Fig 2: Expression levels of claudins 1 and 5 in leukocytes from MS and T1D. (A) Claudin 1 protein levels in PBLs of healthy controls (n = 13), OND controls (n = 14), T1D controls (n = 14), MS patients in remission (n = 13) and MS patients in relapse (n = 10). (B) The expression levels of CLDN5 RNA (2ΔCT) relative to reference genes were determined by real- time RT-PCR of PBL RNA derived from MS patients in relapse (n = 33), MS patients in remission (n = 39), and healthy controls (n = 18). (C) Claudin 5 protein levels in the PBLs of healthy controls (n = 13), OND controls (n = 14), T1D controls (n = 14), MS patients in remission (n = 11), and MS patients in relapse (n = 17). Box plot and data points display the relative protein levels determined by Western blot analysis. The relative intensity of claudin 1 or claudin 5 proteins compared to β-actin signals was determined by densitometry. Medians are depicted by horizontal bars.

Fig 3: Brain macrophage heterogeneity in multiple sclerosis brain tissues. a Workflow of the experiment. b UMAP depicting 56,522 cells. Colors depict unsupervised clustering. c UMAP where colors depict donor of origin (left) or tissue region of origin (right). d UMAPs where color scales depict module scores of microglia- and CAM-enriched gene sets (top) or expression levels of S100A9 and PLP1 (bottom). e Bar plots depicting cluster distribution among donor groups with standard error. Black circles depict individual samples. C = NACT; W = NAWM; L = WML. f Dot plot depicting marker genes of each cluster. Size of the circles depicts the fraction of cells expressing the gene, color scale depicts average expression level. g Heatmap depicting enrichment scores of literature-derived gene-sets in the scRNAseq dataset. h Immunofluorescent images of stainings for FCGR2B (orange; Hs8); CLDN5 (magenta; blood vessels), Hoechst (cyan; nuclei) and IBA1 (green; macrophages) in an active and inactive lesion. Left panels: overview image. Scale bar = 25 µm. Center panel: Zoom in on parenchymal macrophages. Scale bar = 10 µm. Right panel: Zoom in on vessel-associated macrophages. i–j Quantification of immunohistochemistry for FCGR2B in parenchyma and near/on blood vessels. In (j) a subset of tissues is presented to enable visualization of differences in samples with low tissue coverage. *: p < 0.05. Abbreviations: NACT = normal appearing cortical tissue; NAWM = normal appearing white matter; WML = white matter lesion, PLWM = perilesional white matter

Fig 4: The role of circ-FoxO3 in alleviation of BBB damage depends on autophagy(A and B) Evans blue extravasation was detected in male mice with circ-FoxO3 knockdown or upregulation that were pretreated with Rapa or CQ for 1 week before MCAO/R. CQ, chloroquine, 5 mg/kg/daily. Rapa, rapamycin, 0.75 mg/kg/daily. Vehicle control, saline containing 5% polyethylene glycol 400 and 5% Tween 80. White arrowheads indicate cerebral hemorrhage. Four of five mice displayed cerebral hemorrhage. Scale bar, 2 mm. n = 5. (C) The level of BBB-related proteins such as ZO-1, Mfsd2a, and Cldn5 in BMECs (bEnd.3 and HBMEC) with circ-FoxO3 knockdown or overexpression was analyzed by immunoblotting when cells were pretreated with CQ (or Rapa) before OGD/R. Rapa, 50 nmol/L; CQ, 30 μmol/L. n = 4. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. MCAO/R, middle cerebral artery occlusion for 2 h followed by reperfusion for 2 h; OGD/R, oxygen-glucose deprivation for 3 h followed by reoxygenation for 3 h. All data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Fig 5: METH induces BBB transport via endothelial caveolae in vivo(A–D) C57BL/6 mice were injected i.v. with Alexa Fluor 594 biocytin (A and B) or FITC-BSA (C and D) followed by i.p. injection of METH (2.5 mg/kg) or saline (NT). After 5 h, animals were sacrificed and perfused with 4% paraformaldehyde (PFA). Fixed sections were then counterstained using IB4 and imaged for the presence of perivascular and parenchymal tracers. Shown are representative images from n = 3 independent experiments. Tracer fluorescence was then quantified in 6 brain areas (see Figure S3A) using projections of optical sections spanning 25 μm and normalized to vascular density (B and D). Scale bars, 100 μm.(E) C57BL/6 mice were injected i.p. with METH (2.5 mg/kg) or saline (NT). After 1 or 5 h, brains were removed, frozen, sectioned, and stained for CLDN5 or VE-cadherin. Shown are maximal projections of confocal sections spanning 27 μm (representative of n = 3 independent experiments). Scale bar, 20 μm.(F) WT or Cav1−/− mice were injected i.v. with EB followed by i.p. injection of METH (2.5 mg/kg) or saline (NT) at time 0 (0–3 and 0–5 h) or 5 h (5–10 h). Animals were sacrificed and perfused at 3, 5, or 10 h, the brains isolated and digested and the EB extracted and quantified. Shown is the brain-to-serum ratio of EB fluorescence per brain weight ± SEM and the individual data points from each animal. ns, non-significant, ∗p < 0.05; ∗∗∗p < 0.001 (ANOVA, Bonferroni post hoc test).