Fig. 3.

RRIMs are increased in the Optn^−/− mouse model of ALS. (A) inDrop isolated spinal cords enriched for CD45⁺ cells of 3.5-mo-old Optn^Fl/Fl and Optn^−/− male mice (n = 2 per group) treated with either a vehicle control or Nec-1s. tSNE plot of cells isolated from all groups and identified based on differentially expressed genes generated using Seurat. n = 6 animals. (B) tSNE plot of microglia from all Optn groups reclustered in Seurat using semisupervised clustering to identify four subpopulations, including HOM, DAM, and RRIM. n = 6 animals. (C) Violin plots of marker genes for RRIM subcluster from all Optn groups generated using Seurat. Values plotted are TPM. n = 6 animals. (D) GO analysis of pathways enriched in marker genes (padj < 0.01, logFC >1.5) for RRIM subcluster analyzed using GSEA. (E) Proportion of RRIM cluster as a percentage of total microglia were calculated for each animal and quantified using two-way ANOVA with multiple comparison testing relative to Optn^−/− Vehicle group. n = 2 animals per group. *P < 0.05; ns, not significant. Data are represented as mean ± SEM. (F) SmartSeq2 prepared scRNAseq of FACS microglia from 3.5-mo-old Optn^−/− untreated male mice (n = 2). tSNE plot of all cells generated using Seurat. (G) Volcano plot of transcriptional alterations between RRIM and HOM subclusters in SmartSeq2 Optn dataset. Significant genes indicated in red as logFC > 1.5 and −Log₁₀P ≥ 10.