Fig. 4: Proteomic and phosphoproteomic analysis of optic nerves at the peak of EAE to validate the effects of C3KO on mTORC1 signaling in the CNS.
From: Myeloid lineage C3 induces reactive gliosis and neuronal stress during CNS inflammation
a Volcano plot of all detected proteins in full proteomic analysis of N = 7 WT (mean score = 2) vs N = 7 C3KO (mean score = 1.8) EAE mouse optic nerves (each group consisted of mice from both sex). Both optic nerves from a mouse were needed to generate sufficient protein to constitute a biological replicate. To validate the findings of reduced ribosomal biogenesis in C3KO mice from our transcriptomic experiments (See supplemental Fig. S11) both small and large ribosomal proteins have been highlighted in red. b Ingenuity Pathway Analysis performed on the differentially abundant proteins from the proteomics experiment. Proteomics confirmed pathway-level decreases in mTOR signaling, glycolysis, mRNA translation, and antigen presentation in C3KO optic nerves, while C3KO mice upregulated energetically efficient processes like oxidative phosphorylation and the TCA cycle as well as reparative programs like the detoxification of reactive oxygen species. All pathways are significant by an adjusted p-value of <0.05. c Pre-ranked GSEA was run on the average relative log2 fold change in protein abundances between EAE C3KO vs WT mice. Proteins that were most upregulated in C3KO mice lie on the left of the ranked protein list, so a positive Normalized Enrichment Score (NES) indicates an upregulated pathway in C3KO mice (scores listed above each plot). d Individual analysis of the abundance of proteins that regulate mTORC1 signaling. Adjusted p-values are listed as numbers above the comparison bars. Note that TSC2 S939 refers to relative abundance of phosphorylation of TSC2 at serine 939, a post-translational modification that inhibits the TSC complex from acting as a GAP for Rheb. All other bar graphs indicate total protein abundance. Note that we compared the 7 WT and 7 C3KO EAE mice to 2 WT and 2 C3KO naïve mice optic nerve proteomes for controls. Data presented as mean +/- SEM, one-way ANOVA adjusted for multiple comparisons. e Diagram depicting results from proteomics and phosphoproteomics experiments on how C3 knockout affects mTORC1 regulation. Figure 4e created in BioRender. Smith, M. (2025) https://BioRender.com/d23a966.
