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Neurobiology of Aging 35 (2014) 309e321

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Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

IPAF inflammasome is involved in interleukin-1b production from astrocytes,induced by palmitate; implications for Alzheimer’s Disease

Li Liu a, Christina Chan b,c,*

aDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USAbDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USAcDepartment of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, USA

a r t i c l e i n f o

Article history:Received 5 February 2013Received in revised form 25 July 2013Accepted 16 August 2013Available online 20 September 2013

Keywords:Alzheimer’s diseaseFatty acidInflammasomeIL-1bIPAF

* Corresponding author at: Michigan State UniversEngineering and Materials Science, 2527 EB, East Lan517 432 4530; fax: þ1 517 432 1105.

E-mail address: krischan@egr.msu.edu (C. Chan).

0197-4580/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2013.08.016

a b s t r a c t

Inflammatory response has been strongly implicated in the pathogenesis of numerous diseases,including Alzheimer’s disease (AD). However, little is known about the molecular mechanisms initiatingthe generation of inflammatory molecules in the central nervous system, such as interleukin-1b (IL-1b).Previously we identified that palmitate can induce primary astrocytes to produce cytokines, causing AD-like changes in primary neurons. Here we investigated and identified that palmitate induced the acti-vation of ice protease-activating factor (IPAF)eapoptosis-associated speck-like protein containing acaspase activation and recruitment domains (CARD) (ASC) inflammasome in astrocytes leading to thematuration of IL-1b, thereby implicating that not only pathogen-related factors can activate the IPAF-ASCinflammasome. Moreover, downregulating IPAF (which was found to be regulated by cAMP responseelement-binding protein) in astrocytes through silencing to decrease IL-1b secretion from the astrocytesreduced the generation of amyloid-b42 by primary neurons. Furthermore, the expression levels of IPAFand ASC were found significantly elevated in a subgroup of sporadic AD patients, suggesting aninvolvement of the IPAF-ASC inflammasome in the inflammatory response associated with AD, and thuscould be a potential therapeutic target for AD.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Interleukin (IL)-1b is a major proinflammatory cytokine thatinitiates and amplifies diverse cellular responses. Elevated IL-1b hasbeen linked to diseases, such as type 2 diabetes, certain cancers,central nervous system (CNS) dysfunction, and dementia (Mayeux,2003; Vandanmagsar et al., 2011). Systemic inhibition of inflam-mation has been suggested to improve type 2 diabetes, enhanceneuroprotection during brain injury, and delay the onset of Alz-heimer’s disease (AD) (Hailer, 2008; Maedler et al., 2009; Weggenet al., 2001; Weiner and Frenkel, 2006). Blocking or neutralizingIL-1b reduced cognitive impairment and decreased AD-like path-ological changes in AD mouse models (Gonzalez et al., 2009;Kitazawa et al., 2011). Similarly, knockout of the IL-1b receptorantagonist in mice increased the neuronal damage induced byamyloid-b (Ab) (Craft et al., 2005). These findings suggest that IL-1bcould be involved in AD pathogenesis.

ity, Department of Chemicalsing, MI 48824, USA. Tel.: þ1

ll rights reserved.

IL-1b is produced by various types of cells, including astrocytes(Liu et al., 2013a; Tuppo and Arias, 2005; Wen et al., 2011). Astro-cytes are resident brain cells that play crucial roles in synapticformation and function, and in neuronal cell survival and death. Onactivation of astrocytes by diverse factors, chemokines and cyto-kines are released, including IL-1b (Dowell et al., 2009; Parpuraet al., 2012; Tuppo and Arias, 2005). As major contributors of IL-1b production in the CNS, activated astrocytes are involved in thepathogenesis of many neurodegenerative diseases, including AD(Tuppo and Arias, 2005). Elevated IL-1b levels have been detected inastrocytes of brains of AD patients and AD animal models (Halleet al., 2008; Hunter et al., 2012; Reilly et al., 2003). The inflamma-tory mediators secreted from activated astrocytes alter normalfunction of neurons and microglial cells, and trigger neurotoxicity,which further exacerbates the neurodegenerative pathology(Khandelwal et al., 2011).

IL-1b is derived from an inactive precursor, proeIL-1b, by themultiprotein complex, inflammasome (Dinarello, 2009). Inflamma-somes generally consist of nucleotide-binding oligomerizationdomain-like receptor (NLR) member and proecaspase-1. On acti-vation, caspase-1 cleavesproeIL-1b resulting in thematurationof IL-1b. The composition of each inflammasome differs and is cell-type

Table 11Patient information

Group Ref# Age, y Sex PMI,h

BraakStage

Front NPcount

NFTcounts

ApoEalleles

MMSE

Control 1132 95 F 3.5 0 3.0 0 3/4 26Control 1159 86 F 3.5 0 0.0 0 3/3 28Control 1165 92 M 3.3 0 10.4 0 3/3 28Control 1206 94 F 2.3 2 0.0 0.4 3/3 29Control 1221 81 M 2.8 2 5.6 0 3/3 27AD 1013 80 M 2.5 5 5.8 1AD 1098 81 F 2.8 5 6.2 2 3/3 15AD 1174 96 M 3.5 5 19.2 4 2/3 22AD 1201 94 M 2.0 5 6.0 0.2 3/3 22AD 1215 91 F 3.0 5 15.2 7.2 3/3 13

Patient information was provided by the University of Kentucky Alzheimer’s diseasecenter tissue bank.Key: AD, Alzheimer’s disease; ApoE, apolipoprotein E; F, female; M, male; MMSE,Mini-Mental State Examination; NFT, neurofibrillary tangle; NP, neuritic plaque;PMI, postmortem interval; Ref#, reference number.

L. Liu, C. Chan / Neurobiology of Aging 35 (2014) 309e321310

and stimulus-specific. The NLR protein 1 (NLRP1) and NLR protein 3(NLRP3) inflammasome can be activated by pathogenic or danger-associated molecules (Masters et al., 2010). Apoptosis-associatedspeck-like protein containing a caspase activation and recruitmentdomains (CARD) (ASC), the adaptor protein, is required for theactivation of NLRP1 and NLRP3 inflammasomes (Masters et al.,2010). The ice protease-activating factor (IPAF) inflammasome(also known as NLRC4) has been demonstrated to play a role in hostdefense mechanisms against pathogen-associated molecules(Jamilloux et al., 2013; Schroder and Tschopp, 2010). Although theASC adaptor protein is not essential for the activation of the IPAFinflammasome, under certain conditions, ASC is required for IPAFinflammasome activation (Martinon et al., 2009).

We previously reported that palmitate (PA) induced IL-1bmaturation and secretion from primary rat astrocytes, whichcontributed to AD-like changes in primary rat neurons (Liu et al.,2013a). PA is the most abundant saturated fatty acid in the diet.Studies have shown that a high-fat diet induces Ab deposition andmemory deficits in APP transgenic mice (Julien et al., 2010;Maesako et al., 2012a, 2012b). PA and its metabolites, such asceramides, regulate many gene expression and immunologicalpathways (Ajuwon and Spurlock, 2005). Elevated ceramide levelshave been consistently reported in AD patients (Cutler et al., 2004;Geekiyanage and Chan, 2011; Han et al., 2002; He et al., 2010).Ceramide can be synthesized from serine and palmitoyl coenzymeA (palmitoyl-CoA) by serine palmitoyltransferase (SPT), the rate-limiting enzyme of de novo ceramide synthesis (Perry, 2000;Perry et al., 2000). We found that the effects of high-fat diets, inparticular PA, on AD-like pathology are mediated by ceramidesthrough SPT (Geekiyanage and Chan, 2011; Geekiyanage et al.,2013; Liu et al., 2013a; Patil et al., 2007). We reported that a high-fat diet containing 60% of the total fat as PA significantlyincreased ceramide and SPT levels in an ADmousemodel comparedwith those fed a control chow diet, and the elevated ceramide andSPT levels mediated the increase in Ab levels (Geekiyanage andChan, 2011; Geekiyanage et al., 2013). Inhibiting SPT in an ADmouse model fed a high-fat diet through subcutaneous adminis-tration of L-cylcoserine, an inhibitor of SPT, significantly down-regulated cortical Ab42 and hyperphosphorlated tau levels(Geekiyanage and Chan, 2011; Geekiyanage et al., 2013).

To elucidate how PA, through ceramides, increased Ab levels, weperformed cell studies. We demonstrated that an upregulation ofbeta-secretase 1 (BACE1) and hyperphosphorylation of tau in pri-mary neurons is mediated through conditioned media (CM) fromastrocytes culturedwith PA (Patil and Chan, 2005; Patil et al., 2006).Interestingly, culturing primary neurons directly with PA did notinduce these AD-like pathologies (Patil and Chan, 2005; Patil et al.,2006). We determined that PA induced the release of cytokines, inparticular interleukin-1b (IL-1b) and tumor necrosis factor-a(TNF-a), from the astrocytes into the media (i.e., CM). As supportingevidence, saturated fatty acid PA also has been shown to induceproinflammatory cytokine release frommicroglia cells (Wang et al.,2012). Inhibiting SPT in the astrocytes to mitigate the elevated cer-amide levels significantly reduced IL-1b release from the astrocytescultured with PA. Congruently, neutralizing IL-1b and TNFa in theastrocyte- CMsignificantly reduced theupregulation of BACE1 in theprimary neurons cultured in the neutralized astrocyte-CM (Liu et al.,2013a). Taken together, the evidence suggest a potential connectionexists between PA and AD pathogenesis mediated by ceramides andthe generation of cytokines, thus we conducted experiments in thisstudy to uncover themolecularmechanismbywhich PA triggers theproduction of IL-1b from primary astrocytes. We demonstrated thatPA activates the IPAF inflammasome in primary rat astrocytes toinduce the maturation of IL-1b. To our knowledge, this is the firstreport in which the IPAF inflammasome is induced by factors other

than pathogen-associated molecules. Moreover, the expression ofIPAF in the astrocytes on exposure to PA is regulated by the tran-scription factor, cyclic adenosine monophosphate (cAMP) responseelement-binding protein (CREB). Downregulating IPAF in primaryrat astrocytes significantly reduced the Ab42 levels produced by theprimary neurons cultured in CM from PA-treated astrocytes. Finally,the levels of IPAF and ASC were found significantly upregulated in asubgroup of sporadic AD patient brains.

2. Methods

2.1. Human samples

AD and control neocortical brain samples were obtained fromthe University of Kentucky Alzheimer’s disease center tissue bank(ADC). Diagnoses were confirmed by neurologists, neuropatholo-gists, and neuropsychologists in the ADC clinic. Most samples hadbeen obtained in less than a 4-hour postmortem interval, and theage for the patients ranged from 88 to 99 years. Table 1 lists thereference number, sex, Braak stage, Mini-Mental State Examinationscores, frontal neuritic plaque numbers, neurofibrillary tanglenumbers, and apolipoprotein E genotype of the individuals. Theinformation was provided by the ADC.

2.2. Isolation and culture of primary rat astrocytes and neurons,and astrocyte-CM

All procedures in cell isolationwere approved by the InstitutionalAnimal Care and Use Committee at Michigan State University. Pri-mary cortical astrocytes were isolated from postnatal day 0e2newborn Sprague-Dawley rats as previously described (Liu et al.,2013a; Patil et al., 2007). Approximately 4 � 104 cells per cm2 wereseeded on poly-L-lysine (Cultrex, Gaithersburg, MD, USA)-coatedplates. Primary astrocytes were maintained in DMEM/F12 supple-mented with 10% fetal bovine serum, 100 mg/mL streptomycin, and100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA). When the as-trocytes reached approximately 60%e70% confluence, the astrocytemediumwas removed and subsequently cultured in cortical mediafor 24hours (see later in text). Thepurityof the astrocytemonolayerswere >90%, determined according to glial fibrillary acidic protein(GFAP) immunoreactivity and flow cytometry (SupplementaryFig. 1).

Cortices from postnatal day 0 Sprague-Dawley rats were usedfor neuronal culture. The primary neurons were plated on poly-L-lysineecoated plates at 2.5�105cells per cm2, and were cultured inneurobasal A medium with B27, 0.5 mM glutamine, and penicillinstreptomycin (PS) for 3e4 days after isolation. Then the neurobasal

L. Liu, C. Chan / Neurobiology of Aging 35 (2014) 309e321 311

A/B27 media was removed and the neurons were cultured incortical media (see later in text) for 24 hours before treatment (i.e.,CM from the astrocytes). The purity of the neurons was >90%,determined using bIII tubulin immunostaining and flow cytometry(Supplementary Fig. 1).

Cortical medium (DMEM 10313 supplemented with 10% horseserum, 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonicacid (HEPES), 2 mM glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin [Invitrogen]) was used to culture primary neurons andprimary astrocytes for 24 hours before treatment (i.e., CM fromastroyctes for treating primary neurons or PA for treating astro-cytes). Fatty acid-free bovine serum albumin (BSA; Millipore, Bill-erica, MA, USA), or 0.4 mM PA (Sigma, St Louis, MO, USA) plus BSAas a carrier protein (molar ratio was 3:1) was added to the corticalmedium and used to incubate the astrocytes for 12 hours. Theseastrocyte-CM (CM-B or CM-P) were used subsequently to treatprimary neurons for 12 hours.

Forskolin and isobutylmethylxanthine (Sigma) at concentrationsof 10 mM and 100 mM, respectively, were used to increase the CREBlevel.

2.3. Total messenger RNA (mRNA) extraction and quantitative real-time polymerase chain reaction

Total mRNA from cells was extracted using the RNeasy Plus kit(QiaGen, Valencia, CA, USA) according to the manufacturer’s in-structions. Total mRNAwas extracted from human brain neocorticesusing TRIzol and RNeasy Plus kits. The total mRNAwas reverse tran-scribed into cDNAusing the cDNA synthesis kit (BioRad, Hercules, CA,USA) as previously described (Wu et al., 2011; Zhang et al., 2011). Thefollowing primer sets (Operon, Huntsville, AL, USA) were usedfor polymerase chain reaction (PCR): rat actin (50-ctcttccagccttccttcct-30 and 50-aatgcctgggtacatggtg-30), rat proeIL-1b (50-gcatccagctt-caaatctc-30 and 50-ggtgctgatgtaccagttg-30), rat proecaspase-1(50-gacaagatcctgagggcaaa-30 and 50-ggtctcgtgccttttccata-30), rat IPAF(50-gcgaaacctgaagaagatgc-30 and 50-aacgctcagcttgaccaaat-30), rat ASC(50-gcaatgtgctgactgaagga-30 and 50-tgttccaggtctgtcaccaa-30), rat CREB(50-tgttcaagctgcctctggt-30 and50-tctttcgtgctgcttcttca-30), humanactin(50- tggacttcgagcaagagatg-30 and 50- aggaaggaaggctggaagag-30), hu-man IPAF (50-agcttgctgaaggcttgttgct-30 and 50-tcacccatctggattgcaca-30). Quantitative real-time PCR was performed using iQSYBR GreenSupermix andReal-TimePCRDetectionSystem(BioRad). The reactionprogram was: cycle 1 (1 time) 95 �C, 15 minutes; cycle 2 (40 times),step 1: 94 �C, 15 seconds, step 2: 57 �C, 30 seconds, step 3: 70 �C, 30seconds; cycle3 (1 time)72 �C,7minutes; andcycle4 (80 times)55 �C,10 minutes. The cycle threshold values were determined using theMyIQ software (version 1.0.410).

2.4. Western blot analysis

Whole cell extracts from cells and from human brain neo-coritices (homogenized) were measured for protein concentrationsusing the Bradford assay. Protein samples (15e30 mg) were sub-jected toWestern blot analysis as previously described (Bilgin et al.,2013; Wu et al., 2013) using proeIL-1b antibody (Biovision, Milpi-tas, CA, USA), caspase-1 (Abcam, Cambridge, MA, USA), IPAF (SantaCruz Biotech, Dallas, TX, USA), ASC (Novus Biologicals, Littleton, CO,USA), CREB and phospho-CREB (pCREB) (Cell Signaling, Danvers,MA, USA), beta-actin, and Goldberg-Hogness box (TATA box)binding protein (Sigma). Anti-mouse and anti-rabbit horseradishperoxidase (HRP)-conjugated secondary antibodies were pur-chased from Thermo Scientific (Asheville, NC, USA), and donkeyanti-goat immunoglobulin G-HRP was purchased from Santa CruzBiotech. The blots were visualized using SuperSignal West Femtomaximum sensitivity substrate (Thermo Scientific).

2.5. Enzyme-linked immunosorbent and Ab42 assays

The levels of IL-1b in the astrocyte cultured supernatants fromvarious treatments were analyzed using an enzyme-linked immu-nosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA)according to the manufacturer’s instructions. The sensitivity of theassay was 5 pg/mL for IL-1b. Ab42 levels were detected using an Ab42ELISA kit (Invitrogen). Optical densities were measured using aSpectra MAX Plus384 plate reader. Each sample concentration wascalculated based on a standard curve of IL-1b or Ab42 standards. Allreadingswerenormalized to the total protein levelsdeterminedusingBradford assay, and the data were then normalized to the control.

2.6. Endotoxin assay and measurement of cytotoxicity

To detect for possible contamination of PA with lip-opolysaccaride, the endotoxin assay was performed using Tox-inSensor, an endotoxin detection system, following themanufacturer’s instructions (GenScript, Piscataway, NJ, USA). Theendotoxin level in the PA-BSA mixture was less than 0.022 ng/mL,which is far below the concentration required to induce astrocyticactivation (Bhat et al., 1998; Lieberman et al., 1989).

The cytotoxicity of the PA treatment was determined using anintracellular and secreted lactate dehydrogenase (LDH) assay ac-cording to the manufacturer’s instructions (Roche, Indianapolis, IN,USA). LDH released into the medium and retained in the cells weredenoted as LDHm and LDHc, respectively. Percentage of release wascalculated with the following equation: LDH release % ¼ LDHm/(LDHm þ LDHc) � 100.

2.7. Caspase-1 fluorometric activity assay and caspase-1 inhibition

Caspase-1 activity in astrocytes was determined using acaspase-1 FLICA kit (Immunochemistry Technologies, Bloomington,MN, USA) according to the manufacturer’s instructions. Briefly, theastrocytes were incubated with BSA or 0.4 mM PA for 12 hours.FAM-FLICA Caspase-1 reagent, FAM-YVAD-FMK, was reconstitutedwith dimethyl sulfoxide (DMSO), and was further diluted withphosphate buffered saline (PBS) to a concentration of 150 mM. FAM-FLICA Caspase-1 reagent (150 mM) was added to the cultured mediaat a final concentration of 5 mM. The cells were incubatedwith FAM-FLICA Caspase-1 reagent for 1 hour at 37 �C. The cells bearing activecaspase-1 coupled to FLICA showed as green. Fluorescence imageswere taken using a confocal microscope, Olympus FluoView 1000,and the green fluorescence signal was detected using a SpectraMAX GEMINI EM plate reader. All readings were normalized to theprotein levels obtained using the Bradford assay. Caspase-1 activityin astrocytes was inhibited using either the general caspase inhib-itor, Z-VAD (R&D Systems) or the specific inhibitor, Z-YVAD, tocaspase-1 (Biovision).

2.8. Transfection

SiTENOME SMARTpool small interfering RNA (siRNA) targetingASC, SPT, IPAF, CREB, and caspase-1 were purchased from Dhar-macon (Pittsburgh, PA, USA), and scramble siRNA was purchasedfrom Ambion. In brief, for 1 well in a 6-well plate, 200e250 pmolsiRNA was diluted to 250 mL Opti-MEM I reduced serum mediumwithout serum (Invitrogen), 5 mL lipofectamine RNAiMAX wasadded to 250 mL Opti-MEM. Diluted siRNA and diluted RNAiMAXwere combined, mixed gently, and incubated for 10e20 minutes atroom temperature to form an siRNA-RNAiMAX complex. When theconfluence of the astrocytes reached 70%e80%, the astrocyte cul-ture medium was changed to 2 mL of astrocyte culture mediumwithout antibiotics. The siRNA-RNAiMAX complexes (500 mL) were

L. Liu, C. Chan / Neurobiology of Aging 35 (2014) 309e321312

added to the 2 mL of astrocyte culture mediumwithout antibiotics.The final concentration of the siRNA in themediumwas 80e100 nMand the media containing siRNA-RNAiMAX complexes was used totreat the astrocytes for 18e24 hours. After silencing, either PA orBSA was used to culture the astrocytes for 12 hours.

The emptyvectorpCMV(cytomegalovirus) and the constitutivelyactive form of CREB (VP16-CREB) were used for overexpression.Briefly, the cells were transfected with 0.8 mg pCMV or VP16-CREBusing Lipofectamine 2000 (Invitrogen) according to the manufac-turer’s instruction. Media were changed after 6 hours and the cellswere incubated in fresh astrocyte media for up to 24 hours beforetreatment with PA.

2.9. Statistical analysis

All experiments were performed at least 3 times, and repre-sentative results are shown. Statistical analysis was performed us-ing an unpaired, 2-tailed Student t test (and analysis of variance),and Mann-Whitney tests were used for human brain samples.

Fig. 1. Pro-IL-1b and IL-1b levels produced by astrocytes with PA treatment. (A) Primary rat aat the indicated concentration for 12 hours. The mature form of IL-1b in cultured media was dastrocytes treated with BSA (ctrl) or 0.4 mM PAwere detected using quantitative real-time PClevel of IL-1b. (E) Representative Western blot result and quantification of Western blots of Sfor 24 hours. (F) IL-1b protein expression level. Wild-type astrocytes or astrocytes silenced wdetected using ELISA and normalized to astrocytes treated with BSA (ctrl) (n ¼ 3). ** p < 0.0Abbreviations: B, BSA; BSA, bovine serum albumin; ctrl, control; ELISA, enzyme-linked immPCR, polymerase chain reaction; Scrsi, scramble siRNA; SPT, serine palmitoyltransferase; si,

3. Results

3.1. ProeIL-1b and the mature form of IL-1b increase in astrocyteson treatment with PA

IL-1b can be produced by monocytes, macrophages, dendriticcells, and astrocytes on stimulation by diverse factors (Dong andBenveniste, 2001; Eder, 2009). PA can induce IL-1b productionfrom several types of cells, including macrophages and microglia(Wang et al., 2012). Fatty acid concentration in normal humanplasma in vivo generally ranges between 0.3 and 1.0 mM (Dole,1956; Shultz, 1991; Tikanoja et al., 1989). We demonstrated that0.2 mM PA can induce IL-1b secretion from primary rat astrocytes(Liu et al., 2013a). Here we confirm that IL-1b secreted from as-trocytes increases in a dose-dependent manner with PA concen-tration compared with the control (BSA) (Fig. 1A). To determinewhether PA promoted cell death and cytotoxicity, primary astro-cytes were incubated with different concentrations of PA for 12hours and LDH release was measured. At concentrations of 0.2 and

strocytes were treated with regular astrocyte-cultured medium (AM), or BSA (ctrl) or PAetected using ELISA assay (n � 3). (B, C, D) The mRNA and protein levels of proeIL-1b inR and Western blot analyses, respectively (n � 3). (E and F) Silencing SPT decreased thePT levels in primary astrocytes treated with scramble siRNA (scbsi) or SPT siRNA (SPTsi)ith scramble siRNA or SPT siRNA cultured with AM, BSA, or PA for 12 hours. IL-1b was1, *** p < 0.001. A line indicates comparison between the 2 bars connected by the line.unosorbent assay; IL, interleukin; mRNA, messenger RNA; P, palmitate; PA, palmitate;siRNA.

L. Liu, C. Chan / Neurobiology of Aging 35 (2014) 309e321 313

0.4 mM PA, cell death was not induced and at 0.7-mM concentra-tions of PA, cell death increased significantly (SupplementaryFig. 2), therefore, 0.2 mM and 0.4 mM PA concentrations wereused in subsequent experiments. In addition to the mature form ofIL-1b, the mRNA and protein levels of precursor IL-1b (proeIL-1b)increased in the astrocytes on culture with 0.4 mM PA and treat-ment time (6 and 12 hours) (Fig. 1BeD). Previously, we reportedthat PA increased ceramide levels in primary astrocytes through SPT(Patil et al., 2007), the rate-limiting enzyme for the de novo cer-amide synthesis pathway (Perry, 2000; Perry et al., 2000). Inhibit-ing SPT to reduce ceramide levels using the specific inhibitorL-cycloserine significantly reduced the upregulation in IL-1b (Liuet al., 2013a), suggesting that SPT is involved in the generation ofIL-1b in primary astrocytes treated with PA. Here we furtherdemonstrated that transient silencing of SPT by siRNA significantlyreduced secreted IL-1b from astrocytes cultured with PA (Fig. 1Eand F).

3.2. Upregulation in caspase-1 is involved in the release of IL-1b

The processing of proeIL-1b usually requires cleavage bycaspase-1, thus the activity of caspase-1 is critical to the inflam-matory response. To determine if caspase-1 is involved in thematuration of IL-1b in astrocytes treated with PA, confocal micro-scopy and caspase-1 fluorometric activity assay were performedand established that the level of activated caspase-1 was elevated(Fig. 2A and B). This is further supported by Western blot analysiswhich showed increased caspase-1 and proecaspase-1 mRNA andprotein levels in astrocytes on PA treatment (Fig. 2CeF). Primaryastrocytes pre- and cotreated, for 30 minutes and 12 hours,respectively, with the general caspase-1 inhibitor Z-VAD, or specificcaspase-1 inhibitor Z-YVAD, along with PA, significantly reducedthe secreted IL-1b (Fig. 2G and H). To further confirm the inhibitionresults, proecaspase-1 was transiently silenced in astrocytes usingsiRNA (Fig. 2IeK), and the proecaspase-1esilenced astrocytes weresubsequently incubated with PA. Silencing caspase-1 reduced theIL-1b secreted by the PA-cultured astrocytes compared with the PA-cultured astrocytes with scramble siRNA (Fig. 2L). Taken together,these results are consistent with caspase-1 involvement in thegeneration of IL-1b from astrocytes cultured with PA.

3.3. PA activates the IPAF inflammasome to produce mature IL-1b

Inflammasomes are multiprotein complexes that recruitproecaspase-1 and trigger the activation of caspase-1. Activation ofcytoplasmic receptors of inflammasomes, which are initiators ofinflammasome assembly, is cell-type and stimulus-dependent. Inthe CNS, NLRP1 was reported to be expressed in neurons but wasnot detected in astrocytes (de Rivero Vaccari et al., 2008). A recentstudy uncovered the IPAF inflammasome in microglial cells(Jamilloux et al., 2013). To date, the involvement of inflammasomereceptors in astrocytes treated with PA has not been reported.Because PA can induce IL-1b secretion from astrocytes (Liu et al.,2013a), we evaluated whether the other 2 receptors, IPAF andNLRP3, are involved in the processing of IL-1b in primary rat as-trocytes. Notably, we found PA increased the mRNA level of IPAFsignificantly (Fig. 3A). To further confirm the involvement of IPAF inthe production of IL-1b by astrocytes, IPAF was transiently silencedwith siRNA (Fig. 3BeD) and the upregulation of IL-1bwas abolishedin the astrocytes cultured in PA (Fig. 3E). In contrast, silencingNLRP3 in primary astrocytes did not reduce the upregulation of IL-1b on culture with PA (Fig. 3F). These results suggest that IPAF,expressed in primary rat astrocytes, is activated by PA, and couldrecruit proecaspase-1 and activate caspase-1 to lead to subsequentmaturation and release of IL-1b.

3.4. ASC is important for the activation of the IPAF inflammasome

ASC is an adaptor protein required for the activation ofthe NLRP1 and NLRP3 inflammasomes, whereas the involve-ment of ASC in the activation of the IPAF inflammasome is stimulus-dependent (Sutterwala and Flavell, 2009). On exposure to bacteria,such as Salmonella typhimurium, Shigella flexneri or Pseudomonasaeruginosa, ASC is required for activation of the IPAF inflammasome(Schroder and Tschopp, 2010), but not for IPAF activation inresponse to Legionella pneumophila (Case et al., 2009). We foundthat the levels of ASC protein increased dramatically in astrocytescultured with PA for 6 and 12 hours (Fig. 4A and B). To determine ifASC is required for the activation of the IPAF inflammasome in as-trocytes cultured in PA, ASC was transiently silenced in astrocytes(Fig. 4CeE) before PA treatment for 12 hours. This abolished theupregulation of IL-1b in the astrocytes cultured in PA (Fig. 4F),implying that ASC is required for the activation of the IPAFinflammasome. In addition to ASC, NLR family, apoptosis inhibitoryprotein 5 (Naip5) is sometimes needed for IPAF activation,depending on the stimulus (Lightfield et al., 2011; Sutterwala andFlavell, 2009). On transient silencing of Naip5 in primary astro-cytes, confirmed by a decrease in its mRNA and protein levels, theELISA analysis indicated, however, that the level of IL-1b was notreduced significantly in the astrocytes cultured in PA(Supplementary Fig. 3). These results suggest that ASC, but notNaip5, is required for the activation of IPAF in primary astrocytestreatedwith PA. Currently themechanisms bywhich ASC and Naip5are involved in the activation of IPAF are unknown.

3.5. The IPAF inflammasome regulates the Ab42 level in primaryneurons

Many in vivo and in vitro studies suggest that inflammation isinvolved in the pathogenesis of AD. Specifically, IL-1b has beenshown to induce tau hyperphosphorylation and enhance neurofi-brillary tangles (Griffin et al., 2006; Salminen et al., 2008). Wepreviously also reported that CM from primary astrocytes culturedin PA contained elevated IL-1b, which induced an upregulation inthe levels of tau hyperphosphorylation and Ab42 in primary ratneurons (Liu et al., 2013b; Patil et al., 2007). This study investigateswhether the increase in the level of IPAF inflammasomes in astro-cytes cultured in PA affects the level of Ab produced by primary ratcortical neurons treated with this astrocyte-CM. IPAF was silencedin primary rat astrocytes for 20 hours, and the astrocytes weresubsequently treated with either BSA or PA for 12 hours, and thenthe astrocyte-CMwas used to culture primary neurons for 12 hours.A significant decrease in the level of extracellular Ab42 was detectedin primary neurons cultured in CM from PA-treated astrocytes withIPAF silenced, compared with PA-treated astrocytes without IPAFsilenced (Fig. 5). This suggests that IL-1b generated from IPAFinflammasomes in the astrocytes is involved in enhancing the levelof Ab42 in the neurons.

3.6. IPAF and ASC levels are elevated in sporadic AD patients

It was shown that the level of IL-1b in the brains of AD patients issignificantly elevated (Cribbs et al., 2012; Griffin et al., 1989).Because the IPAF inflammasome in the astrocytes, induced by PA, isinvolved in upregulating Ab42 levels in primary rat neurons throughthe astrocyte-CM, wemeasured the level of IPAF in the frontal braincortices of a subgroup of sporadic AD patients and found that themRNA and protein levels of IPAF were increased significantlycompared with the control (Fig. 6A, D, and F, Table 1). Furthermore,the mRNA and protein levels of ASC were also significantly elevated(Fig. 6B, D, and F) and Naip5 did not change significantly in this

Fig. 2. The upregulated caspase-1 increased the IL-1b level. The primary rat astrocytes were treated with BSA or 0.4 mM PA for 12 hours and the caspase-1 was stained withFAM-YVAD-FMK (green fluorescence). (A) Images taken using a confocal microscope. (B) Fluorescence fold change and the fluorescent signal detected using the fluorescent platereader. (C and D) Primary astrocytes treated with BSA or PA for the indicated time. The mRNA of proecaspase-1 (ProCASP1) was monitored using real-time PCR and normalizedto astrocytes treated with BSA (ctrl), and the protein expression was determined using immunoblot analysis. Quantification of Western blot analysis results are shown (E and F).(G and H) Astrocytes pre- and cotreated with the inhibitor, ZVAD or ZYVAD, and with PA for 12 hours at the indicated concentration. The expression of IL-1b was monitoredusing ELISA and normalized to astrocytes treated with BSA (ctrl). (I and J) Astrocytes cultured with scramble siRNA (scbsi) or siRNA targeting CASP1 (CASPsi) for 24 hours.Representative Western blot results of CASP1 and the quantification of Western blot results are shown (n ¼ 3). (K) The mRNA level of silenced CASP1 was confirmed using real-time PCR (n ¼ 3). (L) The mature form of IL-1b in wild-type or silenced astrocytes. The astrocytes were silenced with scbsi or CASP1 siRNA for 24 hours, or wild-type astrocytes

L. Liu, C. Chan / Neurobiology of Aging 35 (2014) 309e321314

Fig. 3. IPAF is involved in IL-1b maturation. (A) The mRNA expression of IPAF in astrocytes treated with BSA or PA for 6 or 12 hours. The mRNA levels in astrocytes treated with PAand were normalized to astrocytes treated with BSA (n ¼ 3). (B, C, and D) mRNA and protein levels of silenced IPAF. Astrocytes were cultured with scramble siRNA (scbsi) or siRNAtargeting IPAF (IPAF siRNA) for 20 hours. The IPAF level was detected using immunoblot and real-time PCR analyses (n ¼ 3). (E and F) IL-1b expression levels. Wild-type astrocytes orastrocytes silenced with scramble siRNA or IPAF siRNA or NLRP3 siRNA were cultured for 12 hours with astrocyte medium (AM), BSA, or PA. ELISA was performed for IL-1b level andthe expression levels of IL-1b were normalized to BSA-treated wild-type or silenced astrocytes (n � 3). ** p < 0.01. A line indicates comparison between the 2 bars connected by theline. Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; IPAF, ice protease-activating factor; IPAFsi, siRNA targeting IPAF;mRNA, messenger RNA; NA, not available; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; PA, palmitate; si, siRNA.

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subgroup of sporadic AD brains compared with normal controlbrains (Supplementary Fig. 4). These results raise the possibilitythat the IPAF inflammasome could be involved in the inflammatoryresponse associated with AD. Another inflammasome, NLRP3, wasalso evaluated and an increase in the mRNA and protein levels ofNLRP3 was not detected in this subgroup of sporadic AD brains(Fig. 6C, E, and F). Similarly in a recent report, elevated NLRP3 levelswere not detected in AD patient brains, nevertheless, Abwas able toactivate NLRP3 to release cytokines in a transgenic animal model(Cribbs et al., 2012; Heneka et al., 2013), suggesting that NLRP3could still play a role in the inflammatory reactions involved in ADpathogenesis.

were treated with astrocyte medium (AM), BSA (ctrl), or PA for 12 hours. ELISA was perfoBSA (ctrl) (n ¼ 3). * p < 0.05, ** p < 0.01, *** p < 0.001. A line indicates comparison betweeCASP1, caspase-1; ctrl, control; ELISA, enzyme-linked immunosorbent assay; IL, interleukmerase chain reaction; si, siRNA.

3.7. CREB regulates IPAF expression in astrocytes treated with PA

The mRNA level of IPAF is significantly increased, more than 5times, in primary rat astrocytes treatedwith PA for 6 hours (Fig. 3A),and is also upregulated in a subgroup of sporadic AD brains (Fig. 6A).Thus far, the regulation of IPAF has not been extensively studied. Thetranscription factor, activator protein 1 (AP-1), has been reported toregulate IPAF in human leukemia cells treated with tumor necrosisfactor a (Gutierrez et al., 2004). Furthermore, several other tran-scription factors have been predicted, including CREB, SignalTransducer and Activator of Transcription, and E2F (Gutierrez et al.,2004), however, there has beenno report on the regulation of IPAF in

rmed to detect IL-1b level and the results were normalized to astrocytes treated withn the 2 bars connected by the line. Abbreviations: B, BSA; BSA, bovine serum albumin;in; mRNA, messenger RNA; NA, not available; P, palmitate; PA, palmitate; PCR, poly-

Fig. 4. ASC is involved in IL-1b production. (AeE) ASC expression levels. (A and B) Representative Western blot results of ASC protein levels in astrocytes treated with PA for theindicated time. Quantification of ASC Western blot analysis (n ¼ 3). (C and D) Representative Western blot results of ASC protein levels in astrocytes cultured with scramble siRNA orsiRNA targeting ASC for 20 hours. Quantification of ASC immunoblot analysis. (E) mRNA level of ASC detected using real-time PCR (n ¼ 3). (F) IL-1b expression levels. Wild-typeastrocytes or astrocytes silenced with scramble siRNA (scbsi) or ASC siRNA (ASCsi) were cultured with astrocyte medium (AM), BSA, or PA for 12 hours. IL-1b was detected us-ing ELISA and was normalized to astrocytes treated with BSA (ctrl) (n ¼ 3). * p < 0.05, ** p < 0.01, *** p < 0.001. A line indicates comparison between the 2 bars connected by the line.Abbreviations: ASC, apoptosis-associated speck-like protein containing a CARD; B, BSA; CARD, caspase activation and recruitment domains; ctrl, control; IL, interleukin; mRNA,messenger RNA; P, palmitate; PA, palmitate; PCR, polymerase chain reaction; si, siRNA.

Fig. 5. IPAF silencing in PA-treated astrocytes reduces neuronal Ab42 levels. Primaryastrocytes treated with scramble siRNA or IPAF siRNA for 20 hours. After silencing,either BSA or 0.4 mM PA was used to culture the astrocytes for 12 hours. Then theastrocyte-conditioned medium: scramble siRNA-BSA (Scr.si CM-B), scramble siRNA-PA(Scr.si CM-PA), or IPAF siRNA-PA (IPAF si CM-PA) was transferred to treat primary ratneurons for 12 hours. The supernatant was collected and the Ab42 levels were moni-tored using ELISA (n ¼ 3). ** p < 0.01. A line indicates comparison between the 2 barsconnected by the line. Abbreviations: Ab, amyloid-b; BSA, bovine serum albumin;ELISA, enzyme-linked immunosorbent assay; IPAF, ice protease-activating factor; PA,palmitate; siRNA, small interfering RNA.

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astrocytes. Therefore,weanalyzed thepromoter region of IPAFusingthe JASPARandTRANSFACdatabases, and found that CREB could be aputative transcription factor of IPAF (Supplementary Fig. 5). pCREBwas detected using immunoblot analysis and the results showed anupregulation of pCREB in astrocytes treated with PA (Fig. 7A and B).Forskolin and isobutylmethylxanthine, well studied chemicals toincrease CREB level, were used to treat primary astrocytes for 6hours. mRNA analysis indicated the level of IPAF increased 5-fold inthe forskolin and isobutylmethylxanthine-treated over the un-treated astrocytes (controls) (Fig. 7C). To further confirm that CREBcould be involved in the regulation of IPAF transcription, transientsilencing of CREB and overexpression of a constitutively active formofCREB (VP16-CREB)wereperformed (Fig. 7DeF).Overexpressionofthe constitutively active CREB significantly increased the IPAFmRNAlevel and silencing CREB significantly reduced the IPAF transcript(Fig. 7G). Similarly silencing CREB in the astrocytes before culturingwith PA significantly reduced, and overexpressing CREB in the as-trocytes enhanced the mRNA level of IPAF on treatment with PA(Fig. 7H), suggesting that CREB could regulate IPAF expression inprimary rat astrocytes. To furthermonitor the effect of CREBon IL-1bexpression in astrocytes cultured in PA, CREB was silenced or over-expressed in primary astrocytes, and the astrocytes were subse-quently incubated in PA for 12 hours. Silencing CREB significantlyreduced the upregulation of IL-1b, and overexpressing CREB furtherenhanced the IL-1b secreted by astrocytes cultured with PA (Fig. 7Iand J), suggesting that CREB could regulate IL-1b expression in PA-treated astrocytes mediated by IPAF.

Fig. 6. IPAF and ASC are upregulated in sporadic AD brain. The neocortical brain samples were analyzed for IPAF, ASC, and NLRP3. (AeC) The mRNA levels of IPAF, ASC, and NLRP3 inneocortical brain samples. The mRNA levels were measured using real-time PCR. (D and E) The protein levels of IPAF, ASC, and NLRP3 in neocortical brain samples. The protein levelswere detected using Western blot analysis; (F) shows the quantification of the Western blot analyses (n ¼ 5). * p < 0.05, *** p < 0.001. Abbreviations: AD, Alzheimer’s disease; ASC,apoptosis-associated speck-like protein containing a CARD; CARD, caspase activation and recruitment domains; IPAF, ice protease-activating factor; mRNA, messenger RNA; NA, notavailable; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; PCR, polymerase chain reaction.

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4. Discussion

We identify that the IPAF inflammasome is involved inmediatingthe effects of astrocytes in determining the outcome of primaryneurons in response to environmental factors (i.e., fatty acids). IL-1bexpression is regulatedat the transcriptional andposttranscriptionalproteolytic processing levels, namely, it requires 2 signals, one toinduce the expression of proeIL-1b and the other to activate theinflammasome (Wen et al., 2011). We found that PA alone can acti-vate both signals, increase the level of the precursor IL-1b protein,and activate the IPAF inflammasome, leading to thematuration of IL-1b in primary rat astrocytes. The regulation of proeIL-1b expressionby transcription factor, nuclear factorkappa-light-chain-enhancerofactivated B cells (NFkB), is well established (Basak et al., 2005), andNFkB is known to be activated by fatty acids, such as PA or a high-fatdiet (Wang et al., 2012). In support of this, we observed that NFkBexpression in the astrocytes is increased on PA treatment(Supplementary Fig. 6) and inhibitingNFkB significantly reduced themRNA level of proeIL-1b (Supplementary Fig. 7). This indicates that

NFkB could be involved in the regulation of proeIL-1b expression inPA-treated astrocytes. Furthermore, treating primary astrocyteswith PA elevates ASC expression in the primary astrocytes (Fig. 4).The regulation of ASC expression is not as well studied, and ananalysis of thepromoter regionofASC identified thatNFkBcouldbeapotential regulator of ASC. Indeed, inhibiting NFkB reduced theupregulation of ASC in astrocytes cultured in PA (SupplementaryFig. 6), suggesting that NFkB could be involved in the regulation ofASC and proeIL-1b expression in PA-treated astrocytes. In additionto proeIL-1b and ASC, the mRNA level of IPAF also increased in PA-treated astrocytes (Fig. 3). Thus far, the regulation of IPAF has notbeen extensively studied. Several transcription factors, CREB, SignalTransducer and Activator of Transcription, and E2F, have been pre-dicted to potentially bind the human IPAF promoter region(Gutierrez et al., 2004). Our results suggest that CREB could tran-scriptionally regulate rat IPAF expression (Fig. 7). Although theactivation of the IPAF inflammsome is not well understood, elevatedlevels of IPAFandASC raise the possibility of enhanced assembly andactivation of the IPAF inflammasome.

Fig. 7. CREB regulates IPAF expression in astrocytes. (A) pCREB protein levels in primary rat astrocytes on treatment with PA for 12 hours, and quantified in (B). TATA binding protein(TBP) was used as loading control. (C) Astrocytes incubated with forskolin and isobutylmethylxanthine (FI) for 6 hours and mRNA of IPAF detected using real-time PCR. (D)Representative Western blot analysis results of silencing and overexpression of CREB in primary astrocytes. TBP was used as a loading control for the Western blot analyses. CREBoverexpression was performed by transfection of the constitutively active VP16-CREB and silencing was achieved using CREB siRNA. pCMV and scramble siRNAwere used as control.Endogenous CREB is denoted as endo-CREB. (E) Quantification of Western blot analyses. mRNA fold changes of (F) CREB and (G) IPAF levels in astrocytes overexpressing or silencingCREB. (H) Astrocytes treated with pCMV, VP16-CREB, scramble siRNA (scbsi), or CREB siRNA, followed by treatment with PA or BSA (ctrl) for 12 hours. IPAF mRNAwas detected usingreal-time PCR. (I) IL-1b expression level of astrocytes silenced with scramble siRNA or CREB siRNA. The cells were cultured for 12 hours with astrocyte medium (AM), BSA, or PA.ELISAwas performed for IL-1b level and the expression levels were normalized to BSA-treated wild-type or silenced astrocytes. (J) IL-1b expression level of astrocytes overexpressedwith pCMV or VP16-CREB. The cells were cultured for 12 hours with AM, BSA, or PA. ELISAwas performed for IL-1b level and expression levels were normalized to BSA-treated wild-type or silenced astrocytes (n ¼ 3). ** p < 0.01, *** p < 0.001. A line indicates comparison between the 2 bars connected by the line. Abbreviations: BSA, bovine serum albumin; cAMP,cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; ctrl, control; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; IPAF, ice protease-activating factor; mRNA, messenger RNA; PA, palmitate; pCMV, cytomegalovirus; PCR, polymerase chain reaction; pCREB, phospho-CREB; siRNA, small interfering RNA; TATA,Goldberg-Hogness box; TBP, TATA box binding protein.

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The activation of inflammasomes is critical for the maturation ofIL-1b. NLRP1 and NLRP3 inflammasomes can be activated bydiverse factors, involving pathogen-associated molecular patterns,such as microbial and viral components, and danger-associatedmolecular factors, such as adenosine-50-triphosphate (ATP), Ab,

and fibronectin (Masters et al., 2010). Previous studies of the IPAFinflammasome showed that bacterial and viral pathogenic factorsare involved (Jamilloux et al., 2013; Pereira et al., 2011), and ourstudy demonstrated that nonpathogen-associated factor fatty acids,namely PA, also can activate the IPAF inflammasome. To date, the

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signals and mechanisms leading to the activation of IPAF inflam-masomes in astroctyes are unclear. Ab activates the NLRP3inflammasome in microglial cells through the phagocytosis-lysosome pathway (Schroder and Tschopp, 2010). A recent studyin mice reported that the double-stranded RNA-dependent proteinkinase physically interacts with the inflammasome, including IPAF,to regulate inflammasome activity (Lu et al., 2012), and the phos-phorylation of IPAF is critical for the activation of inflammasomes(Qu et al., 2012). Thus, diversemechanismsmight be involved in theactivation of inflammasomes.

Many types of inflammasomes have been found in the CNS(Kummer et al., 2007). NLRP1 has been shown to be expressed inneurons, and NLRP3 and IPAF have been detected in microglial cells(Jamilloux et al., 2013; Masters et al., 2010). We found that the IPAFinflammamsome is also expressed in primary astrocytes (Fig. 3),suggesting that in the CNS the same type of inflammasome could beexpressed in different cell types and a single cell type could expressdifferent inflammasomes. Astrocytes, acting as sentinels, and highlyreactive to the microenvironment in their support of neurons andthe bloodebrain barrier, are important contributors to the releaseof IL-1b (Dinarello, 2009). However, the composition of theinflammasome expressed in PA-treated astrocytes had not beendetermined before this study. The composition of NLRP1 and NLRP3inflammasomes require ASC. For example, in spinal cord injury, ASCwas shown to be required in the activation of NLRP1 inflammasomein the neurons of rat spinal cord (de Rivero Vaccari et al., 2008;Silverman et al., 2009). However, the composition of IPAF is cell-type and stimulus-specific (i.e., under some but not all conditionsASC is required for its activation) (Martinon et al., 2009). In additionto ASC, it was reported that activation of the IPAF inflammasomecould require Naip5 (Schroder and Tschopp, 2010). In this study, wefound that ASC, but not Naip5, is required for the activation of theIPAF inflammasome in the astrocytes treated with PA. Currently themolecular mechanism and function of ASC and Naip5 in the acti-vation of IPAF inflammasomes remains unclear, although ASC hasbeen suggested to stabilize or facilitate caspase-1 recruitment(Martinon et al., 2009).

Inflammation is an important characteristic of AD and has beenimplicated in the etiology of this neurodegenerative disease (Glasset al., 2010). Specifically, an elevated IL-1b level has been reportedin plasma, brains, and cerebrospinal fluid of patients with AD andmild cognitive impairment. Blocking IL-1b rescued cognition,reduced Ab, and attenuated tau pathology in AD transgenic mice(Kitazawa et al., 2011). In addition, the IL-1 receptor antagonistknockout mice studies resulted in increased mortality and neuro-inflammation to Ab-induced neuropathology (Craft et al., 2005).Wepreviously showedPA inducedAD-likepathological changes, suchasBACE1, Ab, and hyperphosphorylated tau, in primary neuronsmediated by the CM fromastrocytes cultured in PA; in particular, weconfirmed the involvement of IL-1b (Liu et al., 2013a; Patil et al.,2007). Silencing IPAF in the astrocytes reduced IL-1b secretion,which significantly diminished the Ab42 level in the neuronsculturedwith the CM from PA-treated astrocytes (Fig. 5).We furtherconfirmed the mRNA and protein levels of IPAF and ASC (but notNaip5) are upregulated in a subgroup of sporadic AD patient’s brains(Fig. 6, Supplementary Fig. 4). In this study, the IPAF inflammasomewas detected in primary rat astrocytes cultured with PA, howeverrecently, the IPAF inflammasome, alongwithNaip5,were reported inmicroglial cells to be involved in IL-1b production (Jamilloux et al.,2013). This suggests that multiple cell types in the CNS have theability to express the same type of inflammasome. Thus, the upre-gulated IPAF level in human AD brains (Fig. 6) could arise from as-trocytes or microglial cells. Abwas reported to trigger the activationof NLRP3 inflammasome in bone marrow-derived dendritic andmicroglia cells (Masters et al., 2010). Although an elevated level of

NLRP3 was not detected in our subgroup of AD patient brains,nevertheless, NLRP3 could still play a role in the inflammatory re-action in AD pathogenesis triggered by other factors, such as Ab(Heneka et al., 2013). Taken together, the results suggest that IPAFcould be involved in the inflammatory response in AD.

Saturated fatty acids have been shown to induce cytokinerelease from several cell types (Wang et al., 2012). Diets rich in fatand brain trauma increase the levels of fatty acids (Jellinger, 2004),such as PA, in the brain. Because fatty acids can freely cross thebloodebrain barrier, they are readily taken up by astrocytes, themajor cell type in the brain that metabolizes fatty acids. Our resultssuggest that PA activates IPAF inflammasomes in primary astrocytesto release IL-1b, which in turn increases Ab42 production in primaryneurons. PA has been reported to induce IL-1b production inmicroglia (Wang et al., 2012), which raises the possibility that fattyacids could contribute to IL-1b production in the brain. IL-1b couldfurther activate other cell types to release inflammatory molecules(e.g., chemotactic and neurotoxic molecules), to initiate and furtherpropagate neuronal dysfunction and eventual neuronal death. Inthe CNS, there are many inflammasomes that could be activated inthe different cell types. For examples, Ab activates the NLRP3-ASCinflammasome in microglia, brain trauma induces the activationof the NLRP1-ASC inflammasome in neurons (de Rivero Vaccariet al., 2008), and we found that PA activates the IPAF-ASC inflam-masome in astrocytes and furthermore is upregulated in a sub-group of AD brains. Regardless of the complexity and whichinflammasome is involved, ASC and caspase-1 are concomitantlyimplicated in the production of IL-1b in the CNS (de Rivero Vaccariet al., 2008; Mawhinney et al., 2011; Silverman et al., 2009). Insupport of this, neutralization of ASC spared the tissue andimproved function in rat neurons exposed to inflammation inducedby the NLRP1-ASC inflammasome (de Rivero Vaccari et al., 2008).Taken together, this suggests that IPAF, ASC, and caspase-1 could bepotential therapeutic targets to decrease inflammation in sporadicAD, and other CNS inflammatory diseases.

Disclosure statement

The authors have no conflicts of interest.Appropriate approval for animal use was obtained.

Acknowledgements

This studywassupported inpart by theNational InstituteofHealth(R01GM079688 and R01GM089866), the National Science Founda-tion (CBET 0941055). The authors thank Dr Peter Nelson and the UKADC NIA P30-AG0-28383 for providing the human autopsy brainsamples, DrDavidGintyat JohnsHopkinsUniversity for providing thepCMV control vector and VP16-CREB plasmids, and Rebecca Martinand Garrett Kohler for assistance with the experiments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.neurobiolaging.2013.08.016.

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