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Blocking IL-1 Signaling Rescues Cognition, Attenuates Tau Pathology, and Restores Neuronal β-Catenin Pathway Function in an Alzheimer's Disease Model This information is current as of September 29, 2021. Masashi Kitazawa, David Cheng, Michelle R. Tsukamoto, Maya A. Koike, Paul D. Wes, Vitaly Vasilevko, David H. Cribbs and Frank M. LaFerla J Immunol 2011; 187:6539-6549; Prepublished online 16 November 2011; Downloaded from doi: 10.4049/jimmunol.1100620 http://www.jimmunol.org/content/187/12/6539 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/11/16/jimmunol.110062 Material 0.DC1 References This article cites 81 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/187/12/6539.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Blocking IL-1 Signaling Rescues Cognition, Attenuates Tau Pathology, and Restores Neuronal b-Catenin Pathway Function in an Alzheimer’s Disease Model

Masashi Kitazawa,*,† David Cheng,*,† Michelle R. Tsukamoto,*,† Maya A. Koike,*,† Paul D. Wes,‡,1 Vitaly Vasilevko,* David H. Cribbs,*,x and Frank M. LaFerla*,†

Inflammation is a key pathological hallmark of Alzheimer’s disease (AD), although its impact on disease progression and neurodegeneration remains an area of active investigation. Among numerous inflammatory cytokines associated with AD, IL-1b in particular has been implicated in playing a pathogenic role. In this study, we sought to investigate whether inhibition of IL-1b signaling provides disease-modifying benefits in an AD mouse model and, if so, by what molecular mechanisms. We report that

chronic dosing of 3xTg-AD mice with an IL-1R blocking Ab significantly alters brain inflammatory responses, alleviates cognitive Downloaded from deficits, markedly attenuates tau pathology, and partly reduces certain fibrillar and oligomeric forms of amyloid-b. Alterations in inflammatory responses correspond to reduced NF-kB activity. Furthermore, inhibition of IL-1 signaling reduces the activity of several tau kinases in the brain, including cdk5/p25, GSK-3b, and p38–MAPK, and also reduces phosphorylated tau levels. We also detected a reduction in the astrocyte-derived cytokine, S100B, and in the extent of neuronal Wnt/b-catenin signaling in 3xTg- AD brains, and provided in vitro evidence that these changes may, in part, provide a mechanistic link between IL-1 signaling and

GSK-3b activation. Taken together, our results suggest that the IL-1 signaling cascade may be involved in one of the key disease http://www.jimmunol.org/ mechanisms for AD. The Journal of Immunology, 2011, 187: 6539–6549.

euroinflammation has been implicated in contributing to patients with AD or mild cognitive impairment, whereas anti- the etiology of Alzheimer’s disease (AD), as well as in inflammatory cytokines are decreased (8–15). Large-scale gene ar- N providing protective mechanisms (1–3). Whether atten- ray studies have also identified significant upregulation of inflam- uation of inflammatory pathways will offer therapeutic benefit for matory-related genes in the brains of AD patients compared with AD remains unclear. Nevertheless, epidemiological and prospec- age-matched cognitively normal individuals (16, 17). Moreover, tive population-based studies show an association between sup- many of the genes that are most significantly associated with the pression of inflammation and reduced risk for AD (4–7). Further- risk of developing AD, including clusterin, complement component by guest on September 29, 2021 more, proinflammatory cytokines, such as IL-1, IL-6, and TNF-a, receptor 1, CD33, CD2AP, IL1A, IL1B, IL8, and TNF, are inflam- are elevated in the plasma, brains, and cerebrospinal fluid of matory genes (18–24). On the basis of these observations, both in vitro and in vivo studies have been conducted to elucidate the role of inflammation *Institute for Memory Impairments and Neurological Disorders, University of Cal- † in the pathogenesis of AD. For example, treatment of a tauopathy ifornia, Irvine, Irvine, CA 92697; Department of Neurobiology and Behavior, Uni- versity of California, Irvine, Irvine, CA 92697; ‡Roche Pharmaceuticals, Palo Alto, mouse model with the immunosuppressant FK506 rescued tau x CA 94304; and Department of Neurology, University of California, Irvine, Irvine, pathology and increased lifespan, supporting the hypothesis that CA 92697 inflammation contributes to disease progression (25). Similarly, 1 Current address: Neuroscience, Bristol-Myers Squibb, Wallingford, CT. inhibition of TNF-a signaling has been shown to attenuate AD- Received for publication March 2, 2011. Accepted for publication October 17, 2011. like pathology and cognitive impairments in transgenic mouse This work was supported by National Institutes of Health/National Institute on Aging models, as well as in AD patients (26–28), whereas upregulation Grants R01AG20335 (to F.M.L.) and AG020241 (to D.H.C.), National Institute of Neurological Disorders and Stroke Grant NS050895 (to D.H.C.), National Institutes of TNF-a has been shown to exacerbate AD pathology. Another of Health Program Project Grant AG00538 (to F.M.L., D.H.C.), National Institutes of proinflammatory cytokine, IL-1b, also appears to play an impor- Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant tant role in AD. IL-1b has been reported to increase the expression K99AR054695 (to M.K.), and Alzheimer’s Association Grant IIRG 91822 (to D.H.C.). Ab peptides and Abs were provided by the University of California Irvine Alzheimer’s of APP in neuronal culture (29, 30), and exposure of primary Disease Research Center, funded by National Institutes of Health/National Institute on neurons to IL-1b exacerbates tau phosphorylation through aber- Aging Grant P50AG16573 and the Institute for Memory Impairments and Neurolog- rant activation of p38–MAPK (31). In transgenic mouse models, ical Disorders. IL-1b or elevated inflammatory responses in the brain increase Address correspondence and reprint requests to Dr. Frank M. LaFerla or Dr. Masashi Kitazawa, Institute for Memory Impairments and Neurological Disorders, Depart- neuronal tau phosphorylation and tangle formation (25, 32, 33). ment of Neurobiology and Behavior, University of California, Irvine, 3212 Biological In contrast, a recent study found that overexpression of IL-1b Sciences III, Irvine, CA 92697-4545 (F.M.L.) or Institute for Memory Impairments reduces Ab-related pathology by modulating innate immune re- and Neurological Disorders, Department of Neurobiology and Behavior, University of California, Irvine, 3208 Biological Sciences III, Irvine, CA 92697-4545 (M.K.). sponses or promoting nonamyloidogenic APP cleavage in a mouse E-mail addresses: [email protected] (F.M.L.) and [email protected] (M.K.) model of AD and in a cell culture model, suggesting that IL-1b The online version of this article contains supplemental material. may play a beneficial role in limiting AD pathology (34, 35). How- Abbreviations used in this article: AD, Alzheimer’s disease; BBB, blood–brain bar- ever, the transgene construct used in the in vivo study bypassed rier; CFC, contextual fear conditioning; Ct, cycle threshold; MWM, Morris water the highly regulated pathway for IL-1b release and was expressed maze. in cells of neuronal lineage (astrocytes), rather than a physiologi- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 cal hematopoietic cell type, such as microglia, and therefore may www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100620 6540 IL-1 SIGNALING AND b-CATENIN PATHWAY IN AD not reflect the physiology role of IL-1b in disease (34). To directly sessions separated by intervals of 25 s under a warming lamp. The training test whether inhibition of IL-1b signaling has the potential for period ended when all groups of mice reached criterion (,25 s mean es- alleviating AD-relevant pathology, we treated a mouse model that cape latency). The probe trial to examine retention memory was assessed 24 h after the last training trial. In the probe trials, the platform was re- exhibits both Ab and tau pathology (3xTg-AD) with an IL-1R moved from the pool, and mice were monitored by a ceiling-mounted blocking mAb (anti–IL-1R) and evaluated the consequences of camera directly above the pool during the 60-s period. All trials were this treatment on pathology and molecular changes. We found that recorded for subsequent analysis. The parameters measured during the anti–IL-1R treatment regulated brain inflammatory responses probe trial included 1) latency to cross the platform location and 2) number of platform location crosses. through the reduction of NF-kB activity and partly reduced fi- The CFC test was performed using the Gemini Avoidance System (San brillar and oligomeric Ab species, albeit without reducing overall Diego Instruments, San Diego, CA) (37). The training trial consisted of Ab plaque burden. Notably, however, neuronal tau pathology was placing a mouse in the illuminated compartment of the device and re- markedly attenuated in the anti–IL-1R–treated animals. The effect cording the time required for it to enter the dark compartment (baseline on tau correlated with reduced activation of cdk5/p25, GSK-3b, latency). Upon entering, the door between the two compartments was closed, and the mouse immediately received an electric shock to the feet and p38–MAPK. We also detected a significant reduction in the (0.15 mA, 1 s). During the retention trial (conducted 24 h after the training levels of S100B, an astrocyte-derived cytokine, and the extent of trial), the mouse was again placed in the illuminated compartment, and the Wnt/b-catenin signaling in neurons. These changes may, in part, latency to enter the dark compartment was recorded. The retention trial explain the mechanistic link between IL-1 signaling and GSK-3b was interrupted if the animal took more than 180 s to cross into the dark compartment. activation. Therefore, the current study provides evidence that ab- rogating IL-1b signaling may offer therapeutic benefit to AD Ab titer patients and begins to elucidate the putative underlying mecha- The titers of mouse anti-rat Igs Abs were measured by ELISA as previously Downloaded from nisms of action for such a treatment. described, with minor modifications (38, 39). Briefly, 96-well plates (Immulon 2HB; Thermo Fisher Scientific, Waltham, MA) were coated Materials and Methods with 1 mM of the rat anti–IL-1R mAb used for passive injection in car- bonate coating buffer (pH 9.6) (Sigma-Aldrich, St. Louis, MO) and in- Animals and treatment paradigm cubated overnight at 4˚C. The wells were washed and blocked with 3% All experiments were carried out in accordance with the Institutional nonfat dry milk for 1 h at 37˚C with shaking. After washing, serial dilu-

Animal Care and Use Committee at the University of California, Irvine tions of all plasma samples, collected at the end of 6 mo of treatment, were http://www.jimmunol.org/ (Irvine, CA) and were consistent with federal guidelines. A total of 200 mg added to the wells, and plates were incubated for 2 h at 37˚C with shaking. rat anti–IL-1R blocking mAb (Roche Pharmaceuticals, Palo Alto, CA) was After washing, HRP-conjugated affinity-purified donkey anti-mouse IgG Abs with minimal cross-reactivity with other species (Jackson Immuno- administered i.p. to 9-mo-old 3xTg-AD mice (Thy1.2–APPswe, Thy1.2– Research Laboratories, West Grove, PA) were added at 1/2000 dilution TauP301L, and PS1M146V-KI) every 8–9 d for 6 mo. The control groups received vehicle (10 mM histidine and 150 mM NaCl [pH 6.5]), whereas for 1 h at 37˚C with shaking, wells were washed, and Ultra-Tetrame- the isotype-matched IgG control group received 200 mg rat IgG in the thylbenzidine ELISA substrate (Pierce Biotechnology) was added for 15 same manner. Each group consisted of an equal number of males and min to develop the reaction. The reaction was stopped by adding 2 N females, and the total number of mice was 12/group. H2SO4 and plates were analyzed on a Synergy HT Spectrophotometer Upon the completion of the treatment period, mice were anesthetized, (Bio-Tek Instruments, Winooski, VT) at 450 nm. The plasma end-point and blood was collected from the right ventricle of heart. Then, mice were titer was defined as the maximal plasma dilution in which the OD for the perfused with ice-cold phosphate buffered saline, and brains were collected. Abs was three times higher than the OD values of the blank wells. by guest on September 29, 2021 One hemisphere of the brains was fixed with 4% paraformaldehyde for Primary astrocyte culture immunostaining. The other hemispheres were first homogenized in tissue protein extraction reagent containing protease/phosphatase inhibitors, Primary astrocytes were isolated from postnatal day 1 (P1) C57BL/6 mice. followed by the centrifugation at 100,000 3 g for 1 h to separate the Briefly, brains were dissected, minced and trypsinized for 20 min at 37˚C. detergent-soluble fraction and insoluble pellets. Pellets were subsequently Tissues were then triturated, and grown in DMEM/F12 supplemented with homogenized in 70% formic acid, and the detergent-insoluble fractions 10% FBS and penicillin and streptomycin for 6 d. When cells were con- were collected. Collected blood was briefly centrifuged to isolate plasma. fluent, astrocytes were purified by shaking at 350 rpm for 24 h at 37˚C. Plasma samples were placed on silicated test tubes and stored at 280˚C Attached cells were trypsinized and cultured on slide chambers to evaluate until further analysis. the purity of astrocytes by staining with glial fibrillary acidic protein (as- For the detection of anti–IL-1R Ab in the brain, we first biotinylated and trocyte marker), Iba1 (microglia marker), CNPase (oligodendrocyte marker), subsequently purified the anti–IL-1R blocking Ab by EZ-LINK Sulfo-NHS- and b-tubulin (neuronal marker) or on 6-well plates for treatments. LC-Biotin kit (Pierce Biotechnology, Rockford, IL), according to the man- Primary astrocytes were exposed to 0.5–3 ng/ml mouse recombinant ufacturer’s instructions. We i.p. injected ∼200 mg biotinylated anti–IL-1R IL-1b (Sigma-Aldrich) for 24 h. Conditioned media were collected for blocking Ab into aged 3xTg-AD mice, and brains were collected at 6 or 24 h treatments of SH-SY5Y cells, and astrocytes were homogenized with mam- after the Ab injection following the method described above. Biotinylated malian protein extraction reagent containing protease and phosphatase Ab was detected using streptavidin–HRP conjugates (for immunoblot) or by inhibitors to collect proteins. Extracted proteins were used for Western streptavidin–Alexa 488 conjugates (for immunofluorescent staining) in the blotting to measure S100B. tissue homogenates or sliced brain sections, respectively. Cell culture studies Cognitive tests Human neuroblastoma SH-SY5Y cells were grown in DMEM/F12 sup- Following 6 mo of Ab treatment, all mice were subjected to cognitive plemented with 10% FBS and penicillin and streptomycin. Cells were evaluation in the Morris water maze (MWM) and the contextual fear exposed to conditioned media collected from primary astrocytes for 24 h, conditioning (CFC) tests (36, 37). MWM primarily measures hippocampal- and cytosolic and nuclear proteins were extracted as follows. Cytosolic dependent cognition, whereas CFC assesses both amygdala and hippo- fractions were collected by lysing cells with buffer containing 10 mM campal function, including the trisynaptic pathway (entorhinal cortex, HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 M DTT, 0.05% Nonidet dentate gyrus, CA3, and CA1). Briefly, the apparatus used for the MWM P-40, and protease and phosphatase inhibitors. Cells were centrifuged at task was a circular aluminum tank (1.2 m diameter) painted white and 3000 rpm for 10 min at 4˚C, and supernatant was used as cytosolic frac- filled with water maintained at 22–24˚C. The maze was located in a room tion. Pellets were resuspended with buffer containing 5 mM HEPES (pH containing several simple visual extramaze cues. Mice were trained to 7.9), 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.3 M NaCl, 26% swim and find a 14-cm-diameter circular clear Plexiglas platform sub- glycerol (v/v), and protease and phosphatase inhibitors. After pellets were merged 1.5 cm beneath the surface of the water and invisible to the mice homogenized, the suspension was left on ice for 30 min and centrifuged at while swimming. On each training trial, mice were placed into the tank at 24,000 3 g for 20 min at 4˚C. Supernatant was used as nuclear fraction. one of four designated start points in a pseudorandom order. Mice were For treatment with S100B protein, SH-SY5Y cells were plated on 6-well allowed to find and escape onto the platform. If mice failed to find the culture plates, and S100B from bovine brain (Sigma-Aldrich) was added to platform within 60 s, they were manually guided to the platform and a final concentration of 0.05–5 mM. After 24 h of incubation, cells were allowed to remain there for 10 s. Each day, mice received four training homogenized with M-PER (Pierce Biotechnology) supplemented with pro- The Journal of Immunology 6541 tease and phosphatase inhibitors and centrifuged to collect total lysates for measuring the areas occupied by plaques. Briefly, three brain sections from immunoblot analysis. All in vitro experiments were performed and analyzed each animal were stained with anti-Ab42 Ab described above, and im- in two to three independent experiments in triplicates or quadruplets. ages were captured using Zeiss Axioskop with Axiocam (Carl Zeiss Microimaging, Thornwood, NY). Ab42-positive plaques were counted in Cytokine assays a 500-mm2 subfield in the hippocampus, subiculum, entorhinal cortex, and amygdala. Plaque burden was calculated by defining approximate areas of Brain or plasma cytokines were quantitatively measured by ELISA (Pierce plaques over the entire areas in the hippocampus, subiculum, entorhinal Biotechnology) or Bio-Plex (Bio-Rad, Hercules, CA), respectively, as cortex, and amygdala using ImageJ software (National Institutes of Health). described previously (40). Briefly, 50 ml brain homogenates were used to determine IL-1b, IL-6, and TNF-a levels by Endogen ELISA kits. For Quantitative analysis of Ab by ELISA Bio-Plex, we used a custom 8-plex detection kit, which measured IL-1a, IL-1b, IL-4, IL-6, IL-10, MCP-1, TNF-a, and IFN-g. We strictly followed Ab40 and Ab42 were detected in both the detergent-soluble and -insoluble the manufacturer’s instructions. Briefly, 10 ml plasma was mixed with 40 fractions by ELISA as described previously (32). ml standard diluent and incubated with a mixture of Abs conjugated with fluorescent beads. Following the detection Ab and streptavidin–PE treat- Real-time PCR ments, levels of each cytokine were measured using the Bio-Plex 200 Total RNAwas isolated from untreated or treated SH-SY5Y cells using trizol System (Bio-Rad). Concentrations were calculated by standard curve and reagent (Molecular Research Center, Cincinnati, OH). Briefly, 1 mgtotal expressed in picograms per milliliter. RNA was used for one-cycle reverse transcriptase reaction to make cDNA by random hexamers using SuperScript III first-strand synthesis system Western blot analysis (Invitrogen, Carlsbad, CA). Resulting cDNA (1 ml) was subjected to a PCR Protein concentrations of detergent-soluble fractions from half-brain (cortex for the detection of S100B using iQ SYBR Green Supermix (Bio-Rad). and hippocampus) were determined by the Bradford protein assay. These Human S100B primers were 59-TGG ACA ATG ATG GAG ACG G-39 fractions were subsequently immunoblotted with the following Abs: HT7 (forward) and 59-ATT AGC ACA ACA CGG CTG G-39 (reverse) (42). Downloaded from (total human tau; Pierce Biotechnology), AT8 (phosphorylated tau at S199/ GAPDH was used for normalizing the S100B expression levels in each S202/T205; Pierce Biotechnology (41)), PHF-1 (phosphorylated tau at treatment, and the primer sequences were 59-AAC TTT GGC ATT GTG S396/S404; Pierce Biotechnology), AT100 (phosphorylated tau at S212/ GAA GG-39 and 59-ACA CAT TGG GGG TAG GAA CA-39. The PCR T214; Pierce Biotechnology), total p65 NF-kB (Cell Signaling Technol- cycle parameters were as follows: denaturing step (95˚C for 30 s), annealing ogy, Beverly, MA), phospho-p65 NF-kB (phosphorylation at S536; Cell step (60˚C for 30 s), and extension step (72˚C for 30 s). The cycle threshold Signaling Technology), YM1 (StemCell Technologies, Vancouver, BC, (Ct) values were determined by MyiQ software (Bio-Rad), and DCt for Canada), arginase-1 (Santa Cruz Biotechnology, Santa Cruz, CA), cdk5 each treatment group was calculated as follows: DCt = Ct (S100B) 2 Ct http://www.jimmunol.org/ (Calbiochem, La Jolla, CA), p35/p25 (Santa Cruz Biotechnology), GSK- (GAPDH). The RQ was then calculated by the following equation: RQ = 2DDCt 3b (BD Transduction Laboratories, San Jose, CA), phospho–GSK-3b 2 , with DDCt = DCt (treatment) 2 DCt (control) (43). (phosphorylation at S9), p38–MAPK, phospho–p38-MAPK (phosphorylation at T180/Y182), Akt, phospho-Akt (phosphorylation at S473; all Statistical analysis from Cell Signaling Technology), S100B (Novus Biologicals, Littleton, All immunoblot and immunohistochemical data were quantitatively ana- CO), b-catenin (Sigma-Aldrich), and phospho–b-catenin (phosphorylation lyzed using Bio-Rad Quantity One software or ImageJ software. Statistics at S552 or S31/S37/T41; Cell Signaling Technology). Membranes were were carried out using one-way ANOVA with post hoc tests or unpaired t reprobed with Ab against b-actin (Sigma-Aldrich), GAPDH (Santa Cruz test, and p , 0.05 was considered to be significant. Biotechnology), and/or nuclear matrix p84 (Abcam, Cambridge, MA) to control for protein loading or to confirm no cross-contamination of each fraction. Band intensity was measured using Quantity One software (Bio- Results by guest on September 29, 2021 Rad) and normalized by corresponding loading control proteins. Blocking IL-1R reduces NF-kB activation in the brain Dot blot analysis was used to quantitatively measure oligomeric species of Ab. Conformation specific anti-oligomer Abs, A11 and OC, were used To suppress IL-1 signaling, we administered anti–IL-1R blocking (a gift from Dr. C. Glabe, University of California, Irvine). Briefly, 3 mg mAb (anti–IL-1R) to 9-mo-old 3xTg-AD mice over the course of brain homogenates was spotted on nitrocellulose membrane. After nonspe- 6 mo using the same paradigm as a prior study with anti-CD40 Ab cific binding was blocked, A11 or OC Ab was applied, and the membrane in PDAPP mice (44). During the 6 mo of dosing, two control mice, was incubated overnight at 4˚C. Subsequently, secondary anti-rabbit HRP-conjugated Ab was used to detect A11 or OC Ab. two IgG-treated mice, and four anti–IL-1R–treated mice died. No clear signs of autoimmune responses or CNS damage were ob- Immunohistochemical analysis served. All data presented in this study were obtained from mice For immunohistochemical analysis, each half-brain was cut into 50-mm slices that survived the entire experimental procedure. Immunohisto- using a Vibratome and stored in TBS. To qualitatively assess Ab plaque chemical studies confirmed that astrocytes, microglia, endothelial burden, free-floating sections were pretreated with 90% formic acid for 7 cells of blood vessels, and, to some extent, neurons were recog- min and incubated with biotinylated Ab against Ab42 (1/400, clone D32 nized by the anti–IL-1R mAb, consistent with reported expression from Drs. V. Vasilevko and D.H. Cribbs) overnight, followed by avidin/biotin complex reagent for 1 h. Sections were washed with TBS and visualized patterns (Supplemental Fig. 1A,1B) (45). by diaminobenzidine, according to the manufacturer’s specifications. IL-1R At the end of the 6-mo treatment, we measured plasma Ab titers expression in the brain was visualized by staining with IL-1R blocking mAb against rat Abs, the species in which anti–IL-1R was generated, (1/100), followed by incubation with biotinylated anti-rat secondary Ab and found that they were 0.516 6 0.049 for untreated mice, 1.420 6 (1/200; Vector Laboratories, Burlingame, CA) and visualized with diamino- 6 benzidine. Sections were counterstained with hematoxylin to visualize nu- 0.354 for IgG control (sham-treated mice), and 1.354 0.330 clei. For phospho-tau qualitative analysis, sections were stained with AT8 for the anti–IL-1R–treated mice, with the titers reaching 1/1600, (1/2000), AT100 (1/2000), and HT7 (1/2000) in the same manner. To assess which suggests an induction of low levels of anti-rat Abs in both microglial activity around Ab plaques, free-floating sections were pretreated IgG- and anti–IL-1R–treated animals. We also examined whether with 90% formic acid for 6 min and then stained for Ab plaques using the IL-1R blocking mAb crossed the blood–brain barrier (BBB). 6E10 (1/400) and microglia with anti–Iba-1 (1/400), CD68 (1/100; Sero- tec, Raleigh, NC), or YM1 (1/100) overnight. To visualize colocalization, It has been reported in numerous studies that Abs administered sections were incubated for 1 h with goat anti-mouse Alexa Fluor 488 and peripherally by i.p. injections cross the BBB and exhibit their ef- goat anti-rabbit Alex Fluor 555, with TOTO3 (1/200) for nuclear staining. fects centrally, particularly in AD research treating transgenic The colocalization of IL-1b in microglia was assessed by staining sections mouse models with Abs against Ab (44, 46–52). In our study, we with anti–IL-1b (1/100) and Iba-1 (1/1000) overnight, followed by secondary Abs donkey anti-goat 488 and donkey anti-rabbit 555. attempted to identify anti–IL-1R mAb binding in the CNS of the treated 3xTg-AD mice using a double immunofluorescent stain- Semiquantitative analysis of plaque burden ing, but the result was inconclusive, although we did observed an Ab plaque burden in hippocampus, subiculum, entorhinal cortex, and increased signal for anti–IL-1R mAb on microglia, astrocytes, and amygdala was quantified by counting plaques (.20 mm in diameter) or neurons at the end of the 6-mo treatment (data not shown). We 6542 IL-1 SIGNALING AND b-CATENIN PATHWAY IN AD then examined the Ab penetration of the BBB at earlier time mice reached criteria (escape latency at 20 s) on the fourth day of points. The anti–IL-1R mAb was first biotinylated and then in- the training and performed significantly better than control or jected i.p. in the 3xTg-AD mice. The presence of the biotinylated sham-treated group (p , 0.05; Fig. 1A). The control or sham- Ab in the brain was measured biochemically and histochemi- treated group reached criteria on the fifth or sixth day, respec- cally after 6 and 24 h postinjection. A total of 50 mg protein tively, and there was no overall statistical difference between these extract from a half-brain was loaded, and the presence of bio- two groups. In the 24-h probe trial to test hippocampal-dependent tinylated anti–IL-1R mAb was detected by a streptavidin–HPR retention memories, we found that blocking IL-1 signaling sig- conjugate. A significant increase of the biotinylated anti–IL-1R nificantly improved both escape latency and the number of plat- mAb (Supplemental Fig. 1C, shown as H and L chains, arrow- form crosses (p , 0.05; Fig. 1B,1C). Similarly, the anti–IL-1R– heads) in the brain was detected in both 6- and 24-h anti–IL-1R– treated mice performed significantly better on this cognitive test treated 3xTg-AD mice by immunoblot, whereas virtually no bio- versus control or sham-treated mice in the CFC test (p , 0.05; tinylated Ab was present in untreated control 3xTg-AD mice. The Fig. 1D). Thus, blocking IL-1 signaling in the 3xTg-AD mice binding of anti–IL-1R mAb in CNS cells was further confirmed by rescued hippocampal-dependent cognitive impairments as mea- a double immunostaining (Supplemental Fig. 1D). A fraction of sured by two independent behavioral tests. neurons and blood vessels in brain sections from untreated control Ab plaque pathology is marginally affected following 3xTg-AD mice were positive by streptavidin–Alexa488 (Supple- suppression of IL-1 signaling mental Fig. 1Di from the CA1 hippocampus and Supplemental Fig. 1Dii from the cortex; arrowheads), suggesting endogenous non- We next assessed the effects of blocking IL-1 signaling in aged specific biotinylated molecules or proteins that were also detected 3xTg-AD mice on Ab pathology. Quantitative Ab ELISA revealed Downloaded from by Western blot (Supplemental Fig. 1C). In contrast, it was clear a significant reduction of detergent-insoluble Ab42, but not Ab40, that the biotinylated anti–IL-1R mAb was present on the surface of in the brains of anti–IL-1R–treated animals (p , 0.05; Fig. 2B). microglia, astrocytes, neurons, and endothelial cells in the brain Interestingly, soluble Ab levels were either unchanged or in- sections from the biotinylated anti–IL-1R–injected 3xTg-AD mice creased with anti–IL-1R treatment (Fig. 2A). Histological analysis (Supplemental Fig. 1Dv–x, arrows). This colocalization was revealed that the number of plaques over a 20-mm diameter in

minimal in the control mice (Supplemental Fig. 1Dii–iv). Taken the hippocampus, subiculum, entorhinal cortex, and amygdala was http://www.jimmunol.org/ together, these data suggest that a small proportion of the sys- significantly reduced with anti–IL-1R treatment when compared temically administered anti–IL-1R mAb crossed the BBB in the with control group, which was in part supported the quantitative 3xTg-AD mice. In addition, brain NF-kB activity, measured by ELISA data described above (p , 0.05; Fig. 2C,2E, Supple- the steady-state levels of phosphorylated p65 at Ser536, was sig- mental Fig. 3). In contrast, the Ab plaque burden failed to show nificantly reduced in the anti–IL-1R–treated mice, further sug- any significant difference among the treatment groups, and it was gesting an antagonizing action of the Ab in CNS (Supplemental in part because of the unevenly distributed plaques within each Fig. 2A). However, it is important to point out that we cannot mouse brain and variability among individual mice within the exclude a possibility that any downstream effects described in this treatment group (Fig. 2C,2D, Supplemental Fig. 3). Similarly, study might be due not only to direct action in the CNS but also to soluble protofibrillar and fibrillar Ab oligomers detected by con- by guest on September 29, 2021 peripheral effects of blocking IL-1R. formational specific Abs, A11 and OC, respectively, showed mixed results. Although protofibrillar Ab oligomers were unaf- Blocking IL-1 signaling rescues AD-related cognitive fected, fibrillar Ab oligomers were markedly reduced in the aged impairments 3xTg-AD mice that received the IL-1R blocking Ab (Fig. 2F, The effect of IL-1 signaling on cognition was assessed by MWM 2G, Supplemental Fig. 2B). APP expression or processing was not and CFC tests. In the acquisition phase, the anti–IL-1R–treated significantly altered by the treatment, because the steady-state

FIGURE 1. Administering an IL-1R blocking mAb rescues hippocampus-dependent cognitive impairments in the 3xTg-AD mice. A, Acquisition curve during training of MWM is expressed as mean 6 S.E.M. No sig- niﬁcant difference was observed among the groups. Escape latency (B) and number of crosses (C) on platform location for 24-h retention trial in MWM. Each bar is expressed as mean 6 SEM; *p , 0.05 compared with control and IgG control groups. D, The freezing index of CFC is expressed as mean 6 SEM. *p , 0.05 compared with control and IgG control groups. The number of mice tested: n = 10 for control and IgG control, and n = 8 for IL-1R blocking mAb treatment. The Journal of Immunology 6543 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Blocking IL-1 signaling and effect on amyloid pathology in the 3xTg-AD mice. Quantitative Ab ELISA in detergent-soluble brain fraction (A) and detergent-insoluble (formic acid soluble) brain fraction (B). Each bar is expressed as mean 6 SEM; *p , 0.05 compared with control and IgG control groups. C, Representative immunohistochemical staining of amyloid plaque burden in the hippcampus and amygdala. Anti–Ab42-specific Ab detects aggregated Ab-containing plaques (scale bars, 500 mm). D, Amyloid burden in the hippocampus, sebiculum, entorhinal cortex, and amygdala is expressed as a bar graph. E, Plaque count (.20 mm in diameter) in 500 mm2 subfield in the hippocampus, subiculum, entorhinal cortex, and amygdala is expressed in the bar graph (mean 6 SEM). *p , 0.05 compared with control. Dot blot analysis of oligomeric Ab species using Ab A11 (F) and Ab OC (G). Each bar is expressed as mean 6 SEM; *p , 0.05 compared with control and IgG control groups. More dot blot data for A11 and OC are found in Supplemental Fig. 2B. H, Immunoblot analysis of APP processing in the brain following the treatment. The densitometric analysis of C99 and C83 fragments was shown in the graph. Each bar is expressed as mean 6 SEM. No statistical significance was obtained. The number of mice tested: n = 10 for control and IgG control, and n = 8 for IL-1R blocking mAb treatment. levels of APP or C-terminal fragments of APP, such as C99 and IL-1R treatment did not completely suppress proinflammatory C83, were not changed among the treatment groups (Fig. 2H). responses in the brain. Double immunofluorescent staining further Collectively, these results suggest that blocking IL-1 signaling may confirmed the reduction of IL-1b production in activated micro- be capable of reducing certain fibrillar or oligomeric Ab species glia around plaques (Fig. 3B). and has marginal effects on blocking maturation or formation of These changes in the brain milieu may affect the phagocytic Ab plaques. abilities of microglia. Double immunofluorescent staining detected that the number of microglia harboring 6E10-positive APP/Ab Anti–IL-1R treatment attenuates microglial activation in fragments was increased, suggesting that these cells were actively 3xTg-AD brains engulfing Ab (control, 4.8/20 cells; sham, 6.2/20 cells; and IL-1R, Despite modest changes in Ab pathology, proinflammatory and 12.2/20 cells, using 340 field, representative figures in Fig. 3C). microglial responses were found to be altered in the brain fol- Additional staining was conducted to further analyze the micro- lowing anti–IL-1R treatment of aged 3xTg-AD mice. The levels of glial phenotype using several markers, CD68, YM1, and arginase- the proinflammatory cytokines, IL-1b and TNF-a, in the brain 1, which have been previously described as phagocytic markers were significantly decreased when IL-1 signaling was blocked (53, 54). Although CD68-positive microglia were uniformly de- (Fig. 3A). IL-6 showed a trend toward reduction but did not tected around plaques in all groups (data not shown), more YM1- achieve significance (Fig. 3A). However, it is important to note positive microglia were observed in the anti–IL-1R–treated mice that the levels of these proinflammatory cytokines were signifi- (Fig. 3D). Quantitative analysis of YM1 and arginase-1 showed cantly higher than age-matched nontransgenic mice even with significant increases in the brain of the anti–IL-1R–treated 3xTg- anti–IL-1R treatment (data not shown), suggesting that the anti– AD mice. Therefore, anti–IL-1R treatment appears to increase the 6544 IL-1 SIGNALING AND b-CATENIN PATHWAY IN AD

FIGURE 3. Blocking IL-1 signaling decreases proinflammatory cytokines and enhances microglial phago- cytosis. A, ELISA analysis of selected proinflammatory cytokines. Each bar is expressed as mean 6 SEM (n = 10 for control and IgG control, and n = 8 for anti–IL- 1R treatment); *p , 0.05 compared with control and IgG control groups. B, IL-1b levels are decreased in Downloaded from animals receiving the anti–IL-1R blocking mAb. Double immunofluorescent staining with IL-1b and microglia. Asterisks, Amyloid plaques (scale bars, 10 mm). C, Suppressing IL-1 signaling promotes the phago- cytosis of Ab by microglia. Representative double- immunofluorescent staining with Ab (6E10) and mi-

croglia (Iba1) in the brain of anti–IL-1R–treated http://www.jimmunol.org/ 3xTg-AD mice. Arrows, Ab within microglial compartment (scale bars, 10 mm). D, Double immunofluorescent staining of YM1 (green) and tomato lectin (red) around Ab plaques in control and anti–IL-1R– treated mice. Arrow indicates activated microglia with high YM1 expression. YM1 fluorescent intensity was measured and plotted in graph (mean 6 SEM). *p , 0.05 or **p , 0.01 compared with IgG control or control, respectively. Scale bars, 10 mm. E, Immu- noblots and densitometric analyses of Ab degrading by guest on September 29, 2021 enzymes, insulin degrading enzyme (IDE), and neprilysin in the brain homogenates (n = 10 for control and IgG control; n = 8 for IL-1R blocking mAb treatment). No statistical significance is detected by densitometric analyses (mean 6 SEM).

phagocytic activation of microglia (Supplemental Fig. 2C). The we next investigated pathological changes in tau following anti– levels of two major amyloid-degrading enzymes that are secreted IL-1R treatment of aged 3xTg-AD mice. Blocking IL-1 signaling from microglia, insulin-degrading enzyme and neprilysin, were, significantly reduced the number of phospho–tau-bearing neurons however, not significantly altered in the brain (Fig. 3E). detected by Abs AT8 (pSer199/Ser202/Thr205) and AT100 (pSer212/ Thr214) in the CA1 subfield of the hippocampus, whereas no clear Tau pathology is significantly attenuated following anti–IL-1R alteration in total tau by the Ab HT7 was detected in the same field treatment of aged 3xTg-AD mice (Fig. 4A). This change was also evident by immunoblotting, be- We and others (25, 32, 53) previously demonstrated that altered cause AT8-, AT100-, or PHF-1 (pSer396/Ser404)-positive phospho- inflammation in the brain significantly influenced the subse- tau levels were significantly reduced in mice after treatment, quent development of tau pathology, independent of Ab pathol- whereas the steady-state levels of total tau remained unchanged ogy, in the 3xTg-AD mice and other mouse models. Consequently, (Fig. 4B, Supplemental Fig. 2D). The Journal of Immunology 6545

The reduction of phospho-tau levels in the brain was accom- panied by a significant reduction of p25 levels (p , 0.01), increased levels of phospho–GSK-3b (at Ser9 position), and a reduction of phospho-p38–MAPK in the brains of the anti–IL- 1R–treated mice, which are predicted to lead to lower activity of cdk5, GSK-3b, and p38–MAPK, respectively (Fig. 4C). The total steady-state levels of cdk5, GSK-3b, and p38-MAPK were not significantly altered among the treatment groups (Fig. 4C). The sum of these results highlights the diverse disease-modifying effects of blocking IL-1 signaling. IL-1 modulates S100B secretion in astrocytes and suppresses b-catenin signaling in neurons It is not clear whether IL-1 signaling directly affected neurons and regulated intraneuronal signaling cascades or whether the pathology was affected by other cell types in the brain or the pe- riphery. Indeed, the role of IL-1 signaling in AD etiology may be pleitropic, and activities of the kinases identified in this study, p38–MAPK, cdk5, and GSK-3b, may influence multiple signaling Downloaded from cascades in multiple cell types. For instance, it has been demonstrated that IL-1 signaling activates p38–MAPK in neurons, leading to tau phosphorylation (31, 53, 55). At the same time, IL-1 signaling promotes production of IL-1b and other proinflammatory molecules by microglia, astrocytes, and peripheral inflamma-

tory cells, in part, via a p38–MAPK cascade (56–58). Therefore, http://www.jimmunol.org/ the reduction of p38–MAPK activation we observe in response to anti–IL-1R treatment may be due to the action of inhibiting IL-1 signaling in peripheral inflammatory cells, microglia, and astrocytes, as well as neurons. The relationship between IL-1 signaling and the activation of cdk5 and GSK-3b are less well characterized. Therefore, we sought to better understand the underlying mechanisms by which activities of these tau kinases were suppressed following anti–IL- 1R treatment. Because IL-1b regulates the expression and secre- by guest on September 29, 2021 tion of a key AD-relevant cytokine, S100B, from astrocytes (59– 61), and because S100B has been shown to promote tau pathology by disrupting a substrate of GSK-3b, b-catenin, in neuronal stem cell culture (62), we hypothesized that S100B might mediate the reduction in GSK-3b activity that we observed. Indeed, we found that S100B was predominantly produced in astrocytes of 3xTg- AD mice and was significantly reduced by anti–IL-1R treatment (Fig. 5A,5B,5F). The levels of S100B showed a trend to be lower in the IgG control group, perhaps because of nonspecific modulation of innate immune responses by IgG, although this difference did not reach significance. We next sought to examine the b-catenin signaling cascade in our mice. In anti–IL-1R–treated mice, the steady-state levels of b-catenin phosphorylated at Ser33/ Ser37/Thr41, sites that are primarily phosphorylated by GSK-3b, showed a trend toward decrease, although failed to reach signifi- FIGURE 4. Suppressing IL-1 signaling attenuates tau pathology in the cance (Fig. 5A,5C). In contrast, we detected a significant increase 3xTg-AD mice. A, Tau pathology is attenuated by blocking IL-1 signal- of b-catenin phosphorylated at Ser552 (Fig. 5A,5D). This partic- ing. Representative immunohistochemical staining with various tau Abs: 199 202 ular phosphorylation event is mediated by Akt, and results in the HT7, total tau, AT8, phosphorylated tau at Ser and Ser , and AT100, phosphorylated tau at Ser212 and Thr214 (scale bars, 500 mm). B, Immu- nuclear translocation of b-catenin and transcriptional upregulation noblot and densitometric analyses of changes in the steady-state levels of of prosurvival genes. Consistent with the increase in phospho- 6 552 phosphorylated tau in the brain. Each bar is expressed as mean SEM Ser –b-catenin, we observed that the active form of Akt (n = 10 for control and IgG control; n = 8 for anti–IL-1R treatment); *p , (phospho-Akt) was significantly elevated in the brains of anti–IL- 0.05 or **p , 0.01 compared with control and IgG control groups. More 1R–treated mice (p , 0.05; Fig. 5A,5E). Collectively, we hy- immunoblot data for phospho-tau are found in Supplemental Fig. 2D. C, pothesized that blocking IL-1 signaling in astrocytes attenuated Suppressing IL-1 signaling results in decreased activations of tau kinases. their proinflammatory responses and S100B secretion, and these Immunoblot and densitometric analyses of the steady-state levels of GSK- changes restored neuronal b-catenin signaling cascades and in- 3b, cdk5/p35/p25, and p38-MAPK. Each bar is expressed as mean 6 SEM hibited GSK-3b activity in the 3xTg-AD mice. Therefore, the (n = 10 for control and IgG control; n = 8 for anti–IL-1R treatment); *p , , pathological effects of IL-1 may be mediated not only by its direct 0.05 or **p 0.01 compared with control and IgG control groups. action on neurons but also in the cross-talk with glial cells and neurons. However, additional studies would be needed to directly 6546 IL-1 SIGNALING AND b-CATENIN PATHWAY IN AD

primary astrocytes with various concentrations of IL-1b (0.5–3 ng/ml) resulted in the increased production of S100B, which was released into the conditioned media. Quantitative RT-PCR showed a 3.2-fold elevation of S100B mRNA even at 0.5 ng/ml IL-1b treatment compared with control (p , 0.05). Conditioned media from the IL-1b–treated primary astrocytes at all concentrations signiﬁcantly reduced phospho-Akt and phospho–GSK-3b levels in SH-SY5Y cells and suppressed the translocation of b-catenin into the nuclear compartment (Supplemental Fig. 4A). The effect was not as great with IL-1b alone, because SH-SY5Y cells directly exposed to IL-1b only showed signiﬁcant (p , 0.05) reductions of cytosolic b-catenin, phospho–GSK-3b and phospho-Akt at the highest concentration (3 ng/ml), whereas no clear changes in nuclear b-catenin was detected (Supplemental Fig. 4B). We therefore used the lowest concentration of IL-1b (0.5 ng/ml) that exerted an effect on SH-SY5Y through primary astrocytes in subsequent experiments to hone in on the astrocyte-mediated pathological mechanisms of IL-1b on neuronal cells. Cotreat-

ment of the IL-1R blocking mAb (0.1 mg/ml) with rIL-1b (0.5 ng/ Downloaded from ml) in primary astrocytes significantly reduced S100B levels (Fig. 6A)andrestoredb-catenin nuclear translocation in SH-SY5Y cells, along with increases in phospho–GSK-3b and phospho- Akt (Fig. 6B). Last, SH-SY5Y cells were treated with purified S100B protein to confirm the effect of S100B on GSK-3b and

b-catenin signaling. Following 24-h incubation, the highest con- http://www.jimmunol.org/ centration of S100B (5 mM) signiﬁcantly reduced total b-catenin levels, and S100B activated GSK-3b in SH-SY5Y cells in a concentration-dependent manner (Fig. 6C). These data corroborate our in vivo ﬁndings that IL-1b modulates S100B production and secretion by astrocytes and affects b-catenin signaling cascades in neurons.

Discussion A chronic administration of anti–IL-1R blocking mAb in aged by guest on September 29, 2021 3xTg-AD mice resulted in a marked change in brain inflammation, a significant attenuation of tau pathology, and a reversal of cognitive deficits. Although our data on central versus peripheral mechanisms of action are still suggestive, a fraction of systemically administered Ab appeared to cross BBB and bound IL-1Rs on the surface of astrocytes, microglia, neurons, and blood vessels in the brain. The presence of background staining or nonspecific bands in all brains made the case difficult confirm or determine the exact amount of the peripherally injected Ab in the brain as well FIGURE 5. S100B and b-catenin signaling are altered in anti–IL-1R as its localization in specific cell types, yet the increased fluo- Ab-treated 3xTg-AD mice. A, Immunoblot analysis of the steady-state rescent signals in astrocytes, microglia, and neurons suggest the levels of phospho–b-catenin at Ser33/Ser37/Thr41. B–E, Densitometric anal- Ab binding to the IL-1R on the cell surface. Therefore, the ben- ysis of the intensity of immunoblots. Each bar is expressed as mean 6 eficial disease-modifying effects observed in this study, including SEM (n = 6 for all groups); *p , 0.05 or **p , 0.01 compared with a reduction of NF-kB activation, increased levels of phagocytic corresponding group. F, Double immunostaining with S100B (green) and markers in microglia, a suppression of S100B secretion by as- glial fibrillary acidic protein (GFAP) (astrocyte marker, red) confirms that trocytes, and subsequent restoration of Wnt/b-catenin signaling S100B is predominantly produced in astrocytes in the brain of 3xTg-AD cascades in neurons, may in part be mediated through a direct mice. TOTO-3 (blue) is used to counterstain nuclei. Scale bars, 20 mm inhibition of IL-1 signaling on these cells. However, it is important (upper panels) and 10 mm(lower panels). to point out that the overall suppression of neuroinflammation in the brain directly or indirectly by the inhibition of IL-1Rs may demonstrate a causal relationship between S100B and b-catenin also exhibit beneficial disease-modifying effects in these mice. signaling. Furthermore, the peripheral effects of anti–IL-1R blocking Ab on immune cells, such as T cells, B cells, and macrophages, as well b S100B-mediated activation of GSK-3 in a cell culture model as their impacts on CNS and AD pathologies, remain to be eluci- We next set out to test whether reduced IL-1b–induced S100B dated. To support the central effect of systemically administered secretion from astrocytes mediated the activation of GSK-3b in Abs, a number of studies has reported penetration of peripherally neurons in vitro, as observed in our in vivo model. We exposed administered Abs or endogenous Abs generated by vaccinations murine primary astrocytes to rIL-1b and collected conditioned into brain tissues to exhibit therapeutic effects (44, 46–52, 63, 64). media after 24 h. The astrocyte-conditioned media were then Although more detailed analyses are required to confirm the direct added to human neuroblastoma SH-SY5Y cells. Treatment of effect of the Ab in the CNS, the primary aim of our study was to The Journal of Immunology 6547 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. IL-1b triggers S100B-mediated alterations in b-catenin signaling in neurons. A, Immunoblot and densitometric analysis (mean 6 SEM) of S100B in primary astrocytes exposed to rIL-1b with or without anti–IL-1R blocking mAb for 24 h. *p , 0.05 or **p , 0.01 compared with control (n = 6). GAPDH is used for a loading control. B, Conditioned media from mouse primary astrocytes exposed to 0.5 ng/ml mouse recombinant IL-1b with or without 0.1 mg/ml anti–IL-1R blocking Ab for 24 h are used to treat SH-SY5Y cells for 24 h, and subseqent changes in b-catenin signaling cascades are detected by immunoblots. Densitometric analyses (mean 6 SEM) show a significant changes in nuclear translocation of b-catenin, cytosolic phospho–GSK-3b (at Ser9), and phospho-Akt (at Ser473) in cotreatment with anti–IL-1R blocking mAb (*p , 0.05 or **p , 0.01, n = 6). GAPDH and nuclear matrix p84 are used to confirm no cross-contamination between cytosolic and nuclear fractions, respectively. C, SH-SY5Y cells are treated with various doses of purified S100B protein for 24 h, and changes in b-catenin and GSK-3b are examined by immunoblot analysis. Densitometric analysis of band intensity (mean 6 SEM) is expressed in a bar graph; *p , 0.05 or **p , 0.01 compared with control (two separate experiments, n = 4/experiment). explore the pathological role of IL-1 signaling in AD neuropa- showing that proinflammatory cytokines ameliorate Ab plaques thology, and it is apparent that IL-1b plays a central role in mod- were conducted in much younger mice. This aging discrepancy ulating and propagating proinflammatory responses in the brain. may partly explain the dichotomouseffectsofproinflamma- The effect of blocking IL-1b signalingonAb plaque pathology tory responses in the brain and subsequent diverse pathological was unexpectedly minimal, although certain Ab species appeared changes. to be reduced by the treatment with the IL-1R blocking Ab. The inflammation- or IL-1b–induced pathological tau devel- Recent studies, however, reported an Ab plaque clearance effect opment has been well documented, and our findings are consis- of proinflammatory cytokines, including IL-1b, through phago- tent with previous studies (25, 31–33, 53, 68). The inhibition of cytic activation of microglia in mouse models of AD (34, 35, 65, IL-1 signaling significantly suppressed the activation of cdk5/p25, 66). Various factors, such as age and strain of mice, and trans- GSK-3b, and p38–MAPK, all major kinases that phosphorylate gene expression, could be involved in showing these dual effects tau in neurons. A recent study demonstrated a direct effect of IL- of IL-1b in the Ab plaque pathology, and more detailed mech- 1b secreted by microglia on neurons and subsequent activation of anistic studies under defined conditions need to be conducted p38–MAPK and accumulation of tau phosphorylation (53). We to fully understand these differential actions of microglia. For believe that the observed reduction of phospho-p38–MAPK in the example, aging itself triggers differential activation in microglia anti–IL-1R–treated mice may be mediated by the blockade of isolated from the APP/PS1 transgenic mouse model (54, 67). IL-1Rs on neurons as well as microglia and astrocytes, or even These studies show that aged microglia are more easily activated peripheral inflammatory cells, resulting in decreased levels of IL- toward proinflammatory/classical activation. In our study, we 1b in the brain and decreased direct IL-1b signaling. In contrast, used relatively old (15-mo-old) 3xTg-AD mice, whereas studies the interplay between IL-1b and GSK-3b or cdk5 has not been 6548 IL-1 SIGNALING AND b-CATENIN PATHWAY IN AD well characterized. Our study examined one possible underlying 10. Galimberti, D., C. Fenoglio, C. Lovati, E. Venturelli, I. Guidi, B. 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Interleukin-1b and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer’s and de novo Parkinson’s disease patients. Neurosci. Lett. 202: including AD, schizophrenia, and autism (76, 77). In AD patients, 17–20. b-catenin levels are significantly reduced, and GSK-3b is upreg- 15. Cacabelos, R., M. Barquero, P. Garcıá, X. A. Alvarez, and E. Varela de Seijas. ulated and accumulates in neurons (78, 79). Glial progenitor cells 1991. Cerebrospinal fluid interleukin-1b (IL-1b) in Alzheimer’s disease and neurological disorders. Methods Find. Exp. Clin. Pharmacol. 13: 455–458. from AD patients exhibit abnormal b-catenin phosphorylation 16. Parachikova, A., M. G. Agadjanyan, D. H. Cribbs, M. Blurton-Jones, V. Perreau, and increased GSK-3b, along with impaired neurogenesis (80). J. Rogers, T. G. Beach, and C. W. Cotman. 2007. Inflammatory changes parallel

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