The following antibodies were purchased from commercial suppliers: anti-C- terminal domain of APP (#18961, Immuno-Biological Laboratories, Gunma, Japan, 1:1000 dilution), anti-C terminus of sAPPβ (#18957, Immuno-Biological Laboratories, Gunma, Japan, 1:1000 dilution), anti-C-terminal domain of BACE1 (#18711, Immuno- Biological Laboratories, Gunma, Japan, 1:1000 dilution), anti-Nct C terminus (N1660, Sigma, MO, USA, 1:1000 dilution), anti-α-tubulin DM1A (T6199, Sigma, MO, USA, 1:2000 dilution), anti-V5 Tag antibody (Thermo Fisher Scientific, MA, USA, 1:1000 dilution), TUJ1 anti-human βIII tubulin (R&D systems, MN, USA, 1:2000 dilution), Clone N103/39 anti-ALDH1L1 antibody (NeuroMab, CA, USA, 1:1000 dilution), 6H7 anti-EPHA4 antibody (Abnova, Taipei, Taiwan, 1:1000 dilution), 4G10 anti- Phosphotyramine-KLH antibody (Merck Millipore, MA, USA, 1:500 dilution).
Peptides and reagents
KYL (H2N-KYLPYWPVLSSL-COOH), biotinylated KYL (H2N- KYLPYWPVLSSLGSGSK-(biotin)-COOH), WDC (H2N-WDCNGPYCHWLG-COOH), and biotinylated WDC (H2N-WDCNGPYCHWLGGSGSK-(biotin)-COOH) were synthesized by BEX CO., LTD. (Tokyo, Japan). Recombinant ephrin-Fc were obtained from the companies as following: mouse ephrin-A1/Fc Chimera and mouse ephrin-B1/Fc Chimera from R&D Systems (MN, USA), ChromPure Human IgG Fc Fragment and AffiniPure goat anti-human IgG Fc fragment specific from Jackson Immuno Research Laboratories (PA, USA). 100 μL of ephrin-Fc (100 μg/ mL) was mixed with 19 μL anti-human IgG and added to cells after 1 hr of incubation at 37℃.
Plasmids preparation and transfection
Epha4-WT plasmid was kindly provided by Dr. Atsuko Sehara at Kyoto University. V5-His was tagged at the C terminus of Epha4-WT. Epha5-WT was cloned using the same vector. Epha4-KD (kinase-dead) has a K653M mutation, leading to the loss of kinase activity as previously reported(22). Epha4-ΔSAM has a deletion range from 908 to 964 amino acid residues, only remains 12 amino acids of SAM domain at the upstream of the PDZ binding motif.
For overexpression of EPHA4 and EPHA4 mutants, Neuro2a (N2a) cells were transfected with a mixture of the plasmid with polyethylenimine (PEI) or FuGENE6 (Promega, WI, USA) solution according to the manufacture’s instruction.
Cell culture and generation of stable cell lines
N2a cells (#CCL–131, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, UT, USA), and 50 unit/mL Penicillin/Streptomycin (Thermo Fisher Scientific, MA, USA). Primary neuron-glia mixed cells were cultured in Neurobasal medium (Thermo Fisher Scientific, MA, USA) supplemented with 2 mM L-Glutamine (Thermo Fisher Scientific, MA, USA), 50 unit/mL Penicillin/Streptomycin and 2% B–27 supplement (Thermo Fisher Scientific, MA, USA). All cell lines were maintained in a 5% CO2, 95% air atmosphere incubator at 37°C. Contamination of mycoplasma is routinely tested by PCR and DNA agarose electrophoresis.
To generate stable cell lines, N2a cells were transiently transfected with plasmids coding murine Epha4-WT or Epha4-mutants using PEI solution and underwent neomycin G418 selection (Millipore Sigma, St. Louis, USA).
For neuron-glial mixed culture and primary neuron-enriched culture, the plate was coated using 250 μL of poly-L-ornithine solution (PLO; SIGMA, MO, USA) overnight. PLO was washed by FBS-free DMEM before collect primary cells. Primary cells were obtained from the fetuses of E18 or E19 pregnant Wistar rat (Japan SLC, Inc., Shizuoka, Japan). All procedure was carried out using cold Hanks’ Balanced Salt Solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The collected brain tissue was incubated with 0.25% Trypsin (Thermo Fisher Scientific, MA, USA), 0.1 μL/mL DNase (Nippon Gene, Toyama, Japan), 0.8 mM MgSO4 (Kanto Chemical, Tokyo, Japan), and 1.85 mM CaCl2 (Kanto Chemical, Tokyo, Japan) at 37°C. After centrifugation, cells were counted for the appropriate amount and plated into the plate. For primary neuron-enriched culture, cells were cultured in Neurobasal medium containing 1 μM Ara-C (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) from DIV1. For primary glia-enriched culture, the plate without pre-coating with PLO was used and cells were cultured in DMEM supplemented with 10% FBS.
For the measurement of the secreted Aβ, conditioned media were collected, and cell debris was removed by the centrifugation at 240 x g for 3 min. For secreted Aβ from primary cells, Aβ levels were analyzed by two-site enzyme-linked immunosorbent assay (ELISA) using Human/Rat β Amyloid (40) ELISA Kit (294–64701, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and Human/Rat β -Amyloid (42) ELISA Kit, High Sensitivity (292–64501, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), as described(23,24). The secreted Aβ from N2a cells was analyzed by ELISA using a homemade Aβ detecting plate based on the same principle of manufacturer’s ELISA Kit(9).
Aβ levels measured by ELISA were then standardized by protein concentrations of the cell lysates and further normalized to the control in each experiment as indicated.
All procedures of immunoblotting were performed as previously described(24). Briefly, for sample preparation, cells were lysed by Laemlli 1X sample buffer (2% sodium dodecyl sulfate (SDS; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 80 mM Tris-HCl with pH 6.8, 15% glycerol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.0025% Brilliant green (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.00625% Coomassie Brilliant Blue G–250 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)) and sonicated (BRANSON, Danbury, CT, USA). Protein concentrations were measured by using BCA protein assay (Takara Bio, CA, USA) following the manufacturer’s instruction. The conditioned medium was collected and diluted using a 5X sample buffer. All samples were added with 1% 2- mercaptoethanol (Millipore Sigma, St. Louis, USA) and boiled at 100°C.
Samples and protein marker, Precision Plus Protein Dual Xtra Standards (BIORAD, CA, USA), were applied to SDS-polyacrylamide gel (7.5–15% Tris-Glycine or Tris-Tris gels) and transferred onto PVDF membrane (Millipore, MA, USA). The immunodetection was used ImmunoStar detection kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) or Supersignal West Femto (Thermo Fisher Scientific, MA, USA), and chemiluminescence was detected using Image Quant LAS4000 (GE Healthcare, IL, USA). The immunoreactive protein bands were digitally captured and quantified using ImageJ (NIH) software.
Biotinylated antagonist binding assay
N2a cells overexpressing EPHA4 or EPHA5 were harvested the day after transfection. Cells were washed with phosphate-buffered saline (PBS; 8 mM Na2HPO412H2O, 2 mM NaH2PO42H2O, 130 mM NaCl), mixed with the appropriate volume of HEPES lysis buffer (10 mM HEPES pH7.4 (DOJINDO, Kumamoto, Japan), 150 mM NaCl, 1% TritonX–100 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 1 mM EDTA, 10% glycerol) and sonicated. All samples were adjusted to contain an equal amount of total protein after quantification by BCA protein assay. Streptavidin Sepharose (Thermo Fisher Scientific, MA, USA) was then added, followed by the rotation at 4°C for 1 hr. After centrifugation at 15,000 rpm for 3 min, the supernatant was collected and a small portion of it was used as an input sample in immunoblotting. The remained supernatant was added with biotinylated KYL or biotinylated WDC with/without non-tagged KYL or WDC at the indicated concentration, and rotated at 4°C overnight. After centrifugation at 15,000 rpm for 3 min, the pellet was washed by lysis buffer and added with sample buffer as pulled down sample in immunoblotting.
Stereotaxic injection of KYL peptide
All experiments using animals in this study were performed according to the guidelines provided by the Institutional Animal Care Committee of the Graduate School of Pharmaceutical Sciences at the University of Tokyo (protocol no.: P26–9). 10 mM KYL peptide/PBS and PBS were injected into the right hippocampus (Anteroposterior: –2.0, Mediolateral: –1.5, Dorsoventral: –1.6) and left hippocampus (Anteroposterior: –2.0, Mediolateral: +1.5, Dorsoventral: –1.6), respectively, of 8 weeks old C57BL/6J male mice (Japan SLC, Inc., Shizuoka, Japan). 8 hrs after injection, both hippocampi were collected, lysed using RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing Complete protease inhibitor cocktail (Millipore Sigma, St. Louis, USA), homogenized, and ultra-centrifuged at 444,000 rpm at 4°C for 20 min. The supernatant was used for Aβ detection by ELISA.
Cells were harvested, washed by cold PBS, and lysed with 1% 3-[(3- cholamidopropyl) dimethylammonio]–2-hydroxypropanesulfonate (CHAPSO; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) / HEPES buffer (10 mM HEPES pH7.4, 150 mM NaCl, Complete protease inhibitor cocktail EDTA free (Millipore Sigma, St. Louis, USA)). After the centrifugation at 15,000 rpm at 4°C for 3 min, the appropriate amount of the supernatant was taken as an input sample. Aliquots of the supernatant were mixed with 30 μL of 50% Protein G agarose (Thermo Fisher Scientific, MA, USA) / Tris-buffered saline (TS) and rotated at 4°C for 1 hr. After the centrifugation at 3,000 rpm for 5 min, the supernatant was added with an anti-EPHA4 antibody, and rotated at 4°C overnight. 30 μL of 50% Protein G agarose/TS was added to all samples on the following day. After the additional rotation at 4°C overnight, samples were centrifuged at 3,000 rpm, 4°C for 5 min. The pellet was washed several times with lysis buffer, and added with sample buffer to each sample, followed by the immunoblotting using 4G10 anti-Phosphotyramine-KLH antibody.
Knockdown by shRNA treatment
For knockdown of Epha4 in the primary neuron, shRNA targeting Epha4 sequence (CCGGggatatgtccaatcaagatgtTTCAAGAGAacatcttgattggacatatccTTTTTG) was cloned into the pLKO.1 puro vector (#8453, Addgene). LentiX–293T cells were transiently co-transfected with the packaging plasmids (pCAG-KGP4.1R, pCAG4-RTR2, and pCAGS-VSVG) and the prepared plasmid using PEI solution. After the collection of the medium including lentivirus particles, it was concentrated using Lenti-X™ concentrator (Clontech, CA, USA). The lentiviral particles were resuspended in 500 μL Neurobasal medium and added at 30–60 μL/well into the primary neuron (DIV3). Medium change with fresh 250 μL Neurobasal medium was done at DIV7. The conditioned medium for detecting Aβ levels was collected after 24 hrs of incubation and the cells were collected by sample buffer for immunoblotting.
Two public RNAseq datasets were obtained from AMP-AD Knowledge Portal (https://www.synapse.org/#!Synapse:syn2580853) as previously described(25): the Mayo sample set(26) and MSBB studies. The Mayo study comprises temporal cortex samples from 164 subjects with the following pathological diagnosis: 84 patients with AD and 80 controls. We assessed EPHA4 expression of the temporal cortex between AD patients and controls by a simple model (syn6090804) adjusting for key covariates: age at death, gender, RIN, source, and flow cell. For the MSBB study, we obtained Clinical information of each subject, RNAseq covariates, and normalized EPHA4 or BACE1 normalized RNA read counts (syn7391833). We selected 201 samples of the parahippocampal gyrus (BM36) from subjects and excluded the samples without the information of the Braak NFT stage. These data were applied and analyzed using RStudio. The comparison of EPHA4 and BACE1 gene expression levels was performed under different set conditions. We divided samples into two categories, healthy control subjects (CT) and AD patients, depending on the NP.1 stage, neuropathology Category as measured by CERAD. As described in the figure legend, we also divided degrees of neuritic plaque density (plaque levels) into five categories depending on the provided plaque mean which is the mean neocortical plaque density across 5 regions, the middle frontal gyrus, orbital frontal cortex, superior temporal gyrus, inferior parietal cortex and calcarine cortex (of plaques/mm2). Suitable statistical analysis, Mann-Whitney U test, Kruskal-Wallis test with Dunn’s post hoc analysis, and Kendall rank correlation were performed to compare the EPHA4 and BACE1gene expression levels under each condition.
Data analyses were carried out from independent cells and were not conducted in a blinded fashion. And we excluded samples only when there is evidence of contamination, cell peeling, or cell death prior to the experiments. Data are presented as mean values and error bars indicate the standard error of the mean (s.e.m.). Suitable statistical analysis, unpaired/paired two-tailed Student’s t-test or ANOVA with Tukey’s or Dunnett’s post hoc test, was performed. A p-value of less than 0.05 was considered to have a statistically significant difference between groups.