Fig. 3: Single nucleus RNA-sequencing from optic nerves reveals that global C3 depletion results in widespread alterations in transcription characterized by a reduction in reactive glial profiles.

a Experimental paradigm. 10 C3KO mice and 10 WT mice were immunized and carried out to the peak of disease (PID 16). 5 mice were pooled per sample, resulting in 10 optic nerves per sample that were flash frozen and from which nuclei were extracted. snRNA-seq analysis was performed on the resulting 4 samples. b Annotated integrated UMAP plot of nuclei captured from optic nerves of N = 2 WT samples and N = 2 C3KO samples (See Fig. S8 for full UMAP and cluster identification markers). c Hierarchical subclustering and reanalysis of four key cell types showing cells from WT samples in orange and cells from C3KO samples in blue. d Specific gene changes in astrocytes and microglia. Gene names in red indicate MIMS and TIC-responsive genes in microglia and astrocytes respectively. Green gene names indicate homeostatic or neuroprotective genes. FDR < 0.1 denoted with an asterisk. e GSEA of DEGs in C3KO vs WT astrocytes (top), microglia (middle), and monocytes/macrophages (bottom). Color scheme for each pathway indicated below the GSEA plots. Pathways with peaks on the left are downregulated in C3KO cells. All shown pathways have FDR < 0.05. f Representative image of immunofluorescent staining of hippocampi from the mice used for the optic nerve snRNA-seq experiment (top is WT, bottom is C3KO). CA-1 region used for quantification highlighted. Inset depicts an example of an IBA1⁺ CD68⁺ Tyrobp⁺ cell from a WT mouse^. Scale bar = 500 μM in low magnification view of hippocampus, 50 μM in high magnification insets, and 5 μM in single cell magnification insets. g Quantification of IBA1⁺ CD68⁺ Tyrobp⁺ cells in the CA-1 region of the hippocampus (N = 5 mice per genotype, WT average final EAE score = 2.3 +/− 0.24; C3KO average final EAE score = 1.9 +/− 0.2; 60% male in both genotypes; p-value from unpaired student t-test, data presented as mean +/− SEM). Source data are provided as a source data file. Figure 3a created in BioRender. Smith, M. (2025) https://BioRender.com/k30u268.