Temporal profiling of male cortical astrocyte transcription predicts molecular shifts from early development to aging

Astrocytes are the most abundant glial cell type in the central nervous system (CNS). Most astrocytes are born during the early postnatal period in the rodent brain and mature alongside neurons, demonstrating remarkable morphological structural complexity, attaining maturity in the second postnatal month. Across the remainder of the lifespan astrocytes participate in CNS homeostasis, support neuronal partners, and contribute to nearly all aspects of CNS functioning. In the present study, we analyzed astrocyte gene expression in rodent wild type cortex across the lifespan (7 days to 18 months). A pairwise timepoint comparison of differential gene expression during early development and CNS maturation (7 days 60 days) revealed four unique astrocyte gene clusters, each with hundreds of genes and demonstrating a unique temporal profile. These clusters distinctively related to cell division, cell morphology, Astrocytes are the most abundant glial cell type in the central nervous system (CNS). Astrocytes are born during the early postnatal period in the rodent brain and mature alongside neurons, demonstrate remarkable morphological structural complexity which is attained in the second postnatal month. Throughout this period of development and across the remainder of the lifespan, astrocytes participate in CNS homeostasis, support neuronal partners, and contribute to nearly all aspects of CNS function. In the present study, we analyzed astrocyte gene expression in the cortex of wild-type rodents throughout their lifespan (postnatal 7 days to 18 months). A pairwise timepoint comparison of differential gene expression during early development and CNS maturation (7 days60 days) revealed four unique astrocyte gene clusters, each with hundreds of genes, and which demonstrate unique temporal profiles. These clusters are distinctively related to cell division, cell morphology, cellular communication and vascular structure and regulation. A similar analysis across adulthood and in the aging brain (3 months to 18 months) identified similar patterns of grouped gene expression related to cell metabolism and cell structure. Additionally, our analysis identified that during the aging process astrocytes demonstrate a bias toward shorter transcripts, with loss of longer genes related to synapse development and a significant increase in shorter transcripts related to immune regulation and the response to DNA damage. Our study highlights the critical role that astrocytes play in maintaining CNS function throughout life, and reveals molecular shifts that occur during development and aging. Three E14.5 testes (control or COUP-TFII KO) were pooled per sample. RNA was extracted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, KIT0204). Briefly samples were dissociated in 100 ml of in extraction buffer with a microtube homogenizer and incubated at 42C for 30 minutes. Posteriorly samples were snap frozen for at least one hour. RNA was precipitated with 100 ml of 70% ethanol and loaded into a pre-conditioned column. Samples were centrifuged and the column was washed with Wash Buffer 1, followed by a step of DNA removal using the RNase-Free DNase Set (Qiagen, #79254). Samples were washed twice with Wash Buffer 2 and the columns were centrifuged to eliminate residual buffer. Samples were eluted in 12 ml of elution buffer.星形胶质细胞（Astrocytes）是中枢神经系统（central nervous system, CNS）中含量最丰富的胶质细胞类型。大部分星形胶质细胞于啮齿类动物脑的出生后早期产生，并与神经元同步成熟，展现出显著的形态结构复杂性，于出生后第二个月达到成熟状态。在其后的整个生命周期中，星形胶质细胞参与中枢神经系统稳态维持、支持神经元伙伴，并几乎参与中枢神经系统功能的所有方面。本研究中，我们分析了整个生命周期（出生后7天至18个月）内啮齿类野生型大脑皮层中星形胶质细胞的基因表达情况。对早期发育与中枢神经系统成熟阶段（7天至60天）的差异基因表达进行两两时间点比较，发现了4个独特的星形胶质细胞基因簇，每个基因簇包含数百个基因，并展现出独特的时间表达谱。 星形胶质细胞是中枢神经系统中含量最丰富的胶质细胞类型，于啮齿类动物脑的出生后早期产生并与神经元同步成熟，展现出显著的形态结构复杂性，于出生后第二个月达到成熟状态。在发育阶段及其后的整个生命周期中，星形胶质细胞参与中枢神经系统稳态维持、支持神经元伙伴，并几乎参与中枢神经系统功能的所有方面。本研究中，我们分析了整个生命周期（出生后7天至18个月）内野生型啮齿类动物大脑皮层中星形胶质细胞的基因表达情况。对早期发育与中枢神经系统成熟阶段（7天至60天）的差异基因表达进行两两时间点比较，发现了4个独特的星形胶质细胞基因簇，每个基因簇包含数百个基因，并展现出独特的时间表达谱。这些基因簇分别与细胞分裂、细胞形态、细胞通讯以及血管结构与调控显著相关。对成年及衰老大脑（3个月至18个月）开展的类似分析，鉴定出了与细胞代谢和细胞结构相关的相似分组基因表达模式。此外，我们的分析发现，在衰老过程中星形胶质细胞倾向于表达更短的转录本：与突触发育相关的较长基因出现表达丢失，而与免疫调控及DNA损伤应答相关的较短转录本则显著增加。本研究强调了星形胶质细胞在终生维持中枢神经系统功能中的关键作用，并揭示了发育与衰老过程中发生的分子转变。 每个样本混合3个E14.5天的睾丸（对照组或COUP-TFII基因敲除（KO）组）。采用Arcturus PicoPure RNA提取试剂盒（Applied Biosystems，货号KIT0204）提取RNA。简要步骤为：将样本置于100 mL提取缓冲液中，使用微量管匀浆器进行解离，随后于42℃孵育30分钟。之后将样本快速冷冻至少1小时。用100 mL 70%乙醇沉淀RNA，并将其加载至预处理层析柱中。对样本进行离心，用洗涤缓冲液1（Wash Buffer 1）冲洗层析柱，随后使用无RNase DNase试剂盒（Qiagen，货号#79254）进行DNA去除步骤。用洗涤缓冲液2（Wash Buffer 2）冲洗样本两次，再离心层析柱以去除残留缓冲液。最后用12 mL洗脱缓冲液洗脱RNA。