Animals
C57BL/6J WT mice (RRID: IMSR_JAX: 000664) were bought from SLC. LOTUS-overexpressing transgenic (LOTUS-tg) mice were created using synapsin-1 promoter, which selectively expresses neuron-specific LOTUS, as previously explained (Hirokawa et al., 2017). These mice were raised in rectangular plastic cages with stainless-steel mesh covers (four mice in each cell at most) in a pathogen-free facility under 12 h/12 h light/dark conditions and free access to autoclaved water and food. During the experimental procedures, all efforts were made to reduce the number of animals used and minimize their suffering. The experimental procedures were approved by the institutional animal care and use ethical committee of Yokohama City University and were conducted in accordance with the approved guidelines. The procedures were approved by the institutional animal care and use ethics committee of Yokohama City University (approval number #T-A-20-002).
Antibodies and reagents
Mouse monoclonal antibodies against rat LOTUS (custom-made, ITM, RRID: AB_2819118), goat polyclonal antibodies against mouse PIR-B (AF2754, R&D Systems, RRID: AB_2249965), rat monoclonal antibodies against mouse PIR-B (550348, BD Biosciences, RRID: AB_393627), mouse monoclonal antibodies against synthetic β-actin (A5316, Sigma-Aldrich RRID: AB_476743), rabbit monoclonal antibodies against cofilin (#5175, CST, RRID: AB_10622000), rabbit monoclonal antibodies against phospho-cofilin (Ser3) (77G2) (#3313, CST, RRID: AB_2080597), mouse monoclonal antibodies against PSD-95 (MA1-046, Thermo Fisher Scientific, RRID: AB_2092361), goat polyclonal antibody against human immunoglobulin (Ig)-like transcript 4 (ILT4)/CD85d (AF2078, R&D systems, RRID: AB_355136), biotin-SP-labeled goat antibodies against mouse IgG (115-065-003, Jackson ImmunoResearch, RRID: AB_2338557), Alexa Fluor 488-labeled donkey antibodies against mouse IgG (715-545-151, Jackson ImmunoResearch, RRID: AB_2341099), Alexa Fluor 596-labeled donkey antibodies against goat IgG (705-585-147, Jackson ImmunoResearch, RRID: AB_2340433), purified rat IgG (6-001-A, R&D Systems, RRID: AB_10144734), peroxidase-conjugated antibodies against mouse IgG (115-035-003, Jackson ImmunoResearch, RRID: AB_10015289), peroxidase-conjugated antibodies against rabbit IgG (111-035-003, Jackson ImmunoResearch, RRID: AB_2313567)
Aβ preparation
Recombinant biotin-LC-Aβ (1–42) peptides (AS-20276, Anaspec Inc) were dissolved in 1% NH₄OH and diluted in phosphate-buffered saline (PBS) to the concentration of 100 µM. The Aβ peptide was incubated at 37℃ for 48 h and stored at − 80℃ until use for Aβ to oligomerize. Successful Aβ oligomerization was confirmed by non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Construction of plasmid vectors
Plasmids encoding full-length mouse LOTUS (NM_145123) and full-length mouse PirB (NM_001357394) were generated as previously explained (Sato et al., 2011; Kurihara et al., 2020). The plasmid encoding enhanced green fluorescent protein (EGFP) was generated by injecting the coding sequence of the EGFP construct (pEGFP-N3, 6080-1, BD Bioscience) into the 3’ region of the CAG promoter in another mammalian exhibition vector. The plasmids encoding full-length human LilrB2 (NM_001080978) and full-length human LOTUS (NM_001206528) construct were generated by subcloning the full-length cDNA of LilrB2 and LOTUS from the human brain cDNA library (9503, TAKARA) into the vector. The expression plasmids of the mutated human Fc-tagged Streptavidin binding protein (SBP) (Fc-SBP), original signal peptide region-, and the transmembrane region-deleted mouse LOTUS fusing Fc-SBP (LOTUS-Fc-SBP), as previously described (Kurihara et al., 2020). Correct alignment of the nucleotides inserted into each plasmid was validated by DNA sequencing.
Ligand-receptor binding assay and immunocytochemistry
Cos-7 cells (RRID: CVCL_0024) cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) (08458-16, Nacalai Tesque) including 10% fetal bovine serum (FBS) (04-001-1A, Biological Industries) and 0.5% penicillin-streptomycin mixed solution (Nacalai Tesque). All cells were operated using a sterile cell culture procedure and incubated at 37℃ with 5% CO2. Cells were seeded at 5.0 × 10⁴ cells/well in 4-well culture plates (176740, Thermo Fisher Scientific). The plasmids encoding mouse LOTUS, mouse PirB, human LOTUS, or human LilrB2 were transfected to the cells using FuGENE 6 (E2691, Promega) and cultured for 44 h. For the binding assay, transfected cells were treated with biotin-LC-Aβ (1–42) at each concentration for 1 h at 37℃ with 5% CO2. Following treatment, the cells were fixed with 4% PFA in PBS containing 2 mM MgCl2 for 1 h at room temperature (RT), rinsed with PBS containing 2 mM MgCl2, and incubated for 1 h at 67℃ to inactivate endogenous alkaline phosphatase (AP). Next, the cells were incubated with AP-conjugated avidin-biotin complex (AK-5000, Vector Laboratories, RRID: AB_2336792) in TBS-T for 1 h at RT. Aβ peptide bound to the cells was visualized with the enzymatic reaction product of nitro blue tetrazolium (NBT) (11383213001, Roche) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (11383221001, Roche). The digital images were captured with a BZ-8100 microscope (Keyence) fitted with a 10× objective lens. For the purpose of quantification, its intensity was detected using pNPP (N2770-50SET, Sigma-Aldrich) by measuring its absorbance at 405 nm using xMark Microplate Absorbance Spectrophotometer (Bio-Rad) and Microplate Manager 6 software (Bio-Rad).
Immunocytochemistry was used to validate the cell surface expression of mouse PirB and human LilrB2 under non-permeabilizing conditions as previously described (Sato et al., 2011; Kurihara et al., 2012, 2014, 2020). Briefly, the transfected and cultured cells were incubated with antibodies against mouse PIR-B (1 µg/ml, AF2754, R&D Systems, RRID: AB_2249965), human ILT4/CD85d (1 µg/ml, AF2078, R&D systems, RRID: AB_355136) and/or LOTUS (1 µg/ml, H24G11-mAb, Sato et al., 2011, RRID: AB_2819119) in the culture medium for 1 h at 37℃ with 5% CO2 and subsequently fixed with 4% PFA in PBS for 1 h at RT. After washing with TBS-T, the fixed cells were incubated with Alexa Fluor 488-labeled donkey antibodies against mouse IgG (0.75 µg/ml, 715-545-151, Jackson ImmunoResearch, RRID: AB_2341099) and Alexa Fluor 596-labeled donkey antibodies against goat IgG (0.75 µg/ml, 705-585-147, Jackson ImmunoResearch, RRID: AB_2340433) in TBS-T for 1 h at RT. Digital images were captured with a BZ-8100 microscope (Keyence) equipped with a 10× objective lens. For quantitative detection of cell surface expression of PirB or LilrB2, the transfected and cultured cells were incubated with antibodies against PIR-B (1 µg/ml, AF2754, R&D Systems, RRID: AB_2249965) or human ILT4/CD85d (1 µg/ml, AF2078, R&D systems, RRID: AB_355136) in the culture medium for 1 h at 37℃ with 5% CO2, fixed with 4% PFA in PBS for 1 h at RT, and heated in PBS containing 2 mM MgCl2 for 1 h at 67℃. The treated cells were then incubated with biotin-SP-labeled donkey antibodies against goat IgG (0.7 µg/ml, 705-065-003, Jackson ImmunoResearch, RRID: AB_2340396) in TBS-T for 1 h at RT and with VECTASTAIN ABC-AP Staining Kit (AK-5000, Vector Laboratories, RRID: AB_2336792) diluted with TBS-T for 1 h at RT, followed by incubation with pNPP for 4 h at RT. The enzymatic reaction product of pNPP was measured using xMark Microplate Absorbance Spectrophotometer (Bio-Rad) and Microplate Manager 6 software (Bio-Rad) with absorbance at 405 nm wavelength.
Primary culture.
Hippocampal neurons obtained from WT or LOTUS-tg mice on embryonic day 17.5 (E17.5) were dissociated with 0.25% trypsin at 37℃ for 12 min and subsequently treated with 100 µg/ml DNase at 37℃ for 5 min. Dispersed cells were seeded in a 24-well dish (Greiner Bio-One) on poly-L-lysine (100 µg/ml, 163-19091, Wako)-coated glass coverslips (φ12 mm; Matsunami) or 6-well dishes at each concentration and incubated in neurobasal medium (21103, Gibco) containing 10% FBS and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes)-NaOH (pH 7.3) for 3 h. Next, the medium was altered to a neurobasal medium containing 1×B-27 (17504-044, Gibco), 1×Glutamax (Gibco), 10 mM Hepes-NaOH (pH 7.3), and the cells were incubated at 37℃ with 5% CO2.
PSD95 expression and cofilin dephosphorylation
Aβ peptide was added to cultured hippocampal neurons (2.5 × 10⁵ cells/well) at each concentration on DIV12 or DIV14 and incubated for 48 h (for PSD95) or 1 h (for cofilin dephosphorylation). For the experiments to examine the PirB antibody effect on the Aβ-induced PirB downstream pathway, the antibody against PirB (550348, BD Biosciences, RRID: AB_393627) or control rat IgG (6–001-A, R&D Systems, RRID: AB_10144734) was added at a final concentration of 3 µg/ml at the same time as Aβ peptide. Following each incubation, cells were rinsed with cold PBS and lysed using a lysis buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA-NaOH (pH 8.0), 1% Nonidet P-40, 1 mM Na3VO4, 0.05 mM (p-amidinophenyl) methanesulfonyl fluoride (Wako, 019-26331), 0.1 U/mL aprotinin (Sigma-Aldrich, A6279) and the lysate was centrifuged at 20,000 × g for 10 min at 4℃. The supernatant was mixed with 4× Laemmli buffer (40% glycerol, 8% SDS, 250 mM Tris-Cl pH 6.8, 0.03% bromophenol blue) and heated at 100℃ for 7 min. The samples in Laemmli buffer were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Immobilon-P Membrane, IPVH00010, Millipore). The membrane was blocked with 5% skim milk in TBS-T for 1 h at RT, incubated sequentially with each primary antibody in TBS-T containing 5% skim milk for 1 h at RT with horseradish peroxidase (HRP)-labeled antibodies against mouse IgG, HRP-labeled antibodies against rabbit IgG in TBS-T containing 5% skim milk or 5% BSA for 1 h at RT, and immersed in chemiluminescent HRP substrate (ECL Western Blotting Detection Reagents, RPN2109, GE Healthcare Life Sciences) (Immobilon Western Chemiluminescent HRP Substrate, WBKLS0100, Millipore). The chemiluminescent signals were detected using ImageQuant LAS 4000 mini apparatus (GE Healthcare Life Sciences) or LAS-4000 multicolor apparatus (Fujifilm) and with ImageQuant TL software (GE Healthcare Life Sciences, RRID: SCR_014246).
Hippocampal neuron transfection and spine imaging
Plasmid encoding EGFP was transfected into cultured hippocampal neurons (5.0 × 10⁴ cells/well) with Viafect (E4981, Promega) on DIV11for visualizing dendritic spine morphology in primary hippocampal neurons to assess for dendritic spine density. Approximately 24 h following transfection, Aβ was applied at each concentration and incubated for 48 h. The Aβ-treated cells were fixed at 4% PFA in PBS for 10 min at 37℃. After washing with PBS, fluorescent images of the dendritic spine were acquired using a confocal microscope (TCS SP8; Leica) equipped with a 63× (NA, 1.4) oil-immersion objective and the LAS X software (Leica). Images were captured at a resolution of 1024 × 1024 pixels with a z-step of 0.3 µm. Cells with no abnormalities, including protrusion of cell membrane or fragmentation of dendrites, were evaluated.
Protein purification
HEK293T cells (RRID: CVCL_0063), within 25 passages, were seeded (9 × 106 cells/dish) on 145 mm cell culture dishes (639160, Greiner Bio-One), cultured in DMEM (08458-16, Nacalai Tesque) containing 10% FBS and 0.5% penicillin-streptomycin solution. Following 48 h culture, plasmid encoding Fc-SBP and LOTUS-Fc-SBP were transfected with lipofection reagent (Polyethylenimine Max, 24765, Polysciences) and cultured for an additional 4 days. The culture medium was ultracentrifuged at 117,000 × g for 1 h. Subsequently, the supernatant was added to streptavidin beads (High Capacity Streptavidin Agarose Resin, 20361, Thermo Fisher Scientific). SBP-fused protein was eluted from the beads with PBS containing 2 mM biotin. These proteins were stored at − 80℃ until use. The protein sample was prepared with 4× Laemmli buffer (40% glycerol, 8% SDS, 250 mM Tris-Cl pH 6.8, and 0.06% bromophenol blue) containing 10% β-mercaptoethanol and boiled for 7 min to determine the concentration of purified proteins. Each sample was electrophoresed on a Tris-glycine SDS polyacrylamide gel, and the gel was incubated with Coomassie brilliant blue R-250 (031-17922, Wako Pure Chemical Industries). The intensity of the stained protein was measured for each concentration using an ImageQuant LAS 4000 mini instrument with ImageQuant TL software.
Human LOTUS-LilrB2 ligand-receptor binding assay
Transfected Cos7 cells were treated with Fc-SBP or human LOTUS-Fc-SBP protein at each concentration and incubated for 1 h at 37℃ with 5% CO2. Following treatment, the cells were fixed with 4% PFA in PBS containing 2 mM MgCl2 for 1 h at RT, washed with PBS containing 2 mM MgCl2, and incubated for 1 h at 67℃ to inactivate endogenous AP. Subsequently, fixed cells were incubated sequentially with antibodies against SBP (0.04 µg/ml, sc-101595, Santa Cruz Biotechnology, RRID: AB_1128239) in 1% skim milk/TBS-T for 1 h at RT and with biotin-SP-labeled goat antibodies against mouse IgG (0.7 µg/ml, 115–065–003, Jackson ImmunoResearch, RRID: AB_2338557) in 1% skim milk/TBS-T for 1 h at RT, AP-conjugated avidin-biotin complex (AK-5000, Vector Laboratories, RRID: AB_2336792) was diluted to 1/5000 with TBS-T for 1 h at RT. The binding of the SBP-fused peptides was visualized with the enzymatic reaction product of NBT (11383213001, Roche) and BCIP, or detected quantitatively with that of pNPP. Digital images were captured using a BZ-8100 microscope (Keyence) equipped with a 10× objective lens. For the quantitative detection of protein binding, absorbance of the enzymatic reaction product of p-nitrophenyl phosphate (pNPP) (N2770-50SET, Sigma-Aldrich) was measured at 405 nm wavelength using the xMark Microplate Absorbance Spectrophotometer (Bio-Rad) and Microplate Manager 6 software (Bio-Rad). As previously reported, immunocytochemistry was used to confirm the cell surface expression of human LilrB2 under non-permeabilizing conditions (Kurihara et al., 2014).