Animals
The 5×FAD mice were purchased from Jackson Laboratory (Stock no. 034848-JAX, Bar Harbor, USA) with five familial Alzheimer’s disease gene mutations on human amyloid precursor protein [K670N/M671L (Swedish) + I716V (Florida) + V717I (London)] and human presenilin 1 (M146L + L286V). The mutated genes are overexpressed in the brain driven by the murine neural specific Thy1 promoter. The animals were housed in groups of no more than five mice per cage. The room temperature was controlled at 22 ± 1°C with standard 12-hour light/dark cycle and the humidity was controlled at 45–65%. All animal protocols were approved by the Ethics Committee of Fujian Medical University and the entire experimental process was carried out in accordance with the guidelines of the Animal Ethics Committee of Fujian Medical University and followed the international guidelines for the ethical use of animals. The number of experimental animals used were minimized in the study.
Vectors and virus production
The full open reading frame complementary DNA clone of human CFH (Cat: HG10714-G) was purchased from Sinobiological (Beijing, China). FHL-1WT, FHL-1V62I, and FHL-1Y402H mutations were generated by cloning. They were then cloned into the lentiviral vector pLVX-CMV-AcGFP1-N1 (Cat: 632154, Clontech, Mountain View, USA) with inserted 3×FLAG and P2A sequence prior to GFP for the preparation of recombinant lentivirus as previously described[16]. All sequences of plasmids were verified by DNA sequencing and the lentiviruses carrying the gene of Control, FHL-1WT, FHL- 1V62I, and FHL-1Y402H were produced by OBiO Technology (Shanghai) Corp in China.
Stereotaxic virus injection
5 months (5M) old 5×FAD mice were anaesthetized with isoflurane and placed on a stereotactic frame (RWD Life Science CO, LTD, China). The lentiviruses carrying the gene of Control, FHL-1WT, FHL- 1V62I, and FHL-1Y402H (1 × 106 viral genomes in 5ul PBS at one site) were bilaterally injected into two spots of the hippocampus (AP: − 2.0 mm; ML: ± 1.5 mm; DV; -1.5 and − 2.0 mm) of the mice (four groups, each 7 male mice) with 10 µl Hamilton syringes connected to an infusion pump (RWD Life Science, China) at a speed of 0.5ul/min. The needle was left in place for an additional 5 min to allow diffusion before removing it.
Cell Culture and infection
Human HEK-293 cell line (Cat: CL-0001) and BV2 microglia cell line (Cat: CL-0493A) were purchased from Procell Company in China. These cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; PAN biotech, Germany) and 1% penicillin/ streptomycin (Solarbio, China) at 37°C in a humidified, 5% CO2 atmosphere. Cells were infected with lentivirus at MOI 10 when the sub-cultured cells grew to 50–60%. The infected cells were then further cultured for 72 hours and processed for the related experiments.
Open field test
Mice were allowed to habituate the experimental room for 30 min before test. Mice were then placed into the center of an opaque box (40x40x40 cm) open field arena and left to explore freely for 10 min. Data including move distance and speed were automatically recorded and measured using ANY-maze video tracking software (Stoelting, CO, USA).
Novel object recognition test
Neutral-colored wooden objects were employed in this test and the animals were monitored by an overhung video camera. On day 1, mice were placed in an opaque box (40x40x40 cm) to freely explore for 10 min without objects. On Day 2, mice were left to freely explore the arena with two identical objects positioned equally for 10 min. The memory test was conducted 24 hour later (day 3). One of the familiar objects was replaced by a novel object different in shape and color, and mice were left to explore both objects for 10 min. Data were automatically collected and analyzed using ANY-maze video tracking software (Stoelting, CO, USA).
Morris water maze test
The Morris water maze test was applied to assess the spatial learning and memory of mice (7 mice per group) based on a previous protocol [17]. A transparent round platform (8 cm in diameter) was placed in the southeast corner in a dark circular pool (1.2 m in diameter and 0.5 m in height). Before testing, the pool was filled with opacified water and the water temperature was set at 22 ± 2°C. During the 5-day training period, each mouse underwent 4 trials each day and the starting of each trial was from a different location. The mice were left to swim freely for 60 second to search the platform and the average value of the 4 trials was calculated as the escape latency time. 24 hours after the last training trial, the probe trial (day 6) was carried out with the platform removed. Each mouse was given 60 second to explore the pool and the swimming performance was recorded and analyzed with ANY-maze video tracking software (Stoelting, CO, USA).
Western blotting
Mice’s brains were perfused with PBS to remove blood. The brain tissues or cell samples were ground and sonicated in a cold RIPA buffer with protease (Cat: K1007, APExBIO, China) and phosphatase (Cat: K1015, APExBIO, China) inhibitor cocktail. The lysates were then centrifuged at 12,000 rpm for 15 min at 4°C and the supernatants were collected. The protein concentration was measured with the BCA protein assay kit (Cat: ab287853, Abcam, USA ) and the samples were heated at 100°C for 10 min. Equal amounts of protein were separated by SDS-PAGE and transferred to 0.45µm PVDF membranes. The membranes were blocked with 1% BSA-TBST at room temperature (RT) for one hour. It was then incubated with primary antibodies diluted in 1% BSA-TBST at 4°C overnight. After washing, the membrane was incubated with a secondary antibody for 1 hour at RT. Immunoblot signals were detected using an ECL substrate (Cat: HRD0815, Heruibio, China). Quantification was processed with the NIH ImageJ software. Applied antibodies were listed as followings: Sheep anti-CFH (1:1000, Cat: ab8842, Abcam, USA), Rabbit anti-C3 (1:1000, Cat: ab200999, Abcam,USA), Rabbit anti-CD68 (1:1000, Cat: ab125212, Abcam, USA), Mouse anti-β-actin(1:5000, Cat: 3700, CST, USA), Goat Anti-Rabbit IgG H&L (HRP, 1:5000, Cat: ab6721, Abcam, USA), Goat Anti-Mouse IgG H&L (HRP, 1:5000, Cat: ab6789, Abcam, USA), Donkey Anti-Sheep IgG H&L(HRP, 1:5000, Cat: ab6900, Abcam, USA).
Immunofluorescence assays (IF)
Mice were perfused transcardially with PBS. The brains were then fixed in 4% paraformaldehyde and cryoprotected as previously described[16]. Free-floating serial coronal sections (30 µm thickness) of the hippocampus were cut with a freezing sliding microtome. Sections were blocked with blocking buffer (0.3% Triton X-100 PBS with 1% BSA and 5% normal donkey serum) at RT for one hour, and then incubated with indicated primary antibodies diluted in blocking buffer at 4°C overnight. The sections were incubated with fluorescent secondary antibodies at RT for two hours. Images were acquired under a Leica TCS SP5 confocal microscope or THUNDER DM6B microscope. The three-dimensional (3D) reconstruction imaging was processed with Leica software. The quantification of the area and optical density of the stained cells were measured with ImageJ software. Plaque-associated astrocytes or microglia were defined by the number of astrocytes or microglia in a plaque-centered circle within 50 µm in diameter. For quantification of microglia-mediated synapse phagocytosis, at least 20 microglia within the hippocampal DG region were randomly chosen for each sample and the individual 3D reconstruction images of double immuno-labelling with Iba1+ and Syn were collected and analyzed with Leica software. The total volume of all engulfed Syn-positive aggregation per microglia was quantified. The antibodies used were shown as followings: Sheep anti-CFH (1:1000, Cat: ab8842, Abcam, USA), Rat anti-C3 (1:100, Cat: ab11862, Abcam, USA), Rabbit anti-GFAP (1:1000, Cat: ab7260, Abcam, USA), Rabbit anti-NeuN (1:1000, Cat: ab177487, Abcam, USA), Rabbit anti-Iba1 (1:1000, Cat: ab178846, Abcam, USA), Mouse anti-β-Amyloid (1:1000, Cat: 803001, Biolegend, USA), Rabbit anti-β-Amyloid Fibrils OC (1:500, Cat: AB2286, Sigma, USA), Goat anti-Iba1(1:200, Cat: ab5076, Abcam, USA), Rabbit anti-Synaptophysin (1:1000, Cat: ab32127, Abcam, USA ), Goat anti-Rat IgG(H + L) Alexa Flour™ 594 (1:250, Cat: A11007, Invitrogen, USA ), Donkey anti-Sheep IgG(H + L) Alexa Fluor™ 488 (1:250, Cat: A11015, Invitrogen, USA), Donkey Anti-Rabbit IgG H&L Alexa Fluor® 405 (1:250, Cat: ab175649, Abcam, USA), Donkey Anti-Mouse IgG (H + L) Alexa Fluor® 594 (1:500, Cat: 715-585-150, Jackson ImmunoResearch, USA), Donkey Anti-Mouse IgG (H + L) Alexa Fluor® 488 (1:500, Cat: 715-545-150, Jackson ImmunoResearch, USA), Donkey Anti-Rabbit IgG (H + L) Alexa Fluor® 594 ( 1:500, Cat: 715-585-152, Jackson ImmunoResearch, USA), Donkey Anti-Rabbit IgG (H + L) Alexa Fluor® 488(1:500, Cat: 711-545-152, Jackson ImmunoResearch, USA), Donkey Anti-Goat IgG (H + L) Alexa Fluor® 488(1:500, Cat: 705-545-147, Jackson ImmunoResearch, USA).
RNA-seq analysis
BV2 cells were infected with lentiviruses carrying the gene of Control, FHL-1WT, FHL- 1V62I, and FHL-1Y402H for 72 hours. The cDNA library was constructed and sequenced using Illumina HiSeq™ X Ten by Novogene, China. The sequencing reads were mapped to the mouse reference genome using HISAT5. FPKM was used to measure gene expression levels and genes with FPKM values ≥ 0.5 were considered to be expressed. Differentially expressed genes (DEGs) between groups were processed for clustering analysis and functional annotation. For genes, a fold change of ≥ 1.0 and a false discovery rate (FDR) of < 0.05 were considered statistically significant. Pathways represented by DEGs were annotated using the GO and KEGG (Kyoto Encyclopedia of Genes and Genomes) database.
Statistical analysis
Various groups of the above experiments were carried out by researchers blinded to the mice groupings. All collected experimental data was presented as means ± SEM and processed with GraphPad Prism (Boston, USA) for statistical and graphing processing. Two-tailed t-test was used to compare the results between two groups, and one-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to compare the results of multiple groups. A value of p < 0.05 was considered statistically significant.