Animals and human non-AD and AD brain samples
All animal studies were conducted in compliance with the guidelines of the Animal Studies Committees of Hokkaido University (Sapporo, Japan), Shiga University (Shiga, Japan), and the National Institute of Biomedical Innovation (Osaka, Japan). Mice were maintained under a 12-h light/12-h dark cycle (lights on, 7:00 A.M.–7:00 P.M.), and provided with food and water ad libitum. X11-/-/X11L+/+, X11+/+/X11L-/-, and X11-/-/X11L-/- mice have already been described [20, 21]. HCN-/- mice (stock number 005034) were purchased from The Jackson Laboratory (Bar Harbor, Maine). Male mice were used for all experiments.
Brain samples containing the superior temporal gyrus of cynomolgus monkeys (Macaca fascicularis) were obtained from Shiga University of Medical Science and the National Institute of Biomedical Innovation. The monkeys were housed in individual cages prior to the experiment and were maintained according to institutional guidelines for experimental animal welfare. Human brain samples containing the superior temporal gyrus (Broadmann area 22) were obtained from the Brain Bank for Aging Research, the Tokyo Metropolitan Institute of Gerontology (Itabashi, Tokyo, Japan). Human temporal cortical specimens for the quantification of proteins were obtained from brains that were removed, processed, and stored at −80°C within 16 h postmortem at the Brain Bank at Tokyo Metropolitan Institute of Gerontology. (Patients were placed in a cold (4°C) room within 2 h of death.) For all brains registered at the bank, written informed consent for their use for medical research was obtained from the patient prior to death or from the patient's family. Brain specimens were collected from Broadmann area 22 (superior temporal gyrus) for 12 AD patients (79.2±4.4 years of age) and 13 control patients (80.4±4.2 years of age) . Detailed descriptions of all subjects, including the relative protein/tubulin ratio for each individual, are shown in Table 1.
Polyclonal rabbit anti-HCN1 antibody  and polyclonal rabbit anti-X11 UT153 antibody  have already been described. Monoclonal mouse anti-tubulin DM1A antibody and polyclonal rabbit anti-c-Fos, rabbit anti-Egr-1, and goat anti-HCN1 antibodies (sc-19706) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Characterization and demonstration of the antigen-specificity of the goat anti-HCN1 antibody (sc-19706) is shown in Additional file 1: Figure S7. Monoclonal mouse anti-X11L/mint2 and anti-PSD95 antibodies were purchased from BD Transduction Laboratories (Lexington, KY, USA). Anti-actin antibody and the anti-HCN1 antibody, AB5884, were purchased from Millipore (Billerica, MA, USA). Anti-FLAG M2 and polyclonal rabbit anti-APP cytoplasmic domain (N-terminus) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the anti-human APP extracellular domain antibody (LN27) was purchased from Zymed (San Francisco, CA, USA). Anti-FLAG M2 affinity gel and FLAG peptide were purchased from Sigma-Aldrich.
Human APP695 (hAPP695) and FLAG-APP695 cDNA were inserted into the pcDNA3 plasmid at the HindIII/XbaI restriction sites to produce pcDNA3-hAPP695 and pcDNA3-FLAG-hAPP695 . The cDNA constructs pcDNA3-hAPPΔcyt (in which amino acids 652–695 of hAPP695 are deleted) and pcDNA3.1-C99-FLAG (in which the signal sequence of hAPP is inserted into the 5’ region of C99) were generated by PCR using pcDNA3-hAPP695 as the template. The generated fragments were ligated into pcDNA3-hAPP695 and pcDNA3.1-FLAG at the BamHI/XbaI restriction site and the HindIII/XbaI restriction site, respectively. The pCI-murine HCN1 vector was a kind gift from Dr. Takahiro M. Ishii .
Murine brain tissue sections were prepared and incubated with primary antibodies as described . The sections were further incubated with goat anti-rabbit IgG antibodies conjugated to biotin (Vector Laboratories, Burlingame, CA, USA), followed by the ABC complex. Peroxidase activity was revealed using diaminobenzidine as the chromogen. Alternatively, sections were incubated with donkey anti-mouse IgG coupled with Alexa Fluor 488, donkey anti-rabbit IgG coupled with Cy3, or donkey anti-goat IgG coupled with Alexa Fluor 633 in phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA) for 2 h at room temperature. Sections were mounted onto slides with Shandon Immu-Mount (Thermo, Pittsburgh, PA, USA) and viewed under a BZ-9000 microscope (Keyence, Woodcliff Lake, NJ, USA).
Immunoblotting and co-immunoprecipitation analysis
The cortices of wild type, X11-/-/X11L+/+, X11+/+/X11L-/-, X11-/-/X11L-/-, HCN1+/-, and HCN1-/- mice, and cynomolgus monkey brains (superior temporal gyrus) and human post-mortem brains (superior temporal gyrus) were homogenized in eight volumes of radioimmune precipitation assay buffer containing 0.5% (w/v) sodium dodecyl sulfate (SDS) and a protease inhibitor mixture (5 μg/ml chymostatin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin). The homogenates were lysed by sonication on ice and centrifuged at 20,000 × g for 10 min at 4°C. The resulting supernatants were used for immunoblot analysis. Proteins (10 μg per lysate) were separated via SDS (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% (w/v) polyacrylamide gels.
The cortex (from one mouse) and EC-rich region (from five mice) from wild type and gene-null mice were homogenized in eight volumes of HBS-T lysis buffer (10 mM HEPES [pH 7.6] containing 150 mM NaCl, 5 mM EDTA, 0.5% [v/v] Triton X-100, 5 μg/ml chymostatin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin A). Homogenates were then centrifuged at 20,000 × g for 10 min at 4°C. N2a cells (~1 × 106) were transiently transfected with 0.8 μg pcDNA3-FLAG-hAPP695, pcDNA3-hAPP695, pcDNA3-hAPPΔcyt, or pcDNA3.1-C99-FLAG and 0.4 μg of pCI-murine HCN1 using Lipofectamine 2000 (Invitrogen) and cultured for 24 h in medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). Cells were harvested, lysed in lysis buffer (PBS containing 1.0% [v/v] Triton X-100, 5 μg/ml chymostatin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin A), and centrifuged for 5 min at 4°C. The resulting supernatants were incubated with anti-FLAG M2, anti-hAPP extracellular domain (LN27), anti-HCN1, anti-X11, or anti-X11L/Mint2 antibody at 4°C for 2 h. Each immunocomplex was recovered with Dynabeads® Protein G (Invitrogen) and washed three times with lysis buffer. The proteins were separated on 7.5% (w/v) polyacrylamide gels, transferred onto nitrocellulose membranes, and analyzed by immunoblotting with the indicated antibodies. The immunoreactants were detected using the ECL plus™ detection system (GE Healthcare, Houston, TX, USA) and quantified using a Versa Doc model 3000 (Bio-Rad, Hercules, CA, USA).
Affinity purification of FLAG-sAPP from N2a conditioned medium and immunoprecipitation of APP-HCN1 complex
N2a cells (~8.8 × 106) were transiently transfected with 5 μg pcDNA3-FLAG-hAPP695 using Lipofectamine 2000 (Invitrogen) and cultured for 24 h in 8 mL of medium (DMEM) containing 10% (v/v) FBS. FLAG-sAPP was collected from the conditioned culture medium by using 50 μL of anti-FLAG M2 affinity gel. The collected FLAG-sAPP that was bound to the gel was washed twice with wash buffer I (20 mM Tris–HCl [pH 8.0], 1 M NaCl, and 0.1% Triton X-100) and twice with wash buffer II (50 mM Tris–HCl [pH8.0], 150 mM NaCl, 1% Triton X-100, 0.05% SDS, and 5 mM EDTA). Collected FLAG-sAPP was then eluted from the affinity gel with 20 μg FLAG-peptide and subjected to immunoblotting and Coomassie brilliant blue (CBB) staining to ascertain the degree of purification.
FLAG-sAPP or FLAG-peptide coupled to anti-FLAG M2 affinity beads were then incubated for 2 h at 4°C with HBS-T-soluble lysates derived from wild type N2a cells or N2a cells transiently overexpressing HCN1. The beads were washed three times with HBS-T lysis buffer. The proteins bound to the beads were separated on 7.5% (w/v) polyacrylamide gels, transferred onto membranes, and analyzed by immunoblotting with the indicated antibodies. The immunoreactants were detected using the ECL plus™ detection system (GE Healthcare) and quantified using a Versa Doc model 3000 (Bio-Rad).
Quantification of Aβ40 and Aβ42
Endogenous murine Aβ was measured as described previously  using cortices dissected from 4-month-old mice. Murine Aβ40 and Aβ42 were measured using a sandwich ELISA (sELISA) system (mouse/rat Aβ40 and Aβ42 assay kit, Immuno-Biological Laboratories (IBL), Fujioka, Japan). N2a cells (~2 × 105) were transiently transfected with 0.2 μg pcDNA3-FLAG-hAPP695 and 0.1 μg pCI-murine HCN1 using Lipofectamine 2000 (Invitrogen) and cultured in medium (DMEM) containing 10% (v/v) FBS. After 24 h, cells were incubated in fresh medium for an additional 4 h with or without 10 μM ZD7288 (Tocris Bioscience, Bristol, UK). Human Aβ40 (hAβ40) and hAβ42 secreted into the culture medium during the 4-h incubation were quantified using the sELISA system.
To obtain free-moving cortical electrocorticogram recordings, recording and reference electrodes were screwed onto the skull over the temporal (anterior = −3.1 mm, lateral = 2.5 mm, relative to bregma) and occipital regions of the murine brain. Recordings were continuously made using a cortical electroencephalogram linked to a telemetry system (Unimec, Usmate Velate, Italy) throughout the experiment .
Ih current recording
All experiments were performed in a blinded manner. Mice (12–14 weeks old) were anesthetized with halothane (Takeda Chemical Industries) and then sacrificed by decapitation. The brain was rapidly removed and immediately placed in a cold (4°C) cutting solution, which contained 234 mM sucrose, 2.5 mM KCl, 1.1 mM NaH2PO4, 10 mM MgSO4, 26 mM NaHCO3, 12 mM glucose, and 0.5 mM CaCl2. Horizontal slices (300 μm thick), which included the EC and the hippocampus, were prepared using a vibratome (VT1000S, Leica, Nussloch, Germany). During recording, individual slices were transferred to a submerged recording chamber and continuously perfused with artificial cerebrospinal fluid (ACSF) maintained at 30–32°C. The ACSF contained 125 mM NaCl, 2.5 mM KCl, 1.1 mM NaH2PO4, 1.0 mM MgSO4, 26 mM NaHCO3, 12 mM glucose, and 2.0 mM CaCl2 and was saturated with 95% O2 and 5% CO2. Whole-cell patch-clamp recordings were obtained from principal excitatory cells in layer II of the EC. The patch pipettes were filled with an intracellular solution containing 30 mM K-methanesulfonate, 6 mM NaCl, 0.2 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na3-GTP, and 10 mM phosphocreatine-Tris (pH 7.3). In layer II cells of the EC, the hyperpolarization-induced and “slowly-activating” inward currents in the voltage-clamp mode mainly consisted of Ih currents . When Ih currents were studied in the voltage-clamp mode, membrane potentials were first held at −65 mV, and then voltage steps with a duration of 7 s were applied from −55 mV to −125 mV (10 mV increments), after which the holding potentials were allowed to return to −65 mV to obtain the tail currents. The amplitudes of the tail currents at 50 ms after the end of the final voltage step were analyzed to obtain the Ih currents. In all electrophysiological analyses, pooled data were represented as the mean ± SEM.
Statistical analyses were performed using a two-tailed Mann–Whitney U-test, a one-way analysis of variance followed by Tukey’s multiple comparison test, or the two-tailed Pearson's correlation coefficient. All analyses were conducted with GraphPad Prism 5 software.