Mice and treatments
All animal procedures were approved by the Institutional Animal Care and Usage Committee and in accordance with the National Institute of Health guidelines. The Tg2576 transgenic mice expressing human APP with the Swedish mutation (K670N/M671L) used in these studies were as previously described . They were genotyped by PCR analysis using tail DNA and kept in a pathogen-free environment, on a 12-hour light/dark cycle and had access to food and water ad libitum. All the experiments presented in this paper were performed with female mice. Starting at 7 months of age, mice were randomized to receive MK-591 (40 mg/kg weight) (n = 11) or vehicle (n = 9) in their chow diet for 8 months until they were 15 months old. Considering that each mouse eats on average 5 g/day of chow diet and the diet is formulated for 320 mg MK-591 per kg diet (Harlan Teklad, WI, USA), the final dose of the active drug was approximately 40 mg/kg weight/day. During the study, mice in both groups gained weight regularly, and no significant difference in weight was detected between the two groups. No macroscopic effect on the overall general health was observed in the animals receiving the active treatment. Post-mortem examination showed no sign of macroscopic pathology in any of the organs considered (spleen, liver, thymus, ileum).
After sacrifice, animals were perfused with ice-cold 0.9% PBS, the brain removed and dissected in two by midsagittal dissection. One was immediately stored at −80°C for biochemistry assays or total RNA extraction, and the other immediately immersed in 4% paraformaldehyde in 0.1 M PBS (pH 7.6) overnight for immunohistochemistry studies.
Immunostaining was performed as reported previously by our group [8, 9]. Serial 6-μm-thick coronal sections were mounted on 3-aminopropyltriethoxysilane-coated slides. Every eighth section from the habenular to the posterior commissure (8 to 10 sections per animal) was examined using unbiased stereological principles. The sections for Aβ were deparaffinized, hydrated, pretreated with formic acid (FA; 88%) and subsequently with 3% hydrogen peroxide in methanol. The sections for glial acidic fibrillary protein (GFAP) and CD45 were deparaffinized, hydrated and treated with 3% hydrogen peroxide in methanol and subsequently antigen retrieved with citrate (10 mM). Sections were blocked in 2% fetal bovine serum before incubation with primary antibodies (4 G8 for Aβ, anti-GFAP, and anti-CD45) overnight at 4°C. Subsequently, sections were incubated with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) and then developed using the avidin-biotin complex method (Vector Laboratories) with 3,3’-diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by Aβ-immunoreactivity using the software Image-Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD, USA). The threshold optical density that discriminated staining from background was determined and kept constant for all quantifications. The area occupied by Aβ-immunoreactivity was measured by the software and divided by the total area of interest to obtain the percentage area of Aβ-immunoreactivity.
Mouse brain homogenates were sequentially extracted first in radioimmunoprecipitation assay (RIPA) for the Aβ soluble fractions and then in FA for the Aβ insoluble fractions as previously described [5, 8, 9]. Aβ1-40 and Aβ1-42 levels were assayed by a sensitive sandwich ELISA kits (WAKO Chem, Richmond, VA, USA.). Interleukin 1-β (IL-1β) levels in brain homogenates were assayed by a specific and sensitive sandwich ELISA kit, following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Supernatants from the cell culture experiments were also assayed for their levels of lactate dehydrogenase (LDH) by a colorimetric assay kit (BioVision, Milpitas, CA, USA). Analyses were always performed in duplicate and in a coded fashion.
Western blot analyses
RIPA extracts from brain homogenates were used for western blot analyses. Samples were electrophoresed on 10% Bis-Tris gels or 3% to 8% Tris-acetate gel (Bio-Rad, Richmond, CA, USA), according to the molecular weight of the target molecule, transferred onto nitrocellulose membranes (Bio-Rad), and then incubated with appropriate primary antibodies as follows: anti-APP N-terminal raised against amino acids 66 to 81 for total APP (22 C11, Chemicon International, Temecula, CA, USA), anti-BACE-1 (IBL America, Minneapolis, MN,USA), anti-ADAM-10 (Chemicon), anti-secreted-APPα (2B3, IBL America), anti-secreted-APPβ (6A1, IBL America), anti-C-terminal fragments (CTF; EMD Biosciences, Inc., Billerica, MA, USA), anti-presenilin1 (PS1; Sigma, St. Lousi, MO,USA), anti-nicastrin (Cell Signaling, Daners, MA, USA), anti-anterior pharynx-defective 1 (APH-1; Millipore, Billerica, MA, USA), anti-presenilin enhancer 2 (Pen-2; Invitrogen, Grand Island, NY, USA), anti-GFAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-5LO (BD Transduction Laboratories, San Jose, CA, USA); anti-Notch intracellular domain (NICD; Cell Signaling), anti-insulin-degrading enzyme (IDE) N-terminal (EMD Biosciences), anti-neprilysin (Santa Cruz Biotechnology, Inc.), anti- apolipoprotein E (Santa Cruz Biotechnology, Inc.), anti-cAMP response element-binding protein (CREB; Cell Signaling) and anti-phosphorylated-CREB (Cell Signaling), anti-Sp1 (Santa Cruz Biotechnology, Inc.) and anti-β actin (Santa Cruz Biotechnology, Inc.).
After three washings with T-TBS (Tween-Tris Buffered Saline), membranes were incubated with IRDye 800CW or IRDye 680CW-labeled secondary antibodies (LI-COR Bioscience, Lincoln, NE, USA) at 22°C for 1 h. Signals were developed with Odyssey Infrared Imaging Systems (LI-COR Bioscience). β-actin was always used as internal loading control.
Real-time quantitative RT-PCR amplification
RNA was extracted and purified using the RNeasy mini-kit (Qiagen, Valencia, CA, USA), as previously described . Briefly, 1 μg of total RNA was used to synthesize cDNA in a 20 μL reaction using the RT²First Strand Kit RT-PCR (Super Array Bioscience, Valencia, CA, USA). Mouse BACE-1, PS1, nicastrin, APH-1 and Pen-2 genes were amplified by using the corresponding primers designed and synthesized by Super Array Bioscience. β-actin was always used as an internal control gene to normalize for the amount of RNA. Real-time PCR was performed in an Eppendorf ep realplex thermal cycler (Eppendorf, Hauppauge, NY, USA). Two microliters of cDNA was added to 25 μL of SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). Each sample was run in duplicate, and analysis of relative gene expression was done by using the 2-ΔΔCt method . Briefly, the relative change in gene expression was calculated by subtracting the threshold cycle (ΔCt) of the target genes from the internal control gene (β-Actin). Based on the fact that the amount of cDNA doubles in each PCR cycle (assuming a PCR efficiency of 100%), the final fold-change in gene expression was calculated by using the formula relative change = 2-ΔΔCt.
N2A cells stably expressing human APP carrying the K670N/M671L Swedish mutation (APPswe) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (Cellgro, Herdon, VA, USA), and 400 μg/mL G418 (Invitrogen), at 37°C in the presence of 5% CO2 as previously described .
For each experiment, equal numbers of cells were plated in six-well plates; 24 h later media were removed and fresh media containing either MK-591 (1 μM, 10 μM or 25 μM) or vehicle were added. After incubation for 24 h, supernatants were collected for Aβ and LDH measurement, and cell pellets harvested in lytic buffer for immunoblot analyses as described in the previous paragraphs.
For transfection studies, N2A-APPswe cells were transfected with 1 μg Myc-tagged mΔE-Notch-1 complementary DNA overnight (a generous gift from Dr. L. D´Adamio, Albert Einstein Medical College, NY, USA) by using Lipofectamine 2000 (Invitrogen). The media were removed and fresh media containing MK-591, L685,458 or vehicle were added. After incubation for 24 h, cells lysates were collected NICD expression levels assayed by western blot analysis.
Data analyses were performed using SigmaStat for Windows version 3.00. Statistical comparisons were performed by unpaired Student’s t-test or the Mann–Whitney rank sum test when a normal distribution could not be assumed. Values represent mean ± standard error of the mean. Significance was set at P <0.05.