First, we tested IL-1β secretion of BV2 microglial cells in response to Aβ (2 µM) ± ATP (1mM) or TDCA (400ng/ml) to optimize experimental settings ( Figure 1A ). IL-1β secretion was increased upon treatment with Aβ alone and was further increased when combined with ATP. However, ATP or TDCA alone did not increase IL-1β secretion. Primary microglia from the brains of C57BL/6 (B6), P2X7R^-/-, or GPCR19^-/- mice were sorted using a magnetic column. The purity was above 95% ( Supplementary Figure 1A ). When stimulated with Aβ (2 µM) and ATP (1 mM), the IC₅₀ of TDCA in inhibiting IL-1β secretion by microglia from B6 mice was 490.6 nM ( Figure 1B ). However, TDCA did not inhibit IL-1β secretion by microglia from GPCR19^-/- or P2X7R^-/- mice when stimulated with Aβ (2 µM) and ATP (1 mM) ( Figure 1B ), suggesting that GPCR19 might regulate P2X7R function. GPCR19 and P2X7R were colocalized in resting primary microglial membranes ( Figure 1C ; Supplementary Figure 1B ). The expression levels of GPCR19 in the frontal cortex were significantly lower in 6- and 9-month-old 5xFAD mice than in 3-month-old 5xFAD mice or B6 mice ( Figure 1D ). In comparison, the expression levels of P2X7R in the frontal cortex were significantly higher in 9-month-old 5xFAD mice than that of 3-month-old 5xFAD mice or B6 mice ( Figure 1D ). Brain tissues from individuals with AD or without AD were analyzed after immunohistochemical staining. GPCR19 and P2X7R were colocalized in the brain cells of non-AD subjects ( Figure 1E ; Supplementary Figure 2 ). Expression of GPCR19 in non-AD subjects (n = 3) was higher than those of AD patients (n = 5). On the contrary, expression of P2X7R in non-AD subjects was lower than those of AD patients. Treatment of BV2 cells with Aβ ± ATP for 1 h significantly downregulated the expression of GPCR19 on the cell surface, while this effect was reversed by TDCA treatment ( Figure 1F ). In contrast, the surface expression of P2X7R was upregulated by Aβ ± ATP and was inhibited by TDCA treatment ( Figure 1F ; Supplementary Figure 3 ). In BV2 cells, GPCR19 (green) and P2X7R (red) showed colocalization (yellow) in resting state ( Figure 1F ). Colocalized area decreased after treatment with Aβ ± ATP, which was upregulated by TDCA treatment ( Figure 1F ).

In the frontal cortex of 5xFAD mice, GPCR19 expression was significantly increased after treatment with TDCA (1 mg/kg, i.p., q.d. for 10 weeks) compared with PBS-treated mice ( Figure 1G ; Supplementary Figure 4 ). However, the expression levels of P2X7R were significantly lower in 5xFAD-TDCA mice than in 5xFAD-PBS mice ( Figure 1G ; Supplementary Figure 4 ). Colocalization areas (yellow) of GPCR19 (green) with P2X7R (red) in the brain were significantly lower in 5xFAD-PBS mice compared with 5xFAD-TDCA mice ( Figure 1G ).

Primary microglia were isolated from the brains of B6, P2X7R^-/-, or GPCR19^-/- mice. In response to P2X7R agonists (ATP or BzATP), Ca⁺⁺ mobilization was significantly decreased in the microglia from P2X7R^-/- or GPCR19^-/- mice compared with microglia from B6 mice ( Figures 2A, B ). When treated with Aβ, Ca⁺⁺ mobilization of BV2 cells was significantly inhibited by TDCA in response to BzATP in a dose-dependent manner ( Figure 2C ; Supplementary Figure 5 ). ATP ( Figure 2D ) or BzATP ( Figure 2E ) increased Ca⁺⁺ mobilization of BV2 microglial cells in the presence of Aβ, and Ca⁺⁺ mobilization was significantly inhibited by TDCA (400 ng/ml).

It was reported that Aβ (2.5~5 µM) could induce caspase-1-mediated IL-1β secretion by microglia (48). When BV2 cells were treated with Aβ (2 µM) ± ATP (1mM), the transcript levels of NLRP3, ASC, pro-caspase-1, and proIL-1β were increased ( Figure 3A ). Although Aβ (2 µM) alone could increase these transcripts, ATP alone did not increase these transcript levels (but proIL-1β), suggesting that Aβ (2 µM) alone could activate priming phase of NLRP3 inflammasome. TDCA significantly inhibited the upregulation of these transcripts increased by Aβ ± ATP. In primary microglial cells, Aβ treatment significantly increased the expression of NLRP3 and ASC ( Figure 3B ). Treatment with Aβ + ATP further increased the expression of NLRP3. Aβ ± ATP treatment increased expression of NLRP3 and ASC in nucleus (white arrows) and in the cytosolic compartment (yellow, Figure 3B ). TDCA treatment significantly suppressed the expression of these two molecules, as well as their colocalization (yellow dots, Figure 3B ; Supplementary Figure 6 ). Interestingly, the levels of nuclear NLRP3 (white arrows) were significantly downregulated by TDCA treatment. Caspase-1 secretion from primary microglia treated with Aβ + ATP was inhibited by TDCA treatment ( Figure 3C ). In vivo, TDCA administration (1 mg/kg, i.p., q.d.) for 10 weeks decreased the expression of NLRP3 and ASC in the frontal cortex of 5xFAD mice compared with PBS-treated 5xFAD mice ( Figure 3D ; Supplementary Figure 7 ).

To elucidate how TDCA inhibits the transcription and expression of N3I components, we analyzed the GPCR19-cAMP-PKA-NF-kB axis after BV2 cells were treated with Aβ ± TDCA (39) ( Figures 3E–I ). TDCA increased cAMP production in BV2 cells irrespective of Aβ treatment ( Figure 3E ). Adenylyl cyclase inhibitor (KH7) blocked TDCA-mediated cAMP production in BV2 cells. BV2 cells increased the production and secretion of IL-1β, IL-18, and TNF-α upon treatment with Aβ ± ATP, while TDCA suppressed the production of these cytokines ( Figures 3F–H ). BV2 cells produced ROS in response to Aβ and was inhibited by TDCA treatment ( Figure 3I ).

NeuN⁺ cells in the brains of 5xFAD mice were stained after treatment with TDCA for 10 weeks (1 mg/kg, i.p., q.d., Figure 4 ). The frontal cortexes and hippocampi (CA1, CA3, and DG) were observed using confocal microscopy ( Figure 4A ). Number of NeuN⁺ cells in the frontal cortex were significantly higher in 5xFAD mice treated with TDCA than in those of 5xFAD mice treated with PBS ( Figure 4B ; Supplementary Figure 8A ). The mean fluorescent intensity (MFI) of the NeuN⁺ area in the hippocampi of 5xFAD mice was significantly higher in the TDCA group than in the PBS group ( Figure 4C ; Supplementary Figures 8B-D ). MFI of TUNEL+ apoptotic cells in the cortex were significantly higher in 5xFAD mice treated with PBS than in 5xFAD mice treated with TDCA ( Figure 4D ).

After i.p. administration of TDCA for 10 weeks, the numbers of Iba-1⁺ reactive microglia in the frontal cortex ( Figure 4E ; Supplementary Figure 9A ) and in the dentate gyrus (DG) ( Supplementary Figure 9B ) of the hippocampus decreased. The number of GFAP⁺ reactive astrocytes also significantly decreased in the frontal cortex ( Supplementary Figure 10A ), DG ( Supplementary Figure 10B ), and CA3 regions ( Supplementary Figure 10C ) of the hippocampus in 5xFAD mice upon treatment with TDCA. The numbers of Aβ plaques and the total area of plaques (white arrows) in the frontal cortex and in the hippocampus of 5xFAD mice decreased after TDCA treatment ( Figure 4F ). In vitro, TDCA treatment increased the phagocytosis of Aβ by BV2 cells ( Figure 4G ). The phagocytosed fluorescent Aβ oligomers (fAβ) colocalized with the LAMP-2 phagosomes marker in BV2 cells ( Figure 4G ; Supplementary Figure 11 ), suggesting fAβ oligomers are in the phagosomes, not on the cell surface.

After administration of TDCA (1 mg/kg, i.p., q.d.) for 10 weeks, the proteomes of brain tissues from 5xFAD mice were analyzed ( Figures 5A, B ). Total 3,259 unique proteins were identified at a protein threshold of a 1.0% false discovery rate (FDR). Among these proteins, 460 proteins showing peptide spectral counts in more than two assays from triplicate assays, with a fold change of more than 2 between PBS- and TDCA-treated groups, are depicted on the heatmap, plotted with the Perseus software platform (http://www.perseus-framework.org). Proteomic analysis indicated that 56 proteins exhibited more than 2-fold changes in the 95% confidence interval ( Figure 5A ), demonstrating two distinct proteome clusters that were upregulated in TDCA-treated groups and downregulated in PBS-treated groups, or vice versa. The functions of these proteins were further analyzed based on QIAGEN’s IPA database ( Figure 5B ). Notably, a canonical pathway, regulation of eIF4 and p70S6K, was enriched by TDCA treatment, suggesting that TDCA plays critical roles in translational regulation followed by calcium signaling, which could exert allosteric regulatory effects on many enzymes and proteins. Based on proteomic analysis, we further analyzed the transcript levels of several proinflammatory cytokines in both the hippocampi and cortices of 5xFAD mice after TDCA treatment for 10 weeks. TDCA downregulated IL-1β, TNF-α, IL-33, IL-12, CCL-11, and CCL-5 transcripts. In contrast, the TDCA treatment increased the transcript levels of IFN-γ, IL-10, CCL-17, GPCR19, CD47, FPR-2, CD36, SRB1, and SRA in the brains of 5xFAD mice ( Figure 5C ).

B6 and 5xFAD mice were administered with PBS or TDCA for 10 weeks (1 mg/kg, i.p., q.d.), and spatial learning and memory were assessed using the Morris water maze (MWM) test ( Supplementary Figure 12A ). There were no significant changes in body weight after TDCA treatment for 10 weeks ( Supplementary Figure 12B ). Escape latency decreased over the 4-day training period in all three groups. Mice in the TDCA group showed a significant reduction in time latency to reach the platform ( Figure 6A ), which was comparable to that of B6 mice. In the probe test on day 5, TDCA-treated 5xFAD mice showed increased numbers of platform crossings ( Figure 6B ). TDCA-treated 5xFAD mice spent more time on the platform than the PBS group ( Supplementary Figure 12C ). TDCA-treated 5xFAD mice remained for longer periods of time in the target quadrant than in the opposite quadrant compared with PBS-treated 5xFAD mice ( Figure 6C ). Times in the target quadrant and on the platform of 5xFAD-TDCA group were comparable to those of B6 mice. The movement of 5xFAD mice treated with PBS were relatively random and disorganized compared with those of B6 mice and TDCA-treated-5xFAD mice ( Figure 6D ).

Spatial learning and memory were also assessed using a Y-maze test and novel object recognition test (NOR). In the Y-maze test, the alternation percentage of the mice in the 5xFAD-PBS group was significantly lower than that of mice in the B6-PBS group and 5xFAD-TDCA group ( Figure 6E ). In the NOR test, exploration time to a new object compared to an old object was significantly higher in B6-PBS and 5xFAD-TDCA mice ( Figure 6F ). 5xFAD-PBS mice did not exhibit a difference in exploration time between old and new objects. The discrimination index for the mice in the 5xFAD-TDCA group was significantly higher than that for mice in the 5xFAD-PBS group ( Figure 6G ).

A total of eight postmortem brain samples were obtained ( Supplementary Table 1 ); five from individuals with clinically and neuropathologically diagnosed AD (Braak stages: V–VI) and three from nondemented controls. Brain sections were provided by the Korea Brain Bank Network (KBBN) operated through the National Brain Bank Project funded by the Ministry of Science and ICT. The project was approved by the Scientific Committee of the Brain Bank.

TDCA was purchased from New Zealand Pharmaceuticals Ltd. (Palmerston North, New Zealand). KH7 (Ann Arbor, Michigan, USA) was dissolved in DMSO. ATP and BzATP (Sigma–Aldrich, St. Louis, MO, USA) were dissolved in PBS.

A lyophilized 0.1 mg vial of amyloid β protein fragments 1-42 (Aβ; Sigma–Aldrich) was dissolved in 50 µl DMSO (Sigma–Aldrich) for 1 h at room temperature with continuous rotation. Oligomeric Aβ_1-42 was prepared by diluting the dissolved Aβ to 100 µM using DMEM/F12 media. The resulting solution was then incubated for 24 h at 4°C with continuous rotation.

Primary microglia were obtained from the cerebral cortices and hippocampi of male and female 1- to 2-day-old B6 mice or adult B6, GPCR19^-/-, and P2X7R^-/- mice (Jackson Laboratory, USA) using a mouse brain dissociation kit (Miltenyi Biotec GmbH, Bergisch, Gladbach, Germany). The tissue was cut into fragments using a scalpel blade (Medicom, Los Angeles, CA, USA) and then homogenized using a gentleMACS(TM) C tube and gentleMACSTM Octo Dissociator (Miltenyi Biotec GmbH). Single-cell suspensions were obtained after passing the tissue homogenates through MACSR Smart Strainers (70 µm; Miltenyi Biotec GmbH). The myelin was depleted using a debris removal solution supplied with the kit. Microglial cells were isolated using CD11b MicroBeads, LS Columns, and a MACS multistand system (all obtained from Miltenyi Biotec GmbH). Isolated microglia were washed with PB buffer (0.5% FBS in PBS) and cultured using DMEM-F12 media with 20% FBS and 10 ng/ml M-CSF (PeproTech, Rocky Hill, NJ) in a 95% air and 5% CO2 atmosphere at 37°C.

For inflammasome activation, primary microglial cells from 1- to 2-day-old B6 mice were seeded on 12-mm microscope cover glasses (Deckglaser, Luda-Konlgshofen, Germany) in 24-well PDL-coated cell culture plates (Corning, Wujiang, Jiangsu, China) at 7.5 x 10⁴ cells/ml per well for 24 h, serum starved for 10 h and treated with Aβ (2 µM), TDCA (400 ng/ml) for 24 h, and ATP (1 mM) for the final hour.

BV2 cells (Murin microglial cell line) were maintained in a 95% air and 5% CO₂ atmosphere at 37°C in DMEM (Dulbecco’s modified Eagle’s medium complete media; Invitrogen, Carlsbad, CA, USA) containing 10% FBS (Fetal Bovine Serum; Invitrogen) and 1% penicillin and streptomycin (Invitrogen). BV2 cells were seeded in 6-well (1.5 x 10⁵ cells/2 ml), 12-well (1 x 10⁵ cells/ml), or 24-well (5 x 10⁴ cells/0.5 ml) PDL-coated cell culture plates for 12 h using DMEM complete media. Cells were serum starved before treatment for 10 h using DMEM/F12 media (Thermo Fisher Scientific, Waltham, MA, USA). Cells were treated with Aβ (2 µM), TDCA (400 ng/ml), and ATP (1 mM) for 1 h in the case of GPCR-19–P2X7R interaction. For inflammasome activation and inhibitor (KH7) assays, cells were treated with Aβ (2 µM), KH7 (4 µM), TDCA (400 ng/ml) for 24 h, and ATP (1 mM) for the final hour. DMEM/F12 medium was used unless stated otherwise. The cell-lysed supernatants were used to check the concentrations of cAMP, and cell culture supernatants were used to analyze the concentrations of IL-1β, IL-18, TNF-α, and caspase-1 using ELISA.

Adult mouse microglial cells isolated from adult B6, GPCR19^-/-, and P2X7R^-/- mice were seeded on 25-mm cover glasses (Deckglaser) in a 6-well plate. After 48 h of culture with DMEM/F12 medium containing 20% FBS and 10 ng/ml M-CSF in a 95% air and 5% CO₂ atmosphere at 37°C, cells with glass coverslips were transferred and loaded with 2 µM Fluo-4/AM for 30 min at 37°C in a physiological external solution consisting of 138 mM NaCl, 5.6 mM KCl, 0.5 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). After loading, cells on the coverslips were transferred to an open perfusion chamber, and fluorescence was measured at 494/506 nm using a fluorescence microscope (Nikon, Tokyo, Japan). The microscope was equipped with an LED lamp (Andover, UK), integrated shutter, and cooled EM-CCD camera. The camera and shutter were controlled using MetaMorph software (Molecular Devices, Foster City, CA). Single cells were defined as regions of interest (ROIs). Sixteen-bit grayscale images with a binning of 1 x 1 were captured every 1 s with a ranging exposure time. Data were processed using OriginPro 8 software (OriginLab) and merged from three independent experiments.

BV2 cells were seeded on 25-mm cover glasses in a 6-well plate. After starvation, cells were treated with Aβ (2 µM) with or without TDCA (400 ng/ml) for 24 h. Cells with glass coverslips were transferred and loaded with 2 µM Fluo-4/AM for 30 min at 37°C in the physiological external solution mentioned above. After loading, the cells were transferred to an open perfusion chamber, and fluorescence was measured at 494/506 nm as previously described.

BV2 cell Ca⁺⁺ sensing was measured using a BD calcium assay kit (BD Bioscience, San Diego, CA, USA) according to the manufacturer’s protocol. In a 6-well plate, 1.5 x 10⁵ cells/2 ml media were treated with Aβ with or without TDCA for 24 h after serum starvation. Cells were harvested from the cell culture plate in a FACS tube, washed with complete RPMI media, loaded with loading dye, and incubated for 1 h at 37°C in a 5% CO₂ atmosphere. Cells were acquired for 1 min for basal signaling, incubated with BzATP (300 µM) for 2 min, and recorded for an additional 4 min using flow cytometry (LSRFortessa, BD Biosciences, San Jose, CA, USA). Relative fluorescent units (RFUs) were calculated using FlowJo version 9.0 (Treestar, Ashland, OR, USA).

Cortices were harvested from 3 sets of B6 (3-month-old) and 5xFAD (3-, 4-, 6-, and 9-month-old) mice, lysed with RIPA buffer (Thermo Fisher Scientific, Meridian Road, Rockford, USA) supplemented with phosphatase inhibitor cocktail 2 (Sigma–Aldrich), and centrifuged to remove cell debris. The concentrations of the prepared protein samples were determined using a BCA kit (Thermo Fisher Scientific). Protein samples were separated by electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred electrophoretically to immobilon, polyvinylidene-difluoride membranes (Merck, Millipore, Billerica, MA, USA). The membranes were incubated at 4°C overnight with anti-GPCR19 polyclonal antibody (Novus Biologicals, Littleton, CO, USA), P2X7R monoclonal antibody (Clone 1F11, Biolegend, San Diego, CA, USA), and anti‐β‐actin (Sigma–Aldrich). The following day, the membranes were washed and then incubated with horseradish peroxidase‐labeled anti‐rabbit or anti‐mouse secondary antibodies for 1 h at room temperature. Subsequently, membrane‐bound horseradish peroxidase‐labeled antibodies were detected using an enhanced chemiluminescence detection system, including the Pierce ECL Western Blotting Substrate kit (Thermo Fisher Scientific). Densitometric quantification of the bands was conducted using ImageJ software (Rasband, W.S., NIH, Maryland, USA). Protein levels were normalized to β‐actin for quantification.

BV2 cells or primary microglial cells isolated from adult mouse brains were seeded on 12-mm microscope cover glasses (Deckglaser) in 24-well plates. Cells were stained with anti-GPCR19 polyclonal antibody (Novus Biologicals) and P2X7R (Clone 1F11, Biolegend) at 4°C overnight, followed by staining with goat anti-rabbit IgG, Alexa Fluor 488 (Thermo Fisher Scientific), and goat anti-rat IgG, Alexa Fluor 546 (Invitrogen) for 1 h at RT.

To stain for inflammatory components, primary microglial cells isolated from the brains of 1- to 2-day-old B6 mice were seeded on 12-mm microscope cover glasses in 24-well plates and treated with Aβ (2 µM), TDCA (400 ng/ml) for 24 h, and ATP (1 mM) for the final hour. The cells were permeabilized and stained with anti-NLRP3 polyclonal antibody (Abcam, Cambridge, United Kingdom) and anti-ASC antibody (Clone B-3, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA) at 4°C overnight, followed by staining with secondary polyclonal antibodies, Alexa Flour 488-labeled goat anti-rabbit IgG, or Alexa Flour 532-labeled goat anti-mouse IgG (Invitrogen) for 1 h at RT.

After rinsing with secondary polyclonal antibodies, cover glasses were placed on DAPI mounting solution (Vector laboratories, Burlingame, CA, USA) on glass slides. Fluorescence imaging was performed using a Confocal Microscope A1 (Nikon, ECLIPSE Ti, New York, USA). The colocalization percentage of NLRP3-ASC was measured using ImageJ software after setting the color threshold caliper to only yellow. NIS-Elements.AR. Ink (version 4.2, Nikon) was used to measure the mean fluorescence intensity (MFI) of the ROIs.

Tissue or cells were harvested after treatment, and total RNA was isolated using an RNeasy Plus Mini kit (QIAGEN, Hilden, Germany). cDNA was prepared from 1 µg of total RNA using a Maxime RT PreMix kit (iNtRON Biotechnology, Gyeonggi-do, South Korea). Thereafter, real-time quantitative PCR (qPCR) was performed using SYBR Green Fast mix (Applied Biosystems, Woolston, Warrington, UK) and primers specific to target genes ( Supplementary Table 2 ) in the StepOnePlus™ Real-Time PCR system (Applied Biosystems, Marsiling Industrial Estate Road, Singapore). The expression levels of target mRNAs were analyzed using the ddCt method and were normalized to the expression levels of mouse GAPDH, a housekeeping gene used as an endogenous control. All fold changes are expressed relative to the control group.

Cell-secreted cytokines were measured from the cell culture supernatant using commercially available ELISA kits for mouse TNF-α, IL-1β/IL-1F2 (R&D Systems, Minneapolis, MN, USA), IL-18 (MBL, Naka-Ku, Nagoya Aichi, Japan), and Caspase-1 (Novus Biologicals) according to the manufacturer’s protocols. BV2 cells were cultured in the presence of Aβ (2 µM), TDCA (400 ng/ml), or KH7 (4 μM) for 24 h. Cells were harvested and lysed with 0.1 M HCl to measure intracellular cAMP using a cAMP assay kit (Abcam) according to the manufacturer’s protocol.

BV2 cells were harvested in a FACS tube after treatment with Aβ (1~4 µM) and TDCA (400 ng/ml) for 24 h. Cells were washed with DPBS and incubated with 5 μM 2′-7′-dichlorofluorescin diacetate (Invitrogen, Eugene, Oregon, US) in DMEM containing 1% penicillin and streptomycin for 30 min at room temperature in the dark. Samples were washed in DPBS, suspended in FACS buffer containing DAPI (0.3 µg/ml), immediately acquired and analyzed using a BD LSR Fortessa flow cytometer (BD Bioscience, San Jose, CA, USA) and FlowJo v9 software.

Primary microglia isolated from 1- to 2-day-old B6 mice were cultured, pretreated with TDCA (400 ng/ml) for 12 h, and then incubated with fAβ (1–42) oligomer (green) for 3 h. After incubation with fAβ, the cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde (PFA). After blocking, the cells were incubated at 4°C overnight with a primary antibody against P2X7R (BioLegend) or control IgG. The cells were then rinsed and incubated with goat anti-mouse IgG (H+L) secondary antibody (Invitrogen) for 1 h at room temperature in the dark. After rinsing, cover glasses were placed on the DAPI mounting solution on the glass slides. Fluorescence imaging was performed using a confocal microscope A1. NIS-Elements.AR. Ink (version 4.2, Nikon) was used to measure the MFI of ROIs.

BV2 cells were seeded on microscope cover glasses in 24-well plates for confocal microscopy. Cells were treated with or without TDCA for 12 h. HilyteFluor™ 488-labeled Aβ (1-42; AnaSpec, Freemont, CA, USA) was used for fAβ oligomers, as mentioned above. After pretreatment with TDCA, the cells were incubated with fAβ oligomers for 3 h, fixed with 4% paraformaldehyde (PFA), and permeabilized with 0.3% Triton X-100. After blocking, the cells were incubated at 4°C overnight with primary antibody for LAMP2 (Clone M3/84, BD Pharmingen Inc., San Jose, CA, USA) or control rat IgG. After rinsing, the cells were incubated with goat anti-rat IgG, Alexa Fluor 546 (Invitrogen) for 1 h at room temperature in the dark. After rinsing, cover glasses were placed on the DAPI mounting solution on the glass slides. Fluorescence imaging was performed using a confocal microscope A1. The colocalization percentage of fAβ-LAMP2 was measured using ImageJ software after setting the color threshold caliper to only yellow.

The 5xFAD mice co-overexpress high levels of APP with three FAD mutations (Swedish (K670N/M671L), Florida (I716V), and London (V717I)) and high levels of presenilin 1 (PSEN1) with two FAD mutations (M146L, L286V), which are specifically overexpressed in the brain and regulated by the neural-specific Thy1 promoter (62). 5xFAD mice were maintained by breeding male 5xFAD mice with female B6 mice. The SJL F1 hybrid was produced by an SJL male and a B6 female. PCR was performed to genotype the mice. 5xFAD or SJL mice were kindly provided by Professor Mook-Jung, In-hee or Professor Sung, Jung-Joon, respectively, of Seoul National University. GPCR19^-/- mice (C57BL/6-Gpbar1^{tm1(KOMP)Vlcg} mice) and P2X7R^-/- mice (C57BL/6. 129P2-P2rx7^tm1Gab/J) used in this study were purchased from Jackson Laboratory. All animal experiments were approved by the institutional animal care and use committees (IACUC) of Seoul National University (SNU-170517-25) and performed in accordance with animal ethics regulations. Mice were maintained in specific pathogen-free conditions at the animal facility of the Wide River Institute of Immunology.

Male and female 5xFAD mice (8 to 10 weeks old) were injected intraperitoneally (i.p.) with either 1 mg/kg TDCA or PBS five times/week for 10 weeks. Age- and gender-matched nontransgenic littermates were used as a control group. Behavioral tests were performed after treatment, and then mice were sacrificed for further experimentation.

The maze was composed of a circular pool (1.5 m in diameter, 80 cm in height) with spatial cues at three different locations. Before testing, the pool was filled with opaque water adjusted to 20 ± 1 °CC. On the first day, mice were allowed to freely swim in the water for 60 sec to find the escape platform located in one quadrant of the pool. When the mice failed to find the platform, they were guided to the platform. Once on the platform, mice were allowed to remain there for 30 sec. From the next day for 4 consecutive days, the same procedure was repeated from three different starting points to train the mouse, and the time to reach the platform was recorded every day. After the 4-day training period, the probe test was performed in the same manner but without the platform. Each mouse was allowed to swim from one starting point for 60 sec, which was recorded using a video camera. The video was analyzed for the movement of mice in the water using tracking software (SMART3.0, Panlab Harvard Apparatus, Barcelona, Spain) to count the number of crossings and the time on the platform and to measure the time spent in each quadrant of the pool.

Mouse functional behavior tests were performed on TDCA- or PBS-treated 5xFAD mice and WT (B6) control mice. The Y-maze test was assessed over the course of four days. On the first 2 days, individual mice were habituated to the task room and experimenter for 5 min. On the third day, after the task room and experimenter habituation, mice were allowed to habituate to the Y-maze for less than 1 min. On the last day, mice entered the middle of the Y-maze and were allowed to move freely within the maze for 8 min. Each mouse movement was recorded using a video camera. The video was analyzed for all mouse entries regarding limbs that pass through each half arm of the maze. Total arm entries and the percentage of alteration were counted for each mouse and compared between groups of mice.

NORT was assessed over the course of 4 days. The apparatus consisted of a white acrylic box (350 mm x 450 mm x 250 mm). The basement of the box was divided into six equal rectangles. On the first 2 days, each mouse was habituated to the box for 10 min. On the third day, two similar cylindrical objects were fixed in the box, and mice were allowed to explore the objects freely for 10 min. On the last day, one cylindrical object was replaced by a similarly sized object, and the mouse explored this for 5 min. Exploration of two cylindrical objects on the third day and exploration of the novel object and cylindrical object on day 4 were recorded for each mouse using a video camera. The total time and frequency of novel and old object exploration were counted for each mouse using video footage. The percent discrimination index of the novel object was calculated from the exploration time of novel and old objects.

Human samples of brain paraffin tissue sections (4 µM) from KBBN were used for cell surface GPCR19 and P2X7R expression. Tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series (100%, 90%, 80%, and 70%). Antigen unmasking was performed by heating the brain sections in citrate-based buffer (pH 6, Vector laboratories, Burlingame, CA, USA). The sections were blocked with a blocking solution of 10% normal donkey serum (Thermo Fisher Scientific) and 1% BSA in PBS for 1 h. Tissue samples were incubated overnight at 4°C with GPCR19 (Novus Biologicals) and P2X7R (Novus Biologicals) primary antibodies. Slices were subsequently incubated for 1 h at room temperature with Donkey anti-Rabbit Alexa 488 (Invitrogen) or Donkey anti-Goat Alexa 555-conjugated (Invitrogen) IgG secondary antibodies and then counterstained with DAPI for 10 min.

TDCA- or PBS-treated 5xFAD or B6 mice were anesthetized and perfused with ice-cold PBS. Brains were harvested and maintained in 10% neutral buffer formalin (Sigma–Aldrich) for 24 h at 4°C and then embedded in paraffin (Lecia, Illinois, USA). The paraffin-embedded brains were cut (3 μM) using a microtome (Thermo Fisher Scientific), deparaffinized in xylene, and rehydrated in a graded ethanol series (100%, 90%, 80%, and 70%). Antigen unmasking was performed by heating the brain sections in citrate-based buffer (pH 6, Vector laboratories, Burlingame, CA, USA). The sections were incubated in 0.3% Triton X-100 for 30 min at room temperature for intracellular staining and then blocked with a blocking solution of 10% normal goat serum (Thermo Fisher Scientific) and 1% BSA in PBS for 1 h. Tissue samples were incubated overnight at 4°C with the following primary antibodies: GPCR19 (Novus Biologicals), P2X7R (Clone 1F11, Biolegend), NLRP3 (Abcam), ASC (Santa Cruz Biotechnology), NeuN (Clone A60, Merck Millipore, Temecula, CA, USA), and GFAP (Thermo Fisher Scientific). Slices were subsequently incubated for 1 h at room temperature with Alexa 488-, 532-, or 546-conjugated IgG secondary antibodies, as appropriate, and then counterstained with DAPI for 10 min.

For apoptotic cell (Tunnel+) detection, frozen tissue sections from TDCA- or PBS-treated 5xFAD mice were fixed using 4% neutral buffer formalin in TBS for 20 min at RT. After washing, the TBS slides were incubated with 0.1% Triton X-100 for 3 min. The TUNEL assay was performed according to the manufacturer’s instructions (Roche, In-Situ Cell Death Detection TMR, red) and then counterstained with DAPI.

For Iba-1 (Wako, Osaka, Japan) staining, a frozen section was prepared from a separate group of TDCA- or PBS-treated 5xFAD or B6 mice. Perfused brains were cryopreserved in a phosphate-buffered 30% sucrose solution for 24 h. The brains were then frozen on dry ice, cut on a cryostat into sequential 3 μm sections, and stored at −80 °C until processed for analysis.

The Aβ core plaque was labeled by treating brain tissues with 1% thioflavin-S (Sigma–Aldrich) in PBS for 10 min at room temperature after being deparaffinized in xylene and rehydrated in a graded ethanol series (100%, 90%, 80%, and 70%). The tissue slides were then washed three times with 70% ethanol following DW three times. Fluorescence imaging was performed using a Confocal Microscope A1 (Nikon). NIS-Elements.AR. Ink (version 4.2, Nikon) was used to measure the MFI of the ROI.

Anesthetized mice were perfused with cold PBS, and the whole brain was extracted, followed by snap-freezing using liquid nitrogen. The frontal cortex and hippocampus region of each mouse were collected and digested using urea- (8 M) based in-solution digestion. After protein quantification using the BCA assay (Micro BCA Protein Assay Kit, Thermo Fisher Scientific, Bremen, Germany), samples were pooled together group by group, and 300 µg of tissue extracts per group was separated via high pH reversed-phase fractionation using an Agilent 1260 HPLC infinity purification system (Agilent Technology, Santa Clara, CA, USA). The fractionated peptide samples were subsequently loaded onto traps (C18, 3 μm, 0.7 cm, Thermo Fisher Scientific) and EASY-Spray columns (C18, 2 µm, 100 Å, 50 cm, Thermo Fisher Scientific). Easy nano II Ultra Performance Liquid Chromatography and Q-Exactive Mass Spectrometry systems (Thermo Fisher Scientific) were used to separate the peptides. For Q-Exactive, a top 10 method was used. The Orbitrap mass analyzer was used to acquire full MS scans (m/z 300–1,600 range; resolution, 70,000). The Trans Proteomic Pipeline (Seattle Proteomic Center, Seattle, WA, USA) was used to convert the mass data files into mzXML files. Peptide masses were searched using a concatenated forward and reverse mouse international protein index (IPI) database (Decoy ipi.MOUSE.v3.80 database, 54285 entries) (63) with the SEQUEST-Sorcerer platform (Thermo Fisher Scientific, Sage-N Research, Milpitas, CA). Sorcerer (Sage-N Research, Milpitas, CA, USA) was used to estimate the FDR. Scaffold Q+ (Proteome Software, Portland, OR, USA) was used to compare spectral counts, validate MS/MS-based peptides, identify proteins (FDR < 1% in at least 2 peptides), and calculate Log2-fold changes (FC) and P values (Student’s t-test). Differentially expressed proteins (FC > 2, and P values < 0.05) were uploaded to the web-based Ingenuity Pathway Analysis (IPA^®) software (Version 26127183; QIAGEN) for functional analysis and protein interaction networks.

The data are expressed as the mean ± SEM and were analyzed using a two-sided, unpaired Student’s t-test. The mean value between groups was compared using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA) unless otherwise indicated. P values < 0.05 were considered statistically significant.