Fig. 7: Restoration of neonatal BBB function through GSK3β activation in BECs normalizes neurological function in adult mice. | Nature Communications

Fig. 7: Restoration of neonatal BBB function through GSK3β activation in BECs normalizes neurological function in adult mice.

From: Endothelial SHANK3 regulates tight junctions in the neonatal mouse blood-brain barrier through β-Catenin signaling

Fig. 7

a–e GSK3βS9A overexpression effect on the BBB permeability in vivo. a Injection of AAV-PHP.v1-CLDN5-GSK3βS9A-HA through STV at P0. b IHC analysis using Shank3f/f:Tek-Cre:Ai-14 triple mutant mice shows specific expression of GSK3βS9A-HA (green) in the tdTomato-positive BECs of P5 brain. c Schematic illustration of BBB permeability assay using AAV-PHP.v1-CLDN5-GSK3βS9A-HA-injected eShank3-KO mice at P5. In vivo BBB permeability was assessed with P5 male (d) and female (e) eShank3-KO and control Tek-Cre mice injected by AAV-PHP.v1-CLDN5-GSK3βS9A-HA (n = 10 for male eShank3-KO, n = 13 for male Tek-Cre, n = 11 for female eShank3-KO, n = 14 for female Tek-Cre) or control AAV-PHP.v1-CLDN5-HA (n = 10 for male eShank3-KO, n = 9 for male Tek-Cre, n = 13 for female eShank3-KO, n = 11 for female Tek-Cre) at P0. The increased BBB permeability in male eShank3-KO pups was normalized by overexpression of GSK3βS9A in the BECs. Created in BioRender. Kim, S. (2025) https://BioRender.com/z54o523. f GSK3βS9A overexpression effect on the neuronal excitability. AAV-PHP.v1-CLDN5-GSK3βS9A-HA or control AAV-PHP.v1-CLDN5-HA) was injected into STV at P0 to express GSK3βS9A in the BECs from the neonatal period. To label the excitatory neurons, AAV-PHP.eB-CaMKIIα-EGFP was injected into the tail vein of adult mice (13–15 weeks old). Patch clamp analysis was then performed on GFP-labeled mPFC neurons of 17–19 week-old mice. Created in BioRender. Kim, S. (2025) https://BioRender.com/r42u905. g–j Analysis of neuronal excitability in adult male (n = 9 for eShank3-KO with HA expression, n = 11 for Tek-Cre with HA expression, n = 10 for eShank3-KO with GSK3βS9A expression, n = 9 for Tek-Cre with GSK3βS9A expression) (g) and female (n = 9 for each group) (i) eShank3-KO and Tek-Cre control mice, with or without GSK3βS9A expression in BECs. Graphs represent the number of evoked APs at the indicated current injection (g, i) and the RMP (h, j) of each group of mice. Data are mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons tests (d, e, h, j) and two-way ANOVA with repeated measures followed by two-stage linear step-up procedure of Benjamini, Kreiger and Yekutieli (false discovery rate) (g, i). Detailed statistical methods and results are described in Supplementary Table 1. Source data are provided as Source Data File.

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