细胞中RNA氧化可引起RNA变异进而导致蛋白质变异；据此我们用细胞模型证明了RNA氧化增加可引起Aβ分泌增加，用动物模型证明了RNA氧化增加可引起小鼠脑组织老年斑形成和认知障碍，表明RNA氧化增加与AD发病相关。本研究我们提出RNA氧化增加导致APP基因的RNA-DNA差异（RNA-DNA difference，RDD）增加是Aβ增加和老年斑形成起始因素的假设。我们拟用CHO-APP-iMTH1细胞模型研究RNA氧化增加所致Aβ分泌量增加与APP基因RDD的相关性；用SAM鼠动物模型研究RNA氧化增加所致SAM鼠老年斑形成和认知障碍与APP基因RDD的相关性，为揭示SAD发病机理提供科学的实验依据。

本研究我们建立了nLC-orbitrap MS对Aβ谱的检测体系、并建立了CHO-APP-iMTH1 细胞模型研究体系，当CHO-APP-iMTH1 细胞模型中参入RNA氧化底物8-oxo-GTP时、用LC-MS/MS检测获得了细胞中的RNA 氧化增加的实验证据、用nLC-orbitrap MS检测获得了Aβ分泌量增加和种类增加的直接实验证据。.本研究我们建立了第二代测序技术的RNA变异检测体系，该体系检测DNA-RNA变异检测敏感度可达到10-6copy/test，并建立MTH1-敲减细胞模型研究体系和变异蛋白质研究体系。当细胞模型中参入RNA氧化底物8-oxo-GTP时、可检测到RNA氧化导致变异蛋白质表达量增高，用第二代测序技术检测获得了RNA 变异率增加的实验证据。.我们用小鼠模型研究了RNA 氧化增加所致的小鼠老年斑形成和认知障碍的相关性。通过该研究获得了RNA 氧化增加可引起APP 基因RNA 变异率增加、RNA变异、Aβ分泌量增加的实验结果。.本研究最重要的研究成果是证明了人体细胞中的RNA氧化增加可以引起RNA链变异率的增加，并以此为模板导致Aβ分泌量的增加和种类增加，提示RNA氧化增加可能是阿尔茨海默病（AD）新的发病机制。该研究成果以本研究承担着为通讯作者在美国科学院院报发表：Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells. Proc Natl Acad Sci U S A. 2018 Apr 17;115(16):4218-4222.