Antibodies and reagents
Anti-neurofilament (NF), anti-MAP2, anti-CHT, anti-Mint1, anti-Cask and 22C11 antibodies were purchased from Chemicon; anti-synaptophysin and anti-SV2 antibodies were from DAKO and the Developmental Studies Hybridoma Bank (DSHB) respectively; anti-HA antibody was obtained from Santa Cruz Biotechnology, and anti-Flag antibody was from sigma. The anti-rabbit monoclonal APP antibody used for immunostaining recognizes the C-terminal sequence of APP containing the YENPTY motif and was available from Epitomics Inc. (clone Y188). The polyclonal anti-APP C-terminal antibody APPc was described previously (Wang et al., 2007
). α-bungarotoxin (α-BTX) was from Molecular Probes.
The human full-length APP (695 form), APPΔC and APPc99 in pcDNA3 vectors were described in Wang et al. (Wang et al., 2007
). APP extracellular deletion constructs were generated from the full length cDNA by PCR and site-directed mutagenesis. Amino acids 22-189 and 289-500 of APP were excised to generate APPΔE1 and APPΔE2 construct, respectively. To generate a glycosyl phosphatidylinositol (GPI) anchored form of APP, the entire APP extracellular sequence was fused with exon5 of mouse acetylcholine esterase (AchE) which encodes the 43 C-terminal amino acid residues including the signal for GPI modification. To construct the APP/SynCAM chimera, the entire cytoplasmic tail of APP (amino acids 649-695) was replaced by the intracellular sequences of SynCAM (amino acids 428-474), which was amplified by RT-PCR of mouse brain RNA.
For constructing lentiviral expression vectors, the following primers were used to amplify the cDNAs from their pcDNA3 based vectors by PCR. The amplified fragments were subsequently cloned into the lentiviral shuttle vector FUGW(cmv)-RBN at NheI and PacI sites (Xu et al., 2008
APP FL and APPc99:
- 5′-ATGCTAGCGCCACCATGCTGCCCGGTTTGGCA-3′ and
Mint1 and Cask constructs kindly provided by Dr. Sudhof. To construct expression vectors for BiFC analysis, the N-terminal 172 amino acid of YFP (nYFP) or the C-terminal 173-238 amino acids of YFP (cYFP) from the Venus-nYFP or Venus-cYFP vectors (Chen et al., 2007
) were amplified by PCR and cloned into the C-terminal end of APP, and both the N-terminus and the C-terminus of Mint1 and Cask as in-frame fusion proteins. APP constructs with YENPTY motif deletion were generated with site-directed mutagenesis kit (Stratagene). The final constructs were verified by DNA sequencing.
Mouse production, breeding and genotyping
The creation of APP conditional allele is described in Supplemental Methods. These mice, as well as all other animals used for this study, have been backcrossed onto C57BL/6J background for 6 generations. The N-dCKO and M-dCKO mice were generated by crossing onto the APLP2 null background and from the following breeding:
The APLP2 genotyping was described in previously (von Koch et al., 1997
). The wild-type (WT) and APP
floxed allele were identified by PCR using primers:
- p1: 5′-GAC CAT CCA GAA CTG GTG CAAG-3′ and
- p2: 5′-TCC CCC AGG CTT GGG ATA CAC ATT A-3′.
The two primers amplify: WT allele: 443 bp; floxed (fl) allele: 503 bp due to insertion of loxP and restriction sites.
The Nestin-Cre and Mck-Cre transgenic mice were identified by PCR using the following primers, which amplifies a 791 bp fragment:
- CRE-F: 5′-GGC GTT TTC TGA GCA TAC CTG GAA-3′
- CRE-R: 5′-CAC CAT TGC CCC TGT TTC ACT ATC-3′
For embryonic analysis, timed mating was set up and the day when a vaginal plug was observed was considered to be embryonic day 0.5 (E0.5).
All animal experiments were performed in accordance with the Baylor College of Medicine Institutional Animal Care and Use Committee and with national regulations and policies.
Whole-mount immunostaining of the diaphragm muscle and quantification of neuromuscular phenotypes were carried out as described (Wang et al., 2005
). For muscle APP immunostaining, the stenomustoid muscles fixed overnight in 4% PFA were isolated, frozen in OCT (Triangle biomedical sciences), and cryosectioned at 20 μm longitudinally. Sections were permeabilized with 0.1% Triton X-100 in PBS (PBST) for 30 minutes, blocked with 3% BSA and 4% goat serum in PBST for 1 hour, and incubated with the APP rabbit polyclonal antibody (1:250) in PBST with 4% goat serum at room temperature for 30 minutes and then continued at 4°C for overnight. Sections were then washed 6 times for 5 min each in PBS and incubated with Alexa 555 conjugated goat anti-rabbit secondary (1:1000) and Alexa 488 conjugated α-BTX (1:1000) in PBST with 2% goat serum for 3 hours. Sections were again washed 6 times for 5 min each in PBS and then mounted with fluoromount-G mounting medium. Confocal images were obtained with a Zeiss 510 laser scanning microscope, and quantification was done using the ImageJ program from NIH.
Synaptic transmission of NMJ was examined at 28°C ± 0.5°C using intracellular recordings on acutely isolated phrenic nerve-diaphragm preparations at P0-2. Muscles were dissected in ice-cold oxygenated normal Ringer's solution containing the following (in mM): NaCl 116, KCl 4.5, MgSO4 1, NaHCO3, 23, NaH2PO4 1, Dextrose 11, CaCl2 2, pinned onto a Sylgard-coated recording chamber, and continuously superfused with oxygenated Ringer's solution at a rate of 2 ml/min. Sharp glass microelectrodes with resistance ranged from 15 to 25 MΩ were filled with 3 M KCl. Miniature endplate potentials (mEPP) were recorded in normal Ringer's solution containing 2.3 μM μ-Conotoxin GIIIB (Bachem) to selectively block muscle voltage-gated sodium channels and therefore, muscle contraction. Data were collected with a MultiClamp 700B amplifier, digitized at 10 kHz and recorded to a computer using pClamp 9 software (Axon Instruments). Offline data analysis was performed using Clamfit 9 (Axon Instruments), MiniAnalysis (Synaptosoft) and OriginPro 7.5 (OriginLab).
Primary hippocampal/HEK293 mixed culture
The preparation of primary hippocampal culture and co-culture for synaptic puncta quantification were performed essentially as described (Biederer and Scheiffele, 2007
). Briefly, primary hippocampal cultures were prepared from postnatal day 0 (P0) of C57BL/6J mice. Dissected hippocampi was suspended in trypsin-EDTA for 15 min at 37°C, washed three times with Ca2+
-free HBSS and triturated with a fire-polished glass pipette to dissociate the cells. Cells were resuspended in serum-free Neurobasal medium with B27 supplement (Invitrogen) and plated onto 12 mm cover-slips pre-treated with poly-D-lysine (5 μg/ml, Sigma) at a density of 20,000 cells/cm2
HEK293 cells were transiently co-transfected with NL1 or APP together with GFP at 10:1 molar ratio. After 24 hours, the transfected cells were seeded at 30,000 cells/cm2 to the above hippocampal neurons cultured for 6-7 days in vitro (DIV). 48 hours later, the cultures were fixed and stained with an anti-synaptophysin antibody (1:500, DAKO) followed by incubation with the Alexa Fluor-594 conjugated secondary antibody. The cells were also counter-stained with an anti-MAP2 antibody (1:1000, Chemicon), and the MAP2 negative synaptic puncta were selected for the quantification of heterologous synapse formation. Synapses were identified by the punctuate structures atop co-cultured HEK293 cells and quantified by confocal microscopy followed by ImageJ analysis.
The production of recombinant lentivirus was done by co-transfection of the expression vector FUGW(cmv)-APP, the packaging vector CMV Δ 8.9 and pVSVG into HEK293T cells by Lipofectamine 2000 (Invitrogen). HEK293 cells were split one day before the transfection and were about 70-80% confluent on the day of transfection. The DMEM culture medium was replaced with neuronal culture medium (Neurobasal containing 1× B27 and 0.5 mM L-Glutamine) before the transfection. For each T75 flask, 10 μg FUGW(cmv)-APP, 7.5 μg CMV Δ 8.9 and 5 μg of pVSVG plasmid were used for transfection. Sixty hours after transfection, the supernatant was collected and filtered with 0.45 μm filter, aliquoted and stored in -80°C. Hippocampal neurons were infected 1-day after plating using unconcentrated viral supernatant (~50-100 μl/ml media). 5-6 days later, half of infected neurons were co-cultured with HEK293 cells expressing APP and GFP as described above. The APP expressing neurons and axons were identified by GFP fluorescence. The other half was used for Western blot analysis of APP expression using the APPc antibody.
Western blotting and biotinylation
Mouse brain, spinal cord or muscle samples or cultured neuron or HEK293 cells were homogenized using RIPA lysis buffer (1% NP40, 50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) containing complete protease inhibitor cocktail (Roche). After 3 sets of 10 pulses of sonication, the homogenates were spun at 14000 rpm for 15 minutes. Protein concentrations were determined using Bio-Rad Dc Protein Assay. 10 μg of protein were loaded on a 10% SDS-PAGE gel run at 100V for 2 hours at room temperature and transferred onto a nitrocellulose membrane (Bio-Rad) at 100V for 1 hour. Membranes were blocked 1 hour using 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBST, Sigma). Primary antibody incubation was done in 5% milk in PBST. After three washes with PBS with 0.1% Tween-20, secondary antibody application was performed at room temperature for 1 hour using 5% milk in PBST followed by three additional washes with PBST. Bands were visualized using Immobilon™ Western ECL system (Millipore).
For biotinylation of surface proteins, HEK293 cells were transiently transfected with the APP constructs. Cells were washed with ice-cold PBS containing 1.0 mm MgCl2 and 0.1 mm CaCl2 (PBS/Ca-Mg) and treated with sulfo-NHS-SS-biotin (1.5 mg/ml, Pierce) for 1 h on ice in PBS/Ca-Mg. Biotinylating reagents were removed by incubating with cold 100 mM glycine in PBS/Ca-Mg for 30 min, followed by three washes with cold PBS/Ca-Mg. Cells were then lysed in 1% CHAPS lysis buffer containing 50 mM Tris pH 7.4, 150 mm NaCl and protease inhibitors (Roche). Biotinylated and non-biotinylated proteins were separated by incubation with Ultralink-Neutravidin bead (Pierce) for 1 hr at room temperature. Samples were subject to Western blot analysis as described above.