A glial circadian gene expression atlas reveals cell type and disease-specific reprogramming in response to amyloid pathology or aging

While circadian rhythm disruption may promote neurodegenerative disease, how aging and neurodegenerative pathology impact circadian gene expression patterns in different brain cell types is unknown. Here, we used translating ribosome affinity purification methods to define the circadian translatomes of astrocytes, microglia, and bulk cerebral cortex, in healthy mouse brain and in the settings of amyloid-beta plaque pathology or aging. Our data reveal that glial circadian translatomes are highly cell type-specific and exhibit profound, context-dependent reprogramming of rhythmic transcripts in response to amyloid pathology or aging. Transcripts involved in glial activation, immunometabolism, and proteostasis, as well as nearly half of all Alzheimer Disease (AD)-associated risk genes, displayed circadian oscillations, many of which were altered by pathology. Amyloid-related differential gene expression was also dependent on time of day. Thus, circadian rhythms in gene expression are cell- and context dependent and provide important insights into glial gene regulation in health, AD, and aging. Mice were sacrificed at specified time points and perfused with cycloheximide. Cortex was immediately isolated and tissue frozen. After tissue processing, astrocyte- or microglia-specific RNA were isolated by incubating tissue with anti-GFP or anti-HA coated beads, respectively. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.RNA-seq reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3-8 software.尽管昼夜节律紊乱可能促进神经退行性疾病的发生，目前尚不清楚衰老与神经退行性病理如何影响不同脑细胞类型的昼夜节律基因表达模式。本研究采用翻译核糖体亲和纯化（translating ribosome affinity purification）技术，在健康小鼠大脑、淀粉样β斑块病理模型及衰老模型中，分别解析星形胶质细胞、小胶质细胞与全大脑皮层的昼夜节律翻译组（circadian translatomes）。研究数据显示，胶质细胞的昼夜节律翻译组具有高度的细胞类型特异性，且在应对淀粉样病理或衰老时，节律性转录本会发生显著的、依赖于环境的重编程。涉及胶质细胞激活、免疫代谢与蛋白质稳态的转录本，以及近半数阿尔茨海默病（Alzheimer Disease, AD）相关风险基因，均呈现昼夜振荡现象，其中多数振荡模式会因病理状态发生改变。淀粉样相关的差异基因表达同样呈现昼夜时间依赖性。综上，基因表达的昼夜节律具有细胞类型与环境依赖性，该发现为健康状态、阿尔茨海默病及衰老过程中的胶质基因调控提供了重要见解。实验小鼠于指定时间点处死，并以环己酰亚胺（cycloheximide）进行灌流。随后立即分离大脑皮层组织并速冻保存。组织处理完成后，分别通过与抗GFP或抗HA包被磁珠孵育，分离得到星形胶质细胞或小胶质细胞特异性RNA。总RNA完整性通过安捷伦生物分析仪（Agilent Bioanalyzer）或4200 TapeStation进行检测。文库构建采用10ng总RNA，要求生物分析仪RIN值大于8.0。双链cDNA的制备严格遵循厂商操作手册，使用Takara-Clontech旗下用于Illumina测序的SMARTer Ultra Low RNA试剂盒完成。利用Covaris E220超声破碎仪对cDNA进行片段化，参数设置为：峰值入射功率18、占空比20%、每循环脉冲数50，超声时长120秒。随后对cDNA进行末端补平，在3'端添加单碱基A，再连接Illumina测序接头。连接产物使用带有独特双索引标签的引物进行12-15个循环的扩增。最终通过Illumina NovaSeq-6000平台开展双端测序，测序读长为150bp。测序得到的RNA-seq读段通过运行版本3.9.3-8软件的Illumina DRAGEN Bio-IT本地服务器，比对并定量至Ensembl第101版初级组装基因组。