Fig 2: Rubicon mutant mice show improved motor recoveryA Number of foot faults before and after injury in wild-type and Rubcn-mutant mice by beam walk test. B Percent of one/two-foot support (left) and three/four-foot support (right) after injury in wild-type and Rubcn-mutant mice by assessed by CatWalk test. C Percentage of ipsilateral cortical damage at 29 dpi between injured wild-type and Rubcn-mutant mice.Data represent mean ± s.e.m. Sample size (n) for A were as follows: WT Sham=11, WT TBI=13, Rubcn Mut. Sham=13, Rubcn Mut. TBI=14. Sample size (n) for B were as follows: n=7–14 mice per group as indicated. Sample size (n) for C were as follows: WT TBI=5, Rubcn Mut. TBI=5. Statistical Analyses: For A-B, mixed-effects analysis with post hoc Tukey’s test; For C, Unpaired Student’s t-test. For all data, significance is represented by * P-value<0.05, ** P-value<0.005.

Fig 3: Reduction in TBI-induced inflammation in Rubcn-mutant mice is temporaryA Heatmap of RT-qPCR data showing expression of inflammatory genes in ipsilateral cortices of wild-type and Rubcn-mutant mice at 1 dpi (TBI D1) and 3 dpi (TBI D3) in comparison to sham. Color coding is based on z-score. B Fold change in expression levels of indicated genes in wild-type and Rubcn-mutant mice corresponding to A. C Heatmap of RT-qPCR data showing expression of inflammatory genes in ipsilateral cortices of wild-type and Rubcn-mutant mice at 29 dpi (TBI D29). D Fold change in expression levels of indicated genes in wild-type and Rubcn-mutant mice corresponding to C.Bars represent mean ± s.e.m. Sample size (n) for A-B were as follows: WT Sham=8, WT TBI D1=5, WT TBI D3=9, Rubcn Mut. Sham=8, Rubcn Mut. D1=7, Rubcn Mut. D3=9. Sample size (n) for C-D were as follows: WT Sham=6, WT TBI D29=6, Rubcn Mut. Sham=7, Rubcn Mut. TBI D29=5. Statistical analyses for all data: Two-way ANOVA with post hoc Tukey’s test and significance is represented by * P-value<0.05, ** P-value <0.005, *** P-value <0.0005, **** P-value <0.0001.

Fig 4: Rubcn-mutant mice have attenuated inflammatory gene expression following TBIA Variant sparse Partial Least Squares-Discriminate Analysis (sPLS-DA) plot demonstrating group separation among ipsilateral cortical samples from wild-type sham, Rubcn-mutant sham, wild-type injured, and Rubcn-mutant injured mouse groups. B Volcano plots highlighting differentially expressed genes (DEGs) between injured and sham mice in wild-type (left) and Rubcn-mutant (right) samples, with red representing significantly upregulated genes and blue representing significantly downregulated genes. C Volcano plots showing DEGs between Rubcn-mutant and wild-type mice in sham (left) and injured samples (right). A data point corresponding to the Rubcn gene has been removed from the right volcano plot to minimize the y-axis. D Heatmap of gene expression across the samples demonstrating top upregulated (top-half) and downregulated (bottom-half) DEGs between Rubcn-mutant and wild-type TBI mice. E Gene Ontology Biological Process (GO:BP) analysis of significant DEGs (P-value<0.01) between Rubcn-mutant and wild-type TBI samples. Top graph shows GO-terms associated with upregulated genes (in orange) and bottom graph shows GO-terms associated with downregulated genes (in blue) in Rubcn-mutant over wild-type TBI samples with FDR<0.05. F Heat map of selected TBI-induced inflammatory DEG expression across wild-type and Rubcn-mutant sham and TBI samples. G Ingenuity Pathway Analysis (IPA) analysis comparing canonical pathways activated following TBI in wild-type versus Rubcn-mutant mice. Orange (activated) and blue (inhibited) indicate significantly enriched canonical pathways based on activation z-score, while gray dots represent pathways detected in the specified dataset that did not meet significance thresholds. H IPA analysis of lipid-related pathways activated following TBI in wild-type versus Rubcn-mutant mice.Sample size (n) were as follows: WT Sham=5, WT TBI D3=5, Rubcn Mut. Sham=4, Rubcn Mut. TBI D3=6.

Fig 5: Rubicon mutation decreases injury-induced autophagy inhibitionA Immunoblot comparing damage-associated marker (α-fodrin) and autophagy markers (P62, LC3II) in ipsilateral cortices from wild-type and Rubcn-mutant mice at 3 dpi to sham. B Densitometric quantification of immunoblot in A. C Immunoblot of ipsilateral hippocampus at 1 and 3 dpi in wild-type and Rubcn-mutant mice probed with autophagy markers. D Densitometric quantification of immunoblot in C. E Immunoblot of ipsilateral hippocampus at 1 dpi (TBI D1) and 3 dpi (TBI D3) in wild-type and Rubcn-mutant mice probed with damage-associated markers. F Densitometric quantification of immunoblot in E. G Immunofluorescence (IF) images of GFP-LC3 (green) coronal sections from wild-type and Rubcn-mutant sham and injured mice at 3 dpi stained for IBA1 (in yellow) and P62 (in magenta). Scale bar represents 50 μm. H Quantification of mean fluorescence intensity of P62 (left) and percent of P62/SQSTM1+ cells within LC3+/IBA+ cells (right) from G.All bars represent mean ± s.e.m. Sample size (n) for A-B were as follows: WT Sham=6, WT TBI D3=6, Rubcn Mut. Sham=6, Rubcn Mut. D3=6. Sample size (n) for C-F were as follows: WT Sham=6, WT TBI D1=6, WT TBI D3=6, Rubcn Mut. Sham=5, Rubcn Mut.D1=5, Rubcn Mut. D3=5. Sample size (n) for G-H were as follows: WT Sham=4, WT TBI D3=4, Rubcn Mut. Sham=4, Rubcn Mut. D3=4. Statistical analyses for all data: Two-way ANOVA with post hoc Tukey’s test and significance is represented by * P-value<0.05, ** P-value<0.005, *** P-value<0.0005, **** P-value<0.0001.