mice were provided by M. Herzig, R. Radde, and M. Jucker (University of Tübingen, Tübingen, Germany) (22
), and FVB.129P2-Abcc1atm1Bor
) mice were purchased from Taconic Farms (24
transgenic mice were crossed to transporter knockout mice for at least 9 generations into the FVB/N background for all strains. All mice were housed in a 12-hour light/12-hour dark cycle at 23°C with free access to food (SNIFF) and water.
For tissue preparation, mice were sacrificed by cervical dislocation and transcardially perfused with PBS. The brain was removed from each mouse, and one hemisphere was stored in buffered 4% PFA for paraffin embedding and immunohistochemistry, while the other hemisphere was snap-frozen in liquid nitrogen and stored at –80°C for biochemical analysis.
ELISA kits (TH40HS, TK42HS: high sensitivity) from the Genetics Company were used for the quantification of Aβ40 and Aβ42 in whole brain hemispheres from which the cerebellum and brain stem had been removed at the level of the midbrain. Hemispheres were homogenized using a PreCellys24 (12 seconds, 6,500 rpm). After addition of carbonate buffer (pH 8.0), homogenates were mixed using the PreCellys (5 seconds, 5,000 rpm) and centrifuged for 90 minutes (4°C) at 24,000 g to separate insoluble from soluble Αβ species. The resulting supernatant (buffer-soluble fraction) was mixed with 8 M guanidine hydrochloride at a ratio of 1:1.6. To extract aggregated Αβ species, the pellet was dissolved in 8 volumes of 5 M guanidine hydrochloride, shaken at room temperature for 3 hours, and centrifuged at 24,000 g for 20 minutes at 4°C. The resulting supernatant represents the guanidine-soluble fraction. Protein contents of all samples were measured in triplicate using a NanoDrop 1000 (Thermo Fisher Scientific). ELISAs were performed according to the manufacturer’s instructions using appropriate dilutions.
Expression analyses of APP and Aβ transporting/cleavage/degrading complex components.
GeneChip Mouse Gene 1.0 Arrays interrogating more than 28,000 murine genes were used to investigate gene expression in APP/PS1×Abcc1–/– and APP/PS1 mice. Array hybridization was performed according to the supplier’s instructions using the Ambion WT Expression Kit (Ambion) plus the GeneChip WT Terminal Labeling and Hybridization Kit and the GeneChip Sample Cleanup Module (Affymetrix). For signal enhancement, an antibody amplification was carried out using a biotinylated antistreptavidin antibody (Vector Laboratories), which was cross-linked by goat IgG (Sigma-Aldrich) followed by a second staining with streptavidin-phycoerythrin conjugate (Molecular Probes; Invitrogen). The microarrays were scanned with the G7 upgraded GeneChip Scanner 3000 (Affymetrix) at 0.7 micron resolution. The data analysis was performed with the Partek Genomics Suite 6.5 software (Partek) using the Robust Multichip Analysis (RMA) algorithm, including an adjustment to the GC content (GCRMA), and employing the quantile normalization method. ANOVA was performed to statistically compare the candidate genes between the 2 groups (APP/PS1 and APP/PS1×Abcc1–/–) of 3 biological replicates each. The data have been deposited at the NCBI Gene Expression Omnibus (GEO GSE30762).
For Western blotting, tissue homogenates were prepared as described by Lesné et al. (37
). Total protein concentrations of the extracts were determined using a BCA assay (Pierce). After electrophoresis of 10 μg total protein per lane, proteins were blotted onto PVDF membranes. After blocking in 5% dry milk in TBST Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature, blots were probed for either APP/Aβ (1:1,000, clone 6E10; Sigma-Aldrich), ADAM10 (1:1,000; Calbiochem), BACE1 (1:1,000; Abcam), IDE (1:500; Abcam), calreticulin (1:1,000; Cell Signaling), caspase-3 (1:1,000; Cell Signaling), COX-IV (1:1,000; Cell Signaling), or β-actin (1:20,000; Sigma-Aldrich) overnight at 4°C. As detection antibodies, anti–mouse-HRP, anti–rat-HRP and anti–rabbit-HRP, respectively, were used. The Amersham ECL Plus Detection Kit and a Roper CoolSnap HQβ Camera (Roper) were used for visualization.
Subcellular fractions of freshly isolated brains were prepared as described by Cox et al. (38
) with slight modifications. Briefly, both hemispheres were dounce homogenized in IH buffer (250 mM saccharose; 10 mM Tris-HCl, pH 7.8; 0.3 mM PMSF; 0.2 mM EDTA; 1 μg/ml leupeptin; 1 μg/ml pepstatin) and centrifuged to pellet nuclei (1,000 g
, 4°C, 10 minutes). The pellet was resuspended in IH buffer and rehomogenized by centrifuging through QIAShredder (QIAGEN) columns. Purification of nuclei was achieved via saccharose gradient centrifugation at 80,000 g
for 40 minutes. Cytosolic, microsomal, and mitochondrial proteins were isolated by centrifugation of the initial supernatant (12,000 g
, 4°C, 15 minutes). Mitochondria in the pellet were isolated using 2 cycles of washes with IH buffer and recentrifugation. The supernatant was centrifuged at 100,000 g
for 60 minutes to pellet microsomes. The supernatant contained pure cytosolic proteins.
Formalin-fixed brains were embedded in paraffin and 4 μm–thick sections were cut. After deparaffinization, the sections were processed using the BOND-MAX Autostainer (Leica Microsystems GmbH/Menarini). Immunostaining was initiated after blocking endogenous peroxidase (5 minutes) and epitope retrieval for 5 minutes with 95% formic acid (for the 6F3D [Dako]), 70% formic acid (for antibody 4G8; Millipore) or 20 minutes with an EDTA buffer at pH 8.5 (for antibody Iba1, Wako; EDTA buffer was supplied by Leica Microsystems GmbH/Menarini). Primary antibodies incubated routinely for 30 minutes at room temperature with dilutions were as follows: Dako clone 6F3D (1:100), 4G8 (1:500), and Iba1 (1:1,000). Primary antibodies were detected with the BOND-MAX Bond Polymer Refine Detection Kit and standard protocol DAB R30. Slides were fully digitized with a resolution of 230 nm using the Mirax Desk and Mirax Midi automated slide scanners, and then semiautomatically analyzed using the AxioVision software package (Zeiss) (23
Assessment of CAA severity.
Brain sections from APPdt mice were stained using the 4G8 antibody. At least 2 nonconsecutive sections were inspected for CAA of meningeal vessels in a blinded fashion. All meningeal vessels were counted manually, and the severity of CAA was categorized as follows: category I, not affected; category II, ≤ 25% of circumference positively stained; category III, 26%–50% of circumference positively stained; category IV, 51%–75% of circumference positively stained; category V, 76%–100% of circumference positively stained (Supplemental Figure 8). The mean number of vessels belonging to each category was calculated relative to the overall number of vessels found.
Mouse brain capillary endothelial cells were prepared as described by Coisne et al. (39
). In brief, at least ten 3- to 4-week-old mice were decapitated and the brains removed. After dissecting the brain stem, white matter, and meninges, tissue was homogenized in 2 volumes of wash buffer B (WBB) (HBSS, 10 mM HEPES, 0.1% BSA) using a 15-ml glass douncer (Wheaton Industries). One volume of 30% dextran solution was added to the homogenate and centrifuged twice at 3,000 g
at 4°C. The pellet containing the vessels was resuspended in WBB, and large vessels were ruptured manually by harsh pipetting of the solution. Vacuum filtration through a 60-μm membrane (SEFAR) was used to separate large vessels from capillaries. After combined collagenase/dispase (HBSS, 10 mM HEPES, 0.15 μg/ml TCLK, 10 μg/ml DNAse-I, 1 mg/ml collagenase/dispase [Roche]) treatment, single-cell suspension was achieved by another harsh pipetting of the solution. Endothelial cells were seeded into Matrigel-coated Transwell inserts (0.4 μm pores, Greiner Bio-One) with a density of 120,000 cells per insert and grown on top of a supportive glial culture. Pe
calculation according to ref. 40
using Lucifer yellow was used to determine paracellular flow during the assays. In brief, the average volume cleared was plotted versus time and the slope was estimated by linear regression analysis. PS is the permeability surface area product (in ml/min), and the calculated slope corresponds to the PSt
. The slope of the clearance curve with the control insert (filter coated with Matrigel without cells) was denoted PSf
. The PS
value for the EC monolayer (PSe
) was calculated as follows: 1/PSe
. The PSe
value was then divided by the surface area of the filters (AO
= 1.13 cm2
) to generate the endothelial permeability (Pe
, in cm/min). Inserts with a Pe
higher than 1.6 × 10–3
cm/min were discarded for Aβ transport analyses. Active Aβ42 transport was calculated as CAβ
)) to control for paracellular flow, where CAβ
refer to the Aβ42 concentration measured and the volume of the luminal chamber, respectively. CLm
corresponds to the amount of Lucifer yellow that passed the Transwell monolayer, and CLi
as the initial amount of Lucifer yellow. For Aβ transport studies, culture medium from the abluminal compartment was replaced with solution containing 10 ng Aβ42 (1.6 nM final concentration). Subsequently, the samples were taken from the luminal compartment after 0.5, 1, 2, 6, and 24 hours. Aβ content was determined by ELISA.
ABCC1 activity assay.
In vitro ABCC1 activity was measured using the SB MRP1 PREDEASY ATPase Kit (Solvo Biotechnology) according to the manufacturer’s instructions. In brief, a provided membrane preparation of Sf9 cells containing human ABCC1 was diluted in assay buffer to provide 8 μg membrane per well. Assay mixtures were prepared either with or without NEM-GS as standard substrate and with or without vanadate to control for nonspecific ATPase activities. Thiethylperazine was diluted in DMSO (Sigma-Aldrich) and added to the appropriate wells (final concentration 1.3 ng/ml). Pipetting was done strictly on ice. After addition of thiethylperazine, the 96-well plate was incubated at 37°C for 10 minutes. The reaction was started by adding 10 μl of 10 mM MgATP. After 10 minutes of incubation at 37°C, the reaction was stopped by adding 100 μl developer. Two minutes later, 100 μl blocking solution was added. The reaction mix was then incubated for a further 30 minutes at 37°C. Finally, 96-well plates were measured using a Paradigm spectrophotometer (Beckman-Coulter) at 610 nm.
The Lilliefors goodness-of-fit test (α = 0.05) was applied to the ELISA data and to the log-transformed ELISA data to distinguish between the assumption of normally distributed sample data and the assumption of log-normally distributed sample data. Despite the small sample size, for both data sets, the null hypothesis was rejected for 5 out of 44 samples (Supplemental Table 4). Consistent with the observation of predominantly positive skew and strict positive sample data, the assumption of normally distributed data was discarded. Mean values and confidence intervals were calculated assuming an underlying log-normal distribution. The Wilcoxon rank-sum test was applied to compare all data from the different mouse strains for each time point (for resulting P values of ELISA measurements, see Supplemental Table 4). P ≤ 0.05 was considered significant.
The experiments were designed to minimize the number of animals used and were approved by local authorities: Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischereiwesen (LALLF) (LALLF M-V/TSD/7221.3-2.3-004/06, LALLF M-V/TSD/7221.3-2.3-003/08, LALLF M-V/TSD/7221.3-1.1-004/11.