Next generation RNA sequencing of Aβ-treated primary cultured mouse astrocytes to study the effect of ODC1 inhibition on AD-like astrocytes

Purpose: The existence and role of the urea cycle has not been well studied in the astrocytes of Alzheimer's Disease (AD). In the past, ODC1-inhibitor DFMO (Diflouromethylornithine) has been shown to have a neuroprotective effect on AD-like pathology, but the biochemical mechanism of this effect remains largely unknown. In this study, we aim to compare the transcriptome of naive and Aβ-treated astrocytes and further study the effect of DFMO treatment in Aβ-treatment condition. Methods: Primary astrocyte culture (from P0-P1 mouse pups) mRNA profiles were generated by high-throughput NGS in 3 treatment conditions in duplicate using HiSeq 2500. Sequence reads were analysed using Partek Genomics Suite, aligned using STAR and then quantified to the mouse genome assembly (mm10 Ensemble transcripts release 99), following which they were normalised to median ratio values and differentially analysed using the DeSeq2 algorithm. Finally, pathway analysis was carried out to study the differential expression of biomolecular pathways, with reference to the KEGG database. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (build mm10) and identified 18783 genes in the dataset. Differential analysis revealed upregulation of autophagy-related genes as well as switching-on of the genes involved in the cyclic metabolism of urea on Aβ treatment of astrocytes. ODC1 inhibition by DFMO induced upregulation of genes involved in the non-amyloidogenic processing of APP and downregulation of amyloidogenic genes. Pathway analysis revealed upregulation of urea cycle and urea metabolic process pathways, as well as selective autophagy and phagosome-lysosome fusion on Aβ treatment. Conclusions: Our study represents the first detailed analysis of mouse astrocyte culture transcriptome on Aβ-treatment, and the effect of ODC1 inhibition on the same, with biological replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. From our results, we can conclude that the urea cycle is turned on in Alzheimer's Disease (AD)-like condition and that ODC1-inhibition can be a suitable therapeutic strategy to reduce the Aβ load in AD. RNA-seq of astrocytes untreated or treated with Aβ or Aβ with DFMO

研究目的：阿尔茨海默病（Alzheimer's Disease, AD）星形胶质细胞中尿素循环的存在及其作用尚未得到充分研究。既往研究表明，ODC1抑制剂DFMO（二氟甲基鸟氨酸，Diflouromethylornithine）对AD样病理具有神经保护作用，但其介导该作用的生化机制仍未明确。本研究旨在比较未处理与Aβ（β淀粉样蛋白，Amyloid β）处理的星形胶质细胞的转录组，并进一步探究DFMO在Aβ处理条件下的作用效果。
实验方法：本研究以P0-P1期小鼠幼崽原代培养的星形胶质细胞为实验材料，采用HiSeq 2500平台开展高通量NGS（下一代测序，Next-Generation Sequencing），设置3种处理条件，每组设置生物学重复。序列读取数据经Partek Genomics Suite进行分析：首先通过STAR软件完成序列比对，以小鼠基因组组装版本mm10（Ensembl转录本版本99）作为参考进行转录本定量；随后采用中位数比率法完成数据标准化，并通过DeSeq2算法进行差异表达分析。最后参考KEGG（京都基因与基因组百科全书，Kyoto Encyclopedia of Genes and Genomes）数据库进行通路分析，以探究生物分子通路的差异表达情况。
实验结果：通过优化的数据分析流程，我们将每个样本约5000万条序列读取比对至小鼠基因组（mm10版本），并在本数据集中鉴定出18783个基因。差异表达分析显示，星形胶质细胞经Aβ处理后，自噬相关基因表达上调，同时参与尿素循环代谢的基因被激活。DFMO对ODC1的抑制作用可诱导APP（淀粉样前体蛋白，Amyloid Precursor Protein）非淀粉样加工通路相关基因的上调，并抑制淀粉样生成相关基因的表达。通路分析结果表明，星形胶质细胞经Aβ处理后，尿素循环、尿素代谢过程通路以及选择性自噬、吞噬体-溶酶体融合通路均出现显著上调。
研究结论：本研究首次通过RNA-seq（RNA测序）技术结合生物学重复，对Aβ处理的小鼠原代星形胶质细胞转录组以及ODC1抑制对其的影响进行了详细分析。本研究报道的优化数据分析流程可为表达谱的比较研究提供参考框架。基于本研究结果，我们可以得出结论：在AD样病理条件下，尿素循环会被激活；而ODC1抑制有望成为降低AD患者Aβ负荷的潜在治疗策略。本数据集涵盖未处理、Aβ处理以及Aβ联合DFMO处理的星形胶质细胞的RNA测序数据。