Methods

Transgene Construction
We created a tetracycline-responsive chimeric mouse/human APP695Swedish/Indiana (swe/ind) vector by replacing the promoter region of the moPrP.XhoI vector (also known as pPrPpE1/E2,3sal [15]) with the tetracycline-responsive promoter of pTetSplice (Life Technologies, Rockville, Maryland, United States), and then ligating mouse APP with a humanized Aβ domain (mo/huAPP695) cDNA into the new vector. We began by cloning the tetracycline-responsive promoter (bp 6–481) from pTetSplice by PCR using primers that added external BamHI and NotI sites to the 5′ end and a BamHI site to the 3′ end, while destroying XhoI and BamHI sites within the promoter (forward: GCC GGA TCC GCG GCC GCC GTC GAG TTT ACC ACT CCC TAT C; reverse: GCC GGA TCC ACT CTA GAA GAT CCC CGG GTA CCG). We then isolated the moPrP.XhoI intron by amplification with primers that added an external BamHI site to the 5′ end of exon 1 and ran through the Asp718 site of exon 2 (forward: GCC GGA TCC GAT CAG CAG ACC GAT TCT GG; reverse: GCC GGT ACC ACT AGG AAG GCA GAA TGC). This 2-kb intron fragment was cloned into a TA cloning vector (Invitrogen, Carlsbad, California, United States), then excised by Asp718 digestion and ligated to the 6.8-kb Asp718 fragment of moPrP.XhoI containing exon 2, exon 3, the 3′ UTR, and pBluescript to generate an intermediate vector with all three exons and a central intron but no promoter. This vector was then opened at the BamHI site introduced by the intron cloning primer, and ligated to the 0.5-kb BamHI-cut tetracycline promoter fragment. This ligation generated a 9.3-kb vector encoding the tetracycline promoter from pTetSplice with two exons, one intron, and the original 3′ UTR of the moPrP.XhoI vector, all carried in the pBluescript cloning vector.
We incorporated the Swedish (KM570/571NL) and Indiana (V617F) mutations into the mo/huAPP695 cDNA (in BS-KS) by PCR using a four-primer strategy: first, two partially overlapping products were generated in separate reactions using primers that encode the desired mutations (Swedish forward: GGA GAT CTC TGA AGT GAA TCT GGA TGC AGA ATT CCG/Indiana reverse: GGG TGA TGA AAA TCA CGG TTG C; Indiana forward: CAA CCG TGA TTT TCA TCA CCC TGG/M13 reverse). The two PCR products were ligated, digested with BglII and ApaI and cloned back into the original mo/huAPP695-BS-KS vector. Finally, the new APP695swe/ind was subcloned into the XhoI site of the moPrP-tetP vector from above to complete the construct.

Pronuclear Injection, Screening of Founders, and Maintenance of the Lines
The moPrP-tetP-mo/huAPP695swe/ind vector was linearized and the pBluescript domain excised by digestion with NotI. The purified vector was injected into the pronucleus of fertilized eggs from C57BL/6J × C3HeJ F1 matings. Founder animals were screened for the presence of the transgene by three-way PCR using the S36 and PrP-S/PrP-AS primers described below. Transgene-positive founders were bred to animals expressing the tetracycline transactivator (tTA) under control of the calcium-calmodulin kinase IIα (CaMKIIα) promoter obtained from Jackson Laboratory [16] (Bar Harbor, Maine, United States; stock # 3010; B6;CBA-TgN[Camk2a-tTA]1Mmay). The colony was thereafter maintained by crossing single transgenic tTA and APP offspring for each of the four APP lines. All mice were provided fresh food and water ad libitum. Animal protocols were approved by both the Johns Hopkins University and the California Institute of Technology Institutional Animal Care and Use Committees.

Doxycycline Administration
Doxycycline (dox) was administered through commercially available dox-containing chow (BioServ, Frenchtown, New Jersey, United States). The chow contained 200 mg/kg of antibiotic; based on estimated consumption of 5 g per mouse per day, the expected dose to each animal was 1 mg dox per day. The average 25-g animal therefore received 40 μg of dox per gram body weight per day. Chow was changed 1–2 times per week to prevent breakdown of the antibiotic.

Genotyping
Offspring were genotyped for the presence of each transgene by PCR amplification of genomic DNA extracted from a 5-mm tail biopsy. Tails were heated to 95 °C for 45 min in 250 μl of 50 mM NaOH, vortexed, then neutralized with an equal volume of 0.5 M Tris-HCl (pH 5.5). Debris was sedimented by centrifugation, and 3 μl of supernatant was used for amplification.
Genotyping for APP and tTA transgenes was performed in the same PCR reaction, using five separate primers. APP was amplified using forward primer S36 located in the 3′ end of the APP cDNA (CCG AGA TCT CTG AAG TGA AGA TGG ATG) and reverse primer PrP-AS-J located in the 3′ UTR of the vector (CCA AGC CTA GAC CAC GAG AAT GC). The tTA transgene was detected using a primer set that amplified across its two subdomains with tet forward located within the Tn10 tetracycline repressor (CGC TGT GGG GCA TTT TAC TTT AG) and tet reverse within the HSV1 VP16 (CAT GTC CAG ATC GAA ATC GTC). All reactions, whether transgene-positive or not, amplified a segment of the endogenous prion protein gene as a control for DNA quality using a forward primer, PrP-S-J, specific to the mouse PrP open reading frame (GGG ACT ATG TGG ACT GAT GTC GG) and a reverse primer, PrP-AS-J, shared by the 3′ UTR of the endogenous PrP gene and the transgene vector. Amplification reactions were run for 37 cycles at 94 °C for 30 s, 64 °C for 1 min, and 72 °C for 1 min. All samples, transgenic and wild-type, gave a 750-bp product from the endogenous PrP gene. The APP transgene yielded an additional band at 400 bp; the tTA product fell in between at 480 bp.

Immunoblotting/Quantitation
Frozen cortical or whole forebrain tissue was homogenized by sonication in five volumes of phosphate-buffered saline (PBS) with 5 mM EDTA and protease inhibitors (Mammalian cell cocktail, Sigma, St. Louis, Missouri, United States), using a probe sonicator set to 50% output (TEKMAR, Cincinnati, Ohio, United States). After dilution with an equal volume of PBS/EDTA/protease inhibitor, the samples were centrifuged briefly and the supernatant used for analysis. Fifty micrograms (6E10 and CT15) or 5 μg (22C11) of brain homogenate was loaded per lane onto 7.5%, 10%–20%, or 4%–20% Tris-HCl PAGE gels (Bio-Rad Laboratories, Hercules, California, United States) and electrophoresed for several hours in 1× Tris-glycine–sodium dodecyl sulfate (1×TG-SDS) buffer (6E10 and 22C11; Amresco, Solon, Ohio, United States) or 1× Tris-tricine-SDS buffer (CT15; Invitrogen, Carlsbad, California, United States). Proteins were transferred overnight to 0.45-μm Optitran nitrocellulose (Schleicher and Schuell, Keene, New Hampshire, United States) in 1× TG buffer (Amresco). Blots were blocked in PBS containing 5% nonfat dry milk powder, and incubated for 3 h at room temperature in blocking solution with one of the following antibodies: mouse monoclonal 22C11 (kind gift of Konrad Beyreuther and Andreas Weidemann; [17]) diluted 1:1,000, mouse monoclonal 6E10 (Signet Laboratories, Dedham, Massachusetts, United States) diluted 1:2,500, rabbit polyclonal anti-superoxide dismutase 1 (m/hSOD1) [18] diluted 1:2,500 to 1:4,000, or rabbit polyclonal CT15 (kind gift of Ed Koo; [19]) diluted 1:1,000. Subsequently, the blots were washed with PBS containing 0.1% Tween-20, and then incubated with either goat anti-mouse– or goat anti-rabbit–HRP conjugated secondary antibody diluted 1:1,000 in blocking solution. After several additional rinses in PBS with 0.1% Tween-20, blots were developed with enhanced chemiluminescence reagent and imaged with the Bio-Rad Molecular Imager FX system.
Staining intensity within each lane was quantified using the Quantity One image analysis software (Molecular Imager FX, Bio-Rad Laboratories). Background was calculated from across the image and subtracted from the entire file. The signal intensity for each band (corrected signal intensity × pixel number) was then calculated using the Volume report tool.

Slot Blot mRNA Analysis
Five micrograms per sample of total RNA extracted from fresh-frozen brain, liver, kidney, heart, lung, spleen, and skeletal muscle was vacuum-filtered through 0.45-μm Optitran nitrocellulose. After several washes through the manifold with 10× SSC, blots were UV-cross-linked and probed with a radiolabeled ∼350-bp BglII–XhoI cDNA fragment of mo/huAPP695 cDNA. After hybridizing overnight at 65 °C in 1% BSA/1 mM EDTA/0.5 M sodium phosphate buffer (pH 7.2)/7% SDS [20], the blots were washed twice at 65 °C for 30 min each in 0.1% BSA/1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/5% SDS before two final 30-min washes at 65 °C with 1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/1% SDS. Blots were wrapped wet and exposed to phosphorscreens overnight at room temperature.

Amyloid Histology
Mice were euthanized by ether inhalation and brains removed for immersion fixation in 4% paraformaldehyde/1× PBS. After 48 h in fixative at 4 °C, brains were transferred to PBS, dehydrated in alcohols, treated with cedarwood oil and methylsalicylate, and embedded in paraffin for sectioning.

Hirano silver stain
Silver impregnation histology was performed on 10-μm paraffin-embedded sections by Hirano's modification of the Bielschowsky method [21]. Briefly, sections were deparaffinized through xylene and alcohols into tap water before being placed into fresh 20% silver nitrate solution for 20 min. After being washed thoroughly with distilled water, slides were immersed in 20% silver nitrate solution titrated with fresh ammonium hydroxide. After 20 min, slides were washed with ammonia water before being individually developed with 100 μl of developer (20 ml of 37% formaldehyde, 100 ml of distilled water, 50 μl of concentrated nitric acid, and 0.5 g of citric acid) added to 50 ml of titrated silver nitrate solution. Slides were then rinsed in tap water, fixed in 5% sodium thiosulfate, and dehydrated through alcohols and xylene.

Thioflavin-S staining
Following deparaffinization of sections through xylene and alcohols, amyloid impregnation with thioflavin-S was performed according to the Guntern modification of the standard protocol. Slides holding 10-μm paraffin sections were washed twice in distilled water, then immersed for 5 min in a 0.25% potassium permanganate solution, followed by 5 min in a 1% potassium metabisulfate/1% oxalic acid solution. After this preparation, slides were placed into a filtered aqueous 0.02% thioflavin-S solution (Chroma-Gesellschaft Schmid, Kongen, Germany) for 8 min. Excess stain was removed by two brief rinses in 80% ethanol, then two in distilled water, after which slides were finished in aqueous mounting medium for florescence photomicrography.

Ubiquitin, glial fibrillary acidic protein, and Aβ immunohistochemistry
Prior to immunostaining, slides were deparaffinized by oven heating followed by immersion in xylene. After rehydration through graded alcohols into tap water, endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in methanol. Slides were microwaved for 5–7 min in water, cooled for 5 min, then washed in TBS. Nonspecific staining was blocked for 1 h with 3% normal goat serum and 0.1% Triton-X 100 in TBS. Slides were then placed into primary antibody (rabbit anti-Aβ peptide polyclonal antibody, Zymed Laboratories, South San Francisco, California, United States; rabbit anti-ubiquitin and rabbit anti–glial fibrillary acidic protein (GFAP) polyclonal antibodies, Dako, Carpinteria, California, United States) diluted 1:500 in TBS with 2% normal goat serum and incubated overnight at room temperature. After being washed of excess primary antibody with several changes of TBS, slides were incubated with either the Vectastain Elite anti-rabbit secondary system (anti-Aβ; Vector Laboratories, Burlingame, California, United States) or peroxidase/anti-peroxidase reagents (anti-ubitquitin and anti-GFAP; Sternberger Monoclonals, Lutherville, Maryland, United States) according to the manufacturers' directions. Antibody binding was visualized with diaminobenzidene, and sections were counterstained with hematoxylin.

Filter Trap Assay
An aliquot of each cortical homogenate used for Western blotting above was partially solubilized by the addition of SDS to a final concentration of 1%. Serial 1:2 dilutions were made with 1× PBS/1% SDS, and 100 μl of each dilution was then vacuum-filtered through a pre-wet 0.22-μm cellulose acetate membrane (Schleicher and Schuell) [22]. Each well was washed several times with PBS, after which blots were incubated overnight with polyclonal anti-Aβ antibody (Zymed Laboratories) diluted 1:600 in a blocking solution of 1× TBS/5% nonfat dry milk powder. After washing the blots three times for 10 min each in 1× TBS/0.1% Tween-20, the membrane was incubated for 1 h with HRP-conjugated protein A (Sigma) diluted 1:5,000 in blocking solution. The membranes were again washed three times with 1× TBS/0.1% Tween-20, before antibody binding was detected with enhanced chemiluminescence (PerkinElmer, Boston, Massachusetts, United States). Digital images of each blot were captured with a Molecular Imager FX gel documentation system, and the intensity of Aβ staining was quantified using Quantity One image analysis software.

Aβ ELISA
An aliquot of cortical homogenate generated for Western analysis described above was subjected to a three-step sequential extraction using PBS, 2% SDS, and 70% formic acid (FA). At each step, the sample was sonicated in appropriate buffer and centrifuged at 100,000g for 30 min (1- to 1.5-mo samples) or 60 min (6- to 12-mo samples) at 4 °C as previously described [23–25]. The supernatant was removed for analysis, and the pellet was sonicated in the next solution in sequence. The 2% SDS extracts were diluted in EC buffer, and the FA extracts neutralized with 1M Tris-phosphate buffer (pH 11) then diluted with EC buffer prior to testing. Human Aβ was measured in each fraction using BAN50 for capture (epitope Aβ1–16) and BA27 and BC05 for detection (Aβ40 and Aβ42, respectively) (Takeda Chemical Industries, Osaka, Japan). Total Aβ (mouse + human; 1- to 1.5-mo samples only) was measured in each fraction using BNT77 for capture (epitope Aβ11–28) and BA27 and BC05 for detection. All values were calculated as picomoles per gram based on the initial weight of cortical tissue.

Activity Monitoring
Daily basal activity was studied in 28 CaMKIIα-tTA × tet-APPswe/ind line 107 mice between 4 and 5 mo of age. Animals were separated into individual cages immediately before the start of each experiment (n = 3–6 per genotype untreated, 2–5 per genotype dox-reared). The cages were placed inside activity-monitoring frames designed to count every time the animal passed through one of three photobeams spanning the width of the cage (San Diego Instruments, San Diego, California, United States). Experiments were started midway through the light phase of the day, and data were collected in 1-h bins for the following 48 h. Testing rooms were maintained on the same 13:11 h day:night cycle as the main animal housing areas and were closed to entry during the experiment.

Statistical Analyses
Statistical analyses of protein expression, ELISA data, and filter trap assays were performed by ANOVA with Tukey's honest significant difference post-hoc test applied to significant main effects or interactions (Statistica 6.0, StatSoft, Tulsa, Oklahoma, United States). In cases of positively skewed data distribution, log10(x + 0.5) transformation was applied to the raw data before submitting them to ANOVA.