Cortical glia in SOD1(G93A) mice are subtly affected by ALS-like pathology. Cortical glia in SOD1(G93A) mice are subtly affected by ALS-like pathology

The role of glia in amyotrophic lateral sclerosis (ALS) is undeniable. Their disease-related activity has been extensively studied in the spinal cord, but only partly in the brain. We present herein a comprehensive study of glia in the motor cortex of SOD1(G93A) mice – a widely used model of ALS. Using single-cell RNA sequencing (scRNA-seq) and immunohistochemistry, we inspected astrocytes, microglia and oligodendrocytes, in four stages of the disease, respecting the factor of sex. We report insignificant motor neuron loss in the cortex, and likewise, minimal changes of glia throughout the disease progression and regardless of sex. Pseudobulk and single-cell analyses revealed subtle disease-related transcriptional alterations at the end-stage in microglia and oligodendrocytes, which were supported by immunohistochemistry. Therefore, our data conclusively prove that the SOD1(G93A) mouse motor cortex does not recapitulate the disease in patients, and we recommend the use of a different model for future studies of the cortical ALS pathology. Overall design: Cerebral cortex of control (CTRL) and SOD1(G93A) mutant mice (SOD1) was isolated at four time points (1, 2, 3 and 4 months of age). Two males and two females were pooled per condition for preparation of cell suspension. The cell suspension was enriched for astrocytes, microglia and oligodendrocytes using FACS and was analysed by scRNA-seq (10X Genomics platform). Please note that transgenic mice expressing high levels of human SOD1(G93A) (JAX Strain: 004435 C57BL/6J-Tg (SOD1*G93A)1Gur/J) and their non-carrier littermates are used in the study.

胶质细胞在肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）中的作用已毋庸置疑。其疾病相关活化状态已在脊髓中得到广泛研究，但在大脑中的相关研究仍较为有限。本文针对广泛使用的ALS模型——SOD1(G93A)小鼠的运动皮层胶质细胞开展了全面研究。本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与免疫组织化学技术，在疾病的四个病程阶段中对星形胶质细胞、小胶质细胞及少突胶质细胞进行了系统分析，并考量了性别因素。
研究发现，小鼠皮层内运动神经元丢失程度无统计学显著性，且在整个病程进程中、无论性别如何，胶质细胞均仅出现极细微的表达变化。伪批量转录组分析与单细胞转录组分析显示，小胶质细胞与少突胶质细胞在疾病终末期仅存在微弱的疾病相关转录组改变，这一结果得到了免疫组织化学实验的验证。综上，本研究数据确凿证明，SOD1(G93A)小鼠的运动皮层无法复现患者体内的ALS病理进程，因此我们建议在未来针对皮层ALS病理的研究中采用其他动物模型。

实验设计：本研究在四个时间节点（小鼠月龄1、2、3、4个月）分别分离了对照（CTRL）组与SOD1(G93A)突变型小鼠的大脑皮层。每个实验组均混合2只雄性与2只雌性小鼠以制备细胞悬液。通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）富集星形胶质细胞、小胶质细胞及少突胶质细胞，随后采用10X Genomics平台完成单细胞RNA测序。

请注意，本研究使用的转基因小鼠为高表达人源SOD1(G93A)的品系（JAX品系编号：004435 C57BL/6J-Tg (SOD1*G93A)1Gur/J），并以其非转基因同窝野生型小鼠作为对照。