Inhibition of ADAM10 promotes the clearance of Aβ across the BBB by reducing LRP1 ectodomain shedding

ADAM10 endothelial KO mice were kindly provided, by Dr. Carl Blobel (Hospital for Special Surgery, New York, NY), Dr. B. De Strooper (VIB Center for the biology of Disease, Leuven, Belgium) and Dr. P. Saftig (Institute of Biochemistry, CAU Kiel, Germany). These mice were generated by crossing ADAM10 flox/flox mice [19] with Tie2-Cre transgenic mice that use the endothelial specific promoter Tie2 to drive the expression of Cre recombinase [20] to produce ADAM10 flox/flox/Tie2-Cre mice. The ADAM10 endothelial KO mice lack ADAM10 expression in the endothelial cells [21]. Wild Type mice on a C57BL/6 background were used as control animals. To evaluate the effect of ADAM10 modulation in an animal model of AD, we utilized transgenic mice overexpressing the human APP695 sw mutation and the presenilin-1 mutation M1 46L (PSAPP) which results in the overproduction of human Aβ [22]. All mice were group housed in a temperature and humidity controlled environment on a 12 h light/dark cycle with free access to food and water. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the Roskamp Institute.

Using a standard protocol to limit aggregation, both human recombinant Aβ42 and fluorescein labeled Aβ42 were solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol to acquire a monomeric/dimeric sample and minimize the formation of β-sheet structures as we previously described [23].

Human Brain Microvessel endothelial cells (HBMECs) (ScienCell, USA) were seeded at 50,000 cells per cm² onto fibronectin-coated 6-well plates as previously described by our group [24]. When approximately 90 % confluent, cells were treated with Aβ42 (2 μM) or both Aβ42 and the ADAM10-selective inhibitor GI254023X (1 μM) (Sigma-Aldrich, USA) and incubated at 37 °C for 48 h. Our previous work demonstrated that 2 μM Aβ significantly induced LRP1 shedding in the absence of any overt cellular toxicity [25]. After the 48 h incubation, the extracellular media was collected and the level of soluble LRP1 was assessed using an ELISA for LRP1 (Cat# SEB010Hu, Cedar Lane Labs, USA).

The impact of ADAM10 on Aβ42 transcytosis was assessed using an in vitro BBB model previously described by our group [24]. HBMECs were seeded onto fibronectin-coated 24-well membrane inserts forming a polarized monolayer representing the BBB. Aβ transcytosis across the BBB was assessed by exposing the basolateral side (“brain”) to 2 μM fluorescein-Aβ42 (rPeptide, USA) and treating the apical side (“blood”) with the ADAM10-selective inhibitor GI254023X (0.1–1 μM). After 60 min of incubation at 37 °C, samples were collected from the apical compartment to assess the basolateral-to-apical transcytosis of fluorescein-Aβ across the BBB model. To ensure Aβ42 or GI254023X treatment did not disrupt the integrity of the BBB monolayer, we examined the effect of Aβ and GI254023X on the permeability of the paracellular marker, 10 kDa Lucifer yellow-dextran.

To determine the role of ADAM10 in the clearance of Aβ42 from the brain to the periphery, we examined the appearance of human Aβ42 in the plasma after intracranial Aβ administration as previously described by our group [24, 26]. Briefly, wild-type and ADAM10 endothelial KO mice (16–24 weeks of age) were anesthetized via inhalation using a 3 % isoflurane/oxygen mix. While under anesthesia, 3 μl of human Aβ42 (1 mM) was bilaterally injected into the caudate putamen of the brain (0.5 mm anterior to the bregma and 2 mm lateral to the midline at a depth of 3 mm below the skull surface). Ten minutes after the intracerebral injections, the mice were euthanized and the plasma and brain tissue were collected. Plasma samples were analyzed for human Aβ42 using an ELISA (Invitrogen, USA). Mouse brains were homogenised in 12 ml of ice cold Hanks Balances Salt Solution (HBSS) with a Dounce Homogenizer. For collection of the soluble brain fraction (i.e., non cell-associated), samples were centrifuged at 6000g for 15 min to remove intact cells, cell associated proteins and non-soluble components. The levels of LRP1 in the soluble fraction were measured in the supernatant using a LRP1 ELISA (Cat# SEB010Hu, Cedar Lane Labs, USA) and normalized to the total protein content in the brain homogenate as determined by the bicinchoninic acid (BCA) protein assay (Thermo Scientific, USA). Soluble LRP1 levels were expressed as ng of LRP1 per mg protein.

PSAPP mice at 35 weeks of age were injected intraperitoneally with 200 mg/kg of GI254023X or vehicle (DMSO) once per day for 5 consecutive days. At this age, PSAPP mice display elevated levels of Aβ peptides in the brain [22]. This treatment regime has previously been shown to effectively inhibit ADAM10 activity in mice and was well tolerated with no adverse effects reported [27]. One hour after the final injection, the mice were euthanatized and plasma and brain tissue were collected. The right hemisphere was used to examine the levels of soluble and insoluble Aβ40 and Aβ42 in the brain. Briefly, the hemispheres were homogenized by sonication in 700 μl lysis buffer [M-PER + 1 % EDTA +0.2 % PMSF (Thermo Scientific, USA)] supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, USA) on ice before centrifugation at 14000g for 30 min at 4 °C. 100 μl of the resulting supernatant was mixed with 5 M guanidine in TRIS buffer resulting in the guanidine soluble (GS) fraction. For the guanidine insoluble (GI) fraction, 100 μl of the guanidine stock was combined with the original tissue pellet. Both GS and GI fractions were subsequently incubated at room temperature for 1 h and were mixed every 15 min. All samples were stored at −80 °C prior to analysis. Quantification of Aβ40 and Aβ42 in the GS, GI, and plasma fractions was determined using an ELISA for human Aβ40 and Aβ42 (Invitrogen, USA) and expressed as ng of Aβ per μg total protein. The left hemisphere was used to examine the levels of soluble LRP1 in the brain. Here, the hemisphere was homogenized using a Dounce homogenizer in ice cold in HBSS using the same procedure as the in vivo LRP1 shedding studies above. LRP1 levels in the soluble brain fraction and plasma were assessed using a mouse ELISA for LRP1 (Cedar Lane Labs, USA). Soluble brain LRP1 was normalized to the total protein content in the brain homogenate as determined by the BCA protein assay (Thermo Scientific, USA). Soluble brain LRP1 levels were expressed as ng of LRP1 per mg protein. Plasma LRP1 levels were expressed as ng/ml. In addition, sAPPα levels were examined in the brain homogenate of these same animals using an ELISA for sAPPα (IBL international, USA). The sAPPα levels were normalized to the total protein content in the brain homogenate as determined by the BCA protein assay and expressed as ng of sAPPα per mg protein.

Statistical analyses were performed using a one-way ANOVA followed by Tukey’s post hoc analysis (GraphPad Prism 5, GraphPad Software Inc., USA). Moreover, where only 2 groups were compared, unpaired t tests were used to evaluate statistical significance.

Exposure of HBMECs to Aβ42 induced a significant increase in the extracellular shedding of LRP1 compared to control conditions (twofold) (Fig. 1). Treatment with the ADAM10 inhibitor GI254023X completely abrogated the effect of Aβ42 on shedding as extracellular LRP1 levels in the presence of GI254023X were the same as that observed under control conditions. The transcytosis of fluorescein-Aβ across an in vitro BBB model was increased following GI254023X treatment in a dose dependent manner (Fig. 2). Significant increases were observed at concentrations higher than 1 μM GI254023X showing effects of 1.25-fold or greater. Of note, no significant difference was observed between control conditions and any of the GI254023X concentrations on the BBB permeability of the paracellular marker Lucifer yellow-dextran, indicating the treatment conditions did not impact BBB integrity (data not shown).

LRP1 levels in the soluble brain fraction of ADAM10 endothelial KO mice were lower than that observed in wild-type animals (Fig. 3a), although this effect did not achieve statistical significance. However, Aβ clearance across the BBB was significantly greater in ADAM10 endothelial KO mice compared to wild-type animals resulting in an increase of approximately 1.75-fold (Fig. 3b).

To evaluate the impact of ADAM10 inhibition on Aβ tissue levels and LRP1 shedding in the brain in an AD animal model, PSAPP mice were treated with the ADAM10-selective inhibitor GI254023X. The level of soluble LRP1 in the brain was significantly lower in the GI254023X-treated mice compared to the vehicle control animals (a reduction of 60 %) (Fig. 4a). In the periphery, LRP1 levels in the plasma were not significantly different when comparing treated and control animals (Fig. 4b). To assess the effect of ADAM10 inhibition on the α-secretase cleavage of APP, the levels of sAPPα in the brain were examined following GI254023X treatment. No significant difference in the level of sAPPα in the brain was detected between GI254023X-treated mice and the control group (Fig. 5). To investigate the impact of ADAM10 inhibition on the levels of Aβ40 and Aβ42 in the plasma and whole brain homogenate of PSAPP mice. GI254023X administration significantly increased the levels of Aβ40 in the plasma (1.45-fold) compared to vehicle-treated mice, while the effect of GI254023X on Aβ42 plasma levels was not significant (Fig. 6a, b). In addition, treatment with the ADAM10 inhibitor reduced both soluble and insoluble Aβ40 (Fig. 7a, b) (1.15- and 1.20-fold respectively) and Aβ42 (Fig. 7c, d) (1.25 and 1.20-fold respectively) levels in the brain compared to vehicle-treated animals, although these values did not reach statistical significance. It should also be noted that this GI254023X treatment regimen was well tolerated as the treated animals did not display any overt changes in appearance, behavior, or weight loss.