DOI: 10.5281/zenodo.13905956
title: Data and Code from: Dysregulation of zebrin-II cell subtypes is a shared feature across polyglutamine ataxia mouse models and human patients

description: 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.