2.1 | Animals
Apoe knockout (KO) mice, APOE3 and APOE4 targeted replacement (TR) mice, which express human apoE isoforms driven by the endogenous murine Apoe promoter, were purchased from Taconic. The mice were maintained at a constant temperature with an alternating 12 hr light/dark cycle. Food and water were available ad libitum. All animal experiments were approved by the Animal Ethics Committee of the Xiamen University and were conducted in compliance with all relevant ethical regulations for animal testing and research.
2.2 | Primary cultures
Brain tissues from C57BL6 mice were dissected for primary glial cell culture. Primary microglial cells were prepared as previously described [40] with modification. Briefly, mixed glial cells from newborn (postnatal 1 to 3 day old) pups were cultured in DMEM (GIBCO) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin in a poly-D-lysine (25 µg/mL) (Sigma)-coated cell culture flasks (Corning, Fisher, USA). The medium was changed within the next day with fresh DMEM medium plus 10% FBS and 12.5 ng/mL GM-CSF (R&D). Microglial cells were harvested by shaking at a speed of 220 rpm for 15 min after 9-10 days culture. The harvested cells were seeded for experimentation or subjected to Apoe knockdown by electroporation.
Primary astrocytes were prepared as described by a previous protocol [41] with modification. Simply, mixed glial cells from newborn (postnatal 1 to 3 day old) pups were cultured in astrocyte culture media (DMEM, high glucose + 10% heat-inactivated fetal bovine serum + 1% penicillin/streptomycin). The medium was changed 2 days after plating of the mixed cortical cells and every 3 days thereafter. At day 9 or day 10, when astrocytes were confluent, mixed cells were shaken at 220 rpm for 30 min to remove the upper microglial cells. Trypsin (Sigma, T2601) was used to split attached astrocytes for further culture or use.
Primary cortical neurons were obtained from 11 to 17 day old embryos of wild-type C57BL/6 mice and cultured in neurobasal medium (GIBCO) supplemented with 0.5 mM GlutaMAX (GIBCO), 2% B27 (GIBCO), and 1% penicillin-streptomycin (Invitrogen) on cover glasses pre-coated with poly-D-lysine solution (50 µg/mL). At day 5 of the in vitro study (DIV5), the neurons were treated with 10 M cytosine arabinofuranoside (Sigma Aldrich) for 2 days to remove glial cells. At DIV7 the culture medium was then replaced with fresh neurobasal medium containing B27 and penicillin-streptomycin. For neuron-microglia co-cultures, microglia were re-suspended in neuronal culture medium and were seeded on top of primary neurons at DIV8 to a final ratio of 1:2 (microglia:neuron).
2.3 | Immunohistochemistry
Cells were fixed in 4% paraformaldehyde and then permeabilized with 0.25% Triton X-100 in PBS. After blocking with 1% BSA in PBS for 30 min, cells were incubated with primary antibody MAP2 (Cell Signaling, Cat#4542, 1:300) overnight at 4℃. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Abcam, ab150077) for 1 hr at room temperature. The nuclei were labeled with DAPI (Abcam, ab104139). Fluorescent signals were detected by confocal laser scanning fluorescent microscopy (Model LSM510 Invert, Carl Zeiss).
2.4 | Apoe knockdown by siRNA
Two different Apoe specific siRNAs (chemically synthesized by Dharmacon) were used to knockdown Apoe in microglia by electroporation using an Amaxa Nucleofector and a glial specific Nucleofector kit (LONZA), according to the manufacturer’s instructions. Each electroporation reaction contained 4 × 106 cells and 300 nM siRNA. Transfected cells were seeded and used for subsequent experiments.
2.5 | Assessment of microglial migration
Microglial cells (5 × 104) in culture medium were added to the upper well of each Transwell insert (Corning), each of which bears an uncoated filter with 8 μm diameter holes. After 48 hr, the medium in the Transwell insert was replaced with 0.2% FBS/DMEM, and DMEM containing 10% FBS, adenosine triphosphate (ATP; 300 nM), or lipopolysaccharide (LPS; 1 µg/mL) (Sigma Aldrich) was added to the lower well to induce migration. After 24 hr, the cell-bearing filters were fixed in 4% paraformaldehyde for 10 min, rinsed with PBS, and the microglial cells remaining on the upper side of each filter removed with a cotton swab. The filters were stained with 0.1% crystal violet (Sigma Aldrich) for 30 min and then rinsed with PBS and water. The number of cells that had migrated to the underside was counted (8 random fields/filter) at 20x or 10x magnification using an Olympus DP71 microscope (Olympus, Tokyo, Japan).
2.6 | Reverse transcription and quantitative real-time PCR
Trizol (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo Research) were used for total RNA extraction according to the manufacturer’s instructions. Reverse transcription was performed using iScript cDNA synthesis kit (Bio-Rad). The relative levels of expression were quantified and analyzed using Bio-Rad iCycler iQ software (Bio-Rad). Relative mRNA levels were calculated by ΔΔCt method with β-actin as a reference. The primer sequences for mouse, and β-actin were as follows:
Il-1β-Forward: 5'-CAGGCAGGCAGTATCACTCATTG-3';
Il-1β-Reverse: 5'-GCTTTTTTGTTGTTCATCTCGGA-3';
Il-6-Forward: 5'-CAATGGCAATTCTGATTGTATG-3';
Il-6-Reverse: 5'-AGGACTCTGGCTTTGTCTTTC-3'
β-actin-Forward: 5'-AGTGTGACGTTGACATCCGTA-3';
β-actin-Reverse: 5'-GCCAGAGCAGTAATCTCCTTC-3'.
2.7 | BrdU incorporation assay
BrdU incorporation assay was performed using BrdU cell proliferation ELISA kit (Abcam) following the manufacturer's protocol. Briefly, microglial cells were seeded at a density of 1.0 x 104 cells/well and BrdU was added to the cells for 48 hr for incorporation. Incorporated BrdU was examined through anti-BrdU antibodies after the cells were fixed, permeabilized and denatured. The cells were then incubated with horseradish peroxidase-conjugated secondary antibodies and the colored reaction product quantified using a microplate spectrophotometer (Varioan Flash; Thermo Fisher Scientific).
2.8 | Western blot analysis
All cells were lysed in RIPA buffer and total protein concentrations were determined via a BCA Protein Assay Kit (Thermo Scientific). Total protein (40 µg) was loaded into 10% SDS-PAGE. Western blot was performed as has been previously described [42]. Briefly, gels were first transferred onto PVDF membranes (Millipore). After which, primary antibodies were incubated overnight at 4°C, followed by secondary antibody incubation. The following antibodies were used in this study: Tubulin (Millipore, Cat # 05829, Host: Mouse), mouse apoE (Santa Cruz M-20, Cat # sc-6384, Host: Goat), ABCA1 (Novus, Cat # NB400-105, Host: Rabbit), ABCG1 (Novus, Cat # NB400-132, Host: Rabbit), human APOE (Meridian Life Science, Cat # K74180B, Host: Goat), GFAP (Millipore, Cat # MAB360, Host: Mouse), and Iba1 (Wako, Cat # 016-20001, Host: Rabbit). Immuno-reactive bands were detected and quantified using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
2.9 | MTT Assay
An MTT assay was performed to assess cell viability via measuring mitochondrial activity following manufacturer’s instructions (Roche). Briefly, cells were plated at a density of 5 x 103 cells in a 96-well plate overnight. Cells were then incubated with MTT reagent for 4 hr at 37°C followed by the addition of developing solution to stop the reaction. The plate was read at 590 nm with a reference wavelength of 650 nm.
2.10 | Fast protein liquid chromatography (FPLC)
Conditioned media were generated by culturing cells in serum-free media for 24 hr in T75 flasks and then collected. Conditioned media were then concentrated 20 fold using a 10-kDa cut-off filter (Millipore) and centrifuged to remove cellular debris before storage at 4°C prior to fractionation. Samples were run through an AKTA FPLC system through a Heparin column (heparin affinity chromatography) or a SuperoseTM 6 column (size exclusion chromatography; GE Healthcare). Fractions were collected and stored at 4°C until further analysis.
2.11 | Cholesterol assay
Cholesterol levels were analyzed using the Amplex Red cholesterol assay kit (Life Sciences) according to the manufacturer’s instructions. Briefly, samples were added into an opaque 96-well plate. Standards and samples were incubated with Amplex Red reagent (300 µM Amplex Red, 2 units/mL cholesterol oxidase, 2 units/mL HRP, and 0.2 units/mL cholesterol esterase) at 37°C for 30 min and fluorescence was measured using excitation in the range of 550 nm and emission detection at 590 nm.
2.12 | ApoE ELISA
A 96-well plate (Fisher Scientific) was coated overnight with WUE4 antibody [43]. After blocking in 1% nonfat milk in PBS, samples of the appropriate dilution were incubated with the detection antibody (K74180B), followed by incubation with streptavidin-poly-HRP antibodies (Fitzgerald). Finally, tetramethylbenzidine (TMB) (Sigma Aldrich) was added to each well and the substrate-peroxidase reaction was stopped by sulfuric acid stop solution. The absorbance was read at 450 nm using a BioTek 600 plate reader. ApoE concentration of samples was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein (Fitzgerald).
2.13 | Analysis of apoE/lipoprotein particles sizes by Native PAGE
48 hr post shaking and seeding of primary cells, the medium was replaced with serum-free medium and the conditioned medium harvested and concentrated. ApoE particles in the concentrated medium were quantified and normalized to apoE3 in the conditioned medium. Then, equal amounts of apoE3 and apoE4 proteins were separated by Native PAGE Novex 4-20% Tris-Glysine gels (Thermo Fisher) under native conditions following the manufacturer’s instructions and transferred to a PVDF membrane (Millipore) at 300 mA for 1.5 hr. Ponceau S staining solution (0.1% (w/v) Ponceau S in 5% (v/v) acetic acid) was used in blotting to visualize the molecular mass markers. Particle sizes were estimated using the Native Mark Unstained Protein Standard (Invitrogen). After washing, the membrane was incubated with goat anti-apoE antibody (K74180B, Meridian Life Science) overnight at 4°C, followed by avidin-labeled donkey anti-goat antibody. Western blot bands were quantified by Image J software.
2.14 | ApoE associated cholesterol assay
Avidin-agarose beads (Pierce) were pre-coupled with biotinylated polyclonal anti-apoE antibody (K74180B, Meridian Life Science) and then incubated overnight with concentrated conditioned media at 4°C. Complexes of bead-antibody-apoE were washed with TBS buffer three times, then apoE associated cholesterol was eluted by 0.1% Triton X-100/TBS. 0.1 M glycine (pH 2.5) was used to elute immunoprecipitated apoE and then neutralized with 1 M Tris (pH 8.5).
2.15 | Quantification of neurite outgrowth and cytokines level in co-culture system
15-25 cells from 5-6 images were analysed and quantified following a method reported previously [44]. Neurite number (neurite initiation sites) and length were counted and measured using Image J. The levels of IL-1β and IL-6 in the co-culture system were examined using Quantikine ELISA kits according to manufacturer’s instructions (R&D Systems, Minneapolis, MN). The absorbance was measured at 450 nm with a 590 nm correction and concentrations were calculated.
2.16 | Statistical analysis
All quantified data is represented as mean ± SEM. Statistical significance was determined with an unpaired t test, one-way ANOVA test, or two-way ANOVA and Tukey’s post hoc test (GraphPad Prism 6). All experiments were performed in at least triplicate and p <0.05 was considered significant.