APLP1 Is a Synaptic Cell Adhesion Molecule, Supporting Maintenance of Dendritic Spines and Basal Synaptic Transmission

Plasmids

Animals

The generation and genotyping of knock-out lines were described previously [APP KO and APLP1 KO (Heber et al., 2000); APLP2 KO (von Koch et al., 1997)]. All mice have been backcrossed at least six times to C57BL/6 mice. The sex of the species used is of either sex. C57BL/6J mice [embryonic day 14 (E14)] were used for the generation of primary cortical neuron cultures. C57BL/6J mice [postnatal day (P0)] were used for the generation of primary hippocampal neuron cultures.

Mice were treated in accordance with the German law for conducting animal experiments and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal housing, breeding, and the sacrifice of mice were approved by the German administration. All experimental protocols were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Analysis of APP family member protein expression levels in mouse brains

Mouse cortices of different postnatal stages were homogenized in lysis buffer (50 mm Tris/HCl, pH 7.5; 1% (v/v) NP-40; 150 mm NaCl; 2 mm EDTA; 1:25 protease inhibitor (complete with EDTA), Roche). After centrifugation for 10 min at 15,000 × g and 4°C, the remaining supernatant (cell lysate) was used for further analysis. The protein concentration of cell lysates was determined via BCA assay (Sigma-Aldrich). Twenty micrograms of mouse brain lysates were separated on 8% Tris/glycine gels. Western blot detection of the APP family members with primary antibodies against the C terminus of APP (Y188; 1:5000; rabbit monoclonal, Abcam), anti-APLP1 (150; 1:2000; rabbit polyclonal; Eggert et al., 2004), anti-APLP1 (57; 1:2000; rabbit polyclonal; Eggert et al., 2004), and anti-APLP2 D2-II (1:5000; rabbit polyclonal; Calbiochem) was followed by incubation with horseradish peroxidase (HRP) coupled secondary antibodies and enhanced chemiluminescence (ECL; Pierce) detection.

Quantification was performed with the program ImageJ. Signals of the APP gene family members were measured and correlated to the signal of the overall protein amount on the nitrocellulose membrane, which was detected via larva purple staining (ServaPurple Stain Solution, Serva; Moritz et al., 2014). Larva purple staining solution was added for 35 min at room temperature (RT) on PVDF membranes after Western blot transfer. Discoloration of the membrane was followed with 15% (v/v) ethanol and 1% (v/v) citric acid. The membrane was dried and the fluorescence was detected via imager (Odyssey Fc Dual-mode-imaging System, Li-Cor).

Analysis of APP family member glycosylation in mouse brains

For deglycosylation, 20 μg of mouse brain lysates were treated with O′-glycosidase, EndoH, and PNGase F (New England BioLabs) according to manufacturer instructions. After incubation for 1 h at 37°C, the samples were shortly centrifuged and then denatured in 4× SDS sample buffer [0.25 m Tris/HCl, pH 6.8; 40% (w/v) glycerol; 8% (w/v) SDS; 0.02% (w/v) Bromophenol Blue; 200 mm DTT]. Equal amounts of protein were separated on 8% Tris/glycine gels. Western blot detection of the APP family members was performed with primary antibodies, as described above.

Synaptosomal preparation and enrichment of the postsynaptic density

Samples were always kept on ice, and all centrifugation steps were performed at 4°C. One mouse brain was homogenized in solution A (0.32 m sucrose; 1 mm NaHCO₃; 1 mm MgCl₂; 0.5 mm CaCl₂) by a Potter S Homogenizer. Centrifugation for 10 min at 800 × g sedimented crude cell fragments [“post-nuclear supernatant” (Post ns)]. The supernatant was centrifuged for 15 min at 9000 × g (smaller cell components stay in the supernatant). The pellet was resuspended in solution A, centrifuged again for 15 min at 10,000 × g, and resuspended in solution B (0.32 m sucrose; 1 mm NaHCO₃) to obtain a raw synaptosomal fraction (Syn crude). Synaptosomal membranes were isolated via hypo osmotic shock by the addition of double-distilled water. The reaction was stopped with 0.5 m HEPES/NaOH, pH 7.4 [fraction “synaptosomes after hypo-osmotic shock” (Syn hyp)]. The solution was centrifuged for 20 min at 25,000 × g, and the pellet was subsequently (dissolved in solution B) loaded on a sucrose gradient (0.5 m; 1 m; 2 M sucrose in 1 mm NaHCO₃). A discontinuous density centrifugation at 83,500 × g was performed for 3 h. The high dense fraction was collected (Syn total) and treated with 2 × LP-buffer (0.32 m sucrose; 12 mm Tris/HCl pH 8.0; 1% (v/v) Triton X-100; 1 mm NaHCO₃). Triton X-100 denatured all proteins of the presynapse, while only the postsynaptic density (PSD) stays intact. A further ultracentrifugation step at 201,800 × g for 20 min pelleted the PSD, which was subsequently resuspended in 5 mm Tris/HCl. To analyze equal amounts of proteins, a BCA test was performed and the samples were loaded on an 8% Tris/glycine gel. The following primary antibodies were used for Western blot detection: PSD-95 (1:2000; mouse monoclonal, Abcam); synaptophysin (1:2000; mouse monoclonal, Sigma-Aldrich); anti-APP C terminus (1:5000; Y188, rabbit monoclonal, Abcam); anti-APLP1 (57, 1:2000, rabbit polyclonal; Eggert et al., 2004); anti-APLP2 (1:1000, rabbit polyclonal; CT12, Calbiochem) followed by secondary antibodies, which were coupled to HRP for detection after Western blotting with ECL (Pierce).

Optical and sciatic nerve preparations

The optical and sciatic nerves were dissected from adult C57BL/6J mice and adult APP KO, APLP1 KO, and APLP2 KO mice. The species studied was of either sex. Sciatic nerves of three mice were collected in 5× volume homogenization buffer (10 mm HEPES, pH 7.3; 1 mm EDTA, pH 8.0; 250 μm saccharose, pH 7.5; Complete Protease Inhibitor, Roche) in a 1.5 ml Eppendorf tube and homogenized mechanically via a micropistil. The homogenate was centrifuged at 1000 × g, and the supernatant was subsequently used for analysis after a BCA assay (Sigma-Aldrich) was performed. Optic nerve preparations were performed according to a protocol from Berg et al. (2000).

Preparation and immunocytochemical staining of transfected primary hippocampal neurons

The hippocampi were dissected from P0–P1 C57BL/6J mice. Preparation of hippocampal neurons followed as described previously (Tyan et al., 2012). After washing three times with buffer [Ca²⁺/Mg²⁺-free HBSS with 1% (v/v) penicillin/streptomycin (Pen/Strep) and 1% (w/v) d-glucose], the hippocampi were incubated for 15 min at 37°C with 0.05% trypsin-EDTA. After centrifugation for 5 min at 447 × g, the hippocampi were resuspended in suspend solution (50% Ham's F-12 and 50% DMEM with 1% Pen/Strep and 0.1% DNAseI) and triturated with a long transfer pipette up to 50 times. Afterwards, the suspension was passed through a disposable cell strainer (70 μm nylon, Falcon). After a further centrifugation step, the neurons were resuspended in growth media [Neurobasal Media with 1% (v/v) sodium pyruvate, 1% (v/v) Pen/Strep, 2 mm l-glutamine, and 2% (v/v) B-27 supplement]. The cells were plated on coverslips (14 mm; Marienfeld) that had been coated with poly-l-lysine in borate buffer previously.

The neurons were transfected on day in vitro 7 (DIV7) via Ca/Pi (Tyan et al., 2012) with APP695 HA pcDNA 3.1(+), APLP1 HA pcDNA 3.1(+), and APLP2-763 HA pcDNA 3.1(+). After 18–22 h, the neurons were fixed for 10 min at 37°C in 4% (w/v) PFA with 4% (w/v) sucrose and permeabilized for 10 min with 0.1% (v/v) NP-40. The APP gene family members were visualized via antibody HA [1:300; rat monoclonal (3F10), Roche). Dendrites were stained with an anti-microtubule-associated protein 2 (MAP2) antibody (1:300; rabbit polyclonal, Santa Cruz Biotechnology) and axons by an anti-Tau1 antibody (1:200; mouse monoclonal, Millipore Bioscience Research Reagents). Secondary antibodies were Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 (1:1000; Invitrogen). Imaging was performed with an Axio Observer Z.1 Microscope (with apotome; Zeiss) and z-stacks were taken in 0.200 μm steps.

The same procedure was performed with DIV7 fixed cortical neurons to analyze endogenous localization of the APP gene family members in axons and dendrites [for preparation of neurons, see Coculture assay (hemisynapse assay)]. For visualization, antibodies anti-APP C terminus (1:5000; Y188, rabbit monoclonal, Abcam), anti-APLP1 C terminus (1:1000; CT-11, rabbit polyclonal, Calbiochem), and anti-APLP2 C terminus (1:1000; CT-12, rabbit polyclonal, Calbiochem) were used.

Coculture assay (hemisynapse assay)

The synaptogenic activity of the APP gene family members was analyzed using a coculture assay with HEK293T (HEK) cells and primary cortical neurons (Scheiffele et al., 2000; Biederer and Scheiffele, 2007; Stahl et al., 2014). HEK293T cells were cultured in DMEM with 10% FBS and 1% Pen/Strep. The primary cortical neurons were prepared on E14 from C57BL/6J mice. The cortices were dissociated with a fire-polished Pasteur pipette after incubation for 15 min at 37°C in 0.05% Trypsin-EDTA and washing five times with ice-cold HBSS supplemented with 10 mm HEPES. The isolated neurons were resuspended in DB1 media [DMEM with 10% (v/v) FCS, 0.79% (w/v) d-glucose, and 2 mm l-glutamine], and 140,000 cells/ml (500 μl/coverslip) were seeded on 14 mm coverslips (Marienfeld) pretreated with poly-l-lysine in borate buffer (20 μg/ml). After 6 h of incubation, the media were replaced by NM media [Neurobasal media with 2% (v/v) B-27 supplement and 2 mm glutamate]. At DIV6 of the neuronal culture, HEK293T cells were transiently transfected with jetPRIME (Polyplus). GFP pcDNA3.1(+) was used as a negative control, and Nlg1 HA pcDNA3.1(+) was used as a positive control. The constructs for the APP gene family, APP695 HA pcDNA3.1(+), APLP1 HA pcDNA3.1(+), APLP2–763 HA pcDNA3.1(+), were also analyzed. Twenty-four hours after transfection, the HEK cells were seeded on the neuronal culture at a density of 400,000 cells/coverslip. After a coculture time of 24 h, the cells were fixed for 10 min at 37°C in 4% (w/v) PFA with 4% (w/v) sucrose and washed three times with 1× PBS. Subsequently, an immunocytochemical staining was performed. The cells were permeabilized with 0.1% NP-40 in 1× PBS for 10 min and blocked in 5% goat serum in 1× PBS. The overexpressed proteins were visualized with an antibody against their HA tag (1:300; rat monoclonal (3F10), Roche) followed by an Alexa Fluor 488-conjugated secondary antibody (1:1000; Invitrogen). For detection of a presynaptic marker, an antibody against synaptophysin (1:200, mouse monoclonal antibody, Sigma-Aldrich; secondary antibody Alexa Fluor 594 conjugated) was used. Synaptophysin staining was the readout of the assay. The synaptophysin puncta at the HEK cells were taken as an indication for presynaptic differentiation in the contacting neuron. For quantification, the number of synaptophysin puncta per cell was counted and the area that was covered by these puncta was determined. To avoid false-positive puncta, the assay was costained with an antibody against MAP2 (1:300; rabbit polyclonal, Santa Cruz Biotechnology; secondary antibody Alexa Fluor 647 conjugated). Only cells that did not contact MAP2-stained dendrites were chosen for quantification to ensure that only so-called hemisynapses between an HEK293T cell and an axon were analyzed. For the staining of axons, an anti-Tau1 antibody (1:200; mouse monoclonal, Millipore Bioscience Research Reagents) was used. The z-stack images were taken with the Axio Observer Z.1 Microscope (with apotome; Zeiss), and quantification was performed via ImageJ analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test (n ≥ 4; *p < 0.05; **p < 0.01; ***p < 0.001). To assess whether APLP1 synaptogenic activity depends on endogenous expression of APP family members, APP^+/−APLP2^−/− heterozygous matings were set up to obtain APP^−/−APLP2^−/− DKO neurons and internal APLP2^−/− single-KO littermate (LM) controls. APLP1 KO and wild-type neurons were obtained from APLP1^+/− matings.

Cell culture and purification of APPex–crystallizable fragment and APLPex–crystallizable fragment

COS-7 cells were stably transfected with APPex–crystallizable fragment (F_C), APLP1ex–F_C, or APLP2ex–F_C. The fusion protein consists of the ectodomain of APP, APLP1, or APLP2 fused to the F_C of human IgG1. The cells were cultured in DMEM containing 10% FBS, the selection antibiotic (Hygromycin B) and penicillin/streptomycin. Conditioned medium of COS-7 cells transfected with APPex–F_C or COS-7 APLP1ex–F_C was collected 3 d after seeding. Cells expressing APLP2ex–F_C were cultured in FBS-containing medium until confluency reached 100%. Afterwards, the medium was replaced by serum-free medium, conditioned for 24 h, and collected. The conditioned medium was sterile filtered (0.45 μm Bottle Top Filtration Unit, VWR), and binding buffer was added (50 mm Tris/HCl, 300 mm NaCl, pH 7.4). APLP2ex–F_C was purified by affinity chromatography using a protein A Sepharose FF 20 ml affinity column (GE Healthcare). After the medium was loaded on the column, it was washed with binding buffer until UV absorption reached baseline levels, and the bound proteins were eluted by adding 100 mm glycine, pH 2.5. Immediately after elution, 1 m Tris/HCl, pH 8.0, was added to restore the pH, and the protein was concentrated and desalted using a PD-10 desalting column (GE Healthcare). For purification of APPex–F_C and APLP1ex–F_C, the medium was first loaded on the protein A Sepharose column, analogous to the purification of APLP2ex–F_C. Additionally, affinity chromatography of the eluted protein from the protein A column was performed using a HiTrap Heparin HP 5 ml affinity column (GE Healthcare) to remove antibodies present in the FBS. After loading the sample on the column, it was washed with 20 mm HEPES and 150 mm NaCl, pH 7.2, until UV absorption reached baseline. The protein was eluted using 20 mm HEPES and 1 m NaCl, pH 7.2. The purity of the sample was examined by SDS-PAGE, and protein concentration was determined via measurement at 280 nm (NanoDrop).

Bead aggregation assay

To analyze the dimerization properties of APPex–F_C, APLP1ex–F_C, and APLP2ex–F_C, 10 μl of magnetic protein A-coated polystyrene beads (Invitrogen) were incubated with 15 μg protein for 2 h at 4°C via end-over-end rotation. Afterwards, the beads were washed three times with binding buffer (20 mm HEPES, 150 mm NaCl, pH 7.2). Subsequently, 400 μl of binding buffer containing 0.1% BSA and 400 μl of 3 d conditioned COS-7 wild-type medium was added to the beads. The beads were sonicated to singularize them and incubated for 2 h at 4°C with end-over-end rotation. For measurements, 200 μl of the suspension was diluted in 10 ml of Isoton II, and aggregates with a diameter of >10 μm were counted on a Beckman Coulter Z2 Counter.

Analysis of proteolytic processing

HEK293T cells were transiently transfected at a confluency of 70% with jetPRIME (PolyPlus) according to manufacturer instructions with APP NT myc pcDNA3.1(+), APLP1 NT myc pcDNA3.1(+), and APLP2 NT myc pcDNA3.1(+), or APP CT HA pcDNA3.1(+), APLP1 CT HA pcDNA3.1(+), and APLP2 CT HA pcDNA3.1(+). The media were conditioned for 2 h (1 ml of media in one well of a 6-well plate). The cells were harvested and lysed in 200 μl lysis buffer [50 mm Tris/HCl, pH 7.5; 150 mm NaCl; 5 mm EDTA; 1% NP-40; 1:25 Complete Protease Inhibitor (with EDTA), Roche] for 20 min. Cell debris was pelleted at 15,700 × g for 10 min at 4°C. For analysis of the CTFs, the same amounts of protein were loaded on a 4–12% Bis-Tris gel (Invitrogen). APP, APLP1, and APLP2 full-length proteins as well as their C-terminal fragments were detected via antibody HA [1:5000; rat monoclonal (3F10), Roche]. To investigate shedding of the APP family members, cell lysates as well as conditioned media were separated on an 8% Tris/glycine gel and visualized via their myc tag (1:1000; rabbit polyclonal, Santa Cruz Biotechnology) followed by secondary antibodies, which are coupled to HRP for detection after Western blotting with ECL (Pierce). Images were analyzed via ImageJ, and the ratio between CTF or secreted proteins and full-length proteins was quantified. Statistical analysis of CTFs was performed using a Kruskal–Wallis test followed by Dunn's multiple-comparison test with Bonferroni's correction (n = 4; *p < 0.05; **p < 0.001; ***p < 0.0001; bars represent the mean ± SEM). Statistical analysis for the secreted fragment was performed using a Kruskal–Wallis test followed by Dunn's multiple-comparison test with Bonferroni's correction (n = 7; *p < 0.05; **p < 0.01; ***p < 0.001; bars represent the mean ± SEM).

Antibody uptake assay

N2a cells were cultured in MEM media with 10% (v/v) FBS, 1% (v/v) Pen/Strep, 1% (v/v) nonessential amino acids, 1% (v/v) sodium pyruvate, and 2 mm l-glutamine. A total of 70,000 cells were plated per 14 mm coverslip (Marienfeld) in a 24-well plate that had been coated with poly-l-lysine in double-distilled H₂O. The cells were transfected the following day with jetPRIME (PolyPlus) according to manufacturer instructions with APP NT myc pcDNA3.1(+), APLP1 NT myc pcDNA3.1(+), APLP2 NT myc pcDNA3.1(+), and APPΔCT 648 NT myc pcDNA3.1(+), which served as a negative control. Seventeen to 24 h after transfection, the cells were placed on ice to stop endocytosis. After washing with OptiMEM (Invitrogen), the cells were incubated for 30 min with the primary antibody c-myc (1:200; 9E10, mouse monoclonal, Abcam) against the myc tag of the transfected APP family proteins. The cells were washed with OptiMEM again to remove the unbound antibody. Subsequently, endocytosis was allowed at 37°C for different time points in prewarmed N2a growth media. Afterwards, the cells were cooled down again to 4°C and fixed with 4% (w/v) PFA with 4% (w/v) sucrose. After permeabilization for 10 min with 0.1% (v/v) NP-40 in 1× PBS, the cells were blocked in 5% (v/v) goat serum in 1× PBS and incubated with Alexa Fluor 594-conjugated secondary antibody (1:400; Invitrogen) to stain the remaining APP/APLPs at the cell surface and the internalized protein. After a further washing step with 1× PBS, the coverslips were embedded in Mowiol. The z-stack images were taken with the Axio Observer Z.1 Microscope (with apotome; Zeiss). Projections of these stacks were performed with the program ImageJ. For each construct and time point, the amount of endocytosed protein was determined by measuring the intensity of the internalized protein and the intensity of the surface protein. The ratio of the endocytosed protein to total intensities (internalized plus cell surface protein) represents the amount of endocytosed protein per cell. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test (n = 4; *p < 0.05; **p < 0.01; ***p < 0.001; bars represent the mean ± SEM).

Coimmunoprecipitation

N2a cells (growth media see above) were transiently cotransfected (jetPRIME) with a CT HA-tagged APP gene family member and one member of the X11 family [X11α CT Flag pcDNA3.1(+), X11β NT Flag pcDNA3.1(+), or X11γ NT Flag pcDNA3.1(+)]. After 17–24 h, the cells were harvested and lysed (see above). Equal volumes of cell lysates containing 800 μg of protein were precleared with 10 μl of protein A Sepharose (GE Healthcare) for 1 h at 4°C. After centrifuging the beads for 2 min at 2000 × g at 4°C, the supernatant was incubated for 2–3 h with 20 μl of HA beads (Roche) at RT. Afterwards, the beads were washed three times with 750 μl lysis buffer [50 mm Tris/HCl, pH 7.5; 150 mm NaCl; 5 mm EDTA; 1% NP-40; 1:25 Complete Protease Inhibitor (with EDTA), Roche)] and sedimented for 2 min at 2000 × g. A last washing step was performed with 10 mm Tris/HCl, pH 7.5. The supernatant was completely removed, and the beads were resuspended in 2× SDS sample buffer (0.125 m Tris/HCl, pH 6.8; 20% glycerol; 4% SDS; 0.01% Bromophenol Blue; 100 mm DTT). The samples were denatured for 5 min at 95°C and loaded onto an 8% Tris/glycine gel. After Western blotting, the X11 family members were detected with an anti-Flag antibody (1:5000; Sigma-Aldrich). Afterwards, the same membrane was incubated with antibody HA (1:5000; 3F10 rat monoclonal antibody, Roche) to detect immunoprecipitated (IP) APP gene family members. Direct loads of the cell lysates were visualized with the same antibodies as the IP samples.

Cell surface biotinylation

A total of 5 × 10⁵ HEK293T cells were seeded on a 6-well plate. The following day the cells were transiently transfected with JetPrime with APP CT HA pcDNA3.1(+), APLP1 CT HA pcDNA3.1(+), or APLP2 CT HA pcDNA3.1(+). After 17–24 h, the cells were rinsed twice with ice-cold 1× PBS and incubated for 30 min at 4°C with 1 ml of EZ-Link Sulfo-NHS-LC-Biotin (2 mg/ml; Pierce) in ice-cold PBS to biotinylate surface proteins. To quench unconjugated biotin, the cells were washed three times with 1× PBS supplemented with 100 mm glycine. Cells were lysed in 1 × RIPA buffer [20 mm Tris/HCl, pH 8.0; 150 mm NaCl; 1% NP-40 (w/v); 0.5% deoxycholate; 5 mm EDTA, pH 8.0; 0.1% SDS; 1:25 Complete Protease Inhibitor (with EDTA), Roche)], and 20 μg of lysate were used for the direct load. Equal protein amounts were incubated with NeutrAvidin Agarose Resin (Pierce) overnight at 4°C. On the following day, the beads were washed with RIPA buffer and boiled at 95°C for 5 min in 2× sample buffer with DTT to recover the biotinylated proteins. Direct load and surface proteins were separated on an 8% Tris/glycine gel and detected with antibody HA (1:5000; 3F10 rat monoclonal antibody, Roche; Stahl et al., 2014).

Golgi–Cox staining

Golgi staining of APLP1 KO mice and wild-type littermates was performed using the Rapid Golgi Staining Kit (FD NeuroTechnologies) according to manufacturer instructions. The 2.5 ml kit solutions A and B were mixed and incubated at RT for 2–3 h. Both hemispheres of each mouse were immersed in 2.5 ml of the mixture with equal parts of kit solutions A and B and were stored at RT for 24 h. The remaining impregnation solution was added, and hemispheres were stored at RT for 2 weeks. Afterwards, brain tissues were transferred into solution C and incubated at RT for 72 h or up to 7 d before sectioning. Solution C was renewed after 24 h. Coronal brain sections (100 μm) from 4% PFA-fixed mouse brains were cut using a vibratome (VT1000S, Leica). Each section was mounted with solution C on an adhesive microscope slide precoated with poly l-lysine (Thermo Scientific) and stained according to the manufacturer protocol, except that Roticlear (Carl Roth) was used instead of xylene. Finally, slides were embedded with Entellan (Merck Millipore).

Images of hippocampal CA1 pyramidal neurons were obtained on an AxioObserver Z1 Microscope (Zeiss) with a 63× oil-objective and a z-stack distance of 0.5 μm under reflected light. The number of spines per micrometer of dendritic length (in total, 100 μm) was determined in second- or third-order dendritic branches of apical dendrites in midapical regions of the stratum radiatum (apical compartment) using Neurolucida software (MicroBrightField). Only dendrites of similar diameter were used for spine density measurements. Basal dendrites of similar thickness within the stratum oriens were at least 20 μm from the soma. At minimum, five animals per genotype and eight neurons per animal were imaged and analyzed blind to genotype.

Golgi–Cox stainings were analyzed using GraphPad Prism (version 6) software. Spine density is expressed as the mean ± SEM. Differences between genotypes were detected with unpaired Student's t test (two-tailed).

Immunohistochemistry

Briefly, 13-month-old APLP1 KO mice and LM controls were perfused in PBS and 4% PFA in 1× PBS. The 30 μm slices (frozen sections) were analyzed. Synaptophysin antibody (guinea pig, Synaptic Systems) was used as a primary antibody (1:300), and Alexa Fluor 488 was used as a secondary antibody (Kullmann et al., 2011).

Preparation of acute hippocampal slices for LTP measurements

In vitro extracellular recordings were performed on acute hippocampal slices of WT littermates and APLP1 KO animals (N = 5). Between recordings, animals were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle and had access to food and water ad libitum. The experimental protocols were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Acute hippocampal transversal slices were prepared from individuals at an age of 4–6 months (young mice cohort) or 11–13 months (aged cohort). Mice were anesthetized with isoflurane and decapitated. The brain was removed and quickly transferred into ice-cold carbogenated (95% O₂, 5% CO₂) artificial CSF (ACSF) containing 125.0 mm NaCl, 2.0 mm KCl, 1.25 mm NaH₂PO₄, 2.0 mm MgCl₂, 26.0 mm NaHCO₃, 2.0 mm CaCl₂, and 25.0 mm glucose. The hippocampus was sectioned into 400-μm-thick transversal slices with a vibratome (VT1200S, Leica) and maintained in carbogenated ACSF at room temperature for at least 1.5 h before being transferred into a submerged recording chamber.

Slices were placed in a submerged recording chamber and perfused with carbogenated ACSF (32°C) at a rate of 1.2–1.5 ml/min. Field EPSPs (fEPSPs) were recorded in stratum radiatum of CA1 region with a borosilicate glass micropipette (resistance, 2–5 MΩ) filled with 3 m NaCl at a depth of ∼150–200 μm. Monopolar tungsten electrodes were used for stimulating the Schaffer collaterals at a frequency of 0.1 Hz. Stimulation intensity was adjusted to ∼40% of maximum fEPSP slope for 20 min of baseline recording. LTP was induced by applying theta-burst stimulation (TBS; 10 trains of four pulses at 100 Hz in a 200 ms interval, repeated three times) and recorded for 60 min.

Basal synaptic transmission properties were analyzed via input–output (IO) measurements, and short-term plasticity was examined via paired-pulse facilitation (PPF). The IO measurements were performed either by the application of defined current values (25–250 μA) or by adjusting the stimulus intensity to certain fiber volley (FV) amplitudes (0.1–0.8 mV). PPF was performed by applying a pair of two closely spaced stimuli in different interstimulus intervals ranging from 10 to 160 ms.

Preparation of acute hippocampal slices for measuring miniature EPSCs

Acute coronal hippocampal slices were prepared from an aged cohort of APLP1 KO mice and wild-type littermates (age, 12–13 months). Animals were decapitated, and the brains were quickly dissected in ice-cold cutting solution containing the following (in mm): 93 N-methyl-d-glucamin, 93 HCl, 30 NaHCO₃, 1.2 NaH₂PO₄, 20 HEPES, 2.5 KCl, 10 MgCl₂, 0.5 CaCl₂, 25 d-glucose, 3 Na-pyruvate, 3 myoinositol, and 5 ascorbic acid. Thereafter, 300-μm-thick slices were obtained using a vibratome (VT1200 S, Leica; HM650V Microtome, Microm International). After sectioning, slices were incubated for 10–12 min at 37°C in the same solution. Thereafter, slices were transferred to ACSF containing the following (in mm): 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 1 MgCl₂, 2 CaCl₂, 10 d-glucose, 2 Na-pyruvate, 3 myoinositol, and 0.44 ascorbic acid, pH 7.4, bubbled with carbogen (95% O₂, 5% CO₂). Finally, slices were held for 60 min at RT (20–24°C) to recover from cutting. To allow for unbiased subsequent patch-clamped recordings, experiments were performed and analyzed blind. Thus, mouse tails were taken for post hoc genotyping. Chemicals were purchased from Sigma-Aldrich or AppliChem, if not stated otherwise.

We used the patch-clamp technique to record miniature EPSCs (mEPSCs) from pyramidal neurons in the hippocampal CA1 region. The recording chamber was placed at an upright microscope equipped with infrared differential interference contrast (60× water-immersion objective, 1.0 numerical aperture; Eclipse FN1, Nikon) and an infrared video camera (XC-ST70CE, Hamamatsu). Whole-cell recordings were performed using a double-patch-clamp EPC10 amplifier and “PatchMaster” software (HEKA Electronic). The pipette solution contained the following (in mm): 140 K-gluconate, 5 EGTA, 10 HEPES, 1 MgCl₂, 2 Na₂ATP, and 0.3 Na₂GTP, pH 7.30. Pipettes were pulled from borosilicate glass capillaries [GB150(F)-8P, [Science Products]] using a horizontal puller (P-87, Sutter Instruments) and had a resistance of ∼7–8 MΩ. For mEPSC recordings, patched neurons were clamped to −70 mV and continuously perfused with 0.5 μm TTX (Biotrend) in ACSF to avoid contamination of recordings by spontaneous neuronal activity. Voltage-clamp recordings were sampled at 20 kHz and filtered at 2.9 kHz. Data were processed and analyzed using IGOR Pro 6.2 Software (WaveMetrics) and MiniAnalysis version 6.0.3 Software (Synaptosoft). Data were analyzed using WinSTAT for Excel (R. Fitch Software). Normally distributed data were assessed by a one-tailed, unpaired (independent) Student's t test, whereas data without normal distribution were assessed with a Mann–Whitney U test (*p < 0.05).