Background
Alzheimer’s disease (AD) is the most common incurable neurodegenerative disease. One of the pathological hallmarks of AD is the extracellular amyloid plaques composed of amyloid-beta peptide (Aβ) which is generated by proteolytic cleavage of amyloid precursor protein (APP). The abnormal accumulation of Aβ is considered to be a critical factor in AD pathogenesis [
1,
2]. The aggregation of Aβ into small oligomers and fibrillar plaques triggers neuroinflammation that contributes to the neuronal loss and cognitive decline [
1,
3]. Although suppression of chronic inflammation has been proposed as a new direction for AD intervention, the therapeutic effects of anti-inflammatory drugs in current clinical trials are far from satisfactory [
4]. Therefore, a novel strategy is necessary to protect neurons from Aβ-induced neurotoxicity and neuroinflammation to preserve memory.
Microglia serve as the first line of host defense in the brain. Microglia activation can be beneficial or detrimental in AD pathogenesis via removing Aβ by phagocytosis or producing pro-inflammatory cytokines that damage neurons [
5,
6]. The activated microglia are classified into M1 inflammatory (classical) and M2 anti-inflammatory (alternative) phenotypes [
7]. The M1 phenotype can be triggered by lipopolysaccharides, interferon-γ, and Aβ. They produce pro-inflammatory cytokines, such as IL-1β and TNF-α [
8,
9], to kill pathogens and induce cytotoxicity [
10]. In contrast, the M2 microglia reduce Aβ plaque deposition and alleviate memory impairments in an AD mouse model [
11]. Therefore, modulation of microglia activation and differentiation is a potential approach to regulate neuroinflammation in AD [
11,
12]. M2 phenotype microglia can be divided into M2a-d subtypes according to the surface and intracellular markers [
13,
14]. The M2a subtype microglia (YM1
+, FIZZ1
+, CCL17
+, arginase-1
+) are induced by IL-4, IL-13, fungal and helminth infections and are capable of suppressing inflammation [
15]. However, the functions of these microglia subtypes are still controversial assayed in different in vitro systems [
16‐
18].
Decoy receptor 3 (DcR3)/TNFRSF6B is a soluble decoy receptor which can neutralize the biological functions of three members of tumor necrosis factor superfamily: Fas ligand (FasL) LIGHT, and TL1A to reduce cell death [
19‐
21]. In addition to its neutralizing effect, DcR3 interacts with heparan sulfate proteoglycans (HSPGs) to promote the differentiation of M2-like macrophages through epigenetic regulation [
22‐
24]. DcR3 upregulates the expression of M2 macrophage markers (mannose receptor/CD206, arginase-I, YM-1, CD86, MMP7 and MMP-9) and downregulates the expression of M1 markers (iNOS, CD80, FcγR, IL-6, and TNF-α) [
24,
25]. DcR3 transgenic mice are resistant to type-I diabetes and Th17-mediated autoimmune diseases [
23]. However, the role of DcR3 in Aβ-mediated neuroinflammation in the brain has not yet been identified.
Given that DcR3 exerts anti-apoptotic and immune-modulatory effects via neutralizing FasL and non-deoy functions we asked whether DcR3 ameliorates AD-like functional deficits and pathological changes using both in vivo and in vitro systems. Here, we demonstrated that Aβ-induced cognitive deficits and neurodegeneration were improved by DcR3 in transgenic mice overexpressing a mutated human APP minigene (hAPP/J20 line). DcR3 skewed microglia differentiation to IL-4+YM1+ M2a-like subtype, modulated neuroinflammation, conserved synaptic density, and reduced Aβ. Our observations suggest that DcR3 may become a promising reagent for the treatment of AD in the future.
Methods
Mice
Hemizygous hAPP transgenic mice (line J20) express an alternatively spliced human APP minigene with the Swedish and Indiana familial AD mutations driven by the PDGF promoter [
26]. Hemizygous DcR3 transgenic mice express human DcR3 driven from the CD68 promoter in macrophages/microglia/monocytes [
25]. Female DcR3 transgene mice were crossed with male APP transgenic mice to obtain wild-type DcR3 single transgenic, APP single transgenic, and APP/DcR3 double transgenic mice. The littermates of these mice were examined in behavioral tests at 6 months of age and sacrificed for pathological examinations at 6 or 12 months of age.
Morris Water Maze
The water maze consisted of a water pool (122 cm in diameter) containing opaque water and a platform (10 cm in diameter) submerged 1 cm below the water surface. The hidden platform test consisted of 10 sessions over 5 days and each session comprised three 60-s trials with 15-min inter-trial intervals. The platform location remained constant during the hidden platform sessions, and the entry points were changed semi-randomly between days. One day after the final day of hidden platform training section, a probe trial was conducted by removing the platform and allowing mice to explore in the pool for 1 min. The quadrant in which the platform was previously located was defined as the target quadrant, and the proportion of time (as a percentage) that the each mouse spent in the target quadrant was used to measure memory retention. The number of platform crossings and swim speed were recorded and analyzed with the EthoVision video tracking system (Version 3.1 Noldus Wageningen, Netherlands).
Fear conditioning
During the day 1 training section mice were habituated in a conditioning box (Graphic State 2.101 Contents, Coulbourn Instruments, PA, USA) for 5 min and then received five pairs of an 8-s tone and a 2-s shock (0.4 mA) followed by a 2-min resting interval. On day 2 testing sections, the trained mice were placed back to the same testing box, and their freezing time was scored for 5 min to measure the contextual conditioned fear response. The cued test was conducted 5 min after the contextual test. Mice were habituated for 5 min in a novel-shaped box and then exposed to three 10-s auditory cues followed with a 2-min resting interval. The freezing times of each mouse were scored during all testing sessions.
Open field
To detect spontaneous locomotor activity mice were placed in an open chamber (24.32 × 24.32 cm2). Their horizontal movement was detected by a 16 × 16 infrared photo-beam arrays placed 1.5 cm above the bottom of the chamber for 15 min (Version 2.0, TRU Scan Photobeam LINC, Coulbourn Instruments, PA, USA).
Elevated plus maze
The elevated plus-shaped maze consisted of two open arms and two closed arms. All mice were individually placed at the center of the maze and allowed to explore for 10 min. The time spent and distances traveled on each arm were calculated with the Etho Vision video tracking system.
Immunofluorescence and thioflavin-S staining
Paraformaldehyde-fixed brains were sliced coronally by using a microtome (Leica SM2010R Heidelberg, Germany) and were stored in cryoprotectant medium (30% glycerol, 30% ethylene glycol in PBS) at -20 °C. For immunohistochemistry (IHC) staining, brain slices were blocked in a TBS-buffered solution containing 1% glycine, 0.4% Triton X-100, 10% FBS (FBL01, Caisson labs, USA), 0.1% sodium azide (13412, Sigma, MO, USA) and 3% serum bovine albumin for 2 h and then incubated for 24 h at 4 °C with anti-Iba1 (019–19741, Wako), anti-YM1 (01404, Stem Cell technology), anti-synaptophysin (04–1019, Millipore), anti-MAP2 (MAB378, Millipore) and anti-Aβ (SIG-39320, 6E10, Covance) to measure the distribution of microglia, M2a activated microglia, pre-synaptic density, neuronal density and the total level of Aβ. After incubation, the slices were incubated for 2 h with Alexa594-labeled (111–585–003, Jackson ImmunoResearch) and Alexa488-labeled secondary antibodies (115–546–003, Jackson ImmunoResearch) at room temperature.
For thioflavin-S staining brain slices were incubated with 0.015% thioflavin-S (T1892; Sigma MO, USA) for 15 min at room temperature. All chemicals unless otherwise stated were purchased from Bio Basic Inc. (Canada).
For immunocytochemistry staining primary cells were fixed with 4% paraformaldehyde to measure the degeneration of primary neurons and the morphology changes of microglia in responses to different treatment conditions. Fixed cells were stained with anti-MAP2 or anti-Iba1 antibody to visualize the structure changes. The stained slices or cells were imaged with a fluorescence microscope (Axio Observer A1; Zeiss Germany) or a confocal microscope (Fluoview FV10i; Olympus USA). Images were analyzed with MetaMorph® Microscopy Automation & Image Analysis Software (Molecular Devices, CA, USA).
To quantify the total or M2a microglia surrounding plaques slices were double stained with 6E10 and anti-Iba1 or anti-YM1 antibodies. Plaque areas were circled to determine the centers. The circles were then enlarged 10 μm in radius from the center, which was considered to be the periphery area for measuring the microglia or secreted YM1 coverage.
Enzyme-linked immunosorbent assays (ELISAs)
For DcR3 measurement up to 500 μl of blood was collected from the facial vein at the submandibular area and was centrifuged at 1,000 g for 15 min to isolate the serum. Serum DcR3 concentrations were measured with a human DcR3 Duo Set (DY142, R&D, USA).
For Aβ measurement the hippocampus of each mouse was homogenized in 5 M guanidine/5 mM Tris (pH 8.0) buffer and diluted with 0.25% casein blocking buffer to a final concentration of 0.5 M guanidine with protease inhibitor (04693116001, Roche, Basel, Switzerland). The levels of total Aβ and Aβ42 were quantified using Aβ ELISA kits (27729 and 27711, IBL, Hamburg, Germany).
For cytokines measurement diluted hippocampus lysates and conditioned media were applied to TNF-α, IL-1β, and IL-6 ELISA kits (555268, 559603, 555240, BD System, NJ, USA). For YM1 measurement, the hippocampus was homogenized in diluting reagent provided by ELISA kit at the concentration of 20 μg tissue/μl, and YM1 concentration were measured using mouse YM1/Chitinase 3-like 3 DuoSet ELISA (DY2446, R&D, USA).
Quantitative real-time PCR (Q-PCR)
The RNA from the hippocampus and the primary microglia were purified using the Total RNA Mini Kit (Geneaid Taiwan) or TRI reagent (T9424, Sigma, MO, USA), and then immediately reverse transcribed into cDNA by MMLV high-performance reverse transcriptase (RT80125K, Epicentre, WI, USA). The mRNA expression levels were analyzed by using primers (listed in Additional file
1: Table S1) mixed with SYBR Green PCR Master Mix (10476600, Roche, Penzberg, Germany). A StepOnePlus Real-Time PCR System (Applied Biosystem, ABI, MA, USA) was used to monitor the changes of fluorescence intensity from PCR products. GAPDH was used as internal control. The data were analyzed using StepOne software version 2.0.
Immunoprecipitation (IP)
Cortexes from 12-month-old mice were homogenized with a pestle at the concentration of 1 μg tissue /9 μl HEPES buffer (1% CHAPS 50 mM HEPES, 10 mM EDTA, 150 mM NaCl, pH 7.4). Tissue lysates were centrifuged at 600 × g for 5 mins and supernatants were collected. 200 μl samples were pre-cleared with 50 μl protein G bead (LSKMAGG02, Millipore, Germany) rotating at room temperature for 30 mins. Pre-cleared lysates were incubated with anti-DcR3 (33302, Biolegend, CA, USA), anti-syndecan-1 (10593–1-AP, ProteinTech, IL, USA), anti-glypican-1 (sc-66910, Santa Cruz biotechnology, TX, USA), or anti-Aβ (6E10, SIG-39320, COVANCE, NJ, USA) antibodies at 4 °C overnight, and were then mixed with protein G beads rotating at room temperature for 1 h. The beads were washed with 0.1% Tween 20 in PBS for 20 mins and were eluted by SDS-sample buffer (87.5 mM Tris-HCl, 1% SDS, 30% glycerol, 0.6 M DTT, 180 μM bromphenol blue, pH 6.8) at 95 °C, 10 mins. The eluted samples and input controls were monitored by Western blot.
Gel electrophoresis and Western blotting analysis
Proteins were separated via 10% or 15% Tris-glycine SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with rabbit anti-PSD95 (3450 Cell Signaling, MA, USA), mouse anti-DcR3 (33302, Biolegend, CA, USA), rabbit anti-syndecan-1 (10593–1-AP, ProteinTech, IL, USA), rabbit anti-glypican-1 (sc-66910, Santa Cruz biotechnology, TX, USA), mouse anti-Aβ (6E10, SIG-39320, COVANCE, NJ, USA), anti-YM1 (01404, Stem Cell technology, Vancouver, Canada), mouse anti-GAPDH (60004-1-Ig, ProteinTech, IL, USA), and mouse anti-actin (MAB1501, Millipore, MA, USA) antibodies. The membranes were washed and probed with the HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (12–349, AP132P, Millipore, MA, USA). Protein signals were developed by using a chemiluminescent substrate ECL detection system (WBKLS0500, Millipore, MA, USA) and quantified by using a luminescence imaging system (LAS-4000, Fujifilm, Japan).
Oligomeric Aβ (oAβ) fibrillar Aβ (fAβ) and DcR3 preparation
HFIP-treated Aβ1-42 peptides (rPeptide Inc. A–1163–2, GA, USA) were dissolved in 10% DMSO at 100 μM and stored at –80 °C. Before the experiment, the stock was aged at 4 °C for 24 h to generate oAβ or aged at 37 °C for 18 days to generate fAβ. A DcR3-SAS and Vector-SAS stable line were generated by transfecting the human DcR3 gene or control vector into SAS cells. Culture medium was collected after 24 h of DcR3-SAS and Vector-SAS growth for the conditioned medium experiment.
Primary neuron and microglia preparation and conditioned medium (CM) stimulation
Primary microglia were prepared from postnatal day 0–5 C57B6/J mice. The cortexes were digested with 100 U papain and 400 U DNase I in HBSS buffer at 37 °C for 30 min. Digested cells were passed through a 70-μm cell strainer (Corning NY, USA). Mixed cortical cells were grown in DMEM-F12. After 21 days incubation, microglia were isolated from a 30:37:70% Percoll (P4937, Sigma. MO, USA) gradient and were seeded in 25-T flasks for 24 h.
Primary cortical neuron cultures were prepared from postnatal day 0–1 C57B6/J mice as primary microglia but seeded at a density of 4x105 per well for 7 days in the neurobasal medium.
To obtain conditioned medium (CM) microglial culture were stimulated with the DcR3-SAS medium before, together or after incubating with Aβ for 72 h and their media were collected as pre-, co- or post-treatment Aβ/DcR3-CM. Aβ-CM was collected from microglial culture stimulated with Vector-SAS medium and Aβ for 72 h, and control-CM was collected from microglial culture stimulated with the vector-SAS medium. For the DcR3 immune-depletion control, the DcR3-SAS medium was incubated with anti-DcR3 (33302, Biolegend, CA, USA) and protein G bead (LSKMAGG02, Millipore, Germany) rotating at 4 °C overnight. DcR3 depleted SAS medium was collected for microglia treatment. For the competition assay, microglia were co-treated with DcR3 and 30 μg/ml heparin sulfate (HS) to interfere the DcR3-HSPG interaction for 8 h and then incubated with Aβ for 72 h. These conditioned media were collected and applied to primary neurons for 72 h.
Cell survival
Neuronal survival rate after different CM treatment was assessed using MTT (3006 Biotium Inc., CA, USA) and propidium iodide (PI) staining assays according to the manufacturer’s instructions. For the MTT assay, formazan was solubilized in lysis buffer (10% SDS and 20 mM HCl), and the concentration was determined according to the optical density at 570 nm with a Sunrise™ absorbance reader with Magellan™ data analysis software (Version 6; Tecan Switzerland).
For PI staining neurons were incubated with 10 μg/ml PI in PBS for 20 min and were fixed in 4% paraformaldehyde for immunofluorescence staining with the MAP2 antibody. The staining results were quantified as the ratio of PI+ neurons to total MAP2+ neurons by using MetaMorph® Microscopy Automation & Image Analysis Software (Molecular Devices, CA, USA).
Mouse cytokine array
The cytokines in the primary microglia conditioned medium were detected by using the mouse cytokine array C1000 (AAM-CYT-1000 RayBiotech, GA, USA). Membranes were incubated with control CM, Aβ-CM or Aβ/DcR3-CM (pre-treatment condition) for 16 h and detected with a Biotin-Streptavidin system. Signals were scanned by using a Fujinon LAS-4000 system and quantified by using Multi Gauge V3.0 software (Fujifilm Corporation, Tokyo, Japan). The level of each cytokine in the control group was set as 100.
Microglial phagocytosis assay
Purified microglia were seeded at a density of 1x105 cells/well on poly-d-lysine coated coverslips. Attached microglia were treated with oAβ or oAβ + DcR3 for 72 h. Their phagocytic ability was examined by incubating with red fluorescent carboxylated microspheres (F8821 1 μm in diameter, Polysciences Life Technologies, USA) coated with fetal calf serum at 37 °C for 30 min. After three PBS washes, microglia were fixed with 4% paraformaldehyde and stained with anti-Iba1 antibody to visualize the number of engulfed microspheres in the microglia.
Statistical analysis
Data are presented as the mean ± s.e.m. from at least three independent experiments and were analyzed using Prism software (GraphPad). Differences between data sets were analyzed by unpaired two-tailed Student's
t-tests or one-way ANOVA followed by the Bonferroni post hoc test. During multiple contrast analysis, the alpha was set as 0.05 (95% confidence intervals). All the precise numbers of samples and their statistical analysis methods of each experiment are listed in Additional file
2: Table S2. A
p value less than 0.05 was considered to be statistically significant.
Acknowledgments
We thank Drs. Ding-I Yang, Young-Ji Shiao, Nien-Jung Chen, Li-Chung Hsu, and Cheng-Chang Lien for the comments on this work. We also thank Po-Han Wei, Chih-Wei Sung, Ming-Ting Huang, Chien-Chun Chen, Yen-Ching Huang, and Yen-Chen Lin for experimental assistance. Behavioral studies were carried out at the Animal Behavioral Core at Brain Research Center, National Yang-Ming University. The technical services of confocal images were provided by Imaging Core Facility of Nanotechnology of the UST-NYMU. We are also grateful to the Transgenic Mouse Model Core Facility of the National Core Facility Program for Biotechnology, National Science Council and the Gene Knockout Mouse Core Laboratory of National Taiwan University Center of Genomic Medicine for technical services.