Dissecting the evolving transcriptional landscape of the remyelinating spinal cord

Demyelination, or the loss of myelin in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and occurs in various forms of CNS injury and neurodegenerative diseases. The replenishing of myelin, termed remyelination, also occurs spontaneously following demyelination. The lysolecithin-induced focal demyelination model enables the investigation into the mechanisms of spontaneous remyelination, providing insight into the molecular basis underlying an evolving remyelinating microenvironment. Here, we present a detailed analysis using high-resolution single-nucleus RNA sequencing to investigate gene expression dynamics across all cell types involved in the remyelination process. We examine three specific time points following focal demyelinating injury. By delineating activation states within the heterogeneous cell populations of demyelinated lesions, we highlight changes in gene expression within subclusters of each cell type from the early stages of injury response to the initiation and maintenance of remyelination. Our findings reveal how shifts in microglial, astrocytic and fibroblast activities within lesions are associated with efficient oligodendrocyte differentiation during remyelination. Overall design: Focal demyeination in the spinal cord, mediated by the injection of 1% lysophosphatidylcholine (LPC, lysolecithin) in the ventral white matter, was performed in 8-12 week old female and male mice. Lesioned tissue, visualized by the injection of 1% neutral red solution (500µL, intraperitoneal) 2 hours preceding euthanasia, was collected at 5, 10, and 20 days post lesion. Rostral, non-lesioned tissue was collected at 5, 10, and 20dpl as an internal control. As another contro, naive, unlesioned spinal cord white matter tissue from a separate cohort of mice was also collected. Single nuclei were isolated from tissue samples and analyzed via snRNAseq.