Neuromol Med (2014) 16:205–215 DOI 10.1007/s12017-013-8274-6
ORIGINAL PAPER
Glial Uptake of Amyloid Beta Induces NLRP3 Inflammasome Formation via Cathepsin-Dependent Degradation of NLRP10 Niamh Murphy • Belinda Grehan • Marina A. Lynch
Received: 4 April 2013 / Accepted: 26 October 2013 / Published online: 7 November 2013 Ó Springer Science+Business Media New York 2013
Abstract The NLRP3 inflammasome forms in response to a diverse range of stimuli and is responsible for the processing and release of interleukin-1b (IL-1b) from the immunocompetent cells of the brain. The pathological peptide of Alzheimer’s disease, amyloid beta (Ab), induces formation of the NLRP3 inflammasome in a manner dependent on the family of proteases, cathepsins; however, the pathway by which cathepsins induce formation of the inflammasome has not yet been elucidated. In this study, we show that Ab treatment of primary rat glial cultures increases cathepsin activation in the cytosol, formation of the NLRP3 inflammasome, caspase 1 activation and IL-1b release. We also show that a second NOD-like protein, NLRP10, is found bound to apoptosis-associated speck-like protein under resting conditions; however, with Ab treatment, both in vitro and in vivo, NLRP10 is decreased. Further to these data, we show that cathepsins are capable of degrading NLRP10 and that treatment of glial cultures with recombinant NLRP10 reduces Ab-induced caspase 1 activation and IL-1b release. We propose that Ab-induced cathepsin released into the cytosol degrades NLRP10, thus allowing dissociation of NLRP3 and formation of the inflammasome. Keywords NLRP3 inflammasome Amyloid beta Cathepsins NLRP10 Glia
N. Murphy (&) B. Grehan M. A. Lynch Department of Physiology, Trinity College Institute of Neuroscience, Trinity College, College Green, Dublin 2, Ireland e-mail: [email protected]
Introduction Neuroinflammation is recognized as the brain’s innate immune response to both acute injury and neurodegenerative changes. It has been identified as a key component in the pathology of Alzheimer’s disease where the degree of inflammation parallels the severity of the disease (Sheng et al. 1997). Alzheimer’s disease has been associated with several markers of neuroinflammation including increased and robust activation of microglia and astrocytes, the clustering of these activated cells around amyloid plaques, increases in the expression of pro-inflammatory cytokines, cell adhesion molecules and chemokines, and induction of inflammatory enzyme systems such as inducible nitric oxide synthase (iNOS) (Heneka et al. 2010; Sardi et al. 2011). One of the key pro-inflammatory cytokines involved in the neuroinflammatory process is interleukin (IL)-1b. IL1b is primarily produced as the precursor, pro-IL-1b, which requires processing to become biologically active. In order for IL-1b to become processed, a group of intracellular proteins, collectively known as the NLRP3 inflammasome, must assemble to form a complex. The NLRP3 inflammasome includes the apoptosis-associated speck-like protein (ASC), the protease caspase 1, and the NOD-like receptor, NLRP3, recruitment of which leads to the proteolytic cleavage of caspase 1 to its active form which then processes pro-IL-1b to active IL-1b. A novel NOD-like receptor, NLRP10 identified in 2004 (Wang et al. 2004), was shown to reduce caspase 1-induced processing and release of IL-1b in HEK293 cells. Subsequent studies have shown that the anti-inflammatory effects of NLRP10 are due to its interactions with, and inhibition of, the actions of NLRP3 and ASC (Imamura et al. 2010).
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In this study, we show that amyloid-b (Ab), the pathogenic peptide associated with Alzheimer’s disease, induced cathepsin B release into the cytosol and that this degrades the anti-inflammatory protein NLRP10 which subsequently results in the activation of the NLRP3 inflammasome and IL-1b release.
Materials and Methods Primary Mixed Glial Cultures Mixed glia were prepared from the cortices of 1-day-old Wistar rats (Trinity College, Dublin, Ireland). Cortical tissue was cross-chopped and incubated for 25 min at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Fisher Scientific, Ireland) supplemented with 10 % fetal bovine serum (Fisher Scientific, Ireland) and 50 U/ml penicillin/streptomycin (Fisher Scientific, Ireland) and plated (2.5 9 105) as previously described (Nolan et al. 2004). Cells from each rat were treated separately. Inflammasome activation requires two signals; signal 1 is induced by Toll-like receptor stimulation, leading to the synthesis of pro-IL-1b. In our system, after 13 days, cells were primed by incubating with lipopolysaccharide (LPS; 1 lg/ml; Sigma, Ireland) for 4 h, which provided ‘signal 1.’ Cells were treated with a cocktail of recombinant human Ab1–40 (4.3 lM, aggregated for 24 h at 25 °C; Invitrogen, Ireland) and Ab1–42 (5 lM, aggregated at 37 °C for 48 h; Invitrogen, Ireland), and incubation continued for a further 24 h. In some experiments, LPS-treated cells were incubated in the presence of an inhibitor of cathepsin Z-FG-NHO-Bz (10 lM; Millipore, Ireland); although the primary target of this cysteine protease inhibitor is cathepsin B (k2/Ki = 8.9 9 103 M-1 s-1), it inhibits cathepsins L and S (k2/Ki = 3.8 9 105 and 4.2 9 104 M-1 s-1) and also the plant-specific protease, papain (k2/Ki = 2.4 9 103 M-1 s-1). The concentration of the inhibitor used in this study was determined following preliminary dose-finding experiments. In other experiments, cells were incubated in the presence or absence of cytochalasin D (3 lM, Millipore, Ireland) or recombinant NLRP10 (150 ng/ml, Origene, Maryland, US) for 1 h prior to addition of Ab cocktail. In all experiments, supernatants were collected and cells were harvested for later analysis. An MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay (Promega, UK) was carried out after all treatments in order to assess cell viability. Isolation of Neuronal, Microglial and Astrocytic Cultures To prepare purified microglia and astrocytes, cells were grown in T25 flasks in DMEM as above. After 12 days, the
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flasks were shaken for 2 h at 110 rpm, at room temperature and tapped several times to remove non-adherent microglia. The supernatants were removed from the flask and centrifuged at 2,000 rpm for 3 min at 21 °C. The pellet was resuspended in DMEM, and the cells were counted. Cells were pipetted into 6-well plates at a density of 1 9 105 cells/ml. To prepare astrocytes, the flasks containing the adherent astrocytes were washed with PBS, and 1 ml of 0/05 % w/v trypsin–EDTA was added at 37 °C until the cells just began to detach, DMEM was then added to the flask to inhibit the action of trypsin. The cells were centrifuged at 2,000 rpm for 3 min. The pellet was resuspended in DMEM, and the cells were plated in 9-well plates at a density of 1 9 105 cells/ml. Primary hippocampal neuronal cultures were prepared from 1-day-old Wistar rats. The hippocampus was dissected in Hanks’ balanced salt solution (HBSS) buffered with 1 mM sodium pyruvate and 1 mM HEPES pH 7.4 and dissociated using papain (15 U/ml; Sigma, UK). Cells were plated at 6 9 104 cells/cm2 on 48-well dishes precoated with poly-D-lysine (50 lg/ml) and maintained in Neurobasal media (NBM) containing 0.5 mM glutamine and B27 supplement. Half the medium was exchanged every 3 days. All media reagents were purchased from Fisher Scientific, Ireland. Treatment of Animals Male Wistar rats aged 3–4 months (Bioresources Unit, Trinity College, Dublin, Ireland) were used in these studies. Animals were housed in groups of 4–6 and were maintained on a 12-h light schedule, at an ambient temperature of 22–23 °C, under veterinary supervision in the Bioresources Unit. The experiments were performed under a license issued by the Department of Health and Children, Ireland, with the approval of the local ethics committee and in accordance with local guidelines. Rats were randomly divided into 2 groups, half of which were treated with a cocktail of recombinant Ab1–40 and Ab1–42, while the other half received the reverse peptide, Ab40–1. Animals were anaesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and implanted with osmotic mini-pumps (model 2004, Alzet, USA). The pump was implanted subcutaneously in the mid-scapular region and was attached via polyvinylchloride tubing (Alzet, 0.69 mm diameter) to a chronic indwelling cannula (Alzet, Infusion Kit II), which was positioned stereotaxically in the ventricle (0.9 mm posterior to bregma, 1.3 lateral to the midline and 3.5 mm ventral to the dura). The cannula was affixed to the skull using cryanoacetate gel and was secured in place by a smooth covering of dental cement (Stoelten, USA). Post-operative care included a subcutaneous injection of the analgesic Rimadil (5 mg/kg). The pumps
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delivered a cocktail of Ab1–40 (26.9 lM) and Ab1–42 (36.9 lM; aggregated for 24 h at 25 and 37 °C for 48 h; Invitrogen) or control peptide Ab40–1 (63.8 lM) intracerebroventricularly at the rate of 0.25 ll/h (±0.05 ll) for 28 days. Analysis of the Ab preparation by the thioflavin T fluorescent assay and gel electrophoresis revealed the presence of low oligomeric species; the predominant form (36 % of the total) was the 13.5-kDa species.
peroxide-conjugated secondary antibodies (Jackson ImmunoResearch, US), and bands were visualized using Supersignal West Pico Chemiluminescent Substrate (Pierce, US). Images were captured using a Fujifilm LAS3000 (Brennan and Co, Ireland). Densitometry was performed using ImageJ software (http://rsb.info.nih.gov/ij/).
Analysis of Cathepsin Activity in the Cytosolic Fraction of Glial Cultures
For immunoprecipitation studies, mixed glial samples containing 500 lg protein were incubated overnight at 4 °C in the presence of ASC-specific antibody (5 lg; rabbit polyclonal; Santa Cruz Biotechnology, US) or IgG isotype control (negative control) (Sigma-Aldrich, Ireland). A/G protein agarose beads (50 ll; Santa Cruz Biotechnology, US) were added, samples were incubated for 2 h, washed in PBS containing NP-40 (0.01 %) and centrifuged. Loading buffer (19) was added to each sample, and samples were boiled. Proteins were separated (see above), and NLRP10 and NLRP3 were visualized by incubating the membrane in the presence of a NLRP10-specific (mouse monoclonal, R&D Systems Europe, UK) or NLRP3-specific (rabbit polyclonal; Santa Cruz Biotechnology, US) antibodies.
The cytosolic fraction was isolated from treated glial cells by centrifugation. Briefly, cells were homogenized in 19 M-SHE buffer (210 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EDTA and 1 mM EGTA: all Sigma, Ireland). Lysates were centrifuged at 1,2009g for 10 min at 4 °C. The supernatants were removed, and the centrifugation was repeated. The supernatants were removed and centrifuged at 10,0009g for 15 min at 4 °C. The supernatant from this last spin was taken as the cytosolic fraction. Cathespin activity in the cytosolic fraction was measured using a cathepsin B-specific fluorometric assay (RR-AFC substrate) and a cathepsin L-specific fluorometric assay (FR-AFC substrate) (Abcam, Cambridge, UK).
Immunoprecipitation
Protein Degradation Assay Analysis of IL-1b, IL-6 and TNFa Concentrations Supernatant concentrations of IL-1b, IL-6 and TNFa obtained from glial cultures were measured using ELISA. Cytokine concentrations in the test samples were evaluated with reference to the standard curves prepared using recombinant cytokines of known concentrations. Analysis of Activity of Caspase 1 Cells were harvested, washed in phosphate-buffered saline and lysed in lysis buffer. Activity of caspase 1 was measured using a fluorometric assay kit (R&D Systems Europe, UK).
Recombinant cathepsin B and cathepsin L (R&D Systems, UK) were reconstituted in activation buffer (50 mM sodium acetate, 2 mM DTT, 2 mM ethylenediaminetetraacetic acid). Some activated cathepsin B was heatdenatured by incubation at 100 °C for 5 min. Activated cathepsin B and L (10 lg/ml) were incubated with recombinant NLRP10 (0.5 lg/ml; Sigma-Aldrich, Dublin, Ireland) for 1 h at 37 °C. The reaction was stopped using 1 9 protease inhibitor (Sigma-Aldrich, Ireland). Sample buffer (2 9 SDS) was added to each sample which were run on a 15 % polyacrylamide gel, and the proteins were visualized by silver staining.
Analysis of Proteins by Western Immunoblotting
Results
Western blotting was performed as previously described (Lyons et al. 2007). Cultured cells were harvested, homogenized in buffer containing Tris–HCl (0.01 M) and EDTA (1 mM), and protein (20 lg) was boiled in gelloading buffer and separated by 12 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and incubated with antibodies against b-actin (mouse monoclonal; Sigma, Ireland) and NLRP10 (mouse monoclonal, R&D Systems Europe, UK). Membranes were incubated with horseradish
Phagocytosis of Ab Induces Cathepsin B and L Activity in the Cytosol, Caspase 1 Activation and IL-1b Release LPS-primed mixed glial cultures were treated with a cocktail of Ab1–40 and Ab1–42 (4.9 and 5.4 lM, respectively) for a period of 24 h. Ab significantly increased active cathepsin B and L in the cytosol (***p \ 0.001; ANOVA; Fig. 1a, b). It has previously been shown that cathepsins are released from damaged lysosomes in response to phagocytosis of Ab (Halle et al. 2008). To assess whether this was the case in our
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Fig. 1 Ab treatment of LPS-primed rat glial cultures induced activation of (a) cathepsin B and (b) cathepsin L in the cytosol (n = 5; ***p \ 0.001; ANOVA; control vs. LPS ? Ab), and both cathepsins were inhibited by cytochalasin D treatment (???p \ 0.001; ANOVA; LPS ? Ab vs. LPS ? Ab ? cytochalasin D). Ab treatment of LPS-primed glial cultures also induced (c) caspase 1 activation and (d) IL-1b release (n = 5; ***p \ 0.001; ANOVA; control vs. LPS ? Ab) which were both
inhibited by a broad-spectrum cathepsin inhibitor and cytochalasin D (???p \ 0.01; ANOVA; LPS ? Ab vs. LPS ? Ab ? cathepsin inhibitor or LPS ? Ab ? cytochalasin D). LPS-induced release of (e) IL-6 and (f) TNFa (n = 5; ***p \ 0.001; ANOVA; Control vs. LPS) which were both further enhanced with Ab treatment (??p \ 0.01; ?p\0.05; ANOVA; LPS vs. LPS ? Ab). The cathepsin inhibitor and cytochalasin D had no effect on the Ab-induced release of either IL-6 or TNFa
system, we pretreated the cells with cytochalasin D, an inhibitor of actin polymerization. Blocking phagocytosis of Ab resulted in a decrease in active cathepsin B and L in the cytosol (???p \ 0.001; Ab-treated LPS-primed cells with vs. without cytochalasin D; Fig. 1a, b). Treatment of LPS-primed mixed glial cells with Ab resulted in activation of caspase 1 (***p \ 0.001; ANOVA; Fig. 1c;), and caspase 1 activation was inhibited
by treatment of cells with cytochalasin D and also with a broad-spectrum cathepsin inhibitor, Z-FG-NHO-Bz (???p \ 0.001; ANOVA; Fig. 1c). We investigated IL-1b release, since active caspase 1 is responsible for its processing and release, and the data show that Ab significantly increased release of IL-1b from LPS-primed mixed glia (***p \ 0.001; ANOVA; Fig. 1d;). IL-1b release was inhibited by pretreating cells with cytochalasin D and
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Fig. 2 a Ab treatment of LPSprimed glial cells resulted in the association of NLRP3 to ASC as seen by immunoprecipitation and b assessed by optical density quantification (n = 5; **p \ 0.01; student’s t test). c Ab treatment of glial cultures resulted in a dissociation of NLRP3 from NLRP10 as seen by immunoprecipitation and d assessed by optical density quantification (n = 5; **p \ 0.01; student’s t test). e NLRP3 did not associate with NLRP10
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Z-FG-NHO-Bz (???p \ 0.001; ANOVA; Fig. 1c, d). We next examined the effects of Ab on two other inflammatory cytokines, IL-6 and TNFa, and show that, in contrast to IL1b, LPS priming significantly increased supernatant concentration of both cytokines (***p \ 0.001; ANOVA; Fig. 1e, f). While Ab induced a further significant release of IL-6 and TNFa (?p \ 0.05; ??p \ 0.01; LPS with vs. without Ab; ANOVA; Fig. 1e), cytochalasin D and cathepsin inhibition did not modulate the effect of LPS or Ab ? LPS on the release of either cytokine. None of the cell treatments adversely affect cell viability (data not shown). Characterization of the Role of NALP10 in Ab-Induced Inflammasome Formation In order to verify formation of the NLRP3 inflammasome in Ab-treated cells, we immunoprecipitated one of the main
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components of the inflammasome, ASC, from control and Ab-treated LPS-primed glial cultures. The immunoprecipitated samples were electrophoresed, transferred and probed for the second component of the inflammasome, NLRP3. In untreated cells, NLRP3 did not associate with ASC; however, after Ab treatment of LPS-primed cells, there was a strong association of ASC with NLRP3 (**p \ 0.01; student’s t test for independent means; Fig. 2a, b). Apoptosis-associated speck-like protein (ASC) has been previously shown to associate also with another NOD-like receptor protein, NLRP10 (Wang et al. 2004), and here, we investigated the association of NLRP10 with ASC in the presence and absence of Ab. The data show that ASC associated with NLRP10 in untreated mixed glial cells; however, in cells treated with Ab, the association of NLRP10 with ASC was significantly reduced (**p \ 0.01; student’s t test for independent means; Fig. 2d). The total expression of NLRP10 in these LPS-primed, Ab-treated
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Fig. 3 a Western blot was used to assess the expression of NLRP10 in microglia, astrocytes and neurons and b quantified using optical density analysis. c Ab treatment of LPS-primed glial cultures resulted in a decrease in the expression of NLRP10, which was attenuated by cytochalasin D and cathepsin inhibitor as seen by Western blot and d assessed by optical density analysis (***p \ 0.001; ANOVA;
Control vs. LPS ? Ab; ???p \ 0.001; LPS ? Ab vs. LPS ? Ab ? cytochalasin D; ??p \ 0.01; LPS ? Ab vs. LPS ? Ab ? cathpesin inhibitor). e Male Wistar rats that were infused with Ab via a mini-pump for 28 days had increased caspase 1 activation in the cortex and the hippocampus. f These animals infused with Ab also have a reduction in NLRP10 expression in both the cortex and hippocampus
cells was also decreased compared with untreated cells (Fig. 2c). NLRP3 did not associate with NLRP10 (Fig. 2e).
We assessed the expression of NLRP10 in LPS-primed mixed glial cultures after treatment with Ab with or without cytochalasin D and the cathepsin inhibitor, Z-FG-NHO-Bz. LPS priming of glial cells had no effect on the constitutive expression of NLRP10 expression; however, subsequent treatment with Ab induced a significant reduction in NLRP10 (***p \ 0.001; ANOVA; Fig. 3c). Pretreatment with cytochalasin D or Z-FG-NHO-Bz inhibited the Abinduced decreased in NLRP10 (??p \ 0.01; ???p \ 0.001; ANOVA; Fig. 3d). In order to assess whether Ab induced this change in NLRP10 in vivo, we prepared hippocampal and cortical tissue from male Wistar rats which received a cocktail of aggregated recombinant Ab1–40 and Ab1–42 or the reverse
NLRP10 is Highly Expressed in Microglia and is Decreased with Ab Treatment Both In Vitro and In Vivo In order to characterize the expression of NLRP10 in different cell types, 3 separate preparations (S1, S2 and S3) of untreated microglia, astrocytes and neurons were isolated from neonatal Wistar rats. Microglial cells had the greatest expression of NLRP10 (Fig. 3a, b). Astrocytes also expressed NLRP10 but to a lesser extent than microglia. Expression of NLRP10 in neurons was very limited.
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non-aggregating Ab40–1 peptide, chronically by means of an implanted osmotic mini-pump. We first investigated formation of the NLRP3 inflammasome in these animals by assessing the levels of active caspase 1 in the cortex and hippocampus. Active caspase 1 was significantly increased in both the cortex and hippocampus of animals that had been infused with the Ab1–40/1–42 cocktail compared with control animals (*p \ 0.05; student’s t test for independent means; Fig. 3e). We also assessed the expression of NLRP10 in these animals and found that there was a significant decrease in NLRP10 protein in the cortex and hippocampus of those animals that had been infused with the Ab1–40/1–42 cocktail compared with control animals (**p \ 0.001; student’s t test for independent means; Fig. 3f).
75-kDa band disappeared after a 1-h incubation but remained in those samples also containing cathepsin inhibitor (Fig. 4b).
NLRP10 is Degraded by Cathepsins Cathepsins are a group of proteases that have been associated with the formation of the NLRP3 inflammasome in response to Ab (Halle et al. 2008). As the NLRP10 protein is decreased in response to Ab treatment, we hypothesized that cathepsins were responsible for its degradation. We assessed this hypothesis using a degradation assay. Activated recombinant cathepsin B or L was incubated with recombinant NLRP10 for 1 h at 37 °C with or without the cathepsin inhibitor. In the sample containing cathepsin B and NLRP10, the representative 75-kDa NLRP10 band disappeared after 1 h (Fig. 4a). However, in the sample containing cathepsin B, NLRP10 and cathepsin inhibitor, the 75-kDa band remained after 1-h incubation (Fig. 4a). Similarly, in samples containing recombinant active cathepsin L incubated with recombinant NLRP10, the
NLRP10 Inhibits Ab-Induced NLRP3 Inflammasome Formation and IL-1b Release Consistent with the data presented in Fig. 1, Ab induced caspase 1 activation in glial cultures (***p \ 0.001; ANOVA; Fig. 5a). Importantly, treatment with recombinant NLRP10 significantly reduced the Ab-induced change (???p \ 0.001; ANOVA; Fig. 4a). Similarly, Ab increased IL-1b release from LPS-primed glial cultures (***p \ 0.001; ANOVA; Fig. 5b), and this release was also attenuated by treatment with recombinant NLRP10 (???p \ 0.001; ANOVA; Fig. 5b). In order to assess whether NLRP10 had any inflammasome-independent antiinflammatory effects, we assessed the release of IL-6 and TNFa from Ab-treated glial cultures. As previously shown in Fig. 1, Ab enhanced the significant LPS-induced release of IL-6 and TNFa from LPS-primed cultures (**p \ 0.01; ***p \ 0.001; control vs. LPS; ?p\0.05;??p \ 0.01; LPS with vs. without Ab; ANOVA; Fig. 5c, d). Treatment with NLRP10 did not alter the effects of LPS or Ab on the release of either of these cytokines. To clarify that NLRP10 acts downstream of cathepsins in this setting, we assessed the activity of cathepsin B and L in response to Ab and recombinant NLRP10 treatment. The activity of cathepsin B and L in the cytosol of LPSprimed, Ab-treated cultures was increased, confirming the data presented in Fig. 1 (**p \ 0.01; ANOVA; control vs. LPS ? Ab; Fig. 5e, f); however, NLRP10 treatment had no effect on the activity of either cathepsin (Fig. 5e, f).
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Fig. 5 a The Ab-induced increase in caspase 1 activation in LPSprimed glial cells (n = 5; ***p \ 0.001; ANOVA; Control vs. LPS ? Ab) was attenuated by treatment with recombinant NLRP10 (???p \ 0.001; ANOVA; LPS ? Ab vs. LPS ? Ab ? NLRP10). b The Ab-induced increase in IL-1b (n = 5; ***p \ 0.001; ANOVA; Control vs. LPS ? Ab) was also attenuated by recombinant NLRP10 treatment (???p \ 0.001; ANOVA; LPS ? Ab vs. LPS ? Ab ? NLRP10). c LPS induced an increase in the release of IL-6 (***p \ 0.001; ANOVA; Control vs. LPS) which was further increased with Ab treatment (??p \ 0.01; ANOVA; LPS vs.
LPS ? Ab); however, NLRP10 had no effect on this release. d TNFa release was induced by treatment with LPS (n = 5; **p \ 0.01; ANOVA; Control vs. LPS) and further by treatment with Ab (?p\0.05; ANOVA; LPS vs. LPS ? Ab) but was unaffected by NLRP10 treatment. e Cathepsin B activity in the cytosol of LPSprimed glia was increased with Ab treatment (n = 5; **p \ 0.01; ANOVA; Control vs. LPS ? Ab) but unaffected by treatment with NLRP10). f Cathepsin L activity in the cytosol of LPS-primed glia was increased with Ab treatment (n = 5; **p \ 0.01; ANOVA; Control vs. LPS ? Ab) but unaffected by treatment with NLRP10)
Discussion
NLRP10 inhibits the assembly of the NLRP3 inflammasome and therefore is a potential target for anti-inflammatory therapies. Interleukin-1b (IL-1b) is a key mediator in the innate immune response in Alzheimer’s disease, and significantly elevated expression of IL-1b has been seen in both the
The significant finding in this study is that a component of the inflammasome, ASC, constituently interacts with NLRP10 in glia and that Ab markedly reduces the interaction between these proteins. The evidence suggests that
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brain and the plasma of Alzheimer’s patients’ postmortem (Ojala et al. 2009). Preclinical data support a particular role for IL-1b and in a triple transgenic mouse model of Alzheimer’s disease; blocking IL-1b signaling has been shown to rescue cognitive impairment associated with the disease. Additionally, deletion of the IL-1Ra gene in mice leads to increased susceptibility to intra-hippocampal injection of Ab with increased neuroinflammation and loss of synaptic markers (Craft et al. 2005), while the inflammatory changes and loss of synaptic plasticity in transgenic mice which overexpress APP and PS1 were attenuated when these mice were crossed with NLRP3-deficient mice {Heneka 2013 #7971}. IL-1b release from microglia has also been shown to increase activity and/or expression of c- and b-secretases, the enzymes responsible for processing of the amyloid precursor protein ultimately leading to further Ab deposition (Sastre et al. 2008). Interleukin-1b is processed and released from glial cells following the assembly of the NLRP3 inflammasome. The inflammasome can be activated by a range of stimuli including millimolar concentrations of extracellular adenosine triphosphate (Murphy et al. 2011), viruses including influenza A (Thomas et al. 2009) and Neisseria gonorrhoeae (Duncan et al. 2009), crystallized endogenous molecules including cholesterol crystals (Duewell et al. 2010) and monosodium urate crystals (Martinon et al. 2006) as well as inorganic particles like titanium dioxide, silicium dioxide and asbestos (Dostert et al. 2008). In 2008, Halle et al. reported that fibrillar Ab was also a stimulus for the formation of the NLRP3 inflammasome (Halle et al. 2008). The authors reported that phagocytosis of Ab triggered an abnormal lysosomal response resulting in leakage of cathepsin B into the cytosol where it acted to stimulate the formation of the inflammasome. In this study, we verify that Ab treatment of glial cells induced increased activity of cathepsin B, but the data also demonstrate that Ab increased the activity of cathepsin L in the cytosol. We show that Ab increased the association of the inflammsome components ASC and NLRP3 and increased caspase 1 activation and IL-1b release. These changes were attenuated by inhibiting phagocytosis by cytochalasin D or by inhibiting cathepsin activity. NOD-like receptors, such as NLRP3, are localized to the cytoplasm and have varying functions in inflammatory and apoptotic processes. In 2004, an additional member of the NOD-like family, NLRP10 (also known as PYNOD or NALP10), was discovered and was shown to be expressed particularly in cardiac and skeletal muscle, but also in brain (Wang et al. 2004). Here, we report that NLRP10 is highly expressed in glia, particularly microglial cells. It was discovered that, like NLRP3, NLRP10 also interacted with ASC, but NLRP10 inhibited the ASC-mediated activation of caspase 1 and IL-1b release in transfected HEK293T-S cells (Wang et al. 2004). NLRP10 differs from NLRP3 due
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to the fact that it does not contain a leucine-rich repeat (LRR) domain. It is thought that the lack of LRR domain prevents ASC, when bound to NLRP10, from binding and activating caspase 1 (Wang et al. 2004). The findings presented here show that ASC constitutively interacts with NLRP10 in glia and that Ab treatment reduces the association of NLRP10 with ASC by approximately 80 %. Amyloid beta (Ab) markedly reduced NLRP10, but the decrease was redressed by inhibiting cathepsins or by blocking phagocytosis using cytochalasin D. These data indicate that it is the phagocytosis of Ab and the subsequent cathepsin release that are responsible for the decreased NLRP10. It has been shown that degradation of NLRP3 occurs through the autophagy–lysosome pathway (Chuang et al. 2013); a similar process may facilitate degradation of NLRP10, though this remains to be demonstrated. We next assessed caspase 1 activation and NLRP10 expression in hippocampus and cortex of rats in which chronic infusion of Ab has been shown to induce Ab deposition (Frautschy et al. 2001) and found that, in parallel with the NLRP3 inflammasome activation in the hippocampus and cortex of these animals, there was a decrease in NLRP10 expression. To our knowledge, this is the first time that changes in the expression of NLRP10 have been reported in response to Ab. The importance of cathepsins in the activation of the NLRP3 inflammasome has been highlighted by several studies. Serum amyloid A (Niemi et al. 2011), Ab (Halle et al. 2008), Neisseria gonorrhoeae (Duncan et al. 2009), Streptococcus pneumoniae (Hoegen et al. 2011), cholesterol-dependent cytolysins (Chu et al. 2009), cytoplasmic RNA (Rintahaka et al. 2011) and chemotherapeutic agents (Bruchard et al. 2012) have all been shown to induce the inflammasome assembly in a cathepsin-dependent manner. In the case of Ab, its phagocytosis by microglial cells has been shown to cause lysosomal damage which, in turn, leads to release of cathepsins into the cytosol (Halle et al. 2008). Although the precise role for cathepsins in the setting of inflammasome activation has yet to be elucidated. Bruchard et al. have recently reported that cathepsin B binds directly to NLRP3 in response to chemotherapeutic agents, and this drives the activation of the inflammasome. Previous studies have focused on cathepsin B, but the present data indicate that both cathepsin B and L are activated in response to Ab. Both cathepsins, which are released into the cytoplasm in Ab-treated glia, are capable of degrading NLRP10, and this is confirmed by the finding that the degradation was prevented by a cathepsin inhibitor, albeit in cell-free system. On the basis of our findings, we propose that cathepsins, released into the cytosol after Ab uptake, degrade NLRP10 that is normally bound to ASC. Free ASC can now associate with NLRP3, thus activating caspase 1 and processing and release of IL-1b.
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Further to this action of NLRP10, we have identified an anti-inflammatory effect of NLRP10 since it inhibits Abinduced production of IL-1b and activation of caspase 1. We are currently investigating the modulatory role of NLRP10 in mouse models characterized by neuroinflammatory changes and specifically confirming its role in attenuating Ab-induced changes using siRNA technology. Interestingly, peritoneal macrophages from transgenic mice, which overexpress NLRP10, are defective in IL-1b production in response to Salmonella typhimurim and are also resistant to LPS-induced endotoxic shock (Imamura et al. 2010). However, contrary to these data, Lautz et al. (2012) reported that NLRP10 contributed to the proinflammatory cytokine release from fibroblasts and epithelial cells that is triggered by the bacterial pathogen Shigella flexneri by influencing the activation of p38 and NF-jB. Similarly, NLRP10 has also been shown to play an essential role in the initiation of adaptive immunity by dendritic cells in response to an array of inflammatory stimuli (Eisenbarth et al. 2012). Clearly, NLRP10 may have different effects on different cell types in response to varying stimuli. These results suggest that targeting the Ab-induced degradation of NLRP10 may prove to be a useful therapeutic in redressing the persistent neuroinflammation that occurs in, and contributes to, the pathology of Alzheimer’s disease. Acknowledgments The authors would like to thank the Irish Research Council and Science Foundation Ireland for funding this work. Conflict of interest of interest.
The authors declare that they have no conflict
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intracerebroventricular infusion of human beta-amyloid. Journal of Neuroinflammation, 2, 15. doi:10.1186/1742-2094-2-15. Dostert, C., Petrilli, V., Van Bruggen, R., Steele, C., Mossman, B. T., & Tschopp, J. (2008). Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science, 320(5876), 674–677. doi:10.1126/science.1156995. Duewell, P., Kono, H., Rayner, K. J., Sirois, C. M., Vladimer, G., Bauernfeind, F. G., et al. (2010). NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature, 464(7293), 1357–1361. Duncan, J. A., Gao, X., Huang, M. T., O’Connor, B. P., Thomas, C. E., Willingham, S. B., et al. (2009). Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome. The Journal of Immunology, 182(10), 6460–6469. doi:10.4049/ jimmunol.0802696. Eisenbarth, S. C., Williams, A., Colegio, O. R., Meng, H., Strowig, T., Rongvaux, A., et al. (2012). NLRP10 is a NOD-like receptor essential to initiate adaptive immunity by dendritic cells. Nature, 484(7395), 510–513. doi:10.1038/nature11012. Frautschy, S. A., Hu, W., Kim, P., Miller, S. A., Chu, T., HarrisWhite, M. E., et al. (2001). Phenolic anti-inflammatory antioxidant reversal of Abeta-induced cognitive deficits and neuropathology. Neurobiology of Aging, 22(6), 993–1005. Halle, A., Hornung, V., Petzold, G. C., Stewart, C. R., Monks, B. G., Reinheckel, T., et al. (2008). The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nature Immunology, 9(8), 857–865. doi:10.1038/ni.1636. Heneka, M. T., O’Banion, M. K., Terwel, D., & Kummer, M. P. (2010). Neuroinflammatory processes in Alzheimer’s disease. Journal of Neural Transmission, 117(8), 919–947. doi:10.1007/ s00702-010-0438-z. Hoegen, T., Tremel, N., Klein, M., Angele, B., Wagner, H., Kirschning, C., et al. (2011). The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release. The Journal of Immunology, 187(10), 5440–5451. doi:10.4049/ jimmunol.1100790. Imamura, R., Wang, Y., Kinoshita, T., Suzuki, M., Noda, T., Sagara, J., et al. (2010). Anti-inflammatory activity of PYNOD and its mechanism in humans and mice. The Journal of Immunology, 184(10), 5874–5884. doi:10.4049/jimmunol.0900779. Lautz, K., Damm, A., Menning, M., Wenger, J., Adam, A. C., Zigrino, P., et al. (2012). NLRP10 enhances Shigella-induced pro-inflammatory responses. Cellular Microbiology, 14(10), 1568–1583. doi:10.1111/j.1462-5822.2012.01822.x. Lyons, A., Downer, E. J., Crotty, S., Nolan, Y. M., Mills, K. H., & Lynch, M. A. (2007). CD200 ligand receptor interaction modulates microglial activation in vivo and in vitro: A role for IL-4. The Journal of Neuroscience, 27(31), 8309–8313. doi:10. 1523/JNEUROSCI.1781-07.2007. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., & Tschopp, J. (2006). Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature, 440(7081), 237–241. doi:10.1038/ nature04516. Murphy, N., Cowley, T. R., Richardson, J. C., Virley, D., Upton, N., Walter, D., et al. (2011). The neuroprotective effect of a specific P2X(7) receptor antagonist derives from its ability to inhibit assembly of the NLRP3 inflammasome in glial cells. Brain Pathology. doi:10.1111/j.1750-3639.2011.00531.x. Niemi, K., Teirila, L., Lappalainen, J., Rajamaki, K., Baumann, M. H., Oorni, K., et al. (2011). Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway. The Journal of Immunology, 186(11), 6119–6128. doi:10.4049/jimmunol.1002843.
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215 Sastre, M., Walter, J., & Gentleman, S. M. (2008). Interactions between APP secretases and inflammatory mediators. Journal of Neuroinflammation, 5, 25. doi:10.1186/1742-2094-5-25. Sheng, J. G., Mrak, R. E., & Griffin, W. S. (1997). Glial-neuronal interactions in Alzheimer disease: Progressive association of IL1alpha? microglia and S100beta? astrocytes with neurofibrillary tangle stages. Journal of Neuropathology and Experimental Neurology, 56(3), 285–290. Thomas, P. G., Dash, P., Aldridge, J. R, Jr., Ellebedy, A. H., Reynolds, C., Funk, A. J., et al. (2009). The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1. Immunity, 30(4), 566–575. doi:10.1016/j.immuni.2009.02.006. Wang, Y., Hasegawa, M., Imamura, R., Kinoshita, T., Kondo, C., Konaka, K., et al. (2004). PYNOD, a novel Apaf-1/CED4-like protein is an inhibitor of ASC and caspase-1. International Immunology, 16(6), 777–786. doi:10.1093/intimm/dxh081.
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ORIGINAL PAPER
Glial Uptake of Amyloid Beta Induces NLRP3 Inflammasome Formation via Cathepsin-Dependent Degradation of NLRP10 Niamh Murphy • Belinda Grehan • Marina A. Lynch
Received: 4 April 2013 / Accepted: 26 October 2013 / Published online: 7 November 2013 Ó Springer Science+Business Media New York 2013
Abstract The NLRP3 inflammasome forms in response to a diverse range of stimuli and is responsible for the processing and release of interleukin-1b (IL-1b) from the immunocompetent cells of the brain. The pathological peptide of Alzheimer’s disease, amyloid beta (Ab), induces formation of the NLRP3 inflammasome in a manner dependent on the family of proteases, cathepsins; however, the pathway by which cathepsins induce formation of the inflammasome has not yet been elucidated. In this study, we show that Ab treatment of primary rat glial cultures increases cathepsin activation in the cytosol, formation of the NLRP3 inflammasome, caspase 1 activation and IL-1b release. We also show that a second NOD-like protein, NLRP10, is found bound to apoptosis-associated speck-like protein under resting conditions; however, with Ab treatment, both in vitro and in vivo, NLRP10 is decreased. Further to these data, we show that cathepsins are capable of degrading NLRP10 and that treatment of glial cultures with recombinant NLRP10 reduces Ab-induced caspase 1 activation and IL-1b release. We propose that Ab-induced cathepsin released into the cytosol degrades NLRP10, thus allowing dissociation of NLRP3 and formation of the inflammasome. Keywords NLRP3 inflammasome Amyloid beta Cathepsins NLRP10 Glia
N. Murphy (&) B. Grehan M. A. Lynch Department of Physiology, Trinity College Institute of Neuroscience, Trinity College, College Green, Dublin 2, Ireland e-mail: [email protected]
Introduction Neuroinflammation is recognized as the brain’s innate immune response to both acute injury and neurodegenerative changes. It has been identified as a key component in the pathology of Alzheimer’s disease where the degree of inflammation parallels the severity of the disease (Sheng et al. 1997). Alzheimer’s disease has been associated with several markers of neuroinflammation including increased and robust activation of microglia and astrocytes, the clustering of these activated cells around amyloid plaques, increases in the expression of pro-inflammatory cytokines, cell adhesion molecules and chemokines, and induction of inflammatory enzyme systems such as inducible nitric oxide synthase (iNOS) (Heneka et al. 2010; Sardi et al. 2011). One of the key pro-inflammatory cytokines involved in the neuroinflammatory process is interleukin (IL)-1b. IL1b is primarily produced as the precursor, pro-IL-1b, which requires processing to become biologically active. In order for IL-1b to become processed, a group of intracellular proteins, collectively known as the NLRP3 inflammasome, must assemble to form a complex. The NLRP3 inflammasome includes the apoptosis-associated speck-like protein (ASC), the protease caspase 1, and the NOD-like receptor, NLRP3, recruitment of which leads to the proteolytic cleavage of caspase 1 to its active form which then processes pro-IL-1b to active IL-1b. A novel NOD-like receptor, NLRP10 identified in 2004 (Wang et al. 2004), was shown to reduce caspase 1-induced processing and release of IL-1b in HEK293 cells. Subsequent studies have shown that the anti-inflammatory effects of NLRP10 are due to its interactions with, and inhibition of, the actions of NLRP3 and ASC (Imamura et al. 2010).
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In this study, we show that amyloid-b (Ab), the pathogenic peptide associated with Alzheimer’s disease, induced cathepsin B release into the cytosol and that this degrades the anti-inflammatory protein NLRP10 which subsequently results in the activation of the NLRP3 inflammasome and IL-1b release.
Materials and Methods Primary Mixed Glial Cultures Mixed glia were prepared from the cortices of 1-day-old Wistar rats (Trinity College, Dublin, Ireland). Cortical tissue was cross-chopped and incubated for 25 min at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Fisher Scientific, Ireland) supplemented with 10 % fetal bovine serum (Fisher Scientific, Ireland) and 50 U/ml penicillin/streptomycin (Fisher Scientific, Ireland) and plated (2.5 9 105) as previously described (Nolan et al. 2004). Cells from each rat were treated separately. Inflammasome activation requires two signals; signal 1 is induced by Toll-like receptor stimulation, leading to the synthesis of pro-IL-1b. In our system, after 13 days, cells were primed by incubating with lipopolysaccharide (LPS; 1 lg/ml; Sigma, Ireland) for 4 h, which provided ‘signal 1.’ Cells were treated with a cocktail of recombinant human Ab1–40 (4.3 lM, aggregated for 24 h at 25 °C; Invitrogen, Ireland) and Ab1–42 (5 lM, aggregated at 37 °C for 48 h; Invitrogen, Ireland), and incubation continued for a further 24 h. In some experiments, LPS-treated cells were incubated in the presence of an inhibitor of cathepsin Z-FG-NHO-Bz (10 lM; Millipore, Ireland); although the primary target of this cysteine protease inhibitor is cathepsin B (k2/Ki = 8.9 9 103 M-1 s-1), it inhibits cathepsins L and S (k2/Ki = 3.8 9 105 and 4.2 9 104 M-1 s-1) and also the plant-specific protease, papain (k2/Ki = 2.4 9 103 M-1 s-1). The concentration of the inhibitor used in this study was determined following preliminary dose-finding experiments. In other experiments, cells were incubated in the presence or absence of cytochalasin D (3 lM, Millipore, Ireland) or recombinant NLRP10 (150 ng/ml, Origene, Maryland, US) for 1 h prior to addition of Ab cocktail. In all experiments, supernatants were collected and cells were harvested for later analysis. An MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay (Promega, UK) was carried out after all treatments in order to assess cell viability. Isolation of Neuronal, Microglial and Astrocytic Cultures To prepare purified microglia and astrocytes, cells were grown in T25 flasks in DMEM as above. After 12 days, the
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flasks were shaken for 2 h at 110 rpm, at room temperature and tapped several times to remove non-adherent microglia. The supernatants were removed from the flask and centrifuged at 2,000 rpm for 3 min at 21 °C. The pellet was resuspended in DMEM, and the cells were counted. Cells were pipetted into 6-well plates at a density of 1 9 105 cells/ml. To prepare astrocytes, the flasks containing the adherent astrocytes were washed with PBS, and 1 ml of 0/05 % w/v trypsin–EDTA was added at 37 °C until the cells just began to detach, DMEM was then added to the flask to inhibit the action of trypsin. The cells were centrifuged at 2,000 rpm for 3 min. The pellet was resuspended in DMEM, and the cells were plated in 9-well plates at a density of 1 9 105 cells/ml. Primary hippocampal neuronal cultures were prepared from 1-day-old Wistar rats. The hippocampus was dissected in Hanks’ balanced salt solution (HBSS) buffered with 1 mM sodium pyruvate and 1 mM HEPES pH 7.4 and dissociated using papain (15 U/ml; Sigma, UK). Cells were plated at 6 9 104 cells/cm2 on 48-well dishes precoated with poly-D-lysine (50 lg/ml) and maintained in Neurobasal media (NBM) containing 0.5 mM glutamine and B27 supplement. Half the medium was exchanged every 3 days. All media reagents were purchased from Fisher Scientific, Ireland. Treatment of Animals Male Wistar rats aged 3–4 months (Bioresources Unit, Trinity College, Dublin, Ireland) were used in these studies. Animals were housed in groups of 4–6 and were maintained on a 12-h light schedule, at an ambient temperature of 22–23 °C, under veterinary supervision in the Bioresources Unit. The experiments were performed under a license issued by the Department of Health and Children, Ireland, with the approval of the local ethics committee and in accordance with local guidelines. Rats were randomly divided into 2 groups, half of which were treated with a cocktail of recombinant Ab1–40 and Ab1–42, while the other half received the reverse peptide, Ab40–1. Animals were anaesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and implanted with osmotic mini-pumps (model 2004, Alzet, USA). The pump was implanted subcutaneously in the mid-scapular region and was attached via polyvinylchloride tubing (Alzet, 0.69 mm diameter) to a chronic indwelling cannula (Alzet, Infusion Kit II), which was positioned stereotaxically in the ventricle (0.9 mm posterior to bregma, 1.3 lateral to the midline and 3.5 mm ventral to the dura). The cannula was affixed to the skull using cryanoacetate gel and was secured in place by a smooth covering of dental cement (Stoelten, USA). Post-operative care included a subcutaneous injection of the analgesic Rimadil (5 mg/kg). The pumps
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delivered a cocktail of Ab1–40 (26.9 lM) and Ab1–42 (36.9 lM; aggregated for 24 h at 25 and 37 °C for 48 h; Invitrogen) or control peptide Ab40–1 (63.8 lM) intracerebroventricularly at the rate of 0.25 ll/h (±0.05 ll) for 28 days. Analysis of the Ab preparation by the thioflavin T fluorescent assay and gel electrophoresis revealed the presence of low oligomeric species; the predominant form (36 % of the total) was the 13.5-kDa species.
peroxide-conjugated secondary antibodies (Jackson ImmunoResearch, US), and bands were visualized using Supersignal West Pico Chemiluminescent Substrate (Pierce, US). Images were captured using a Fujifilm LAS3000 (Brennan and Co, Ireland). Densitometry was performed using ImageJ software (http://rsb.info.nih.gov/ij/).
Analysis of Cathepsin Activity in the Cytosolic Fraction of Glial Cultures
For immunoprecipitation studies, mixed glial samples containing 500 lg protein were incubated overnight at 4 °C in the presence of ASC-specific antibody (5 lg; rabbit polyclonal; Santa Cruz Biotechnology, US) or IgG isotype control (negative control) (Sigma-Aldrich, Ireland). A/G protein agarose beads (50 ll; Santa Cruz Biotechnology, US) were added, samples were incubated for 2 h, washed in PBS containing NP-40 (0.01 %) and centrifuged. Loading buffer (19) was added to each sample, and samples were boiled. Proteins were separated (see above), and NLRP10 and NLRP3 were visualized by incubating the membrane in the presence of a NLRP10-specific (mouse monoclonal, R&D Systems Europe, UK) or NLRP3-specific (rabbit polyclonal; Santa Cruz Biotechnology, US) antibodies.
The cytosolic fraction was isolated from treated glial cells by centrifugation. Briefly, cells were homogenized in 19 M-SHE buffer (210 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EDTA and 1 mM EGTA: all Sigma, Ireland). Lysates were centrifuged at 1,2009g for 10 min at 4 °C. The supernatants were removed, and the centrifugation was repeated. The supernatants were removed and centrifuged at 10,0009g for 15 min at 4 °C. The supernatant from this last spin was taken as the cytosolic fraction. Cathespin activity in the cytosolic fraction was measured using a cathepsin B-specific fluorometric assay (RR-AFC substrate) and a cathepsin L-specific fluorometric assay (FR-AFC substrate) (Abcam, Cambridge, UK).
Immunoprecipitation
Protein Degradation Assay Analysis of IL-1b, IL-6 and TNFa Concentrations Supernatant concentrations of IL-1b, IL-6 and TNFa obtained from glial cultures were measured using ELISA. Cytokine concentrations in the test samples were evaluated with reference to the standard curves prepared using recombinant cytokines of known concentrations. Analysis of Activity of Caspase 1 Cells were harvested, washed in phosphate-buffered saline and lysed in lysis buffer. Activity of caspase 1 was measured using a fluorometric assay kit (R&D Systems Europe, UK).
Recombinant cathepsin B and cathepsin L (R&D Systems, UK) were reconstituted in activation buffer (50 mM sodium acetate, 2 mM DTT, 2 mM ethylenediaminetetraacetic acid). Some activated cathepsin B was heatdenatured by incubation at 100 °C for 5 min. Activated cathepsin B and L (10 lg/ml) were incubated with recombinant NLRP10 (0.5 lg/ml; Sigma-Aldrich, Dublin, Ireland) for 1 h at 37 °C. The reaction was stopped using 1 9 protease inhibitor (Sigma-Aldrich, Ireland). Sample buffer (2 9 SDS) was added to each sample which were run on a 15 % polyacrylamide gel, and the proteins were visualized by silver staining.
Analysis of Proteins by Western Immunoblotting
Results
Western blotting was performed as previously described (Lyons et al. 2007). Cultured cells were harvested, homogenized in buffer containing Tris–HCl (0.01 M) and EDTA (1 mM), and protein (20 lg) was boiled in gelloading buffer and separated by 12 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and incubated with antibodies against b-actin (mouse monoclonal; Sigma, Ireland) and NLRP10 (mouse monoclonal, R&D Systems Europe, UK). Membranes were incubated with horseradish
Phagocytosis of Ab Induces Cathepsin B and L Activity in the Cytosol, Caspase 1 Activation and IL-1b Release LPS-primed mixed glial cultures were treated with a cocktail of Ab1–40 and Ab1–42 (4.9 and 5.4 lM, respectively) for a period of 24 h. Ab significantly increased active cathepsin B and L in the cytosol (***p \ 0.001; ANOVA; Fig. 1a, b). It has previously been shown that cathepsins are released from damaged lysosomes in response to phagocytosis of Ab (Halle et al. 2008). To assess whether this was the case in our
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Fig. 1 Ab treatment of LPS-primed rat glial cultures induced activation of (a) cathepsin B and (b) cathepsin L in the cytosol (n = 5; ***p \ 0.001; ANOVA; control vs. LPS ? Ab), and both cathepsins were inhibited by cytochalasin D treatment (???p \ 0.001; ANOVA; LPS ? Ab vs. LPS ? Ab ? cytochalasin D). Ab treatment of LPS-primed glial cultures also induced (c) caspase 1 activation and (d) IL-1b release (n = 5; ***p \ 0.001; ANOVA; control vs. LPS ? Ab) which were both
inhibited by a broad-spectrum cathepsin inhibitor and cytochalasin D (???p \ 0.01; ANOVA; LPS ? Ab vs. LPS ? Ab ? cathepsin inhibitor or LPS ? Ab ? cytochalasin D). LPS-induced release of (e) IL-6 and (f) TNFa (n = 5; ***p \ 0.001; ANOVA; Control vs. LPS) which were both further enhanced with Ab treatment (??p \ 0.01; ?p\0.05; ANOVA; LPS vs. LPS ? Ab). The cathepsin inhibitor and cytochalasin D had no effect on the Ab-induced release of either IL-6 or TNFa
system, we pretreated the cells with cytochalasin D, an inhibitor of actin polymerization. Blocking phagocytosis of Ab resulted in a decrease in active cathepsin B and L in the cytosol (???p \ 0.001; Ab-treated LPS-primed cells with vs. without cytochalasin D; Fig. 1a, b). Treatment of LPS-primed mixed glial cells with Ab resulted in activation of caspase 1 (***p \ 0.001; ANOVA; Fig. 1c;), and caspase 1 activation was inhibited
by treatment of cells with cytochalasin D and also with a broad-spectrum cathepsin inhibitor, Z-FG-NHO-Bz (???p \ 0.001; ANOVA; Fig. 1c). We investigated IL-1b release, since active caspase 1 is responsible for its processing and release, and the data show that Ab significantly increased release of IL-1b from LPS-primed mixed glia (***p \ 0.001; ANOVA; Fig. 1d;). IL-1b release was inhibited by pretreating cells with cytochalasin D and
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Fig. 2 a Ab treatment of LPSprimed glial cells resulted in the association of NLRP3 to ASC as seen by immunoprecipitation and b assessed by optical density quantification (n = 5; **p \ 0.01; student’s t test). c Ab treatment of glial cultures resulted in a dissociation of NLRP3 from NLRP10 as seen by immunoprecipitation and d assessed by optical density quantification (n = 5; **p \ 0.01; student’s t test). e NLRP3 did not associate with NLRP10
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Z-FG-NHO-Bz (???p \ 0.001; ANOVA; Fig. 1c, d). We next examined the effects of Ab on two other inflammatory cytokines, IL-6 and TNFa, and show that, in contrast to IL1b, LPS priming significantly increased supernatant concentration of both cytokines (***p \ 0.001; ANOVA; Fig. 1e, f). While Ab induced a further significant release of IL-6 and TNFa (?p \ 0.05; ??p \ 0.01; LPS with vs. without Ab; ANOVA; Fig. 1e), cytochalasin D and cathepsin inhibition did not modulate the effect of LPS or Ab ? LPS on the release of either cytokine. None of the cell treatments adversely affect cell viability (data not shown). Characterization of the Role of NALP10 in Ab-Induced Inflammasome Formation In order to verify formation of the NLRP3 inflammasome in Ab-treated cells, we immunoprecipitated one of the main
− −
+ +
components of the inflammasome, ASC, from control and Ab-treated LPS-primed glial cultures. The immunoprecipitated samples were electrophoresed, transferred and probed for the second component of the inflammasome, NLRP3. In untreated cells, NLRP3 did not associate with ASC; however, after Ab treatment of LPS-primed cells, there was a strong association of ASC with NLRP3 (**p \ 0.01; student’s t test for independent means; Fig. 2a, b). Apoptosis-associated speck-like protein (ASC) has been previously shown to associate also with another NOD-like receptor protein, NLRP10 (Wang et al. 2004), and here, we investigated the association of NLRP10 with ASC in the presence and absence of Ab. The data show that ASC associated with NLRP10 in untreated mixed glial cells; however, in cells treated with Ab, the association of NLRP10 with ASC was significantly reduced (**p \ 0.01; student’s t test for independent means; Fig. 2d). The total expression of NLRP10 in these LPS-primed, Ab-treated
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Fig. 3 a Western blot was used to assess the expression of NLRP10 in microglia, astrocytes and neurons and b quantified using optical density analysis. c Ab treatment of LPS-primed glial cultures resulted in a decrease in the expression of NLRP10, which was attenuated by cytochalasin D and cathepsin inhibitor as seen by Western blot and d assessed by optical density analysis (***p \ 0.001; ANOVA;
Control vs. LPS ? Ab; ???p \ 0.001; LPS ? Ab vs. LPS ? Ab ? cytochalasin D; ??p \ 0.01; LPS ? Ab vs. LPS ? Ab ? cathpesin inhibitor). e Male Wistar rats that were infused with Ab via a mini-pump for 28 days had increased caspase 1 activation in the cortex and the hippocampus. f These animals infused with Ab also have a reduction in NLRP10 expression in both the cortex and hippocampus
cells was also decreased compared with untreated cells (Fig. 2c). NLRP3 did not associate with NLRP10 (Fig. 2e).
We assessed the expression of NLRP10 in LPS-primed mixed glial cultures after treatment with Ab with or without cytochalasin D and the cathepsin inhibitor, Z-FG-NHO-Bz. LPS priming of glial cells had no effect on the constitutive expression of NLRP10 expression; however, subsequent treatment with Ab induced a significant reduction in NLRP10 (***p \ 0.001; ANOVA; Fig. 3c). Pretreatment with cytochalasin D or Z-FG-NHO-Bz inhibited the Abinduced decreased in NLRP10 (??p \ 0.01; ???p \ 0.001; ANOVA; Fig. 3d). In order to assess whether Ab induced this change in NLRP10 in vivo, we prepared hippocampal and cortical tissue from male Wistar rats which received a cocktail of aggregated recombinant Ab1–40 and Ab1–42 or the reverse
NLRP10 is Highly Expressed in Microglia and is Decreased with Ab Treatment Both In Vitro and In Vivo In order to characterize the expression of NLRP10 in different cell types, 3 separate preparations (S1, S2 and S3) of untreated microglia, astrocytes and neurons were isolated from neonatal Wistar rats. Microglial cells had the greatest expression of NLRP10 (Fig. 3a, b). Astrocytes also expressed NLRP10 but to a lesser extent than microglia. Expression of NLRP10 in neurons was very limited.
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(a)
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Fig. 4 Representative silver-stained acrylamide gels indicating that a recombinant cathepsin B and b recombinant cathespin D both degrade recombinant NLRP10 when incubated together for 1 h.
Silver-stained gels also indicate that the degradation of NLRP10 was inhibited by a cathepsin inhibitor
non-aggregating Ab40–1 peptide, chronically by means of an implanted osmotic mini-pump. We first investigated formation of the NLRP3 inflammasome in these animals by assessing the levels of active caspase 1 in the cortex and hippocampus. Active caspase 1 was significantly increased in both the cortex and hippocampus of animals that had been infused with the Ab1–40/1–42 cocktail compared with control animals (*p \ 0.05; student’s t test for independent means; Fig. 3e). We also assessed the expression of NLRP10 in these animals and found that there was a significant decrease in NLRP10 protein in the cortex and hippocampus of those animals that had been infused with the Ab1–40/1–42 cocktail compared with control animals (**p \ 0.001; student’s t test for independent means; Fig. 3f).
75-kDa band disappeared after a 1-h incubation but remained in those samples also containing cathepsin inhibitor (Fig. 4b).
NLRP10 is Degraded by Cathepsins Cathepsins are a group of proteases that have been associated with the formation of the NLRP3 inflammasome in response to Ab (Halle et al. 2008). As the NLRP10 protein is decreased in response to Ab treatment, we hypothesized that cathepsins were responsible for its degradation. We assessed this hypothesis using a degradation assay. Activated recombinant cathepsin B or L was incubated with recombinant NLRP10 for 1 h at 37 °C with or without the cathepsin inhibitor. In the sample containing cathepsin B and NLRP10, the representative 75-kDa NLRP10 band disappeared after 1 h (Fig. 4a). However, in the sample containing cathepsin B, NLRP10 and cathepsin inhibitor, the 75-kDa band remained after 1-h incubation (Fig. 4a). Similarly, in samples containing recombinant active cathepsin L incubated with recombinant NLRP10, the
NLRP10 Inhibits Ab-Induced NLRP3 Inflammasome Formation and IL-1b Release Consistent with the data presented in Fig. 1, Ab induced caspase 1 activation in glial cultures (***p \ 0.001; ANOVA; Fig. 5a). Importantly, treatment with recombinant NLRP10 significantly reduced the Ab-induced change (???p \ 0.001; ANOVA; Fig. 4a). Similarly, Ab increased IL-1b release from LPS-primed glial cultures (***p \ 0.001; ANOVA; Fig. 5b), and this release was also attenuated by treatment with recombinant NLRP10 (???p \ 0.001; ANOVA; Fig. 5b). In order to assess whether NLRP10 had any inflammasome-independent antiinflammatory effects, we assessed the release of IL-6 and TNFa from Ab-treated glial cultures. As previously shown in Fig. 1, Ab enhanced the significant LPS-induced release of IL-6 and TNFa from LPS-primed cultures (**p \ 0.01; ***p \ 0.001; control vs. LPS; ?p\0.05;??p \ 0.01; LPS with vs. without Ab; ANOVA; Fig. 5c, d). Treatment with NLRP10 did not alter the effects of LPS or Ab on the release of either of these cytokines. To clarify that NLRP10 acts downstream of cathepsins in this setting, we assessed the activity of cathepsin B and L in response to Ab and recombinant NLRP10 treatment. The activity of cathepsin B and L in the cytosol of LPSprimed, Ab-treated cultures was increased, confirming the data presented in Fig. 1 (**p \ 0.01; ANOVA; control vs. LPS ? Ab; Fig. 5e, f); however, NLRP10 treatment had no effect on the activity of either cathepsin (Fig. 5e, f).
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Fig. 5 a The Ab-induced increase in caspase 1 activation in LPSprimed glial cells (n = 5; ***p \ 0.001; ANOVA; Control vs. LPS ? Ab) was attenuated by treatment with recombinant NLRP10 (???p \ 0.001; ANOVA; LPS ? Ab vs. LPS ? Ab ? NLRP10). b The Ab-induced increase in IL-1b (n = 5; ***p \ 0.001; ANOVA; Control vs. LPS ? Ab) was also attenuated by recombinant NLRP10 treatment (???p \ 0.001; ANOVA; LPS ? Ab vs. LPS ? Ab ? NLRP10). c LPS induced an increase in the release of IL-6 (***p \ 0.001; ANOVA; Control vs. LPS) which was further increased with Ab treatment (??p \ 0.01; ANOVA; LPS vs.
LPS ? Ab); however, NLRP10 had no effect on this release. d TNFa release was induced by treatment with LPS (n = 5; **p \ 0.01; ANOVA; Control vs. LPS) and further by treatment with Ab (?p\0.05; ANOVA; LPS vs. LPS ? Ab) but was unaffected by NLRP10 treatment. e Cathepsin B activity in the cytosol of LPSprimed glia was increased with Ab treatment (n = 5; **p \ 0.01; ANOVA; Control vs. LPS ? Ab) but unaffected by treatment with NLRP10). f Cathepsin L activity in the cytosol of LPS-primed glia was increased with Ab treatment (n = 5; **p \ 0.01; ANOVA; Control vs. LPS ? Ab) but unaffected by treatment with NLRP10)
Discussion
NLRP10 inhibits the assembly of the NLRP3 inflammasome and therefore is a potential target for anti-inflammatory therapies. Interleukin-1b (IL-1b) is a key mediator in the innate immune response in Alzheimer’s disease, and significantly elevated expression of IL-1b has been seen in both the
The significant finding in this study is that a component of the inflammasome, ASC, constituently interacts with NLRP10 in glia and that Ab markedly reduces the interaction between these proteins. The evidence suggests that
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brain and the plasma of Alzheimer’s patients’ postmortem (Ojala et al. 2009). Preclinical data support a particular role for IL-1b and in a triple transgenic mouse model of Alzheimer’s disease; blocking IL-1b signaling has been shown to rescue cognitive impairment associated with the disease. Additionally, deletion of the IL-1Ra gene in mice leads to increased susceptibility to intra-hippocampal injection of Ab with increased neuroinflammation and loss of synaptic markers (Craft et al. 2005), while the inflammatory changes and loss of synaptic plasticity in transgenic mice which overexpress APP and PS1 were attenuated when these mice were crossed with NLRP3-deficient mice {Heneka 2013 #7971}. IL-1b release from microglia has also been shown to increase activity and/or expression of c- and b-secretases, the enzymes responsible for processing of the amyloid precursor protein ultimately leading to further Ab deposition (Sastre et al. 2008). Interleukin-1b is processed and released from glial cells following the assembly of the NLRP3 inflammasome. The inflammasome can be activated by a range of stimuli including millimolar concentrations of extracellular adenosine triphosphate (Murphy et al. 2011), viruses including influenza A (Thomas et al. 2009) and Neisseria gonorrhoeae (Duncan et al. 2009), crystallized endogenous molecules including cholesterol crystals (Duewell et al. 2010) and monosodium urate crystals (Martinon et al. 2006) as well as inorganic particles like titanium dioxide, silicium dioxide and asbestos (Dostert et al. 2008). In 2008, Halle et al. reported that fibrillar Ab was also a stimulus for the formation of the NLRP3 inflammasome (Halle et al. 2008). The authors reported that phagocytosis of Ab triggered an abnormal lysosomal response resulting in leakage of cathepsin B into the cytosol where it acted to stimulate the formation of the inflammasome. In this study, we verify that Ab treatment of glial cells induced increased activity of cathepsin B, but the data also demonstrate that Ab increased the activity of cathepsin L in the cytosol. We show that Ab increased the association of the inflammsome components ASC and NLRP3 and increased caspase 1 activation and IL-1b release. These changes were attenuated by inhibiting phagocytosis by cytochalasin D or by inhibiting cathepsin activity. NOD-like receptors, such as NLRP3, are localized to the cytoplasm and have varying functions in inflammatory and apoptotic processes. In 2004, an additional member of the NOD-like family, NLRP10 (also known as PYNOD or NALP10), was discovered and was shown to be expressed particularly in cardiac and skeletal muscle, but also in brain (Wang et al. 2004). Here, we report that NLRP10 is highly expressed in glia, particularly microglial cells. It was discovered that, like NLRP3, NLRP10 also interacted with ASC, but NLRP10 inhibited the ASC-mediated activation of caspase 1 and IL-1b release in transfected HEK293T-S cells (Wang et al. 2004). NLRP10 differs from NLRP3 due
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to the fact that it does not contain a leucine-rich repeat (LRR) domain. It is thought that the lack of LRR domain prevents ASC, when bound to NLRP10, from binding and activating caspase 1 (Wang et al. 2004). The findings presented here show that ASC constitutively interacts with NLRP10 in glia and that Ab treatment reduces the association of NLRP10 with ASC by approximately 80 %. Amyloid beta (Ab) markedly reduced NLRP10, but the decrease was redressed by inhibiting cathepsins or by blocking phagocytosis using cytochalasin D. These data indicate that it is the phagocytosis of Ab and the subsequent cathepsin release that are responsible for the decreased NLRP10. It has been shown that degradation of NLRP3 occurs through the autophagy–lysosome pathway (Chuang et al. 2013); a similar process may facilitate degradation of NLRP10, though this remains to be demonstrated. We next assessed caspase 1 activation and NLRP10 expression in hippocampus and cortex of rats in which chronic infusion of Ab has been shown to induce Ab deposition (Frautschy et al. 2001) and found that, in parallel with the NLRP3 inflammasome activation in the hippocampus and cortex of these animals, there was a decrease in NLRP10 expression. To our knowledge, this is the first time that changes in the expression of NLRP10 have been reported in response to Ab. The importance of cathepsins in the activation of the NLRP3 inflammasome has been highlighted by several studies. Serum amyloid A (Niemi et al. 2011), Ab (Halle et al. 2008), Neisseria gonorrhoeae (Duncan et al. 2009), Streptococcus pneumoniae (Hoegen et al. 2011), cholesterol-dependent cytolysins (Chu et al. 2009), cytoplasmic RNA (Rintahaka et al. 2011) and chemotherapeutic agents (Bruchard et al. 2012) have all been shown to induce the inflammasome assembly in a cathepsin-dependent manner. In the case of Ab, its phagocytosis by microglial cells has been shown to cause lysosomal damage which, in turn, leads to release of cathepsins into the cytosol (Halle et al. 2008). Although the precise role for cathepsins in the setting of inflammasome activation has yet to be elucidated. Bruchard et al. have recently reported that cathepsin B binds directly to NLRP3 in response to chemotherapeutic agents, and this drives the activation of the inflammasome. Previous studies have focused on cathepsin B, but the present data indicate that both cathepsin B and L are activated in response to Ab. Both cathepsins, which are released into the cytoplasm in Ab-treated glia, are capable of degrading NLRP10, and this is confirmed by the finding that the degradation was prevented by a cathepsin inhibitor, albeit in cell-free system. On the basis of our findings, we propose that cathepsins, released into the cytosol after Ab uptake, degrade NLRP10 that is normally bound to ASC. Free ASC can now associate with NLRP3, thus activating caspase 1 and processing and release of IL-1b.
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Further to this action of NLRP10, we have identified an anti-inflammatory effect of NLRP10 since it inhibits Abinduced production of IL-1b and activation of caspase 1. We are currently investigating the modulatory role of NLRP10 in mouse models characterized by neuroinflammatory changes and specifically confirming its role in attenuating Ab-induced changes using siRNA technology. Interestingly, peritoneal macrophages from transgenic mice, which overexpress NLRP10, are defective in IL-1b production in response to Salmonella typhimurim and are also resistant to LPS-induced endotoxic shock (Imamura et al. 2010). However, contrary to these data, Lautz et al. (2012) reported that NLRP10 contributed to the proinflammatory cytokine release from fibroblasts and epithelial cells that is triggered by the bacterial pathogen Shigella flexneri by influencing the activation of p38 and NF-jB. Similarly, NLRP10 has also been shown to play an essential role in the initiation of adaptive immunity by dendritic cells in response to an array of inflammatory stimuli (Eisenbarth et al. 2012). Clearly, NLRP10 may have different effects on different cell types in response to varying stimuli. These results suggest that targeting the Ab-induced degradation of NLRP10 may prove to be a useful therapeutic in redressing the persistent neuroinflammation that occurs in, and contributes to, the pathology of Alzheimer’s disease. Acknowledgments The authors would like to thank the Irish Research Council and Science Foundation Ireland for funding this work. Conflict of interest of interest.
The authors declare that they have no conflict
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