Animal and surgery
Adult male Tg2576 mice (APPsw, K670N/M671L) were purchased from Taconic (Hudson, NY, USA). Plaque onset in Tg2576 nice occurs around 9 month of age in the cortex and hippocampus (Hsiao et al., 1996
; Sarsoza et al., 2009
). Therefore, unilateral naris-occlusion was performed on 6 month-old animals, which would allow investigations on whether or not deprivation might affect the timing of plaque onset as well as the progress of age-dependent plaque development. The left or right nostril was cauterized under anesthesia with sodium pentobarbital (50 mg/kg, i.p.). Occluded animals were allowed to survive until they were 7 (n=3), 8 (n=3), 9 (n=4), 12 (n=4), 18 (n=7, including n=3 for assessing β-site APP cleavage activity and ELISA) and 24 (n=7, including n=3 for western blots) month-old.
Animal use was in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Animal Care and Use Committee of Southern Illinois University at Carbondale.
Mice were perfused transcardially with 4% paraformaldehyde in 0.01M phosphate-buffered saline (pH 7.4, PBS) under overdose anesthesia (sodium pentobarbital, 100 mg/kg, i.p.). The brains were carefully dissected out, postfixed in the perfusion fixative overnight at 4 °C, and then cryoprotected with 30% sucrose. The forebrains were cut either perpendicular to the long axis of the bulbs (7-8 month-old mice), or in parallel to the ventral surface of the bulbs (9-24 month-old mice). The latter preparation was especially suitable for concurrent analyses of labelings in the bulb and piriform cortex. Twelve sets of 30 μm sections across the entire bulb (together with the cortex) were collected in order in PBS in cell culture plates. For double immunofluorescence, 4 sets of 8 μm sections around the middle bulb levels in each brain were also collected by thaw-mounting on Superfrost Plus slides (VWR, West Chester, PA, USA). Before section collection, small needle punches were made around the center of the bulb, piriform cortical white matter or the striatum contralateral to the occluded nostril. These fiducial markers were used to determine the orientation of the sections (i.e., the side of the bulb or cerebral cortex). For biochemical studies, animals were briefly perfused with cold PBS to remove blood, and the forebrains were dissected out. The left and right bulbs were separated, weighed, snap-frozen with liquid nitrogen and stored at −70 °C. Bulbs from 18 month-old mice were used for measuring enzymatic activity of β-site APP cleavage and soluble Aβ concentration, and bulbs from 24 month-old mice were used for western analyses.
Two out of the 12 sets of sections from each brain were immunostained for BACE1 (#1, #7) and Aβ (#2, #8, using the rabbit anti-Aβ antibody, Ter-40) with the DAB-peroxidase method for systematic quantitative analyses. The remaining sets were used for Nissl stain (#3, #9) and immunolabelings for some other Aβ antibodies (see ). Sections were first treated with 1% H2O2 in PBS for 30 minutes, and pre-incubated in 5% normal goat or horse serum with 0.3% Triton X-100 for 1 hour. Antigen retrieval techniques were used for BACE1 (50% formamide and 50% 2XSSC at 65 °C for 1 hour) and Aβ antibody (50% formic acid in PBS for 30 minutes at room temperature) labelings before H2O2 treatment. Sections were incubated overnight at 4 °C with BACE1 and Aβ antibodies diluted in PBS containing appropriate blocking sera (see for antibody sources and dilutions). The sections were then reacted with biotinylated goat anti-rabbit or horse anti-mouse IgGs at 1:400 for 2 hours, and subsequently with the ABC reagents (1:400) (Vector Laboratories, Burlingame, CA, USA) for an additional hour. Immunoreactivity was visualized using 0.003% H2O2 and 0.05% diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO, USA). Immunostained sections were mounted on slides, allowed to air-dry, and coverslipped. Some sections were lightly counterstained with cresyl violet to verify the laminar distribution of the immunolabeling.
Primary antibodies used in the present study
Double immunofluorescence was carried out by incubating sections in PBS containing 5% donkey serum and a pair of primary antibodies from different animal species (), followed by a 2 hour reaction with Alexa Fluor® 488 and Alexa Fluor® 594 conjugated donkey anti-mouse, rabbit and goat IgGs (1:200, Invitrogen, Carlsbad, CA, USA). After fluorescent immunolabeling, sections were counter-stained with bisbenzimide (Hoechst 33342, 1:50000), washed 3 times in PBS and mounted with anti-fading medium. Initial antibody specificity tests included preabsorption of primary antibody with neutralizing peptide or omission of primary antibody in the incubation buffer. These controls yielded no specific labeling in brain sections.
NADPH-diaphorase and CO histochemistry
Sections were incubated in 0.05 M Tris-HCl buffered saline (pH 8.0, TBS) containing 0.3% Triton X-100, 1 mM nicotinamide adenine dinucleotide phosphate diaphorase (β-NADPH-d), 0.8 mM nitroblue tetrazolium and 5% dimethyl sulfoxide for 45 minutes at 37 °C. Selected sections were further immunostained for BACE1 using the DAB-peroxidase method to assess colocalization. Cytochrome c oxidase (CO) histochemical stain was processed by incubating the sections in 0.05% DAB, 0.02% cytochrome c oxidase, and 4% sucrose in 0.1 M phosphate buffer (pH 7.4) at 37°C in the dark for 3-5 hours, followed by several rinses with PBS at room temperature. All histochemical reagents were from Sigma-Aldrich (St. Louis, MO, USA).
Left and right bulbs were homogenized with a sonication device in T-PER buffer (10x w/v) (Pierce, Rockford, IL, USA) containing a cocktail of protease inhibitor (Roche Products, Welwyn Garden City, UK) at 4°C. Tissue extracts were centrifuged at 100,000 X g. Supernatants were collected and protein concentrations determined by DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amount of proteins (25-50 μg) were run on 20% (for Aβ40 and Aβ42) and 12% (for other protein products) SDS-PAGE gels (Hoefer Scientific Instruments, San Francisco, CA, USA). The polypeptides were electrotransferred to Tans-Blot® pure nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Non-specific binding was blocked using 5% nonfat milk in PBST (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, and 0.1% Tween-20). Full-length and proteolytic fragments of APP, BACE1 and reference protein β-tubulin-III were blotted with primary antibodies as listed in . Nitrocellulose membranes were incubated with HRP-conjugated second antibodies for 1 hour (1:20000, Bio-Rad), and protein bands visualized with an ECL Plus™ western blotting detection kit (GE healthcare, Pittsburgh, PA, USA), and images captured in a UVP Biodoc-it™ system (UVP, Inc, CA, USA).
β-Site APP cleavage activity and ELISA for Aβ40 and Aβ42
Frozen bulbs were homogenized in T-PER buffer (5x v/w) containing protease inhibitors (Roche Products) and 1% Triton X-100. Samples were then centrifuged and supernatants collected, followed by determination and normalization of protein concentrations. Beta-site APP cleavage activities were measured in 96-well transparent flat-bottom plates using a commercial kit (#565785, Calbiochem, La Jolla, CA, USA) following manufacturer’s instruction. Triplicated loadings (20 μg protein/well) were applied for each sample in a given experiment, and assay was repeated at least once. Levels of soluble Aβ40 and Aβ42 were assayed using commercial kits according to manufacturer’s instruction (Catalog #88-348 and KHB3441, Invitrogen, Carlsbad, CA, USA). Triplicated loadings of 50 μg protein (normalized to 50 μl sample solution) were assayed for each bulb sample. Enzyme activity and ELISA signals were captured in a Bio-Rad microplate reader (Model 3550).
Imaging, densitometry, statistical testing and figure preparation
Sections were examined on an Olympus (BX60) fluorescent microscope equipped with a digital camera and image analysis system (Optronics, Goleta, CA, USA). Double immunofluorescent images were superimposed using the Optronics software. For densitometry, digital images were captured using a 4X objective lens with a numerical aperture of 0.1, such that both sides of the forebrain structures were included within the same image. Higher magnification images were taken using 10X, 20X and 40X objectives with numerical apertures of 0.3, 0.7 and 0.85, respectively. All original images contained 1200×1600 pixels. An identical photo-exposure setting was used for all sections. Optical densities (o.d.), expressed as digital light units (DLU)/mm2, over areas of interest were obtained in equally-spaced (~180 μm apart) and level-matched (5-7 levels/brain, i.e., with the right and left bulb sections attached together) sections using an irregular interconnecting selection tool (OptiQuant 4.1, Parkard Instruments, Meriden, CT, USA), by an experimenter who was blinded to the treatment.
BACE1, CO or tyrosine hydroxylase (TH) labelings were measured over the glomerular layer (GL) and/or external plexiform layer (EPL) of the bulb, or layer I and II/III of the piriform cortex. Data were normalized to the averaged mean from the non-deprived side (defined as 100%). Plaque-related BACE1 and Aβ labelings were measured over the granule cell layer (GCL) and the subependymal zone (SEZ) of the bulb, layers I-III of the piriform cortex together with the lateral olfactory tract (LOT), and layers I-VI in the frontal cortex (see Supplemental-Fig. 1A-F
). To calculate specific (or plaque-related) BACE1 and Aβ immunoreactivities, optic densities over the plaque-free striatum (both sides) were also measured (Supplemental-Fig. 1A-D)
. Measured optic densities (row data) were exported into Excel spreadsheets, and the averaged striatal density was used as the cut-off threshold to define specific densities [i.e., specific BACE1 or Aβ density (in the bulb or cortex) = measured BACE1/Aβ o.d. ─ average of the measured BACE1/Aβ o.d. in the striatum]. Means of specific densities were calculated for each brain and each surviving group, and data were statistically analyzed using student-t
test or ANOVA together with Bonferroni posttests between paired means (Prism GraphPad, San Diego, CA, USA). The minimal significant level of difference between comparing means was set at p
Specific β-site APP cleavage activity was calculated by subtracting non-specific signal from the total signals in individual samples. The non-specific signal level was defined in the same experiment by including excessive enzyme inhibitor provided by the manufacturer in the assay buffer. Specific enzyme activities in individual bulb samples were normalized to the averaged mean (defined as 100%) from the non-deprived bulbs. For ELISA data, Aβ40 and Aβ42 concentrations were calculated according to a standard curve generated using serially-diluted synthetic peptides provided by the manufacturer. The maximal signal in the samples was in the linear range of the standard curve. Figure panels were assembled using Photoshop 7.1 and converted to a final TIFF file, with contrast/brightness adjusted as needed.