Background
Alzheimer disease (AD), the most common cause of dementia in the elderly, is characterized by alteration of cognitive functions, coexistence in the brain of senile plaques and neurofibrillary tangles, and neuronal loss [
1].
Over the past decade, deposition of amyloid-β (Aβ) peptide in senile plaques has been shown to be the driving force of a strong and chronic inflammatory response [
2,
3], characterized by the release of proinflammatory cytokines by reactive microglial cells and astrocytes surrounding amyloid deposits. This neuroinflammation plays a critical role in the pathogenesis of AD, by inducing neuronal toxicity leading to cognitive deficits [
4,
5], but also contributes to protective mechanisms, since glial reactivity increases phagocytosis and clearance of Aβ deposits [
6].
Interleukin-1β (IL-1β) is detected in astrocytes and microglial cells in the brains of AD patients as well as in animal models of AD [
7‐
12]. Since IL-1β lacks a signal peptide, it is produced as the inactive precursor pro-IL-1β and requires processing by the cysteine protease caspase-1 to be active [
13,
14]. Cytosolic multiprotein complexes called “inflammasomes” tightly control the activity of caspase-1. The inflammasome is an oligomeric protein complex organized as a tripartite structure: (1) a cytosolic danger sensor from the NLR (NOD-like receptor) family such as NALP3 (NACHT, LRR, and PYD domains-containing protein 3) also known as NLRP3 (NOD-like receptor family, pyrin domain containing 3) and NALP1 or IPAF (ice protease-activating factor), (2) the proteolytic effector caspase-1, and (3) ASC (apoptosis-associated speck-like protein containing a CARD domain), an adaptor protein needed to recruit NLR and to stabilize caspase-1/NLR complexes [
15].
It was recently demonstrated that inflammasome containing NALP3 can be activated by Aβ peptide in vitro, leading to inflammation and tissue damage [
7]. Moreover, NALP3 deficiency in the APP/PS1 AD mouse model results in a decrease in Aβ deposition and rescue of memory deficits [
16]. Although most of the studies focused so far on the possible implication of microglial inflammasome in AD, recent studies suggest that astrocytic inflammasome can also play a role. Downregulation of astrocytic IPAF inflammasome reduces Aβ42 generation by primary neurons, and expressions of IPAF and ASC are significantly increased in a subgroup of sporadic AD patients [
17].
In the present study, we investigated the role of ASC-mediated activation of the inflammasome on phagocytic activity of astrocytes and its implication in a mouse model of AD. We found that ASC was essential for IL-1β release from primary astrocytes exposed to Aβ42. Interestingly, we showed that the heterozygous expression of ASC in ASC+/− astrocytes was responsible for an increased phagocytic activity linked to the release of CCL3 (C-C motif ligand 3). In vivo, we showed, in the 5xFAD (familial Alzheimer disease) mouse model, that heterozygous ASC expression led to a decrease in amyloid load with a rescue of long-term spatial memory measured in the Morris water maze (MWM).
Methods
Animals
All animal procedures used in the study were carried out in accordance with institutional and European guidelines as certified by Animal Ethics Committee.
ASC knockout mice on the C57Bl6/J genetic background [
18] were purchased from Charles River Laboratory (Brussels, Belgium).
We used 5xFAD transgenic mice [
19] overexpressing mutant human APP695 carrying EOFAD mutations K670N/M671L (Swedish) + I716V (Florida) + V717I (London) and mutant human PS1 harboring 2 EOFAD mutations (M146L and L286V) driven by the thymocyte differentiation antigen 1 (ThyI) promoter (JAX Mice and Services, Bar Harbor, ME, USA).
We crossbred hemizygous 5xFAD mice with ASC mice. Experiments on 5xFAD ASC mice and age-matched control were performed at 7–8 months of age.
All mice were genotyped by PCR analysis of tail biopsies. Animals were housed on a 12-h light/dark cycle in standard animal care facilities with access to food and water ad libitum.
Primary cultures of astrocytes from newborn mice and treatments
All cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Primary glial cultures were prepared from newborn ASC mice (P0-1). Newborn mice were euthanized by decapitation and genotyped. The brains were removed, and the cortico-hippocampal regions were dissected in phosphate-buffered saline. Cells were then mechanically dissociated in Dulbecco’s Modified Eagle’s Medium (DMEM) (GlutaMAX)/penicillin-streptomycin (PS, 50 mg/ml), and the supernatant was collected and centrifuged at 280×g for 5 min. Cells were suspended in DMEM-GlutaMAX/PS supplemented with 10 % fetal bovine serum (FBS) and PS. Cells were seeded in 72-cm2 culture flasks and then incubated in a humidified 5 % CO2 atmosphere at 37 °C. The medium was changed every 5 days, and cultures were maintained for 15 days.
At day 15, non-astroglial cells such as microglia and oligodendrocytes were removed by shaking 5 h at 200 rpm/37 °C in an orbital shaker. Remaining adherent cells were then trypsinized (trypsin-EDTA 0.05 %) and plated at 104 cells/cm2 in appropriate non-coated wells. Immunofluorescent staining using antibodies against CD68 (microglia) (AbdSerotec) and GFAP (glial fibrillary acidic protein for astrocytes) (Dako) revealed that >98 % of cells were astrocytes.
Astroglial cultures were used at day 4 after subculture. Cells were primed with lipopolysaccharide (LPS) from
Escherichia coli 026:B6 (Sigma-Aldrich), at 1 μg/ml for 3 h, washed with fresh medium and then treated for 3 h with 20 μM nigericin or 10 μM Aβ42 (Bachem), previously suspended in milliQ water, incubated 72 h at 37 °C for aggregation [
20], and diluted in serum-free medium. Cathepsin B inhibitor Ca074 at 25 μM, or 10 μM cytochalasin D, was added 15 min before Aβ42.
Total RNA extraction was performed according to TriPure Isolation Reagent manufacturer’s instructions (Roche). Reverse transcription was carried out from 1 μg of extracted RNA resuspended in DEPC-treated water, using the iScript cDNA synthesis Kit (Bio-Rad). Quantitative real-time polymerase chain reaction (qRT-PCR) conducted with the iCycler IQ
TM multicolor Real-Time PCR Detection System (Bio-Rad) was carried out from 2 ng of cDNA template, specific primers at 0.3 μM and the IQ
TM SYBR® Supermix 1x. Murine primers (Sigma-Aldrich) used for
CCL3,
GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and for
IL-1β are presented in the Table
1.
Table 1
List of primers used and size of PCR products
CCL3
| Forward: 5′-ATGAAGGTCTCCACCACTGC-3′ | 196 |
Reverse: 5′-TCAGGAAAATGACACCTGGCT-3′ |
IL-1β
| Forward: 5′-ATGAAGGGCTGCTTCCAAAC-3′ | 186 |
Reverse: 5′-GAAGGTGCTCATGTCCTCATC-3′ |
GAPDH
| Forward: 5′-ACCCAGAAGACTGTGGATGG-3′ | 172 |
Reverse: 5′-ACACATTGGGGGTAGGAACA-3′ |
ASC
| Forward: 5′-CTAGTTTGCTGGGGAAAGAAC-3′ | 394 |
Reverse: 5′-CTAAGCACAGTCATTGTGAGCTC-3′ |
The PCR protocol consisted of 40 amplification cycles with the following steps: 95 °C for 30 s, 60 °C for 45 s, and 79 °C for 15 s. The result for each sample was normalized with the relative expression of
GAPDH, and relative amplifications were calculated by the 2-ΔΔCt method. For standard PCR, 10 μl was used and amplification was performed using 0.125 μl of
Taq DNA Polymerase (Thermo Fisher Scientific) and murine primers for
ASC (Table
1). Cycling was performed in a TProfessional Thermocycler (Biometra), and the amplification protocol consisted of 35 amplification cycles with the following steps: 95 °C for 30 s, 57 °C for 1 min, and 72 °C for 2 min. Aliquots of 8 μl of each PCR products were loaded on a 1.2 % agarose gel with the Midori Green Advanced DNA Stain (Nippon Genetics Europe GmbH) as nucleic acid stain.
ECLIA
IL-1β and CCL3 were quantified in culture media from astrocytes by ECLIA (electro-chemiluminescence immunoassay) on a MSD SECTORTM Imager 2400 (Meso Scale Discovery), following manufacturer’s instructions. The lower limit of detection (LLOD) for IL-1β was 0.6 pg/ml, whereas no LLOD was given by the manufacturer for CCL3 as it was a prototype kit.
Phagocytosis assay and CCL3 neutralization
Phagocytosis assay was performed following supplier’s instructions with pHrodo™ Red Zymosan A BioParticles® (Life Technologies) conjugate for phagocytosis. Briefly, LPS-primed astrocytes plated in 96 wells were treated with 10 μM Aβ42 for 3 h. Bioparticles (0.5 mg/ml) were added 1 h after Aβ42. CCL3-neutralizing antibodies (R&D systems) were added 15 min before bioparticles at a concentration of 0.1 μg/ml, corresponding to the manufacturer’s estimated ND50 range for CCL3-detected levels in our model. Primary astrocytes were also exposed to mouse recombinant CCL3 (R&D systems) at concentrations ranging from 1.5 to 10 ng/ml, to evaluate a specific effect of the chemokine in astrocytic phagocytic activity. After 2 h of incubation with bioparticles, pictures were taken using a digital inverted fluorescence microscope (EVOS-xl; Life Technologies) with a ×4 lens. An ECLIA for CCL3 was performed to evaluate neutralization efficacy after LPS priming and Aβ42 treatment.
Thioflavine S staining
Mice were transcardially perfused with ice-cold PBS to remove blood. Brains were extracted, and the right cerebral hemisphere was fixed by immersion in a 4 % paraformaldehyde solution for 24 h at 4 °C. Forty micrometer-sagittal sections were cut on a vibrating HM650V microtome (Thermo Scientific) and were preserved in PBS/azide 0.1 %. Staining with thioflavin-S (ThioS; Sigma-Aldrich), a specific β-sheet strand intercalant, was performed on brain sections as described previously [
21]. Image acquisition was performed using a digital inverted fluorescence microscope (EVOS-xl; Life Technologies) with a ×4 lens. Plaques were quantified using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA) by measuring the area of ThioS staining in a well-defined selected area of the hippocampus.
Behavioral analysis
The MWM with a mild learning paradigm was used for studying spatial learning and memory. The MWM was essentially performed as previously described [
21]. Age-matched mice (7 months old) were used. A circular polypropylene pool (113 cm in diameter), filled with white-opaque water (24 ± 1 °C) for hiding the submerged escape platform (9.5 cm diameter), was used for testing. Mice were trained to locate the hidden platform in three trials during one training day with an inter-trial interval of 1 h. Training over six consecutive days was performed. Twenty-four hours after the last training session on day 7, the platform was removed for a 60-s probe trial. Mouse behavior in the pool was videotaped and tracked using Ethovision camera and software (EthoVision 6.1 Noldus, Wageningen, The Netherlands).
Statistical analysis
The number of samples or animals is specified in the caption for each experiments. Results are expressed as the mean ± SEM. Statistical analysis was performed by either one-way ANOVA with post Bonferroni multiple comparison test or one-way ANOVA for repeated measures for the training in MWM. All analyses were performed using GraphPad Prism software (GraphPad Software Inc). Statistical significance was defined as P < 0.05.
Discussion
Molecular platforms called inflammasomes are activated upon cellular infection or stress, triggering the maturation of proinflammatory cytokines such as pro-IL-1β to engage innate immune defenses [
32]. It has been shown that aberrant inflammasome signaling contributes to pathology in a large number of infectious and autoimmune diseases [
33]. However, their roles in CNS disorders have not been extensively studied, although recent literature suggests that inflammasomes are activated and participate to neurological diseases including infections, acute sterile brain injury, and chronic neurodegenerative diseases [
34].
There is a high sensitivity of the CNS to IL-1β since multiple cerebral cell types express its receptor, which, upon activation, potentiates proliferation and activation of microglial cells and astrocytes [
35,
36].
Previous studies have shown that the Aβ peptide of AD can activate the NALP3 inflammasome in microglial cells and induce release of inflammatory molecule IL-1β in vivo and in vitro [
16,
24]. Aβ-mediated inflammasome activation in astrocytes remains, however, elusive.
In the present study, using primary culture of murine astrocytes, we show that LPS-primed cells are able to produce and release IL-1β under Aβ42 treatment, depending on ASC expression, as ASC−/− astrocytes did not produce significant amount of IL-1β. Activation of inflammasome in astrocytes was recently debated, from the absence of expression of NALP3, ASC, and caspase-1 in astrocytes [
37] to colocalization of the GFAP astrocytic marker with NALP3 and ASC in a murine model of ALS [
38]. We demonstrate here an activation of astrocytic ASC-dependent inflammasome by synthetic Aβ42, confirming thereby that, as microglial cells, astrocytes are able to produce IL-1β.
Phagocytosis of Aβ fibrils by macrophages or microglial cells was proposed to be the first step for the formation of the inflammasome complex, due to a leakage of cathepsin B from lysosomes into the cytosol [
7]. Using cytochalasin D to inhibit phagocytosis or cathepsin B inhibitor, we show a strong attenuation of IL-1β release, indicating that Aβ42 triggers inflammasome activation through a lysosomal pathway, as is the case for microglial cells or macrophages. The mechanisms by which cathepsin B can activate the inflammasome are not clear. It was recently suggested that the Aβ-mediated release of cathepsin B leads to the degradation of the anti-inflammatory protein NLRP10 involved in inhibition of formation of the NALP3 inflammasome by interacting with ASC. Degradation of NLRP10 allows ASC-NALP3 interaction, caspase-1 activation, and IL-1β release [
39].
As microglial cells, astrocytes express many potential phagocytic receptors in the CNS, like RAGE, CD36, or TLRs, known to have Aβ among their ligands [
40]. But the role of astrocytes as phagocytic cells is less studied, particularly in AD. Addition of astrocytes on brain sections prepared from an AD mouse model decreases Aβ levels [
26]. In APPswe/PS1dE9 mice, internalization of Aβ was observed in transplanted astrocytes [
41]. These data confirm the role of astrocytes in amyloid clearance.
In the present study, we measured phagocytosis by astrocytes using fluorescent bioparticles. We show an increase in phagocytosis by ASC heterozygous astrocytes primed by LPS and treated with Aβ42. This increase was explained by the secretion of soluble factors since exposure of naive ASC+/+ or ASC−/− astrocytes to conditioned medium from ASC+/− cells induced an increase in phagocytosis.
Chemokines are critical inflammatory molecules inducing recruitment of responsive cells such as monocytes/macrophages, microglia, or astrocytes. Among them, CCL3, a CC chemokine, is known to bind receptors on murine astrocytes with a high affinity, stimulating chemotaxis. Three receptors are known to bind CCL3, and CCR1 and CCR5 are expressed at the membrane of astrocytes [
29]. Therefore, we measured secretion of CCL3 in astrocytes in which inflammasome was activated by Aβ. Increase in CCL3 release was observed in all genotypes, with significantly higher levels with ASC+/− cells compared to ASC+/+ and ASC−/−. It was previously reported that astrocytes produce high amount of several chemokines and particularly CCL3 under stimulation by a mix of IL-1β and TNFα [
42]. In our model, there was an inverse correlation between TNFα and IL-1β secretion (Additional file
3). Consequently, co-secretion of both TNFα and IL-1β is optimal in ASC+/− astrocytes, resulting in higher CCL3 production in these cells. It is important to notice that CCL2, another chemokine known to be produced by astrocytes, was expressed at a very high level in control condition and thus displayed a low rate of increase after cytokine stimulation. On the other hand, ASC downregulation using siRNA or shRNA has been shown to reduce MAPK pathway activation in the THP1 cell line [
43] and to activate NF-κB in murine macrophages [
44]. Modifications in activation of these pathways, known to be involved in the expression of cytokines and chemokines, could also contribute to modulation of the inflammatory environment.
Neutralization of released CCL3 by a specific neutralizing antibody induced a significant decrease in phagocytic activity of ASC+/− astrocytes. Moreover, treatment of astrocytes with mouse recombinant CCL3 alone induced a significant increase of astrocytic phagocytosis in each ASC genotype. Involvement of CCL3 in the phagocytic activity of various cell types, including macrophages, has been demonstrated. Macrophages stimulated with recombinant CCL3 displayed an increase phagocytic index against
Pseudomonas aeruginosa [
45]. Moreover, phagocytic activity of alveolar macrophages from CCL3−/− mice toward
Klebsiella
pneumoniae was dramatically decreased [
46]. In vivo, CCL3 KO mice or WT mice treated with anti-CCL3 monoclonal antibodies were more prone to sepsis, suggesting a significant protective role for CCL3 [
45].
We extended our analysis in vivo by crossing the very well-known 5xFAD mouse model with ASC deficient mice. Interestingly, we observed a very significant increase in
CCL3 mRNA levels in the brain of FAD/ASC heterozygous mice as compared to FAD/WT mice. In addition, a significant decrease in amyloid plaques was observed in FAD/ASC heterozygous mice at 7–8 months of age, with a concomitant rescue of long-term memory deficits. These results are in line with the study of Heneka et al. [
16] showing the same effects in APP
swe/PS1dE9 mice KO for NALP3. It is important to notice that NALP3 KO mice displayed a 50 % decrease in IL-1β production, corresponding to our in vitro situation in ASC+/− astrocytes. It seems, however, that high levels or minimal production of IL-1β, as observed in ASC+/+ and ASC−/− astrocytes could decrease their phagocytic activity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JC designed and performed experiments, analyzed data, and wrote the paper. ICS participated in behavioral experiments and thioflavin S staining. OS participated in behavioral study design and setup. FH provided his expertise in inflammasome regulation and phagocytosis experiments. NP, ID, and PKC participated in the conception of the study and have been involved in revising the manuscript critically for important intellectual content. JNO conceived and coordinated the study and wrote the manuscript. All authors read and approved the final manuscript.