Experimental Design and Statistical Analysis
Sample sizes (i.e., animal numbers, brain capillary sample size, number of liver tissue samples) were based on power analyses of preliminary data and past published work (9, 22, 23, 25). Sample size and the number of repetitions are reported in the Results section and the corresponding figure legends.
Results are presented as mean ± SEM, or percent change, as indicated. One-way analysis of variance, with factor treatment group, was used to assess mean differences in outcomes. Tukey’s HSD post-hoc test was used to preserve the family-wise Type 1 error rate at 5%. Post hoc tests were not performed when the omnibus F statistic was not significant. Statistical analyses were completed using SAS 9.4® (SAS Institute, Inc.; Cary, NC, USA).
Animals
The Institutional Animal Care and Use Committee at the University of Minnesota (Protocol #1110A05865, principal investigator; PI: Hartz, AMS) approved all animal experiments, which were conducted in accordance with AAALAC regulations, the Guide of the Care and Use of Laboratory Animals of the NIH, and the US Department of Agriculture Animal Welfare Act.
Male wild type mice (WT; RRID: IMSR_TAC:2789; n = 10) and male transgenic hAPP-overexpressing mice (Tg2576 strain; 129S6.CgTg(APPSWE2576Kha;RRID:IMSR_TAC:2789; n = 45) were acquired from Taconic Farms (Germantown, NY, USA). Mice were received at age 8–12 weeks and were housed individually in an AAALAC-accredited temperature-and-humidity-controlled vivarium (23 °C, 35% relative humidity, 12 h light-dark cycle) and allowed to habituate to their environment for two weeks prior to experiments. Mice had ad libidum access to tap water and standard rodent feed (Harlan Teklad Chow 2918, Harlan Laboratories Inc., Indianapolis, NJ, USA). Mice used for experiments were 10–14 weeks old and had a mean body weight of 27.4 ± 1.1 g (mean ± SD) and 27.6 ± 2.7 g (mean ± SD) for WT and hAPP strains, respectively.
Chemicals
Antibodies against β-actin (ab8226; RRID: AB_306371), human Aβ40 (ab12265; RRID:AB_298985), human Aβ42 (ab12267; RRID:AB_298987), as well as cyclosporin A (CSA; ab120114) were purchased from Abcam (Cambridge, MA, USA). Modified Dulbecco’s phosphate buffered saline (DPBS) with 0.9 mM Ca2+ and 0.5 mM Mg2+ was purchased from HyClone (Logan, UT, USA). Complete™ protease inhibitor was purchased from Roche (Mannheim, Germany). C219 antibody against P-gp was purchased from ThermoFisher (MA126528; RRID:AB_795165; Waltham, MA, USA). Fluorescein-hAβ42 [fluorescein-Aβ(1− 42)] was purchased from rPeptide (Bogart, GA, USA). [N-(4-nitrobenzofurazan-7-yl)-D-Lys8]-cyclosporin A (NBD-CSA) was custom-synthesized by R. Wenger (Basel, Switzerland; (26)). PSC833 was a kind gift from Novartis (Basel, Switzerland). Nocodazole, the ALT Assay Kit (MAK052), CelLytic™ M, Ficoll® PM 400, bovine serum albumin and all other chemicals were purchased at the highest grade from Sigma-Aldrich (St. Louis, MO, USA).
NCZ Dosing
Mice were randomly divided into four treatment groups (Group 1: WT mice treated with vehicle, n = 10; Group 2: hAPP mice treated with vehicle, n = 15; Group 3: hAPP mice treated with nocodazole (NCZ), n = 15; and Group 4: hAPP mice treated with cyclosporin A (CSA) and NCZ, n = 15). Mice were treated for a total of 14 days; the dosing regimens for NCZ and CSA are shown in Table 1. Briefly, mice in Groups 1 and 2 were treated daily with vehicle (i.p.) every three days for the entire duration of the experiment. Mice in Groups 3 and 4 were treated with 5 mg/kg of NCZ (i.p.) every 3 days (days 1, 4, 7, 10, and 13). On days when no NCZ was given, mice in Group 3 were treated with vehicle (p.o.) and mice in Group 4 were treated with 25 mg/kg CSA (p.o.).
Table 1
Treatment Schedule.
Blood and Tissue Collection
At the end of the 14-day treatment period, all mice were euthanized by CO2 inhalation and decapitated; brain tissue, trunk blood, and liver were collected from each animal. Blood was stored in heparinized tubes and plasma was extracted from blood by centrifugation at 5,000 g for 5 min at 4 °C. Brain and liver tissue were frozen in liquid nitrogen at the time of collection and stored at -80 °C until further analysis.
ALT Assay
Alanine aminotransferase activity (ALT) in liver tissue lysate was measured using an ALT activity kit (MAK052; Sigma-Millipore, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, from each mouse 40 mg of collected liver tissue were homogenized in 300 µl of ALT Assay Buffer using a Power Gen 125 tissue homogenizer (Thermo Fisher Scientific, Hampton, NH, USA). Homogenized liver samples were centrifuged at 15,000 g for 15 min at 4 °C. Supernatant containing ALT from each liver sample was diluted 1:100 for ALT activity determination. 20 µl of the diluted samples was pipetted in duplicates onto a 96-well microplate. Wells were treated with ALT Master Mix solution. Changes in colorimetric intensity was measured at 570 nm over 30 min (5 min reading intervals, 37ºC) using a Synergy™ H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT, USA).
The amount of generated pyruvate, a measure of ALT activity, was determined using a standard curve. ALT activity was calculated using Eq. (1).
ALT activity is reported as nmole/min/ml = milliunit/mL (mU/ml), where one milliunit (mU) of ALT is defined as the amount of enzyme that generates 1.0 nmole of pyruvate per minute at 37°C.
Tissue Harvest and Brain Capillary Isolation
Brain capillaries were isolated as previously described (22, 23, 27). Following euthanasia with CO2, brains were collected, dissected, and cleaned of meninges. Frontal cortex tissue (~ 10 mg/brain) was collected, snap frozen in liquid nitrogen, and stored at -80 °C for Aβ analysis. The remaining brain tissue was homogenized in Ca2+/Mg2+-containing DPBS supplemented with 5 mM D-glucose and 1 mM sodium pyruvate. The brain homogenate was mixed with a 15% Ficoll→ PM 400 solution and centrifuged at 5,800 g for 20 min at 4 °C to yield capillary-containing pellets. The pellets were separated and resuspended in 1% BSA-DPBS solution and the capillary suspension was filtered through a 300 µm nylon mesh and then passed through a glass bead column (glass bead diameter: 0.45–0.5 mm) with 1% BSA-DPBS. Capillaries were washed off the glass beads with 1% BSA-DPBS. The resulting capillary suspension was centrifuged at 1,500 g for 3 min at 4ºC and the pellet was washed three times with DPBS. Isolated capillaries were used for transport experiments, isolation of capillary crude membranes, or stored for later analysis at -80ºC.
Capillary Crude Membrane Isolation
Capillary crude membranes were obtained from freshly isolated brain capillaries as described in previous studies (27). Isolated brain capillaries were homogenized in a cell lysis buffer (CellLytic™ M, Sigma-Aldrich, St. Louis, MO, USA) containing Complete™ protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The capillary homogenate was centrifuged at 10,000 g for 15 min at 4 °C to separate organelles and cellular debris. The supernatant was then centrifuged at 100,000 g for 90 min at 4 °C to obtain a pellet containing brain capillary crude membranes. Crude membranes were resuspended in buffer and stored at -80 °C.
Western Blotting
Protein expression levels were determined by Western blot analysis as described in previous studies (9, 22). Protein concentrations of brain capillary crude membrane samples and brain lysate samples were determined with the Bradford assay. The Invitrogen NuPage® Bis-Tris electrophoresis and blotting system was used to perform all Western blots. Following protein transfer, blotting membranes were blocked and incubated overnight with the primary antibody (P-gp: C219, 1 µg/ml; hAβ40: 1 µg/ml; hAβ42: 1 µg/ml; β-Actin: 1 µg/ml). Blotting membranes were washed and incubated for 1 hour with horseradish peroxidase-conjugated ImmunoPure secondary IgG antibody (1:5000, 0.15 µg/ml; Thermo Fisher Scientific, Waltham, MA, USA). Proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were visualized using a Bio-Rad ChemiDoc XRS + gel documentation system. Optical density and digital molecular weight analyses were performed using Image Lab 5.0 software from Bio-Rad Laboratories (RRID:SCR_014210) and a molecular weight marker (RPN800E; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Linear adjustments of contrast and brightness were applied to entire Western blot images. Nonlinear adjustments were not applied.
P-gp Transport Activity and P-gp-Mediated hAβ42 Transport
P-gp transport activity levels and P-gp-mediated Aβ transport were determined as previously described (9, 28, 29). To determine P-gp transport activity, freshly isolated brain capillaries were incubated with 2 µM of the P-gp-specific substrate NBD-cyclosporin A (NBD-CSA) in DPBS for 1 hour. To assess P-gp-mediated hAβ42 transport activity, brain capillaries were incubated with 5 µM fluorescein-hAβ42 in DPBS for 1 hour. Images of 10 capillaries per treatment group were captured by confocal microscopy using the 488 nm line of an argon laser of a Leica TCS SP5 confocal microscope with a 63 × 1.2 NA water immersion objective (Leica Instruments, Wetzlar, Germany). NBD-CSA fluorescence in the capillary lumen was measured in each image using Image J v.1.48v (Wayne Rasband, NIH, USA; RRID:SCR_003070). Specific, luminal NBD-CSA or fluorescein-hAβ42 fluorescence were measured as the difference between total luminal fluorescence and fluorescence in the presence of 5 µM PSC833, a P-gp-specific inhibitor (9, 28, 29).
Aβ Immunostaining of Brain Capillaries
Freshly isolated brain capillaries were immunostained for hAβ40 and hAβ42 using a method described in previous studies (9). Mouse brain capillaries were fixed with 3% paraformaldehyde/0.25% glutaraldehyde for 30 min at room temperature and subsequently washed with PBS. Fixed capillaries were treated with 0.5% Triton X-100 for 30 min, washed with PBS and blocked with 1% BSA/DPBS for 60 min. Capillaries were incubated overnight using a 1:250 dilution (4 µg/ml) of primary rabbit polyclonal antibody to human Aβ1–40 (hAβ40; ab12265, Abcam, Cambridge, MA, USA; RRID:AB_298985) or primary rabbit polyclonal antibody to human Aβ1–42 (hAβ42; ab12267, Abcam, Cambridge, MA, USA; RRID:AB_298987). Capillaries were washed with 1% BSA/PBS for 60 min and incubated with secondary Alexa-Fluor 488-conjugated goat anti-rabbit IgG (1:1000, 1 µg/ml; Invitrogen, Carlsbad, CA, USA; RRID:AB 2576217) for 1 h at 37 °C. Nuclei were counterstained with 1 µg/ml DAPI (MilliporeSigma, Burlington, MA USA;RRID:SCR_014366). Immunofluorescence of hAβ40 and hAβ42 was visualized by confocal microscopy (Leica TCS SP5 confocal microscope, 62 × 1.2 NA water objective, Leica Instruments, Weltzar, Germany). Brain capillary plasma membrane immunofluorescence of hAβ40 and hAβ42 was measured for each capillary with ImageJ software v1.48 as previously described (9, 23). A 10 × 10 grid was superimposed on each image and fluorescence measurements of capillary membranes were taken between intersecting grid lines. The fluorescence intensity for each capillary was the mean of three measurements per capillary.
hAβ40 and hAβ42 ELISA
Human Aβ40 and Aβ42 levels in plasma and brain samples were determined by ELISA following the manufacturer’s protocols (KHB3482 (sensitivity: < 6 pg/ml) and KHB3442 (sensitivity: < 10 pg/ml); Invitrogen, Camarillo, CA, USA). Plasma samples were centrifuged at 5,000 g for 5 min at 4 °C, and then diluted with standard diluent buffer provided in the kit (hAβ40: 1:50 dilution; hAβ42: 1:4 dilution). Brain samples were homogenized in guanidine Tris-HCl buffer (5M, pH 8) and diluted in DPBS containing 5% BSA and 0.03% Tween-20 (hAβ40: 1:20 dilution; hAβ42: 1:5 dilution). Diluted samples were centrifuged at 16,000 g for 20 min at 4 °C; the supernatant was analyzed by ELISA. Absorbance (450 nm) was measured using a Synergy™ H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT, USA). A standard curve was plotted using Gen5™ software v2.07 to determine the concentration of hAβ40 and hAβ42 in plasma and brain samples; values obtained at 450 nm were corrected for background absorbance; four parameter logistic ELISA curve fitting was used.