Data and Code from: Dysregulation of zebrin-II cell subtypes is a shared feature across polyglutamine ataxia mouse models and human patients

Abstract Spinocerebellar ataxia type 7 (SCA7) is a genetic neurodegenerative disorder caused by a CAG- polyglutamine repeat expansion. Purkinje cells (PCs) are central to the pathology of ataxias, but their low abundance in the cerebellum underrepresents their transcriptomes in sequencing assays. To address this issue, we developed a PC enrichment protocol and sequenced individual nuclei from mice and patients with SCA7. Single-nucleus RNA sequencing in SCA7-266Q mice revealed dysregulation of cell identity genes affecting glia and PCs. Specifically, genes marking zebrin-II PC subtypes accounted for the highest proportion of DEGs in symptomatic SCA7-266Q mice. These transcriptomic changes in SCA7-266Q mice were associated with increased numbers of inhibitory synapses as quantified by immunohistochemistry and reduced spiking of PCs in acute brain slices. Dysregulation of zebrin-II cell subtypes was the predominant signal in PCs of SCA7-266Q mice and was associated with the loss of zebrin-II striping in the cerebellum at motor symptom onset. We furthermore demonstrated zebrin-II stripe degradation in additional mouse models of polyglutamine ataxia and observed decreased zebrin-II expression in cerebellum of patients with SCA7. Our results suggest that a breakdown of zebrin subtype regulation is a shared pathological feature of polyglutamine ataxias. Data and Code Availability Here you will find data and code associated with our manuscript "Dysregulation of zebrin-II cell subtypes is a shared feature across polyglutamine ataxia mouse models and human patients", Bartelt et al., Sci. Trans. Med. 16, eadn5449 (2024). The data file labeled "HuCb_filtered.rds" is a processed and annotated single-nucleus RNA-seq Seurat object, containing the gene-level count data for the multiplexed snRNA-seq experiment performed on post-mortem human cerebellar tissues from patients with SCA7 and unaffected controls. Data obtained from WT and SCA7-266Q mice as described in our paper can be accessed in the NIH Gene Expression Omnibus under accession number GSE269430. There are three code files numbered 00 through 02 which contain analysis code for snRNA-seq data applied to both the mouse and human datasets. These files are sequential and will take the user from CellRanger output, to filtered and annotated Seurat objects, and include details for subclustering analysis as well as our pseudobulk DEseq2 differential expression approach. There are places where the user may need to modify the code based on their computer system, version of R or Seurat, and whether they are processing the 5 week, 8 week, or human data sets; these locations in the code are marked with comments. The first file, 00_Preprocessing_MULTIseq, begins with CellRanger filtered_feature_barcode_matrix output, extracts cell barcodes, utilizes the MULTIseq deMULTIplex software to match cell barcodes to oligo barcodes from MULTIseq fastq files, and annotates the Seurat file with metadata. Cell type identification and annotation also takes place in this file. Note: the deMULTIplex step will likely need to be run on a high performance compute cluster. The second file, 01_Seurat_Analysis, uses the filtered and annotated Seurat file to calculate useful QC metrics, investigate disease signals, and perform cell type subclustering analyses. The third file, 02_Pseudobulk_DEseq2, contains custom analysis code to extract raw counts for each cell type and each animal from the Seurat file, and uses the DEseq2 package to calculate DEGs, taking into account biological replicates, and raw read count differences between control and SCA7 animals.# 摘要 7型脊髓小脑共济失调（Spinocerebellar ataxia type 7, SCA7）是一类由CAG-多聚谷氨酰胺重复扩增引发的遗传性神经退行性疾病。浦肯野细胞（Purkinje cells, PCs）是共济失调病理进程的核心受累细胞，但因其在小脑中丰度极低，常规测序实验难以完整捕获其转录组特征。为解决这一难题，我们开发了一套浦肯野细胞富集方案，并对SCA7模型小鼠及患者的单个细胞核进行了测序。对SCA7-266Q模型小鼠开展的单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）分析显示，影响胶质细胞与浦肯野细胞的细胞身份基因存在表达失调现象。具体而言，标记zebrin-II阳性浦肯野细胞亚型的基因在有症状SCA7-266Q模型小鼠的差异表达基因（differentially expressed genes, DEGs）中占比最高。SCA7-266Q模型小鼠的上述转录组学改变，与免疫组织化学定量检测到的抑制性突触数量增加，以及急性脑片实验中浦肯野细胞放电活动减弱相关。zebrin-II细胞亚型调控紊乱是SCA7-266Q模型小鼠浦肯野细胞的主要分子特征，且与运动症状发作时小脑zebrin-II条带缺失现象相关。我们进一步证实，在其他多聚谷氨酰胺共济失调小鼠模型中同样存在zebrin-II条带降解现象，并观察到SCA7患者小脑组织中zebrin-II表达水平降低。本研究结果表明，zebrin亚型调控紊乱是多聚谷氨酰胺共济失调的共同病理特征。 # 数据与代码获取说明 本文关联的数据与代码来自论文"Dysregulation of zebrin-II cell subtypes is a shared feature across polyglutamine ataxia mouse models and human patients"，作者为Bartelt等，发表于*Science Translational Medicine* 16, eadn5449 (2024)。 标注为`HuCb_filtered.rds`的数据文件为经处理与注释的单细胞核RNA测序Seurat（Seurat）对象，包含针对SCA7患者及未受影响对照的死后人类小脑组织开展的多重单细胞核RNA测序实验的基因水平计数数据。本研究中使用的野生型（wild type, WT）及SCA7-266Q模型小鼠相关数据，可在美国国立卫生研究院基因表达综合数据库（Gene Expression Omnibus, GEO）中通过登录号GSE269430获取。 本研究附带3份编号为00至02的代码文件，包含针对小鼠与人类数据集的单细胞核RNA测序数据分析代码。这些文件按顺序执行，可引导用户从CellRanger（CellRanger）输出文件出发，完成过滤与注释后的Seurat对象构建，并涵盖亚聚类分析以及基于伪批量样本的DESeq2（DESeq2）差异表达分析流程。代码中存在若干需用户根据自身计算机系统、R或Seurat版本，以及处理5周龄、8周龄小鼠数据集或人类数据集的需求进行修改的位置，这些位置均已通过注释标记。 1. 首个代码文件`00_Preprocessing_MULTIseq`以CellRanger（CellRanger）输出的`filtered_feature_barcode_matrix`为起始，提取细胞条形码，利用MULTIseq（MULTIseq） deMULTIplex（deMULTIplex）软件将细胞条形码与MULTIseq快速测序文件中的寡核苷酸条形码进行匹配，并为Seurat（Seurat）文件添加元数据。该文件同时完成细胞类型的鉴定与注释。注意：deMULTIplex（deMULTIplex）步骤需在高性能计算集群上运行。 2. 第二个代码文件`01_Seurat_Analysis`利用经过滤与注释的Seurat文件计算关键质控指标，探究疾病相关信号，并开展细胞类型亚聚类分析。 3. 第三个代码文件`02_Pseudobulk_DEseq2`包含自定义分析代码，用于从Seurat文件中提取每种细胞类型与每只动物的原始计数，并借助DESeq2（DESeq2）包计算差异表达基因，同时纳入生物学重复以及对照组与SCA7模型动物间的原始读数差异。