Uninterrupted Mutant Huntingtin CAG-repeat but Not Polyglutamine Length Predicts Striatal Nuclear Pathogenesis and Transcriptionopathy in HD Mice

Huntington's disease (HD) is caused by CAG repeat expansion at the N-terminal region of huntingtin (HTT) gene and characterize by neuronal cell loss and neuroinflammation transcriptional profile change in striatum, cortex and other brain regions. Here we reported generation and characterization of BAC-CAG, a novel full length human HD mouse model host over 120 uninterrupted CAG repeats. we performed RNA-seq analysis of the striatum and cortex of BAC-CAG mice at 2m, 6m and 12m of age (N=6 per genotype, sex balanced). We observed an age-dependent transcriptionopathy in the striatum of BAC-CAG mice, with only 7 and 36 significant DE genes in 2m and 6m striatum respectively (FDR<0.1), but 820 DE genes (538 downregulated and 282 upregulated genes) at 12m of age (FDR<0.1). The transcriptomic deficits in this model are highly striatum-selective, as we only detected 4, 3 and 14 DE genes (FDR<0.1) at 2m, 6m and 12m respectively in BAC-CAG cortices compared to WT controls. Remarkably, we found a highly significant positive transcriptome-wide correlation (r=0.62) of DE Z statistics between BAC-CAG vs WT at 12m and Q140 vs WT at 6m (p<1e-200). This result demonstrates a high concordance of both upregulated and downregulated genes in the BAC-CAG striatum compared to that of allelic series KI mice; but RNA-seq studies of BACHD mice at 12m and found only 50 genes significantly differentially expressed (DE) genes (FDR<0.1) in the striatum at 12m of age compared to WT littermates, 31 downregulated and 19 upregulated genes. In the cortex at 12m of age, BACHD only showed 6 significantly downregulated genes and 7 upregulated genes comapred to WT controls (FDR<0.1). Three males and three females BAC-CAG mice and their WT littermates from 2, 6 and 12m age groups, respectively, were perfused with PBS and striatum, cortex and cerebellum were dissected out immediately. The samples were fresh frozen and kept at -80°C. Total RNA was extracted using Qiagen (Valencia, CA) RNeasy kit with QIA shredder columns. cDNA libraries were generated using Illumina TruSeq RNA Library Prep Kit v2 and sequenced using an Illumina Illumina HiSeq 4000 sequencer with strand-specific, paired-end, 69-mer sequencing protocols to a minimum read depth of 37.5 million reads per sample. In another experiment, 4 BACHD and 4 WT littermate control mice (both 2 males and 2 females) at 12 months age were used. Their cortical and striatal tissues were dissected and assayed as above.亨廷顿舞蹈症（Huntington's disease, HD）是由亨廷顿（huntingtin, HTT）基因N端区域的CAG重复扩增所引发，其特征为纹状体、皮层及其他脑区出现神经元丢失与神经炎症相关转录谱改变。本研究报道了BAC-CAG模型的构建与表征：这是一种携带超过120个连续CAG重复序列的全新全长人类HD小鼠模型。我们对2月龄、6月龄和12月龄的BAC-CAG小鼠（每组基因型n=6，性别均衡）的纹状体与皮层进行了RNA测序分析。结果显示，BAC-CAG小鼠的纹状体存在年龄依赖性转录异常：2月龄和6月龄纹状体中分别仅检出7个和36个显著差异表达基因（differentially expressed, DE）（错误发现率<0.1，FDR<0.1），而12月龄时则有820个差异基因（其中538个下调、282个上调，FDR<0.1）。该模型的转录组缺陷具有高度纹状体选择性：与野生型（wild type, WT）对照相比，BAC-CAG小鼠皮层在2、6、12月龄时分别仅检出4、3和14个差异基因（FDR<0.1）。值得注意的是，我们发现12月龄BAC-CAG小鼠与野生型对比的差异表达Z统计量，与6月龄Q140小鼠与野生型对比的该统计量存在极显著的全转录组正相关（r=0.62，p<1e-200）。此结果表明，BAC-CAG小鼠纹状体的差异表达基因谱与等位基因系列敲入（knock-in, KI）小鼠具有高度一致性；但此前针对12月龄BACHD小鼠的RNA测序研究显示，其纹状体与野生型同窝对照相比仅存在50个差异基因（31个下调、19个上调，FDR<0.1）。在12月龄皮层中，BACHD小鼠与野生型对照相比仅分别有6个下调基因和7个上调基因（FDR<0.1）。本研究中，分别取自2、6、12月龄的3只雄性和3只雌性BAC-CAG小鼠及其野生型同窝对照，经磷酸盐缓冲液（phosphate buffered saline, PBS）灌流后，立即剥离纹状体、皮层与小脑组织，样本经速冻后保存于-80℃。总RNA提取采用Qiagen（美国加利福尼亚州瓦伦西亚）RNeasy试剂盒搭配QIA shredder柱。cDNA文库构建使用Illumina TruSeq RNA Library Prep Kit v2，采用链特异性双端69-mer测序策略，使用Illumina HiSeq 4000测序仪进行测序，每个样本的最小测序深度为3750万条读段。另一项实验中，我们使用了4只12月龄的BACHD小鼠和4只同月龄野生型对照小鼠（雌雄各2只），对其皮层与纹状体组织进行了如上所述的解剖与检测。