Protecting P-glycoprotein at the blood-brain barrier from degradation in an Alzheimer's disease mouse model

Abstract

Background: Failure to clear Aβ from the brain is partly responsible for Aβ brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aβ across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp). In AD, P-gp levels are reduced, which contributes to impaired Aβ brain clearance. However, the mechanism responsible for decreased P-gp levels is poorly understood and there are no strategies available to protect P-gp. We previously demonstrated in isolated brain capillaries ex vivo that human Aβ40 (hAβ40) triggers P-gp degradation by activating the ubiquitin-proteasome pathway. In this pathway, hAβ40 initiates P-gp ubiquitination, leading to internalization and proteasomal degradation of P-gp, which then results in decreased P-gp protein expression and transport activity levels. Here, we extend this line of research and present results from an in vivo study using a transgenic mouse model of AD (human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576).

Methods: In our study, hAPP mice were treated with vehicle, nocodazole (NCZ, microtubule inhibitor to block P-gp internalization), or a combination of NCZ and the P-gp inhibitor cyclosporin A (CSA). We determined P-gp protein expression and transport activity levels in isolated mouse brain capillaries and Aβ levels in plasma and brain tissue.

Results: Treating hAPP mice with 5 mg/kg NCZ for 14 days increased P-gp levels to levels found in WT mice. Consistent with this, P-gp-mediated hAβ42 transport in brain capillaries was increased in NCZ-treated hAPP mice compared to untreated hAPP mice. Importantly, NCZ treatment significantly lowered hAβ40 and hAβ42 brain levels in hAPP mice, whereas hAβ40 and hAβ42 levels in plasma remained unchanged.

Conclusions: These findings provide in vivo evidence that microtubule inhibition maintains P-gp protein expression and transport activity levels, which in turn helps to lower hAβ brain levels in hAPP mice. Thus, protecting P-gp at the blood-brain barrier may provide a novel therapeutic strategy for AD and other Aβ-based pathologies.

Keywords: Alzheimer’s disease; Amyloid beta; Blood–brain barrier; Brain capillaries; P-glycoprotein; Ubiquitin-proteasome system.

Fig. 1

Effect of NCZ on P-gp protein expression and transport activity. a Western Blot for P-gp showing bands for brain capillary membranes isolated from vehicle-treated wild-type (WT), vehicle-treated hAPP mice, hAPP mice dosed with 5 mg/kg NCZ and hAPP mice dosed with a combination of NCZ and CSA (5/25 mg/kg). β-Actin was used as protein loading control; pooled tissue (WT: 10 mice; hAPP: 15 mice; hAPP-NCZ: 15 mice, hAPP-NCZ/CSA: 15 mice). Data were normalized to β-Actin. b Representative confocal images showing accumulation of P-gp-specific substrate NBD-CSA in isolated brain capillaries from WT, hAPP, hAPP-NCZ and hAPP-NCZ/CSA mice after a 1 hour incubation (steady state; 2 µM NBD-CSA). c Data after digital image analysis using ImageJ. Specific NBD-CSA fluorescence is the difference between total luminal NBD-CSA fluorescence and NBD-CSA fluorescence in the presence of the P-gp-specific inhibitor PSC833 (5 µM). Statistics: Data per group are given as mean ± S.E.M. for 10 capillaries from one preparation [pooled tissue: WT (10 mice), hAPP (15 mice), hAPP-NCZ (15 mice), hAPP-NCZ/CSA (15 mice)]. Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, p < 0.001

Fig. 2

Effect of NCZ on P-gp-Mediated hAβ42 transport. a Representative confocal images of isolated brain capillaries showing accumulation of fluorescein-hAβ42 in capillary lumens after a 1 hour incubation (steady state; 5 µM fluorescein-hAβ42). b Data after digital image analysis using ImageJ. Specific fluorescein-hAβ42 fluorescence is the difference between total luminal fluorescein-hAβ42 fluorescence and fluorescein-hAβ42 fluorescence in the presence of the P-gp-specific inhibitor PSC833 (5 µM). Statistics: Data per group are given as mean ± S.E.M. for 10 capillaries from one preparation (pooled tissue: WT (10 mice), hAPP (15 mice), hAPP-NCZ (15 mice), hAPP-NCZ/CSA (15 mice)). Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, p < 0.001

Fig. 3

NCZ shows no effect on ALT activity levels. ALT activity levels (mU/ml) in liver samples from WT, hAPP, hAPP-NCZ and hAPP-NCZ/CSA mice. Data are given for each animal: WT (n = 10), hAPP (n = 15), hAPP-NCZ (n = 15), hAPP-NCZ/CSA (n = 15). Statistics: ANOVA; Data between groups are not significantly different

Fig. 4

NCZ treatment has no effect on hAβ40 and hAβ42 plasma levels in hAPP mice. a hAβ40 levels (pg/ml) and b hAβ42 levels (pg/ml) in plasma samples from WT, hAPP, hAPP-NCZ and hAPP-NCZ/CSA mice. Data are given for each animal: WT (n = 10), hAPP (n = 15), hAPP-NCZ (n = 15), hAPP-NCZ/CSA (n = 15). Statistics: ANOVA; Data between groups are not significantly different

Fig. 5

NCZ lowers Aβ levels in brain capillaries. Representative confocal images of isolated brain capillaries immunostained for a hAβ40 and b hAβ42. Data after digital image analysis of membrane-associated c hAβ40- and (D) hAβ42-immunofluorescence using ImageJ. Statistics: Data per group are given as mean ± S.E.M. for 10 capillaries from one preparation [pooled tissue: WT (n = 10), hAPP (n = 15), hAPP-NCZ (n = 15), hAPP-NCZ/CSA (n = 15)]. Shown are arbitrary fluorescence units (scale 0–255). For hAβ40 p < 0.001; ***, significantly lower than control. For hAβ42, data between groups are not significantly different

Fig. 6

NCZ lowers Aβ brain levels. a Western Blot showing hAβ40 and hAβ42 protein expression levels in brain tissue samples from vehicle-treated WT, vehicle-treated hAPP mice, hAPP mice dosed with 5 mg/kg NCZ and hAPP mice dosed with a combination of NCZ and CSA (5/25 mg/kg). β-Actin was used as protein loading control. b hAβ40 and c hAβ42 levels (pg/ml) in brain tissue samples from hAPP, hAPP-NCZ and hAPP-NCZ/CSA mice determined by ELISA. Statistics: ***, significantly lower than control, p < 0.001; **, significantly lower than control, p < 0.01

Fig. 7

Proposed mechanism. Based on our data presented here and our previously published data [22, 23], we propose that blocking Aβ-induced internalization protects P-gp from proteasomal degradation, which preserves P-gp protein expression and transport activity and helps to reduce Aβ brain levels in hAPP mice