Single-cell RNA-seq analysis of the brainstem of mutant SOD1 mice reveals perturbed cell types and pathways of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons throughout the brain and spinal cord progressively degenerate resulting in muscle atrophy, paralysis and death. Recent studies using animal models of ALS implicate multiple cell-types (e.g., astrocytes and microglia) in ALS pathogenesis in the spinal motor systems. To ascertain cellular vulnerability and cell-type specific mechanisms of ALS in the brainstem that orchestrates oral-motor functions, we conducted parallel single cell RNA sequencing (scRNA-seq) analysis using the high-throughput Drop-seq method. We isolated 1894 and 3199 cells from the brainstem of wildtype and mutant SOD1 symptomatic mice respectively, at postnatal day 100. We recovered major known cell types and neuronal subpopulations, such as interneurons and motor neurons, and trigeminal ganglion (TG) peripheral sensory neurons, as well as, previously uncharacterized interneuron subtypes. We found that the majority of the cell types displayed transcriptomic alterations in ALS mice. Differentially expressed genes (DEGs) of individual cell populations revealed cell-type specific alterations in numerous pathways, including previously known ALS pathways such as inflammation (in microglia), stress response (ependymal and an uncharacterized cell population), neurogenesis (astrocytes, oligodendrocytes, neurons), synapse organization and transmission (microglia, oligodendrocyte precursor cells, and neuronal subtypes), and mitochondrial function (uncharacterized cell populations). Other cell-type specific processes altered in SOD1 mutant brainstem include those from motor neurons (axon regeneration, voltage-gated sodium and potassium channels underlying excitability, potassium ion transport), trigeminal sensory neurons (detection of temperature stimulus involved in sensory perception), and cellular response to toxic substances (uncharacterized cell populations). DEGs consistently altered across cell types (e.g., Malat1), as well as cell-type specific DEGs, were identified. Importantly, DEGs from various cell types overlapped with known ALS genes from the literature and with top hits from an existing human ALS genome-wide association study (GWAS), implicating the potential cell types in which the ALS genes function with ALS pathogenesis. Our molecular investigation at single cell resolution provides comprehensive insights into the cell types, genes and pathways altered in the brainstem in a widely used ALS mouse model.

肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）是一类神经退行性疾病，患者脑与脊髓内的运动神经元会进行性退变，最终引发肌肉萎缩、瘫痪乃至死亡。既往利用ALS动物模型开展的研究表明，脊髓运动系统的多种细胞类型（如星形胶质细胞与小胶质细胞）参与了ALS的发病机制。为明确调控口部运动功能的脑干中ALS的细胞易感性及细胞特异性致病机制，本研究采用高通量Drop-seq技术开展了平行单细胞RNA测序（single cell RNA sequencing, scRNA-seq）分析。研究人员分别于出生后第100天，从野生型与突变SOD1症状性小鼠的脑干中分离得到1894个与3199个细胞。我们捕获到了已报道的主要细胞类型与神经元亚群，包括中间神经元、运动神经元、三叉神经节（trigeminal ganglion, TG）外周感觉神经元，以及此前未被表征的中间神经元亚型。研究发现，多数细胞类型在ALS模型小鼠中均出现了转录组学改变。单个细胞群体的差异表达基因（differentially expressed genes, DEGs）分析显示，多条通路存在细胞特异性改变，其中既包含既往已知的ALS相关通路：如小胶质细胞的炎症应答、室管膜细胞与未表征细胞群的应激反应、星形胶质细胞、少突胶质细胞及神经元的神经发生、小胶质细胞、少突胶质前体细胞与神经元亚型的突触组织与传递功能，以及未表征细胞群的线粒体功能异常；同时还发现突变SOD1小鼠脑干中其他细胞类型特异性的生物学过程改变：包括运动神经元的轴突再生、介导细胞兴奋性的电压门控钠钾通道与钾离子转运功能、三叉神经感觉神经元的温度刺激感知功能，以及未表征细胞群的有毒物质应答过程。本研究鉴定出了在多种细胞类型中均发生改变的差异表达基因（如Malat1），以及细胞类型特异性的差异表达基因。值得注意的是，不同细胞类型的差异表达基因既与文献中报道的已知ALS致病基因存在重叠，也与现有人类ALS全基因组关联研究（genome-wide association study, GWAS）的顶尖关联位点相重合，这提示了ALS致病基因参与ALS发病过程的潜在细胞类型。本研究在单细胞分辨率下开展的分子分析，为这一广泛使用的ALS小鼠模型的脑干内异常细胞类型、基因及通路提供了全面的解析。

搜集汇总

背景与挑战

背景概述

该数据集通过单细胞RNA测序技术，分析了突变SOD1小鼠脑干在肌萎缩侧索硬化症（ALS）模型中的细胞类型和通路变化。研究包括野生型和突变型小鼠的脑干细胞样本，识别了多种细胞类型（如运动神经元、胶质细胞）的转录组差异，并揭示了与炎症、应激反应和线粒体功能相关的通路扰动，为ALS的病理机制提供了单细胞水平的见解。