Circadian disruptions and time-of-day-dependent memory deficiency with long noncoding RNA expression profiling changes in Presenilin1/2 conditional knockout mice

Alzheimer's disease (AD) patients exhibit sleep and circadian disturbances before cognitive decline, and these disruptions worsen with disease severity. However, the molecular mechanisms of sleep and circadian disruptions in AD patients are poorly understood. In this study, we investigated the sleep pattern and circadian rhythms in Presenilin-1/2 conditional knockout mice (DKO mice). Assessment of EEG and EMG recording showed that DKO mice exhibit sleep disorders from 2 months of age that worsen at the age of 6 months. The actogram of wheel running activity of DKO mice manifested movement splitting both in Dark and Light phases during the 24-hour light/dark cycle compared to WT mice. Notably, DKO mice also displayed increased NREM sleep time during the dark phase compared to WT mice at the age of two months; however, REM sleep showed no change in either WT or DKO littermates in the dark phase. When sleep and wakefulness were examined at the age of 6 months, the DKO mice showed increased wakefulness time and decreased total time spent in NREM and REM sleep. Analysis of the circadian modulation of memory revealed that the recall for contextual fear memory trained at ZT2 in WT mice was greater at ZT2 than at ZT14; however, the DKO mice had the same freezing responses with respect to both ZT2 and ZT14. Long noncoding RNAs (lncRNAs) are typically defined as transcripts longer than 200 nucleotides, and they have rhythmic expression in mammals. However, no study has investigated rhythmic lncRNA expression in Alzheimer's disease so we applied RNA-seq technology to profile the expression of lncRNAs in the hippocampus of DKO mice in the light (resting) phase and dark (active) phase. Furthermore, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the biological process, cell component and molecular function of these differentially expressed lncRNAs (DE-lncRNAs). These results provide a useful resource for studying lncRNAs in circadian disruptions in Alzheimer's disease. Mice were deeply anesthetized with sodium pentobarbital (30 mg/kg) and sacrificed at 9 a.m. or 9 p.m. The hippocampal tissues were rapidly removed on ice and then transported into liquid nitrogen followed by storage at -80 prior to RNA extraction. RNA extraction and RNA-seq were performed by Shanghai OE Biotech Co., Ltd.阿尔茨海默病（Alzheimer's disease, AD）患者在认知衰退前即出现睡眠与昼夜节律紊乱，且此类紊乱随疾病严重程度加重而加剧。然而目前学界对阿尔茨海默病患者睡眠与昼夜节律紊乱的分子机制尚缺乏深入认知。本研究针对早老素1/2条件性敲除小鼠（Presenilin-1/2 conditional knockout mice, DKO小鼠）的睡眠模式与昼夜节律展开探究。通过脑电（Electroencephalogram, EEG）与肌电（Electromyogram, EMG）记录评估显示，DKO小鼠自2月龄起即出现睡眠障碍，并于6月龄时症状进一步加重。相较于野生型小鼠（Wild Type, WT），DKO小鼠的转轮活动节律图显示，在24小时明暗循环条件下，其黑暗相与光照相均出现活动分裂现象。值得注意的是，2月龄时，DKO小鼠在黑暗相的非快速眼动（Non-Rapid Eye Movement, NREM）睡眠时长较WT小鼠有所增加；但无论是WT还是DKO同窝小鼠，其快速眼动（Rapid Eye Movement, REM）睡眠时长在黑暗相中均无显著变化。当对6月龄小鼠的睡眠与觉醒状态进行检测时发现，DKO小鼠的觉醒时长增加，而NREM与REM睡眠总时长均有所减少。对记忆的昼夜节律调控分析表明，在授时因子时间（Zeitgeber Time, ZT）2时段训练的情境恐惧记忆，WT小鼠在ZT2时段的回忆效果优于ZT14时段；而DKO小鼠在ZT2与ZT14时段的僵立反应无明显差异。长链非编码RNA（Long noncoding RNAs, lncRNAs）通常被定义为长度超过200个核苷酸的转录本，在哺乳动物中具有节律性表达特征。但目前尚无针对阿尔茨海默病中节律性lncRNA表达的相关研究，因此本研究借助RNA测序（RNA-sequencing, RNA-seq）技术，对DKO小鼠海马组织在光照（休息）相与黑暗（活动）相中的lncRNA表达谱进行了分析。此外，本研究还对这些差异表达长链非编码RNA（differentially expressed lncRNAs, DE-lncRNAs）的生物学过程、细胞组分与分子功能进行了基因本体（Gene Ontology, GO）富集分析以及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析。本研究结果可为阿尔茨海默病昼夜节律紊乱相关lncRNA的研究提供宝贵的参考资源。小鼠经戊巴比妥钠（sodium pentobarbital, 30 mg/kg）深度麻醉后，于上午9时或晚上9时实施安乐死。海马组织迅速于冰上剥离，随后置于液氮中速冻，并于-80℃冰箱保存直至RNA提取。RNA提取与RNA-seq测序服务由上海欧易生物科技有限公司（Shanghai OE Biotech Co., Ltd.）完成。