Fig. 3. Astrocytes with the LRRK2 G2019S mutation fail to support the formation of a functional BBB.

A Representative images of 4.4 kDa dextran-TMRE (red, top images) or rhodamine (red, bottom images) in BBB^G2019S vs. BBB^CTL chips after a 40-min incubation, prepared using two independent LRRK2 G2019S iPSC pairs. B, C Quantification of 4.4 kDa dextran-TMRE (B) and rhodamine (C) apparent permeability (P_app) values in chips prepared using three independent iPSC pairs. D Confocal images illustrating two cross-sections of immunostained vessels expressing ZO-1 (green) and DAPI nuclear stain (blue). White arrows indicate areas of low or absent immunoreactivity, white stars point to the localization of guides imprinted on the microchip. E Quantification of 4.4 kDa dextran-TMRE permeability in a transwell system, results for isogenic and non-isogenic line 1 are combined into a single graph. F Representative immunoblots depicting protein levels of tight junction markers VE-cadherin, ZO-1, claudin-5 and loading control GAPDH in vessels. G–I Quantifications of VE-cadherin (G), ZO-1 (H), and claudin-5 (I) protein levels in vessels were normalized to GADPH. Data are shown as the fold change of BBB^G2019S levels compared to BBB^CTL. J Representative images of immunostained BBB^CTL and BBB^G2019S vessels depicting ZO-1 expression in BMEC-like cells. K, L Quantification of control and LRRK2 G2019S BMEC-like cell mean surface area (K) and frequency distribution (L). M Graph representing vessel width in BBB^G2019S chips normalized to BBB^CTL. Data are from three (E, G), four (H, I, M), five (B) or six (C) biological replicates; data in (K, L) are sampled from >900 individual cells from three independent biological replicates; error bars represent mean + SEM. Outliers were identified using Grubbs’ test with an alpha value set at 0.05 and removed from analysis. Statistical analysis was performed using two-tailed unpaired Student’s t test with equal s.d. Violin plot in (K) shows the median (red line) and quartile (red dotted line) values. Scale bar: 600 µm (A), 100 µm (D), 15 µm (J). The BBB^CTL and BBB^G2019S nomenclature refers to the presence of either control or LRRK2 G2019S astrocytes in the brain compartment of the BBB chip. cm centimeter, iso isogenic iPSC line, kDa kilodalton, min minutes, non-iso non-isogenic iPSC line, s seconds. Source data are provided as a Source Data file.