*2.2. P-gp Mediates Aβ<sup>42</sup> Transport from Brain to Capillary Lumen Ex Vivo*

Freshly isolated brain capillaries provide a unique ex vivo model of the BBB, which can be used to study endogenous transport processes across the endothelium. P-gp is expressed on the luminal (blood-facing) membrane of the BBB, where it exports substrates from the endothelial cells into the blood [33].

To provide a physiological perspective to the binding and transport assays studied in Section 2.1, we compared the accumulation of fluorescent labelled human Aβ<sup>42</sup> (HiLyteTMhAβ42; 5 µM) at steady state in brain capillaries isolated from wild-type (WT) versus P-gp-knockout (KO) mice, using confocal microscopy combined with quantitative image analysis. The Aβ<sup>42</sup> isoform was selected as it displayed a greater capacity for stimulating ATP-hydrolysis and therefore was deemed a higher affinity substrate of P-gp than Aβ<sup>40</sup> (Figure 1e). Accumulation of HiLyteTM-hAβ<sup>42</sup> was lower in the lumen of capillaries isolated from P-gp KO mice compared to capillaries isolated from WT mice (Figure 2a,b). Image analysis revealed that luminal HiLyteTM-hAβ<sup>42</sup> fluorescence was significantly (*p* < 0.001) reduced in capillaries from P-gp KO (60.1 ± 4.4 (r.f.u.)) versus WT mice (127.8 ± 5.9 (r.f.u.)), indicating that P-gp is necessary for active Aβ transport from the bath to the vascular space. In concordance, luminal HiLyteTM-hAβ<sup>42</sup> fluorescence was significantly (*p* < 0.001) reduced in WT capillaries treated with the P-gp-specific inhibitor PSC833 (51.9 ± 2.4 (r.f.u.)) versus untreated capillaries (127.8 ± 5.9 (r.f.u.)), whereas fluorescence levels remained comparable between treated (60.1 ± 4.4 (r.f.u.)) versus untreated KO capillaries (55.5 ± 3.9 (r.f.u.)) (Figure 2c). The residual fluorescence present in PSC833-treated capillaries is due to nonspecific binding of the HiLyteTM-hAβ42 primarily to the endothelial cell surface. Figure 2c shows specific luminal NBD-CSA fluorescence that was taken as the difference between total luminal fluorescence and fluorescence in the presence of PSC833, which represents the P-gp-specific component of HiLyteTM-hAβ42 transport. Together, these data indicate that the observed differences in fluorescence accumulation are specific to P-gp-mediated transport. Western immunoblot analysis confirmed high P-gp protein expression in capillary membranes from WT mice and lack of P-gp expression in capillary membranes from P-gp KO mice. In contrast, low-density lipoprotein receptor-related protein 1 (LRP-1), the receptor at the abluminal (brain-facing) membrane of the capillaries responsible for Aβ uptake into the endothelial cells, was detected in capillaries from both WT and P-gp KO mice (Figure 2d).

These results confirm our previous findings showing Aβ transport at the BBB is an active and ATP-dependent two-step process, involving LRP-1-mediated Aβ uptake from the brain into capillary endothelial cells, followed by P-gp-mediated Aβ transport from the endothelium into the capillary lumen [18,34].

β β β μ β β β μ β β **Figure 2.** P-gp-mediated human Aβ<sup>42</sup> (hAβ42) transport in isolated brain capillaries. (**a**), (**b**) Representative confocal images showing accumulation of HiLyteTM-hAβ<sup>42</sup> in capillary lumens isolated from wild-type (WT) mice, but not in capillaries from P-gp knockout (KO) mice after a 1-h incubation (steady state; 5 µM HiLyteTM-hAβ42). (**c**) Data after digital image analysis using ImageJ. Specific fluorescence refers to the difference between total luminal HiLyteTM-hAβ<sup>42</sup> fluorescence and HiLyteTM-hAβ<sup>42</sup> fluorescence in the presence of the P-gp-specific inhibitor PSC833 (5 µM). (**d**) Western blot showing P-gp protein expression in isolated capillaries from WT mice, but not in capillaries isolated from P-gp KO mice. In contrast, LRP-1 is expressed in isolated capillaries from both WT mice and P-gp KO mice. β-actin was used as the loading control. Statistics: data per group are given as mean ± SEM for 10 capillaries from one preparation (pooled tissue: WT (*n* = 10 mice), P-gp KO (*n* = 10 mice)). Shown are relative fluorescence units ((r.f.u.) scale 0–255). *\*\*\** Significantly lower than control, *p* < 0.001.
