82 Chapter 4 expression alterations were considered significant when the adjusted p value was equal to or lower than 0.05. Bulk RNA-seq data preparation and WGCNA network analysis RAWRNA-seq fastq files for publicly available datasets were downloaded fromSRA. Four public human dataset accession are: GSE100382, GSE55536, EMTAB7572, GSE57494 and mouse dataset accession are: GSE103958, GSE62641, GSE82043, GSE58318, E_ERAD_165. The GEO accession ID for the in-house human RNA-seq data is awaiting. Both human and mouse RNA-seq analysis was then performed using nf-core/rnaseq v.1.4.2 pipeline48. Human RNAseq data was aligned to Homo sapiens genome GRCh38 and Mus musculus genome mm10 respectively. Raw count data was first transformed using variance stabilizing transformation (VST) from R package ‘DESeq2 v. 1.26.0’. Genes with an expression value of 1 count in at least 50% of the samples were included in the analysis. Batch correction across datasets were then performed on VST-transformed data using removeBatchEffect function from R package ‘Limma v. 3.42.2’ using the dataset ID as the batch. Batch-corrected normalised data was then used for co-expression network analysis using the R package ‘WGCNA v. 1.69’49. The power parameter ranging from 1-20 was screened out using the ‘pickSoftThreshold’ function. A suitable soft threshold of 6 was selected, as it met the degree of independence of 0.85 with the minimum power value. We generated a signed-hybrid network using Pearson correlation with a minimummodule size of 30. Subsequently, modules were constructed, and following dynamic branch cutting with a cut height of 0.95. Functional enrichment analysis of the gene modules was performed using the R package ‘WebGestaltR v. 0.4.3’50 using default parameters and ‘genome_protein-coding’ as the background geneset. Human Brain Tissue The rapid autopsy regimen of the Netherlands Brain Bank in Amsterdam (coordinator Prof I. Huitinga) was used to acquire the samples. Human tissue was obtained at autopsy from the spinal cord (cervical, thoracic, lumbar levels) from 12 ALS patients, 7 with short disease duration (SDD; <18 months survival; mean survival 11.1 ± 3.4 months) and 4 with medium disease duration (MDD; >24 months survival; mean survival 71.5 ± 31.5 months). Tissues for controls were collected from 10 age-matched cases with no neurological disorders or peripheral inflammation (Table S1). The hippocampal region was collected from 5 AD patients with Braak stage 6, and 5 aged-matched controls that had no cognitive impairments prior to death (Table S2). Active MS lesions were obtained from 5 MS cases as well as white matter from age-matched controls (Table S3). All tissue was collected with the approval of the Medical Ethical Committee of the Amsterdam UMC. All participants or next of kin had given informed consent for autopsy and use of their tissue for research purposes. Generation and details of mouse and marmoset models Mouse EAE Spinal cord tissue from mice with EAE was obtained from Biozzi ABH mice housed at Queen Mary University of London, UK (originally obtained from Harlan UK Ltd, Bicester, UK). The mice were raised under pathogen-free conditions and showed a uniform health status throughout the studies. EAE was induced via injection of mouse spinal cord homogenate in complete Freund’s adjuvant (CFA) into mice of 8-12 weeks or 12 months of age as described previously51,52. Immediately, and 24 h after injection mice were given 200ng Bordetella pertussis toxin (PT). Age-matched control groups were immunized with CFA and PT. Table
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