Generation of APP transgenic AD mice with deletion of TNFR1
knockout mice (TNFR1−/−
) were constructed on a C57BL/6 background as previously described (Peschon et al., 1998
). APP23 transgenic mice were provided by Novartis Institute for Biomedical Research; these mice express mutant human βAPP (Swedish double mutation, KM670/671NL) under the control of a brain- and neuron-specific murine Thy-1 promoter element. APP23 transgenic mice develop senile plaques in the cerebral cortex and hippocampus and show neuronal loss at 12–18 mo of age; this pathology is most evident in area CA1 of the hippocampus (Sommer and Staufenbiel, 1998
). APP23 mice were also constructed on a C57BL/6 background.
APP23 and TNFR1−/− mice were crossed and their progeny were genotyped. An APP23/TNFR1+/− mouse was backcrossed with TNFR1−/− mice to produce APP23/TNFR1−/− mice. To maintain the heterozygous APP transgene in our mice, we crossed APP23 mice with wild-type C57BL/6 mice. For APP23/TNFR1−/− mice, we crossed APP23/TNFR1−/− with TNFR1−/− mice for three to five generations. Therefore, both APP23 and APP23/TNFR1−/− mice were APP23+/−. We used APP23/TNFR1−/− mice of the F3–F5 generation in our experiments.
Mice homozygous for the TNFR1
targeted mutation (formerly TNFR1
, p55 deficient) show defects in resistance to intracellular pathogens and are resistant to the lethal effects of lipopolysaccharide administration in conjunction with D-galactosamine. Pulmonary inflammatory responses are diminished in p55-deficient mice. There are also defects in splenic architecture, formation of germinal centers, and liver regeneration. TNFR1
- deficient mice display increased susceptibility to atherosclerosis when maintained on a high-fat diet (Peschon et al., 1998
). No observations regarding any syndromes of the central nervous system have been made.
APP23, APP23/TNFR1−/−, and wild-type mice (n = 10 per group) were killed at 12 and 24 mo of age, and one hemisphere of the brain was homogenized in homogenization buffer (250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 1 mM EGTA). An aliquot of the homogenate was dissolved in formic acid and neutralized with a neutralization buffer (1 mM Tris and 0.5 M Na2HPO4). Protein concentration was measured by protein assay (Bio-Rad Laboratories). For total Aβ ELISA, the capture antibody was monoclonal anti-Aβ antibody 4G8 (Chemicon), and the detection antibody was biotinylated monoclonal antibody anti-Aβ 6E10 (AbD Serotec). Aβ40 and Aβ42 were measured with an Aβ40 and Aβ42 ELISA kit (Biosource International). The ELISA system has been extensively tested and no cross-reactivity between Aβ40 and Aβ42 was observed. Data are presented as means ± SD of four experiments.
BACE1 protein levels were measured by ELISA as described previously (Yang et al., 2003
). The capture antibody was anti-BACE1 polyclonal antibody P1 (Yang et al., 2003
) and the detection antibody was biotinylated anti-BACE1 polyclonal antibody P2 (Yang et al., 2003
). TMB substrate was used to visualize the reaction product, which was read at OD450
with a microplate reader (Sigma-Aldrich). BACE1 protein (Amgen) was used as a standard. Data are presented as means ± SD of four experiments.
Aliquots of brain homogenates from APP23, APP23/TNFR1−/−, and wild-type mice were further lysed with 1× RIPA buffer, and 50–150 μg of total protein was subjected to SDS-PAGE (8–12% acrylamide). Separated proteins were then transferred onto polyvinylidene fluoride membranes. The blots were probed with the following antibodies: anti-BACE1 monoclonal antibody (R&D Systems), anti-Aβ (1–17) monoclonal antibody (clone 6E10, 1:2,000; Chemicon), anti-IDE polyclonal antibody (Oncogene Research Products), anti-NEP polyclonal antibody (Chemicon), and anti– β actin antibody (Sigma-Aldrich).
Western blotting for Aβ was performed as described previously (Wiltfang et al., 1997
). To detect minute levels of Aβ, formic acid–dissolved brain tissue was immunoprecipitated with anti-Aβ polyclonal antibody (Zymed Laboratories) and subjected to SDS-PAGE using 10% acrylamide gels containing 8 M urea. Separated proteins were transferred onto polyvinylidene fluoride membranes. Aβ40 and Aβ42 were detected with monoclonal anti-Aβ antibody 6E10. Synthetic Aβ40 and Aβ42 (Biosource International) were used as standards.
BACE1, IDE, and NEP activity
An aliquot of brain homogenates from APP23, APP23/TNFR1−/−, and wild-type mice was further lysed with a lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10% glycerol, and 0.5% Triton X-100). BACE1 enzymatic activity assays were performed by using synthetic peptide substrates containing BACE1 cleavage site (MCA-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-[Lys-DNP]-OH; Biosource International). BACE substrate was dissolved in DMSO and mixed with a 50-mM Hac and 100-mM NaCl, pH 4.1, reaction buffer. An equal amount of protein was mixed with 100 μl of substrate, and fluorescence intensity was measured with a microplate reader (BioTek) at an excitation wavelength of 320 nm and an emission wavelength of 390 nm.
IDE enzyme activity was measured as described previously (Song et al., 2003
). In brief, brains were homogenized in 50 mM potassium phosphate buffer, pH 7.3, containing 200 μm PMSF and a proteinase inhibitor mix (Sigma-Aldrich). Samples were centrifuged and the supernatant fraction was used for IDE activity measurement. The hydrolysis of fluorogenic substrate peptides (2 μm Abz-GGFLRKHGQED-Dnp as substrate in 20 mM potassium phosphate buffer, pH 7.3) was measured by following an increase in fluorescence (excitation at 318 nm and emission at 419 nm) that occurred upon peptide bond cleavage. The max velocity of IDE activity was calculated by the first 20 min and indicated as fluorescence unit/min microgram protein.
For the in vitro NEP activity assay, mouse brains were homogenized in 100 mM MES buffer (pH 6.5) with proteinase inhibitors (Sigma-Aldrich). Homogenate was centrifuged at 20,000 g
for 45 min to separate the membrane fraction and the supernatant was removed. The membrane pellet was resuspended in MES buffer and directly used in NEP activity assay as previously described (Li and Hersh, 1995
To compare BACE1 expression levels, we used the following primers for RT-PCR: mouse BACE1 forward primer, 5′-AGACGCTACACATCCTGGTG-3′, and backward primer, 5′-CCTGGGTGTAGGGCACATAC-3′. The amplified BACE1 fragment was 146 bp. Mouse s18 was used as a loading control: forward primer, 5′-CAGAAGGACGTGAAGGATGG-3′, and backward primer, 5′-CAGTGGTCTTGGTGTGCTGA-3′. The amplified mouse s18 fragment was 159 bp. Total RNA was extracted from the brains of 12-mo-old APP23 and APP23/TNFR1−/− mice (n = 5) using an RNA mini column kit (Invitrogen). RT-PCR was performed using a One- Step RT-PCR kit (Invitrogen) and the following PCR cycles: 50°C for 30 min, 94°C for 2 min, followed by 25 cycles at 94°C for 15 s, 49°C for 30 s, and 68°C for 1 min.
Cell transfection and luciferase assay
We transfected 293 cells with pB1P-A vector containing a BACE1
promoter (−1941 to +292) upstream from a luciferase reporter gene (Christensen et al., 2004
) using lipofectamine (Invitrogen). After transfection, cells were treated with different concentrations of TNFα (R&D Systems), extracellular domain of TNFR1 (R&D Systems), or NF-κB inhibitor 6-amino-4(4-phenoxyphenylethylamino) quinazoline (Calbiochem; Tobe et al., 2003
). Cells were collected 12 h after treatment, and a luciferase assay (Promega) was performed, according to the manufacturer's instructions. Luminescence intensity was measured with a microplate reader, normalized according to protein amount, and plotted as relative luminescence units per milligram of protein.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed as previously described (Matsuoka et al., 2001
). In brief, paraformaldehyde-fixed brains were quickly frozen, and then sectioned at 30 μm. Sections were incubated with either anti-Aβ (6E10 clone or 4G8 clone, 1:1,000; Chemicon), anti-NeuN (MAB377, 1:400; Chemicon), anti-CD11b (MCA711, 1:500; AbD Serotec) and CD45 (MCA1388, 1:500; AbD Serotec), anti-α-smooth muscle actin (α-SM actin, A2547, 1:400; Sigma-Aldrich), or anti-vWF (AB7536, 1:200; Chemicon). Secondary antibodies were applied with horse anti–mouse (for 6E10, NeuN detection, 1:1,000) and goat anti–rat (for CD45 or CD11b, 1:1,000) followed by a DAB substrate (Vector Laboratories). For immunofluorescence, fluorescent-labeling 488 (green) or 594 (red) secondary antibodies against rabbit IgG or mouse IgG were used (1:1,000; Invitrogen). A microscope (DMLS; Leica) with a 10× N PLAN and 20× and 40× PL FLUOTAR was used. Digital images were captured and processed by digital camera (Optronics) and MagnaFire software (version 2.1C; Optronics).
Quantitation of immunoreactive structures
30-μm serial sagittal sections through the entire rostrocaudal extent of the hippocampus were cut on a cryostat. Every 10th section was immunostained with anti-NeuN antibody. On all sections containing the hippocampus, we delineated the pyramidal cell layer CA1. The total number of neurons were obtained using unbiased stereology (Casas et al., 2004
; Schmitz et al., 2004
) and a microscope equipped with a digital camera (DEI-470; Optronics). For each section, we delineated a 400-μm2
area in CA1 and in the entorhinal cortex and counted all NeuN-immunoreactive cells within that 400-μm2
box. The mean sum of neurons was counted per animal (n
= 10). We used the same method to count Aβ-immunoreactive plaques (stained with 6E10) in the hippocampus and entorhinal cortex in a double blind test. We also measured the diameter of each counted plaque. Differences between groups were tested with Image-pro Plus Analysis (Media Cybernetics).
Hole-board memory task
As previously reported (Dodart et al., 2002
), this task measured a mouse's ability to remember which one out of four equidistant holes was baited with food. Two photobeam apparatuses were used with a hole board for assessing directed exploration in mice for behavioral tests. A tested mouse (n
= 10 for each group) was placed in the center of the hole-board and the number of nose pokes was automatically registered for 5 min. After 20 min, each animal was placed in a corner of the hole board and allowed to freely explore the apparatus for 5 min. The number of head dips, time spent head-dipping, and the number of rearings were recorded. A comprehensive cognitive performance was determined by calculating the mean number of correct pokes per trial that mouse made each day. Cognition was expressed as the percentage of correct pokes. The measurements in the hole-board test were analyzed by unpaired t
test. In all cases the significance level was considered to be P < 0.05, and the very significant level was considered to be P < 0.01.
Object recognition task
The day before training, an individual mouse (n = 10 for each group) was placed into a training apparatus (a box the same size as described for the hole-board test) and allowed to habituate to the environment for 15 min. Training was initiated 24 h after habituation. A mouse was placed back into the training box containing two identical objects A and B (die or marble) and allowed to explore these objects. Among experiments, training times varied from 3.5 to 20 min. For each experiment, the same set of animals was used repeatedly with different sets of objects for each repetition. Five repetitions were performed on each set of mice. Each mouse was trained and tested no more than once per week, with a 1-wk interval between testing. Moreover, each experimental condition was replicated independently four times. In each experiment, the experimenter was blinded to the subjects during training and testing. To test memory retention, mice were observed for 10 min, 6 h, and 24 h after training. Mice were presented with two objects, one that was used during training, and thus was “familiar,” and one that was novel. The test objects were divided into 10 sets of “training” plus “testing” objects, and a new set of objects was used for each training session. A recognition index was calculated for each mouse, expressed as the ratio (100TB)×(TA + TB), where TA and TB are the time spent during the second trial on subject A and subject B, respectively. To ensure that the discrimination targets did not differ in odor, the apparatus and the objects were thoroughly cleaned with 90% ethanol, dried, and ventilated for a few minutes after each experiment.
In general, analysis of variance models (ANOVA) were used to analyze behavioral data. Typically, the statistical models included two between-subjects variables, the genotype of mice (APP23 vs. APP23/TNFR1−/−) and age, and one within-subjects variable, such as blocks of trials. When ANOVAs with repeated measures were conducted, the Huynh-Feldt adjustment of α levels was used for all within-subjects effects containing more than two levels to protect against violations of the sphericity/compound symmetry assumptions underlying this ANOVA model.