Restoring blood-brain barrier P-glycoprotein reduces brain amyloid-beta in a mouse model of Alzheimer's disease

Abstract

Reduced clearance of amyloid-beta (Abeta) from brain partly underlies increased Abeta brain accumulation in Alzheimer's disease (AD). The mechanistic basis for this pathology is unknown, but recent evidence suggests a neurovascular component in AD etiology. We show here that the ATP-driven pump, P-glycoprotein, specifically mediates efflux transport of Abeta from mouse brain capillaries into the vascular space, thus identifying a critical component of the Abeta brain efflux mechanism. We demonstrate in a transgenic mouse model of AD [human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576 strain] that brain capillary P-glycoprotein expression and transport activity are substantially reduced compared with wild-type control mice, suggesting a mechanism by which Abeta accumulates in the brain in AD. It is noteworthy that dosing 12-week-old, asymptomatic hAPP mice over 7 days with pregnenolone-16alpha-carbonitrile to activate the nuclear receptor pregnane X receptor restores P-glycoprotein expression and transport activity in brain capillaries and significantly reduces brain Abeta levels compared with untreated control mice. Thus, targeting intracellular signals that up-regulate blood-brain barrier P-glycoprotein in the early stages of AD has the potential to increase Abeta clearance from the brain and reduce Abeta brain accumulation. This mechanism suggests a new therapeutic strategy in AD.

Fig. 1.

P-glycoprotein mediates hAβ42 transport in mouse brain capillaries. A, representative confocal images of brain capillaries isolated from wild-type mice. Capillaries were incubated with 5 μM fluorescein-hAβ42 for 1 h alone (control) or with 5 μM fluorescein-hAβ42 plus PSC833 (P-glycoprotein inhibitor), NaCN (metabolic inhibitor), RAP (LRP1 inhibitor), FTC (BCRP inhibitor), or LTC₄ (MRP inhibitor). B, capillary luminal fluorescein-hAβ42 fluorescence after image analysis. Residual fluorescence is caused by nonspecific binding (Hartz et al., 2008). Data represent mean ± S.E.M. for 10 capillaries from one preparation (pooled tissue from 10 wild-type mice). Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, P < 0.001. C, proposed two-step mechanism of blood-brain barrier Aβ efflux involving the Aβ receptor LRP1, on the abluminal membrane and the efflux transporter, P-glycoprotein, on the luminal membrane of brain capillaries.

Fig. 2.

P-glycoprotein expression and transport activity are reduced at the blood-brain barrier of hAPP mice. A, representative images of brain capillaries isolated from 12-week-old wild-type and hAPP mice. Capillaries were incubated with 2 μM NBD-CSA, a fluorescent P-glycoprotein-specific substrate, for 1 h alone or with PSC833. B, specific (PSC833-sensitive) luminal NBD-CSA fluorescence after image analysis of brain capillaries. C, luminal fluorescein-hAβ42 fluorescence in brain capillaries from wild-type and hAPP mice. D, luminal fluorescence of the MRP-specific, fluorescent substrate, sulforhodamine 101, in brain capillaries alone (control) or with mannitol (osmotic tight junction disruptor), LTC₄ (MRP inhibitor), NaCN (metabolic inhibitor), PSC833 (P-glycoprotein inhibitor), RAP (LRP1 inhibitor), or FTC (BCRP inhibitor). Data in B–D are mean ± S.E.M. for 10 capillaries from one preparation (pooled tissue from 10–20 mice per group). Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, P < 0.001. E and F, Western blots for P-glycoprotein (P-gp) (E) and LRP1, RAGE, and GLUT-1 (F) of brain capillary membranes from wild-type and hAPP mice. β-Actin was used as protein loading control (pooled tissue from 20 mice per group).

Fig. 3.

PXR activation in hAPP mice restores P-glycoprotein expression and transport of NBD-CSA. A, P-glycoprotein (P-gp) Western blot of brain capillary membranes isolated from vehicle-treated wild-type and hAPP mice and hAPP mice dosed with 25 mg/kg PCN once a day for 7 days. β-Actin was used as protein loading control (pooled tissue from 20 mice per group). B, accumulation of NBD-CSA in brain capillaries from vehicle-treated wild-type and hAPP mice and PCN-treated hAPP mice. Capillaries were incubated with 2 μM NBD-CSA for 1 h alone or with PSC833. C, data after capillary image analysis. Data are mean ± S.E.M. for 10 capillaries from one preparation (pooled tissue from 20 mice per group). Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, P < 0.001.

Fig. 4.

PXR activation in hAPP mice restores P-glycoprotein-mediated hAβ42 transport. A, representative images of brain capillaries isolated from wild-type, hAPP, and PCN-treated hAPP mice. Capillaries were incubated with 5 μM fluorescein-hAβ42 for 1 h. B, data after capillary digital image analysis. Data are mean ± S.E.M. for 10 capillaries from one preparation (pooled tissue from 20 mice per group). Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, P < 0.001.

Fig. 5.

Western blots for indicated proteins of capillary membranes from vehicle- and PCN-treated hAPP mice (pooled tissue from 20 mice per group).

Fig. 6.

Restoring P-glycoprotein in hAPP mice reduces Aβ levels in brain capillaries. A and B, representative images of hAβ40-immunostained (A) and hAβ42-immunostained (B) brain capillaries from vehicle- and PCN-treated hAPP mice. C and D, data from membrane hAβ-immunofluorescence analysis. Data are mean ± S.E.M. for 10 capillaries (pooled tissue from 20 mice per group). Shown are arbitrary fluorescence units (scale 0–255). *, significantly lower than control, P < 0.05; **, significantly lower than control, P < 0.01. E, hAβ40 and hAβ42 Western blots of brain capillary membranes.

Fig. 7.

Restoring P-glycoprotein in hAPP mice reduces Aβ brain levels. A, hAβ40, hAβ42, and hAPP Western blots of total brain from vehicle- and PCN-treated hAPP mice. B and C, hAβ40 (B) and hAβ42 (C) ELISA analysis of brain tissue from vehicle- and PCN-treated hAPP mice (pooled tissue from 19 vehicle-treated hAPP mice and 20 PCN-treated hAPP mice). ***, significantly lower than control, P < 0.001.