Single-cell profiling of remyelination in the LPC model

Remyelination is reparative process by which axons that have lost their supportive insulation are re-encased in myelin. Multiple cell types are known to participate in this process, but their transcriptional dynamics from initial injury to full repair remain poorly characterized. Here, we combined high-throughput single-nucleus RNA-seq with Slide-seq, a high-resolution spatial transcriptomics technology, to densely reconstruct the cellular processes that coordinate remyelination after lysolecithin injection. First, we found extensive transcriptional diversity of glia and monocyte-derived macrophages from damage to repair. Second, we identified a population of infiltrating peripheral immune cells-- mostly CD8 T-cells and natural killer cells--that are enriched specifically during remyelination. Thirdly, we identified a concerted Interferon-response signature that is shared across several glial cell types--microglia, astrocytes, and oligodendrocyte lineage--just prior to reestablishment of myelin To model successful remyelination we used the lysophosphatidylcholine (LPC) injection model, delivering a demyelinating agent into the corpus callosum. This results in myelin loss and remyelination in a rapid and stereotyped manner over 3 weeks, enabling dense reconstruction of this reparative process. We chose four time points representing distinct phases of repair: initial myelin damage (3 days post injection, dpi), oligodendrocyte differentiation (7dpi), early (12 dpi) and late (18dpi) remyelination. To profile the gene expression of cells during remyelination we developed an optimized extraction protocol to maximize the yield of nuclei from small white matter samples and performed single-nucleus RNA sequencing (snRNAseq) at all four time points with matched saline-injected controls (n=3 per time point and condition) (Fig. 1a), avoiding isolation aftefacts.