Fig 2: Inhibition of stress-induced increased hdac1 expression promotes resilience and rescues cldn5 expression. (A) Following 10 d of CSDS, hdac1 expression is down-regulated in the NAc endothelial cells of RES mice when compared to SS mice (one-way ANOVA, *P = 0.0301, n = 4 to 5 mice/group). (B and C) Higher stress-induced hdac1 level in the NAc of SS mice was confirmed by quantitative PCR and negatively correlated with SI (SI ratio: one-way ANOVA: F2,34 = 44.27; ***P < 0.0001; hdac1 fold change: one-way ANOVA: F2,29 = 6.122; **P = 0.0061, Pearson’s correlation between SI ratio and hdac1 fold change: **P = 0.0068, n = 10 to 16 mice/group). (D) Mice were screened with the SI test following 10 d of CSDS and split equally in two groups then administered vehicle or MS-275. (E) A second SI test was performed after 10 d of treatment revealing increased social interactions in the MS-275–treated group (unpaired t test: ***P < 0.0001) in line with higher cldn5 expression (Pearson’s correlation: **P = 0.0019, n = 11 mice/group). One-way ANOVAs statistical test were significant, thus Bonferroni posttests were performed with **P < 0.01; ***P < 0.001.

Fig 3: Drug resistant temporal lobe epilepsy induces blood-brain barrier leakage and Cldn5 downregulation in the hippocampus.a Representative dynamic contrast-enhanced MRI (DCE-MRI) images from 4 patients with temporal lobe epilepsy (TLE, top panel). Colour bar represents slope of contrast agent. b T2 weighted images (middle panel) and (c) fluid attenuated inversion recovery (FLAIR) images (bottom panel). d Claudin-5 (CLDN5; green) and CD31 (red) staining in non-diseased autopsy control (CNTRL) and TLE cases. Scale bars, 20 μm. e Collagen-IV (ColIV; green) and IgG (red) staining in CNTRL and TLE cases. Scale bars, 50 μm. f Levels of CLDN5 were significantly decreased (**p = 0.0042) and g IgG was significantly increased (****p < 0.0001) in the brains of TLE cases vs CNTRL. Data represent means ± s.e.m.; each datapoint represents one patient. Number of subjects (n) is indicated on graphs. Two-sided Mann–Whitney test for immunohistochemical and western blot analysis. **p < 0.01; ****p < 0.0001. Source data are provided as a Source Data file.

Fig 4: Cld5 is a crucial component of the blood–thymus barrier. Thymi from Cldn5+/+, Cldn5+/– and Cldn5–/– mice were analyzed at E18.5. (A) Enzymatically digested total thymic cells from Cldn5+/+ and Cldn5–/– mice were stained with anti-CD45, Ter119, CD31 and EpCAM antibodies and analyzed by FCM. The percentages of endothelial and epithelial cells in the CD45–Ter119– fraction are indicated. Horizontal bars indicate median values. Statistical significance was analyzed by using the Mann–Whitney test; N.S., not significant. (B) Total thymocyte numbers and percentages of PI+ dead cells in total thymocytes were analyzed. (C) TJ morphologies in the cortical capillaries were examined by using electron microscopy. (D) The distribution of an i.v.-injected biotin tracer was detected by Avidin staining. The data are representative of at least five independent experiments.

Fig 5: Comparison of cell morphology and localization and expression of claudin-5 and Pgp in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCECs. a Claudin-5 was indirectly stained (hCMEC/D3-WT and pBCECs) or visualized by YFP tag in the stably transduced hCMEC/D3-Cldn5-YFP cell line (for better visualization both claudin-5 and Cldn5-YFP are depicted in green). F-Actin is shown in red and cell nuclei are counterstained in blue by DAPI. b An xz scan of the cell layer revealed interendothelial (junctional) localization of claudin-5 (green) in the two cell lines and pBCECs. Scale bars: 10 µm. c Phase contrast micrographs of hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and primary pBCEC cultures. d Purity of pBCEC isolation, evaluated by fluorescent staining for the endothelial cell marker CD31 (green). Cell nuclei were counterstained with DAPI (blue). e Length and width of hCMEC/D3 and pBCECs. f Representative Western blot showing claudin-5 (Cldn5) expression in hCMEC/D3-WT (7 days after seeding), hCMEC/D3-Cldn5-YFP (7 and 21 days after seeding) and pBCEC cultures (7 days after seeding). ß-actin was used as a loading control. g Quantification of Western blot bands and normalization of claudin-5 expression to ß-actin. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (*P < 0.05). h Representative Western blot showing Pgp expression in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCEC cultures. ß-actin was used as a loading control. i Quantification of Western blot bands and normalization of Pgp expression to ß-actin. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (*P < 0.05)