Inhibition of mTOR protects the blood-brain barrier in models of Alzheimer's disease and vascular cognitive impairment

Abstract

An intact blood-brain barrier (BBB) limits entry of proinflammatory and neurotoxic blood-derived factors into the brain parenchyma. The BBB is damaged in Alzheimer's disease (AD), which contributes significantly to the progression of AD pathologies and cognitive decline. However, the mechanisms underlying BBB breakdown in AD remain elusive, and no interventions are available for treatment or prevention. We and others recently established that inhibition of the mammalian/mechanistic target of rapamycin (mTOR) pathway with rapamycin yields significant neuroprotective effects, improving cerebrovascular and cognitive function in mouse models of AD. To test whether mTOR inhibition protects the BBB in neurological diseases of aging, we treated hAPP(J20) mice modeling AD and low-density lipoprotein receptor-null (LDLR^-/-) mice modeling vascular cognitive impairment with rapamycin. We found that inhibition of mTOR abrogates BBB breakdown in hAPP(J20) and LDLR^-/- mice. Experiments using an in vitro BBB model indicated that mTOR attenuation preserves BBB integrity through upregulation of specific tight junction proteins and downregulation of matrix metalloproteinase-9 activity. Together, our data establish mTOR activity as a critical mediator of BBB breakdown in AD and, potentially, vascular cognitive impairment and suggest that rapamycin and/or rapalogs could be used for the restoration of BBB integrity. NEW & NOTEWORTHY This report establishes mammalian/mechanistic target of rapamycin as a critical mediator of blood-brain barrier breakdown in models of Alzheimer's disease and vascular cognitive impairment and suggests that drugs targeting the target of rapamycin pathway could be used for the restoration of blood-brain barrier integrity in disease states.

Keywords: blood-brain barrier; brain endothelium; cerebrovasculature; mammalian/mechanistic target of rapamycin; rapamycin.

Fig. 1.

Mammalian/mechanistic target of rapamycin (mTOR) inhibition prevents blood-brain barrier (BBB) breakdown in hAPP(J20) mice. A: representative two-photon images from transgenic hAPP(J20) mice and nontransgenic [wild-type (WT)] littermates treated with vehicle or rapamycin-supplemented diet showing extravasation of intravenous FITC-dextran at 5−30 min after injection to indicate BBB breakdown. Scale bar = 100 μm. B: quantitative analyses of rhodamine-dextran extravasation, calculated as background intensity at 30 min postinjection as a percentage of background intensity at 5 min postinjection. *Different from WT + vehicle by Tukey’s multiple-comparison post hoc test [q(10) = 5.01, P = 0.023] applied to a genotype × treatment interaction [F(1,10) = 4.80, P = 0.05]. Values are means ± SE; n = 3–4 mice/group.

Fig. 2.

Mammalian/mechanistic target of rapamycin (mTOR) inhibition attenuates blood-brain barrier (BBB) breakdown in high-fat diet (HFD)-fed low-density lipoprotein receptor-null (LDLR^−/−) mice modeling atherosclerosis and vascular cognitive impairment. A: representative double-immunofluorescence microscopy images of cortical vasculature (illuminated with tomato lectin and fibrinogen, a BBB-impermeable serum protein) from wild-type (WT) and LDLR^−/− mouse brains. Rapa, rapamycin. Magnification: ×40. B: quantitative analyses of fibrinogen extravasation, measured as increased fibrinogen-positive area relative to tomato lectin-positive area. Results were normalized to WT controls. **Different from WT and LDLR^−/− by Tukey’s multiple-comparison post hoc test [q(15) > 6.14, P < 0.003 for both comparisons]. ns, Not significant. *Significant difference between LDLR^−/− + HFD + vehicle (Veh) and LDLR^−/− + HFD + rapamycin by Tukey’s multiple-comparison post hoc test [q(15) = 4.55, P = 0.03]. Post hoc tests were applied to a significant omnibus ANOVA [F(3,15) = 10.12, P = 0.0007]. Values are means ± SE; n = 4–6 mice/group.

Fig. 3.

Mammalian/mechanistic target of rapamycin (mTOR) attenuation increases junctional adhesion molecule A (JAM-A) in cultured endothelial cells and preserves JAM-A expression in cerebromicrovasculature isolated from hAPP(J20) and low-density lipoprotein receptor-null (LDLR^−/−) mice. A–C: increased expression of JAM-A, but not claudin-5 or zonula occludens-1 (ZO-1), in confluent cultures of endothelial cells treated for 24 h with rapamycin. Representative Western blots are shown below graphs. *Significantly different from control [q(50) = 4.13, P = 0.014] by Tukey’s post hoc test applied to a significant one-way ANOVA [F(2,50) = 6.82, P = 0.002]. Values are means ± SE; n = 17–18 mice/group. D: trend toward reduced JAM-A expression in the brain microvasculature of hAPP(J20) mice treated with vehicle (Veh) [P = 0.06, t(26) = 2.45] indicated by Sidak’s post hoc comparisons with a control [wild-type (WT) + vehicle] group. Values are means ± SE; n = 7–8 mice/group. E: increased cerebromicrovascular JAM-A expression in brains of rapamycin (Rapa)-treated compared with vehicle-treated high-fat diet (HFD)-fed LDLR^−/− mice. *q(27) = 4.59, P = 0.016 by Tukey’s post hoc test applied to a significant one-way ANOVA [F(3,27) = 3.65, P = 0.025]. Values are means ± SE; n = 7–8 mice/group. F: JAM-A dimerization may be restored by rapamycin treatment in HFD-fed LDLR^−/− mice compared with vehicle treatment. ^t(10) = 2.45, P = 0.034 by Student’s t-test for comparison of contrasts of interest; however, one-way ANOVA failed to reach significance [F(3.20) = 1.94, P = 0.16]. Data were normalized to the control group. Values are means ± SE; n = 6 mice/group. In representative Western blots, the last two conditions are reversed compared with the graphs above.

Fig. 4.

Hyperactivation of mammalian/mechanistic target of rapamycin (mTOR) induces blood-brain barrier (BBB) dysfunction in vitro. A: in vitro barrier function is established at day 7 in culture. B: in vitro endothelial cell barrier disruption is induced by overactivation of mTOR with excess leucine. ***Significantly different from control [q(92) = 6.39, P = 0.0001]. ####Significantly different from rapamycin [q(92) = 9.24, P < 0.0001]. Attenuation of mTOR activity with rapamycin produced a trend toward improved barrier function compared with control, as indicated by P = 0.11, with q(92) = 3.21. Tukey’s multiple-comparison post hoc tests were applied to a significant omnibus ANOVA [F(3.92) = 14.66, P < 0.0001]. Values are means ± SE; n = 21–27 mice/group. C: mTOR activity modulates ribosomal protein S6 (rpS6) phosphorylation. ****Significant reduction of rpS6 phosphorylation by mTOR inhibition with rapamycin vs. control [q(12) = 20.59, P < 0.0001]. *Significant increase in rpS6 phosphorylation by mTOR activation with leucine vs. control [q(12) = 4.60, P = 0.18]. Tukey’s post hoc tests were applied to a significant ANOVA [F(2,12) = 180.0, P < 0.0001]. Values are means ± SE; n = 5 mice/group.

Fig. 5.

Matrix metalloproteinase (MMP)-9 activity is implicated in amyloid-β (Aβ)- and leucine-induced in vitro blood-brain barrier (BBB) breakdown. A: rapamycin-induced attenuation of mammalian/mechanistic target of rapamycin (mTOR) decreases pro-MMP-9 expression in cultured endothelial cells. OD, optical density. *Significant decrease vs. control [q(15) = 3.95, P = 0.034] by Tukey’s post hoc test on a significant one-way ANOVA [F(2,15) = 8.58, P = 0.003]. Values are means ± SE; n = 6 mice/condition. B: MMP-9 mediates Aβ-induced BBB breakdown in vitro as assessed by FITC-dextran leakage. Three-way ANOVA revealed significant main effects: MMP-9 [F(1,168) = 50.5, P < 0.0001], Aβ [F(1,168) = 30.2, P < 0.0001], and mTOR [F(2,168) = 14.6, P < 0.0001] activity. Holm-Sidak’s multiple-comparison post hoc test revealed that Aβ induced BBB permeability in control cells (****t = 4.96, P < 0.0001). mTOR overactivation with leucine was sufficient to induce barrier disruption (**t = 4.1, P = 0.0029) and exacerbate Aβ-induced barrier permeability (****t = 6.67, P < 0.0001). Attenuation of mTOR with rapamycin treatment prevented Aβ-induced disruption of barrier function (t = 2.92, P = 0.15). Furthermore, MMP-2/MMP-9 inhibition prevented barrier disruption by Aβ in control cells (^^^t = 4.58, P = 0.0005), barrier permeability induced by mTOR overactivation in leucine-treated cells (^t = 3.33, P = 0.044), and exacerbation of barrier permeability by Aβ in the presence of hyperactive mTOR (^^^^t = 7.06, P < 0.0001). Data were normalized to the control group and are expressed as fold change. Values are means ± SE; n = 15 mice/condition.