Fig 2: CYM-5442 protects against BBB disruption after TBI by reducing vesicle transcytosis. A, B, C The protein levels of albumin (B) and caveolin-1 (C) as measured by WB. n = 4/group. D, E Representative immunofluorescence images of CAV-1 protein expression. n = 4/group. Bar = 100 μm. F, G TEM images of transcytosis vesicles (black arrows) in ECs (ECs) after TBI. Scale bar = 500 nm (F). Quantification of the transcytosis vesicular density (G). n = 4/group. H, I HRP-filled vesicles (white arrowheads) (H) were observed in ECs and quantified (I). n = 4/group. Scale bar = 200 nm. J Quantitative analyses of the degree of EB dye extravasation in the ipsilateral cortices of each group on day 3 after TBI. n = 6/group. K Brain water content in the whole cerebrum in each group on day 3 after TBI. n = 10/group. All data was analyzed by one-way ANOVA and Tukey’s multiple comparisons test, all values are presented as the mean ± SD. (-): TBI only; (Ctr): control shRNA; (Vec): vehicle; (OE): overexpression of Mfsd2a

Fig 3: Pathway analysis and differential expression validation.a Dot plots of important signaling pathways whose functions include vascular and BBB regulation, immune/cytokine signaling, respiratory electron transport chain, and glucose/energy metabolism pathways with significant enrichment in the aged brain for upregulated (left panel) and downregulated (right panel) EC DEGs. b Dot plots showing the differential expression profile of selected upregulated (left panel) and downregulated (right panel) genes in aged brain ECs associated with enriched pathways shown in a in the different EC subtypes. c Schematics of validation experiments by quantitative PCR and western blot for selected DEGs found by scRNA-seq (including Flt1, Klf6, Lef1, Smad7, Mfsd2a, and Slc2a1), in whole-brain ECs isolated by immunopanning. d Expression changes of selected genes in immunopanned ECs by quantitative PCR (for each gene, quantified as fold change relative to young adult group mean, error bars represent S.E.M.; Flt1: P = 0.036; Klf6: P = 0.011; Lef1: P = 0.28; Smad7: P = 0.012; Mfsd2a: P = 0.022; Slc2a1: P = 0.73; asterisks in the plot indicates P < 0.05 for easy visualization, two-sided unpaired t-test without adjustment for multiple comparisons; for each group, samples from n = 3 mice were pooled and measured in duplicate, resulting in two measurement values for each gene). e Western blot assays of the encoded proteins of a subset of aged brain EC-upregulated (LEF1, SMAD7) and downregulated (MFSD2A) genes in immunopanned ECs (samples pooled from three mice for each group). Source data underlying d, e are provided as a Source Data file.

Fig 4: Profiling mouse brain endothelial cell transcriptomes across age.a t-SNE visualization of single-cell transcriptomes from young adult (2–3 months old, n = 31,555 cells from five mice, brown) and aged (18–20 months old, n = 12,367 cells from five mice, blue) mouse brains. b Primary cell type clusters identified based on marker gene expression patterns. Cell type abbreviations: EC endothelial cell, SMC smooth muscle cell, PC pericyte, MG microglia, AC astrocyte, NRP neuronal restricted precursor, OPC oligodendrocyte precursor cell, OLG oligodendrocyte, mNeur mature neuron, imNeur immature neuron, EPC ependymocyte, CPC choroid plexus epithelial cell, Hb_EC hemoglobin-expressing vascular cell, MAC macrophage, TNC tanycyte, MNC monocyte. c t-SNE visualization of EC subtypes, including two arterial (aEC1, n = 1141 cells and 526 cells; aEC2, n = 998 and 679 cells from young adult and aged groups, respectively), capillary (capEC, n = 1587 and 697 cells), venous and capillary (vcapEC, n = 3122 and 801 cells), venous (vEC, n = 798 and 412 cells), and arterial/venous (avEC, n = 293 and 229 cells) subtypes. Subsequent differential expression analyses for each EC subtype presented were based on these cells (i.e. sharing the same sample sizes). For visualization, different colors are used for the subtypes, and 6000 cells were subsampled and shown for better clarity. d Expression patterns of arterial (Bmx, Vegfc), capillary (Mfsd2a), venous (Slc38a5, Nr2f2), and arterial/venous (Vwf) marker genes in ECs visualized by t-SNE. For clarity, 6000 cells were subsampled and shown. e Proportions of EC subtypes obtained from each age group. Scale bar: normalized expression level. f Venn diagram showing the overlap of significant differentially expressed genes (DEGs) (FDR-adjusted P-value < 0.05 and |lnFC | > 0.1) between five EC subtypes. g Numbers of significant upregulated (red dots) and downregulated (blue dots) DEGs for each EC subtype. h Heatmap of shared (significant in ≥3 EC subtypes) upregulated (upper panel) and downregulated (lower panel) DEGs in the aged brain. Remarks: asterisks denote transcription factor/regulatory genes, and hashtags denote stress response genes by the Gene Ontology annotation. i Heatmap of EC subtype-specific upregulated (upper panel) and downregulated (lower panel) DEGs (i.e. significant differential expression with adjusted P-value < 0.05 and |lnFC| > 0.1 in only one EC subtype). Up to eight are shown for each EC subtype.

Fig 5: Mfsd2a protein expression was decreased after TBI, and Mfsd2a deficiency resulted in a decreased concentration of S1P in the brain parenchyma. A, B Mfsd2a protein expression at different time points after TBI as measured by Western blotting. n = 4/group. C Immunofluorescence staining was performed with antibodies for Mfsd2a (green) and CD31 (red), and nuclei were stained with DAPI (blue). Scale bar = 10 μm. D, E Immunofluorescence staining revealed differential Mfsd2a protein expression (red) in brain ECs (green) (D). The corresponding statistics are shown in (E). n = 4/group. Scale bar = 50 μm. F, G WB analyses of the protein levels of Mfsd2a in brain tissues on day 3 after TBI. n = 4/group. H Concentrations of total S1P in the brain ECM on day 3 after TBI. n = 8/group. All data was analyzed by one-way ANOVA and Tukey’s multiple comparisons test, *P < 0.05 compared with the Sham group. All values are presented as the mean ± SD. (-): TBI only; (Ctr): control shRNA; (Vec): vehicle; (OE): Mfsd2a overexpression