RNA-sequencing of mouse knockout models for Cnp, Plp1, and Ugt8 in the frontal cortex and cerebellum

Oligodendrocytes (OLs) and myelin are critical for normal brain function and they have been implicated in neurodegeneration. Human neuroimaging studies have demonstrated that alterations in axons and myelin occur early in Alzheimer's Disease (AD) course. However, the molecular mechanism underlying the role of OLs in AD remains largely unknown. In this study, we systematically interrogated OL-enriched gene networks constructed from large-scale genomic, transcriptomic, and proteomic data in human AD postmortem brain samples. These robust OL networks were highly enriched for genes associated with AD risk variants, including BIN1. We corroborated the structure of the AD OL coexpression and gene-gene interaction networks through ablation of genes identified as key drivers of the networks, including UGT8, CNP, MYRF, PLP1, NPC1, and NDGR1. Perturbations of these key drivers not only caused dysregulation in their associated network neighborhoods, but also mimicked pathways of gene expression dysregulation seen in human AD postmortem brain samples. In particular, the OL subnetwork controlled by the AD risk gene PSEN1 was strongly dysregulated in AD, suggesting a potential role of PSEN1 in disrupting the myelination pathway towards the onset of AD. In summary, this study built and systematically validated the first comprehensive molecular blueprint of OL dysregulation in AD, and identified key OL- and myelination-related genes and networks as potential candidate targets for the future development of AD therapies. The mouse knockout models have been previously described for each of Ugt8 (Coetzee et al., 1996), Cnp (Lappe-Siefke et al., 2003), and Plp1 (Klugmann et al., 1997). For each of the two conditions studied (control and homozygous knockout mice), five mice of either sex were sacrificed at postnatal day 20 and brains were flashed-frozen until analysis. The frontal cortex (FC) and cerebellum (CBM) were dissected out and individually processed. RNA was isolated using Trizol reagent and processed using Ribo-Zero rRNA removal. RNA-sequencing was performed using the Illumina HiSeq2000 with 100 nucleotide paired-end reads. RNA-sequencing reads were mapped to the mouse genome (mm10, UCSC assembly) using Bowtie (version 2.2.3.0), TopHat (version 2.0.11), and SamTools (version 0.1.19.0) using a read length of 100. Reads were converted to counts at the gene level using HTSeq on the BAM files from TopHat2 using the UCSC known genes data set.少突胶质细胞（Oligodendrocytes, OLs）与髓鞘对正常脑功能至关重要，且已被证实与神经退行性病变密切相关。人类神经影像学研究表明，阿尔茨海默病（Alzheimer's Disease, AD）病程早期即可出现轴突与髓鞘的异常改变。然而，OLs在AD发病过程中发挥作用的分子机制仍尚未明确。 本研究针对人类AD死后脑样本中的大规模基因组、转录组及蛋白质组数据构建的OL富集基因网络开展系统性解析。这些稳定可靠的OL基因网络高度富集与AD风险变异相关的基因，其中包括BIN1。研究人员通过敲除被鉴定为网络关键驱动因子的基因（包括UGT8、CNP、MYRF、PLP1、NPC1及NDGR1），验证了AD相关OL共表达网络与基因-基因互作网络的结构特征。上述关键驱动因子的扰动不仅会导致其所在网络邻域内的基因表达失调，还可模拟人类AD死后脑样本中观察到的基因表达失调通路。尤为关键的是，受AD风险基因PSEN1调控的OL子网络在AD患者脑中呈现显著失调，提示PSEN1可能通过破坏髓鞘形成通路参与AD的发病进程。综上，本研究构建并系统性验证了首个AD中OL失调的综合性分子蓝图，并鉴定出关键的OL及髓鞘相关基因与网络，作为未来AD治疗药物开发的潜在候选靶点。 此前已有文献对Ugt8（Coetzee等，1996）、Cnp（Lappe-Siefke等，2003）及Plp1（Klugmann等，1997）的小鼠敲除模型进行了描述。本研究设置了对照组与纯合子敲除小鼠两组实验条件，每组各选取5只不限性别的小鼠于出生后第20天处死，取脑后快速冷冻以待后续分析。研究人员解剖分离出额叶皮层（frontal cortex, FC）与小脑（cerebellum, CBM）并分别进行样本处理。使用Trizol试剂提取总RNA，并通过Ribo-Zero核糖体RNA去除试剂盒去除核糖体RNA。采用Illumina HiSeq2000平台进行RNA测序，生成100碱基长度的双端测序读段（reads）。将测序reads比对至小鼠参考基因组（mm10，UCSC组装版本），使用Bowtie（版本2.2.3.0）、TopHat（版本2.0.11）及SamTools（版本0.1.19.0）进行比对，比对参数设置读长为100。使用HTSeq工具基于TopHat2生成的BAM文件，结合UCSC已知基因数据集，将测序reads计数至基因水平。