CREB3 gain of functions variants protect against ALS

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly evolving neurodegenerative disease arising from the loss of glutamatergic corticospinal neurons (CSN) and cholinergic motoneurons (MN). Here, we performed comparative cross-species transcriptomics of CSN using published snRNA-seq data from the motor cortex of ALS and control postmortem tissues, and performed longitudinal single cell RNA-seq data on male Sod1G86R mice. We report that CSN undergo ER stress and altered mRNA translation, and identify the transcription factor CREB3 and its regulatory network as a resilience marker of ALS, not only amongst vulnerable neuronal populations, but across all neuronal populations as well as other cell types. Using genetic and epidemiologic analyses we further identify the rare variant CREB3R119G (rs11538707) as a positive disease modifier in ALS. Through gain of function, CREB3R119G decreases the risk of developing ALS and the motor progression rate of ALS patients. Full length cDNA was obtained using the SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Clontech, CA) according to the manufacturer’s instructions. 11 cycles of cDNA amplification were performed using the Seq-Amp polymerase. 600ng of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT kit (Illumina) followed by 12 cycles of library amplification. Following purification using Agencourt AMPure XP beads (Beckman Coulter), the size and concentration of the libraries were assessed on an Agilent 2100 Bioanalyzer. The libraries were then loaded in the flow cell at a concentration of 3nM, clusters were generated by using the Cbot and sequenced on the Illumina HiSeq 4000 system as paired-end 2x50-base reads, following Illumina's instructions. Raw reads were mapped to the mouse reference genome GRCm38 with STAR version 2.7.0 61 and default parameters using Ensembl gene annotations (version 87). Gene-level abundance estimates were estimated using the option–quantMode geneCount in STAR.

肌萎缩侧索硬化症（Amyotrophic lateral sclerosis, ALS）是一种致命且进展迅速的神经退行性疾病，其发病机制为谷氨酸能皮质脊髓神经元（corticospinal neurons, CSN）与胆碱能运动神经元（cholinergic motoneurons, MN）的丢失。本研究利用已公开的ALS患者及对照死后运动皮层的单细胞核RNA测序（single nucleus RNA-seq, snRNA-seq）数据开展跨物种比较转录组学分析，并对雄性Sod1G86R小鼠进行纵向单细胞RNA测序分析。本研究发现，CSN会发生内质网应激及mRNA翻译调控异常，并鉴定出转录因子CREB3及其调控网络可作为ALS的耐受标志物，该标志物不仅在易受损的神经元群体中存在特异性表达，还可在所有神经元群体及其他细胞类型中被检测到。通过遗传学与流行病学分析，我们进一步确认罕见变异CREB3R119G（rs11538707）是ALS的正向疾病修饰因子。功能获得性实验证实，CREB3R119G可降低ALS的发病风险并减缓ALS患者的运动功能进展速率。实验流程方面，本研究参照制造商说明书，使用SMART-Seq v4超低起始量RNA测序试剂盒（Clontech，美国加利福尼亚州）获取全长cDNA；采用Seq-Amp聚合酶进行11轮cDNA扩增。取600ng预扩增cDNA作为起始样本，使用Nextera XT试剂盒（Illumina）进行Tn5转座子标签化，随后开展12轮文库扩增。经Agencourt AMPure XP磁珠（Beckman Coulter）纯化后，使用Agilent 2100生物分析仪对文库的片段大小与浓度进行质检。将文库以3nM的浓度加载至测序流动槽，通过Cbot生成测序簇，随后按照Illumina官方操作指南在Illumina HiSeq 4000系统上进行双端2×50碱基读长测序。原始测序reads使用STAR v2.7.0 61版本及默认参数，并结合Ensembl基因注释（版本87）比对至小鼠参考基因组GRCm38。基因水平的丰度估计通过STAR的--quantMode geneCount参数完成。