Fig. 6. In vitro test of amylin-induced impairment of Aβ efflux across the BBB.

a Cartoon representation of the in vitro BBB model (ECs monolayer - luminal chamber; astrocytes - abluminal chamber) used in Aβ transcytosis experiments. b Transendothelial electrical resistance (TEER) in EC monolayers (n = 20 preparations) as a function of days in culture. c Representative Western blot and densitometry quantification of LRP1 in lysates from primary rat brain microvascular vascular ECs treated with vehicle or various concentrations of human amylin (500 nM, 1 µM, 5 µM, and 10 µM) for 24 h (n = 3 preparations/test). d Percent cell viability from the MTS assay in ECs treated with amyloid-forming human amylin (500 nM, 1 µM, 5 µM, and 10 µM) or vehicle, for 24 h. e The Aβ₄₂ transcytosis quotient (TQ) across the in vitro BBB, in vehicle- and human amylin-treated EC monolayers. f LRP1 mRNA levels (fold difference using 2^−ΔΔCt method) measured with qRT-PCR in lysates from ECs treated with vehicle, human amylin or rat amylin. g LRP1 mRNA levels measured by qRT-PCR in brain capillary lysates from same rats as in Fig. 5h. h, i miRNA (miR)-103 and miR-107 expression levels measured by qRT-PCR in lysates from ECs treated with vehicle or human amylin (same as in Fig. 5h), and in brain capillary lysates from same rats as in Fig. 5h. j TargetScan schematic showing consensus regions for miR-205, miR200bc-3p/429, and miR-103 and miR-107. Western blot analyses of LRP1 from miRNA (miR) 103 and miR-107 treated ECs compared to miR-control (n = 3 preparations/group) (k), as well as of LRP1 (l) and P-gp (m) from antagomir (amiR) 103 and amiR-107 treated ECs compared to amiR-control treated cells (n = 3 preparations/group). Data are mean ± SEM. one-way ANOVA with Dunnett’s post hoc (F). unpaired t test for the other panels.