Fig. 5: eSHANK3 depletion leads to aberrant ZO1/Claudin5 expressions and structural abnormalities in the junctions between BECs.

a–h eShank3 deletion effects on ZO1 and Claudin5 in BECs. ICC analysis showed discontinuous and patched (arrowheads) expressions of ZO1 and Claudin5 in both eShank3-KO bEnd.3 BEC line (a, b) and primary BECs from eShank3-KO mice (e, f) compared to controls. Blue: DAPI. Scale bars, 20 μm. WB analysis showing reduced expressions of ZO1 and Claudin5 in the eShank3-KO bEnd.3 BEC line (c, d) and primary BECs from eShank3-KO mice (g, h) compared to WT controls (n = 3 per group). Band intensities were normalized with β-Actin. i–l Ultrastructure of junctional area between BECs in the PFC. Representative electron micrographs of BEC junctions observed in the brain capillaries of P5 male (i) and female (k) eShank3-KO mice and control Tek-Cre mice (n = 3 mice per group). Arrowheads indicate cell-cell junctions along the adjacent BECs. Arrows indicate abnormal high electron densities in the cleft between BEC membranes. Insets are magnified views of the junctional areas. j Electron density scanning across the junction between BECs revealed elevated electron densities in the cleft of P5 male eShank3-KO mice. l However, this phenotype was not significant in female group. Data are mean ± SEM. Statistical analysis was performed using two-tailed unpaired t-tests (d, h) and mixed-effects analysis followed by Šídák’s multiple comparisons test (j, l). Detailed statistical methods and results are described in Supplementary Table 1. Source data are provided as Source Data File.