Isobavachalcone ameliorates Alzheimer disease pathology by autophagy-mediated clearance of amyloid beta and inhibition of NLRP3 inflammasome in 5XFAD mice

doi:10.21203/rs.3.rs-3762830/v1

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Isobavachalcone ameliorates Alzheimer disease pathology by autophagy-mediated clearance of amyloid beta and inhibition of NLRP3 inflammasome in 5XFAD mice

https://doi.org/10.21203/rs.3.rs-3762830/v1

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The complex pathology of Alzheimer disease (AD) has led to failure of multiple drugs in clinical trials. We hypothesized that targeting multiple aspects AD pathology may yield better results. Therefore, we identified isobavachalcone (IBC) as a natural compound with dual activity against AD pathology. IBC caused AMPK phosphorylation through CAMKK2 to induce autophagy and inhibit NLRP3 inflammasome in primary astrocytes. The inhibition of NLRP3 inflammasome by IBC was completely reversed when autophagy was inhibited by siAMPK or bafilomycin A1. Further, the primary astrocytes treated with IBC showed a significant intracellular clearance of amyloid beta, which was added externally to the culture. However, when autophagy was inhibited by siRNA-mediated downregulation of AMPK, the clearance of amyloid beta was significantly reduced. Moreover, the inflammatory phenotype of astrocytes also displayed a sharp decline, as indicated by the reduced levels of GFAP and IL-1β. We validated the anti-Alzheimer effect of IBC by treating transgenic 5XFAD mice for two months with IBC. The 5XFAD mice showed a significant improvement in brain health as indicated by improved memory behavior in the radial arm maze test, along with better performance in the open field and rotarod tests. The data revealed that IBC upregulated the autophagic proteins, which led to decreased levels of amyloid beta in the brain and plasma, which further led to reduced levels of neuroinflammation and improved brain health. This study highlights the importance of autophagy in the simultaneous clearance of amyloid beta and inhibition of NLRP3 inflammasome to ameliorate AD pathology.

Alzheimer disease

AMPK pathway

amyloid-beta

autophagy

neuroinflammation

NLRP3 inflammasome

Alzheimer disease (AD) is the most frequently occurring age-related neurodegenerative disorder. The AD symptoms include cognitive decline and behavioral and psychological instability (Kumar et al., 2023). The disease progresses with unregulated deposition of amyloid-beta (Aβ) protein plaques, which facilitate the formation of neurofibrillary tangles (NFTs) by causing tau protein hyperphosphorylation. These pathological anomalies in the brain lead to the innate immune response by glial cells, causing neuroinflammation (Rajmohan and Reddy, 2017; Al-Ghraiybah et al., 2022). The aberrant activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome by Aβ plaques, tau fibrils, and neuronal death is also the major contributor to AD pathogenesis (Van Zeller et al., 2021).

The dysfunctions in the autophagy-lysosomal pathway, the major cellular pathway for clearance of accumulated damaged proteins and organelles, are conducive to the progression of AD (Lee et al., 2022). The Aβ deposition and NLRP3 inflammasome activation can be regulated by inducing macroautophagy/autophagy in the glial cells (Wen et al., 2019; Mishra et al., 2021). By eliminating the sensor NLRP3 ligands like reactive oxygen species (ROS) generating damaged mitochondria and particulate matter from the cell, autophagy is known for regulating NLRP3 inflammasome over-activation. Enhancing autophagy induction can also effectively reduce the Aβ burden from the cell. Therefore, inducing autophagy and inhibiting NLRP3 inflammasome can be a promising therapeutic target for AD.

NLRP3 inflammasome is the component of innate immunity that is activated in response to varied endogenous and exogenous ligands. The NLRP3 inflammasome complex comprises three proteins: NLRP3, the adaptor ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and the protease pro-caspase-1 (Kelley et al., 2019). The assembly formation of NLRP3 inflammasome completes with self-activation of pro-caspase 1 into effector caspase-1 (CASP1), which thereby causes cleavage and secretion of pro-inflammatory cytokines, interleukin-1β (IL-1β) and interleukin-18 (IL-18). The IL-1β produced by NLRP3 inflammasome has detrimental effects on AD patients. The secretion of neurotoxins by NLRP3 inflammasome activation results in neuronal degeneration, decreased Aβ clearance and expedited tau tangle formation. It can mediate a positive feedback loop, which amplifies disease symptoms (Saresella et al., 2016; Ising et al., 2019; Luciunaite et al., 2020).

In this study, we have identified naturally occurring chalcone, isobavachalcone (IBC) as the potent autophagy inducer, which, by inducing AMPK (5’ adenine monophosphate-activated protein kinase) dependent autophagy can inhibit NLRP3 inflammasome and Aβ deposition. IBC (Fig. 1A) can be procured from the seeds of the Psoralea corylifolia plant and is rich in varied pharmacological properties, including anti-inflammatory, anti-cancer, neuroprotective, and antioxidative effects (Wang et al., 2021). Our data suggests that IBC and other compounds that simultaneously target two critical pathways involved in disease progression can act as effective candidates for drug development against AD. This approach of pharmacologically targeting NLRP3 inflammasome-induced neuroinflammation and Aβ deposition can alleviate AD manifestation.

Chemicals and Reagents

Chemicals- Dulbecco’s modified eagle medium (Sigma-Aldrich, D1152), RPMI-1640 (Sigma-Aldrich, R6504), Streptomycin (Sigma-Aldrich, S6501), Penicillin (Sigma-Aldrich, P3032), Triton X-100 (Sigma-Aldrich, T8787), Sodium bicarbonate (Sigma-Aldrich, S5761), Phosphate buffer saline (Sigma-Aldrich, D5652), Lipopolysaccharide (Sigma-Aldrich, L3129), ATP (Sigma-Aldrich, A6419), EDTA (Invitrogen, 15575038), Fetal Bovine Serum (GIBCO, 10270106), Acrylamide (MP Biomedical, 193982), Glycine (MP Biomedical, 194825), Albumin Bovine Fraction V (MP Biomedical, 160069), Phenylmethylsulfonyl fluoride (MP Biomedical, 195381), Skimmed milk (Himedia, GRM1254), Hanks’ Balanced Salt Solution (Sigma-Aldrich, H6648), Strataclean resin (Agilent, 400714-61), bafilomycin A₁ (Sigma-Aldrich, B1793), Rapamycin (Sigma-Aldrich, R8781), Resveratrol (Sigma-Aldrich, R5010), H₂DCFDA (Sigma-Alrdrich, D6883), DAPI (Sigma-Aldrich, D9542), glycerol (Sigma-Aldrich, G5516), Tween 20 (Sigma-Aldrich, P7949), HEPES (Sigma-Aldrich, H3375), Paraformaldehyde (Sigma-Aldrich, P6148), MTT (Sigma-Aldrich, M5655), Trizma (Sigma-Aldrich, T6066), SDS (Sigma-Aldrich, L3771), Uric acid sodium salt (Sigma-Aldrich, U2875), Suberic acid (Sigma-Aldrich, S1885), OPTI-MEM media (Gibco, 11058-021), β-Amyloid (Anaspec, AS-60479).

Antibodies- BECN1 (Santa Cruz Biotechnology, SC-48341), ATG5 (Santa Cruz Biotechnology, SC-133158), ASC (Santa Cruz Biotechnology, SC-22514), CASP1 (Santa Cruz Biotechnology, SC-56036), ATG7 (Santa Cruz Biotechnology, SC-33211), BCL-2 (Santa Cruz Biotechnology, SC-7382), LAMP1 (Santa Cruz Biotechnology, SC-20011), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, SC-2354), AMPK (Cell Signaling Technology, 2532S), pAMPK (Cell Signaling Technology, 2535S), NLRP3 (Cell Signaling Technology, 15101S), siRNA AMPK (Cell Signaling Technology, 6620S), MTOR (Cell Signaling Technology, 2972S), pMTOR (Cell Signaling Technology, 5536S), pCAMKK2 (Cell Signaling Technology, 12818S), pULK1 (Cell Signaling Technology, 14202S), FIP200 (Cell Signaling Technology, 12436S), GFAP (Cell Signaling Technology, 80788S), IBA1 (Cell Signaling Technology, 17198S), NeuN (Cell Signaling Technology, 12943S), HRP-linked anti-rabbit IgG (Cell Signaling Technology, 7074S), HRP-linked anti-mouse IgG (Cell Signaling Technology, 7076S), anti-mouse IgG Alexa flour 488 (Cell Signaling Technology, 4408S), anti-mouse IgG Alexa flour 555 (Cell Signaling Technology, 4409S), anti-rabbit IgG Alexa flour 488 (Cell Signaling Technology, 4412S), anti-rabbit IgG Alexa flour 555 (Cell Signaling Technology, 4413S), (anti-mIL-1β (R & D biotechnology, AF-401-NA), anti-ACTB (Sigma-Aldrich, A3854), ANTI-LC3B-II (Sigma-Aldrich, L7543), Anti-SQSTM1/p62 (Sigma-Aldrich, P0067).

Kits and other reagents- PVDF Membrane (Millipore, ISEQ00010), ECL-kit (Millipore, WBKLS0500), Precision plus protein markers (Bio-Rad, 161–0375), Bradford reagent (Bio-Rad, 500-0006), FuGENE HD (Promega, E2313), IL- 1β ELISA kit (Invitrogen, 88-7013-88), IL- 18 ELISA kit (Invitrogen, 88-50618-88), Human Aβ42 ELISA kit (Invitrogen, KHB3441)

Preparation, fractionation and Isolation of Isobavachalcone from Psoralea corylifolia extracts

The seeds of the plant Psoralea corylifolia were collected from the local market of Jammu, J&K, India. The dried seeds (2.7 kg) of the plant were coarsely powdered by grinding and extracted with a mixture of DCM (dichloromethane): MeOH (methanol) (1:1, 15 L) at room temperature for 24 h and filtered. The marc was reextracted twice with the same solvent mixture using the same volume ratio of material to solvent. The filtrates were combined and concentrated on a rotary evaporator under reduced pressure at 40^°C to yield 322.5 g crude extract with an extractive value (EV) of 11.9%.

DCM: MeOH (1:1) extract was fractionated sequentially with three different solvents, viz hexane, dichloromethane and methanol by liquid-liquid partition chromatography to yield an enriched fraction of isobavachalcone. 300 g of the dried extract was dissolved in 1.5 L methanol and partitioned with 2 L hexane and this whole procedure was repeated two more times. After separating the upper hexane layer, 1.5 L DCM and 150 mL distilled water were added to the remaining methanolic layer for partitioning and the process was repeated two more times for maximum enrichment. All three fractions; hexane (108.4 g), DCM (119.7 g), and aqueous methanol (69.8 g) thus obtained were monitored by thin layer chromatography (TLC). TLC analysis concluded that the DCM fraction had the highest concentration of isobavachalcone followed by the hexane fraction.

Preferentially, the DCM fraction (100 g) was charged on a silica-gel column (60–120 mesh) and eluted with a gradient of n-hexane-ethyl acetate (95:5–50:50, v/v) which provided several subfractions. These subfractions were further analyzed by TLC using solvent system n-hexane/ethyl acetate (70:30). Based on the identification of isobavachalcone on TLC plates, the subfractions are pooled together and charged on silica gel column (60–120) to furnish the desired compound that is isobavachalcone (2.1 g).

Primary astrocytes culture

Primary astrocytes were cultured from mice pups with approval from the Institutional Animal Ethics Committee (IAEC) of CSIR-IIIM, Jammu, under the IAEC number 287/80/2/2022 and 305/81/8/2022.

Astrocytes were isolated from the brain of 3–4 days old C57BL/6J mice pups and were grown in 10% FBS-supplemented DMEM media. Briefly, pups were sacrificed in a CO₂ chamber and the brain was dissected. The cortical region separated using a fine blade was collected in incomplete media and cut into small pieces. After that, these cortices were incubated at 37˚C in 0.25% trypsin for 30 min, with shaking after every 10 min. Trypsin activity was neutralized with complete media and cells were collected using a 70-micron cell strainer followed by centrifugation at 300 g for 5 min. The pellet was dissolved in fresh media and cells were grown in cell culture flasks and were maintained at 37˚C in a CO₂ incubator. Microglia and oligodendrocytes were separated from the underlying astrocyte layer after 6–7 days by shaking the flask for 6 h.

NLRP3 inflammasome activation

Primary astrocytes were first treated with microglia conditional media to induce astrogliosis (Liddelow et al., 2017). For this, N9 cells were treated with 1 µg/ml LPS, and supernatant was collected after 24 h. Seeded astrocytes were then treated with this supernatant for 24 h. Thereafter, the treatment for NLRP3 inflammasome activation was given, including priming with fresh 1 µg/ml LPS for 4 h, followed by washing and IBC or MCC950 treatment in incomplete media (media without fetal bovine serum). ATP at 5 mM concentration was used as secondary signal for 30 min.

The experiment was terminated by collecting the cell supernatant.

Cytokines measurement by ELISA

After the termination of experiments, the supernatant was collected and analyzed for levels of cytokines. For measuring IL-1β and IL-18, supernatant collected after NLRP3 inflammasome activation as mentioned above, was subjected to ELISA. The manufacturer’s protocol was followed for measuring the cytokines. The levels of measured cytokines were normalized with the total protein content of the cells. For total protein content of cell pellet lysed with lysis buffer (0.2 N NaOH and 1% Triton-X 100) was measured.

ASC oligomer formation

Following treatment for NLRP3 inflammasome activation in primary astrocytes, cells were lysed with ice-cold buffer (KCl [150 mM], HEPES-KOH [20 mM, pH 7.5], NP-40 [1%], sodium orthovanadate [1 mM], PMSF [0.1 mM] and protease inhibitor cocktail [1%]). Cells were centrifuged at 300 g for 10 min. The supernatant was collected for western blot analysis of total ASC protein. The pellet was washed with cold PBS and resuspended in 500 µL cold PBS. For the crosslinking of ASC, 2 mM of suberic acid was added to the resuspended pellet and incubated at 37°C for 30 min in water bath. After centrifugation, the supernatant was discarded, and 2X laemmli buffer was added to the pellet. The samples were heated at 95˚C for 5 min and subjected to western blotting for analyzing ASC oligomers.

Autophagy flux measurement

For measuring autophagy flux astrocytes were treated with different concentrations of IBC for 24 h, with or without autophagy inhibitor, bafilomycin A₁. Bafilomycin A₁ was added 3 h prior to the termination of the experiment at 20 nM concentration. Levels of LC3B-II and SQSTM1 proteins were measured in cell lysates using an immunoblotting assay.

Immunoblotting

Levels of cleaved IL-1β and caspase-1 were analyzed in cellular supernatant. The supernatant collected after the end of the experiment was incubated with startaclean resin for 1 h at 4˚C. Samples were kept on rotation so that the resin could bind properly with proteins. The concentrated resin pellet was obtained by centrifuging samples at 300 g for 10 min. 2X laemmli buffer was added to the resin pellet, heated at 95˚C for 10 min and subjected to western blotting.

Cellular lysates were prepared using radio immuno precipitation assay (RIPA) buffer for protein analysis. Briefly, the cell pellet was dissolved in RIPA buffer containing sodium fluoride, sodium orthovanadate, phenyl methyl sulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail for 1 h with vortexing sample every 15 min. The supernatant was collected after high-speed centrifugation of samples for 20 min at 4˚C. Total protein was measured using Bradford assay. Proteins were separated using SDS-PAGE and then transferred from gel to PVDF membrane. The protein of interest was labeled with a specific primary antibody overnight at 4˚C. Further, the blot was incubated with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. The band were visualized in the GenaxyChemiDoc imaging system (Make: Syngene, Maryland, USA; Model: G: BOX, XT-4). Densitometry analysis of all the immunoblots was performed by using Image J software. Band density of proteins were normalized with β-actin. The phosphorylated proteins were normalized with their total forms.

Confocal microscopy

Cells were seeded on coverslips in 6 well plates. After treatments, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.1% Triton-X 100 for 7 min. Blocking was done for 30 min with 2% BSA, followed by overnight incubation with primary antibody. Alexa flour conjugated secondary antibody incubation was given to cells for 1 h for labeling. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) (1µg/ml) for 10 min. Following staining, the coverslips were mounted on slides using mounting media (PBS and glycerol at a ratio of 1:9). Cells were washed thrice with PBS after each incubation. The images were acquired in a CQ1 high-throughput imaging system.

Aβ clearance assay

Aβ₄₂-HiLyte flour488 peptide was prepared following the manufacturer’s protocol (Anaspec Inc.). Primary astrocytes seeded on coverslips were treated with IBC or rapamycin. After 12 h, 2 µg/ml fluorochrome tagged (Hilyte flour488) Aβ₄₂ protein was added to cells for another 12 h. Bafilomycin A₁ was given 3 h prior to experiment termination. After the end of the assay, cells were washed with PBS and cells were fixed with 4% PFA, permeabilized with Triton-X 100, and the nucleus was stained with DAPI, as mentioned above. Similarly, slides were prepared, and images were taken and analyzed in the CQ1 high throughput imaging system.

Transfection with siAMPK

AMPK expression was knocked down from the cells by using siRNA against AMPK using FuGENE HD for 24 h. Primary astrocytes seeded in 6 well plate was incubated in OPTI-MEM media for 30 min prior to transfection. The transfection mixture was prepared and kept in sterile condition for 20 min before being added to the cells. Following this, cells were treated for NLRP3 inflammasome activation as described above.

Animals and Ethical Clearance

Balb/c male mice weighing 22–25 grams were used for performing in-vivo inflammation studies. Animals were kept at 25˚C in a 12-h light-dark cycle. 50–60% relative humidity was maintained. All experiments were carried out after ethical clearance by the Institutional Animal Ethics Committee (IAEC) of CSIR-IIIM, Jammu, under the IAEC number 305/81/8/2022. Animals were euthanized by CO₂ inhalation.

Pharmacokinetic analysis

C57BL/6J male mice were used for pharmacokinetic analysis of IBC. Mice were randomly sorted into 9 groups (n = 5). 30 mg/kg oral dose of IBC (1% DMSO + 70% PEG-200 + 29% water) was given to mice. Blood or brain samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h. The plasma (100 µL) sample was processed with ethyl acetate (200 µL) containing IS (150 ng/mL) and centrifuged at 300 g for 10 min. The organic layer was separated and dried in a vacuum, reconstituted with acetonitrile, and analyzed by LC-MS/MS [SRM transition of IBC and IS (Phenacetin) is 325.2 & gt; 149.0 and 180.2 & gt; 110.2 in Positive mode]. Brain homogenate was prepared using the weighted amount of brain tissue in water at the concentration level of 350 mg/mL and processed similarly to plasma samples.

Dosing in 5XFAD mice

Six months old 5XFAD mice were randomly divided into three groups (n = 5). IBC was orally (OD) given to two groups at different doses, 25 mg/kg (1% DMSO, 2% Tween 80, 19% PEG200 and 78% distilled water) and 50 mg/kg (1% DMSO, 5% Tween 80, 19% PEG200 and 75% distilled water).

Behavioral Analysis

To check the effect of IBC on the behavior of 5XFAD mice, open field test (OFT), rotarod (Make; Ugo Basile, Accelerrota-rod for mice 7650) and radial arm maze tests were performed. To check the effect of IBC on motor coordination and balancing rotarod test was performed. After training mice to run on rotarod for five days, their latency to fall was calculated on the day of analysis. OFT was performed to assess exploratory and locomotor activity in a white box (45 X 60 cm). Distance traveled, speed, time spent in corner and center zones and mobile episodes were recorded through an automated video tracking system using AnyMaze software. For checking the effect of IBC on spatial memory of mice radial arm maze test was done. Mice were trained for five days to locate baited arms with visual clues and on the day of analysis, several parameters were checked using AnyMaze software.

Immunohistochemistry

Mice’s hippocampus tissue was fixed in 10% formalin. Dehydrated tissue slices were made and placed on slides. For analysis, tissue was rehydrated following subsequent incubation in xylene, xylene: ethanol, 100% ethanol, 90% ethanol, 70% and 50% ethanol. After tissue rehydration, cells were permeabilized using 0.4% Triton-X100 for 10 min. Blocking was done in 2% BSA, followed by incubation with primary and secondary antibodies. Images were taken in a CQ1 high throughput imaging system and analyzed using cell pathfinder software.

Aβ estimation in plasma

After the end of the experiment, blood was collected from the retinal orbital plexus of mice in EDTA-coated vials. Samples were centrifuged, and plasma was separated. The levels of Aβ in the plasma were measured by using the protein-specific ELISA kit.

Detection of IL-1β in mice cortex

Mice cortices were isolated and homogenized in RIPA buffer. The levels of IL-1β were analyzed through ELISA of the homogenates by following the given protocol.

Western blot analysis of proteins in mice hippocampi

The hippocampi isolated from mice brains was immediately washed with saline and stored in liquid nitrogen. For lysates preparation, tissue was homogenized in RIPA buffer substituted with sodium fluoride, sodium orthovanadate, phenyl methyl sulfonyl fluoride and 1% protease inhibitor cocktail. Total protein was measured by Bradford assay. The isolated protein supernatant was diluted with 2X laemmli buffer and subjected to western blot assay as described above for protein analysis.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8 software. All in-vitro experiments were performed thrice. The response of independent mice from the group was noted for animal experiments and the mean was calculated. The data presented here represents the mean ± SD. Statistical significance of the experiment was calculated using one-way ANOVA analysis and Bonferroni method was used as a post-hoc test. p value less than 0.05 was taken as significant.

IBC induced autophagy and inhibited NLRP3 inflammasome in primary astrocytes

AMPK is a primary regulator of autophagy and NLRP3 inflammasome-mediated inflammation, and both mechanisms are dysregulated in AD. With this information, we identified IBC as an inducer of AMPK-mediated autophagy and an inhibitor of NLRP3 inflammasome. To prove the effect of IBC on these processes, we used mouse primary astrocytes for all the in vitro experiments. The purity of the culture was tested by using markers of astrocytes (glial fibrillary acidic protein; GFAP), neurons (neuronal nuclear protein; Neu N) and microglial cells (ionized calcium-binding adapter molecule 1; IBA1) (Figure S1A and S1B). To analyze the effect of IBC on AMPK phosphorylation at Thr 172, the cells were treated for 24 h with IBC. The western blot analysis revealed a concentration-dependent increase in the expression of AMPK (Thr 172) (Fig. 1B and S1C). We further found that the cells treated with IBC showed a significant increase in the levels of autophagy marker LC3B-II (Light chain 3). The induction of autophagy by IBC was also supported by reducing levels of another autophagy marker, SQSTM1 (sequestosome 1), with the increase of IBC concentration (Fig. 1C, S1D and S1E). Further, to confirm the completion of autophagy induced by IBC, we checked the autophagy flux by treating the cells with end-stage autophagy inhibitor bafilomycin A₁ (Baf. A1) in the presence and absence of IBC. We found that IBC increased the levels of LC3B-II and decreased the levels of SQSTM1. However, the level of both these proteins was significantly increased in the presence of bafilomycin A₁, thus showing their accumulation and signifying the increased flux of autophagy (Fig. 1D, 1E, and 1F).

After confirming the induction of autophagy by IBC, we proceeded to know whether the ability of IBC to induce AMPK-mediated autophagy can lead to the inhibition of NLRP3 inflammasome. We found through ELISA that the primary astrocytes primed with media from LPS-stimulated microglial N9 cells, when activated with LPS (lipopolysaccharide) and ATP (5’ adenosine triphosphate), led to the release of IL-1β and IL-18. The release of both IL-1β and IL-18 was significantly inhibited with IC50 values 2.95 µM and 2.75 µM, respectively, when the cells were treated with IBC for 1.5 h, which clearly indicated the inhibition of NLRP3 inflammasome (Fig. 1G & 1H). We further confirmed the inhibition of NLRP3 inflammasome by measuring the cleaved and active forms of IL-1β and CASP1 from the supernatant of cells treated with IBC by using western blotting. The data clearly indicated the reduced release of IL-1β and CASP1 from the cells and, thus, the inhibition of NLRP3 inflammasome, though there was no change in the expression of NLRP3 protein (Fig. 1I, S1F, S1G and S1H). Further, to check the effect of IBC on NLRP3 inflammasome complex formation, we analyzed the levels of ASC protein oligomers. Results showed that IBC-treated cells inhibited ASC monomer, dimer, and oligomer formation (Figure S1I and S1J). The ASC oligomers can also be visualized as ASC specks. The IBC treatment at 5 µM and 10 µM concentrations reduced the speck formation in cells, in comparison to ATP induced sample (Figure S1K and S1L).

IBC-induced autophagy by CAMKK2-mediated activation of AMPK pathway

The activation of AMPK is regulated by two main kinases, LKB1 (liver kinase B1) and CAMKK2 (calcium/calmodulin-dependent protein kinase kinase). To ascertain the cause of AMPK activation, we checked the phosphorylation and activation of these kinases after treating primary astrocytes with IBC for 24 h. Interestingly, there was no change in the levels of pLKB1 (Ser 428), whereas pCAMKK2 (Ser 511) was found to be significantly upregulated after concentration-dependent treatment with IBC (Fig. 2A and S2A). However, we found no change in MTOR (mechanistic target of rapamycin kinase) levels. ULK1 (unc-51-like kinase 1), which is a direct target of AMPK, was found to be phosphorylated at Ser 317, marking its activation; its activation was also confirmed indirectly through increased phosphorylation of FIP200, another protein present in the ULK1 complex (Fig. 2A and S2A). Further, the downstream protein BECN1 (beclin 1, autophagy related) did not show any change in the expression. However, BCL2 (B-cell lymphoma), which is known to interact with BECN1 and downregulate autophagy, showed a marked decline, thereby tilting the ratio in favor of BECN1 activation (Fig. 2A and 2B). The level of proteins ATG5 (autophagy related 5) and ATG7 (autophagy related 7), involved in the maturation of the autophagosome, was also found to be upregulated, thus confirming the induction of autophagy by IBC in primary astrocytes (Fig. 2A and S2A). To confirm the fusion of autophagosome with lysosome, we did a confocal microscopy analysis for the co-localization of LC3B and LAMP1 (lysosomal-associated membrane protein 1), membrane marker proteins for autophagosome and lysosome, respectively. Data showed proximity of stated proteins after treatment of primary astrocytes with IBC (Fig. 2C, 2D and S2B). In all these experiments, rapamycin (Rap) at a concentration of 250 nM was used as a standard drug for the induction of autophagy.

IBC inhibited NLRP3 inflammasome through autophagy induction in primary astrocytes

After confirming the induction of autophagy by IBC, we wanted to know if it plays any role in inhibiting NLRP3 inflammasome in primary astrocytes. In our earlier assays, autophagy was checked after 24 h of treatment, and the inhibition of NLRP3 inflammasome was analyzed after 90 min of treatment with IBC. Therefore, we first checked whether IBC could induce autophagy under the conditions used for the NLRP3 inhibition assay. Interestingly, cells treated with IBC in the presence of LPS and ATP showed significantly enhanced phosphorylation of AMPK at Thr 172, along with a marked increase in the level of autophagy marker LC3B-II was observed (Fig. 3A and S3A). Further, the additional autophagy marker SQSTM1 levels also showed a significant decline after treatment with IBC (Fig. 3A and S3A). To confirm the involvement of autophagy in NLRP3 inhibition by IBC, we inhibited the autophagy by end-stage autophagy inhibitor bafilomycin A₁ and checked its effect on NLRP3 inflammasome mediated release of IL-1β. We first checked the inhibition of autophagy by analyzing the expression of LC3B-II, which showed an accumulation of the protein in the presence of bafilomycin A₁, thus confirming the inhibition of autophagy. The inhibition of autophagy completely reversed the inhibitory effect of IBC on the release of IL-1β, which was almost equal to the levels in the cells treated with LPS + ATP as confirmed by both ELISA and western blotting (Fig. 3B, 3C and S3B). These results clearly indicated the autophagic regulation of IBC-mediated inhibition of NLRP3 inflammasome.

Our earlier data showed that AMPK plays an essential role in autophagy induced by IBC. Therefore, we wanted to know if the knockdown of AMPK by using siRNA can affect the inhibition of NLRP3 inflammasome by IBC. We found that under the conditions of NLRP3 inhibition assay, the siRNA-mediated inhibition of AMPK completely reversed the inhibitory effect of IBC on two important proteins (IL-β and CASP1) of the NLRP3 inflammasome complex. The release of both these proteins after treatment with IBC in the presence of siAMPK was almost similar to that of astrocytes treated with LPS + ATP (Fig. 3D and S3C). Interestingly, there was no change in the expression of NLRP3 protein in the presence or absence of siAMPK, whereas its negative effect on autophagy marker LC3B-II was clearly visible (Fig. 3D and S3C). The effect of siAMPK on the assembly proteins involved in NLRP3 complex formation also showed a similar trend. Treating astrocytes with IBC reduced the co-localization of CASP1 and NLRP3 compared to those treated with LPS + ATP. However, in the presence of siAMPK, the cells treated with IBC did not affect the co-localization of CASP1 and NLRP3 as observed through confocal microscopy (Fig. 3E, 3F and S3D). Western blotting and confocal microscopy confirmed the knockdown effect of siAMPK on the AMPK (Thr 172) in all the experiments (Figure S3E, S3F and S3G).

Autophagy induced by IBC promotes the clearance of Aβ in primary astrocytes

After confirming the dual pharmacological activity of IBC, we further wanted to determine whether the autophagic activity of IBC can help glial cells to clear Aβ. Therefore, we treated the astrocytes with IBC for 24 h and added Aβ₄₂-HiLyte fluor488 to the culture 12 h before the termination of the experiment. The confocal image analysis revealed that the cells with IBC showed significantly low fluorescence compared to the cells with Aβ alone, indicating reduced intracellular levels of Aβ in the IBC treated cells. However, when we inhibited the autophagy with bafilomycin A₁, the levels of Aβ were found to be increased in the treated cells, which confirmed the involvement of autophagy in the clearance of Aβ by IBC (Fig. 4A and 4C). These results were further consolidated when we inhibited autophagy at an early stage by using siRNA against AMPK. The cells treated with IBC displayed a marked decline in intracellular levels of Aβ compared to Aβ control. The cells, which were treated with siAMPK, showed a complete reversal of the IBC effect on the clearance of Aβ (Fig. 4B and 4D).

IBC crossed the blood-brain barrier and improved the memory behavior in 5XFAD mice

Based on in vitro data, we decided to investigate the effect of IBC on the pathology of AD in the transgenic mouse model (5XFAD). Before going for a long-term study to analyze the effect of IBC on AD pathology, we first confirmed the penetration of IBC into the brain after a single dose (p.o.). Therefore, we compared the plasma and brain pharmacokinetic (PK) profile of IBC after a single dose of 30 mg/kg (p.o). The blood samples collected at different time points through 24 h showed plasma C_max value of 208 ± 46 ng/ml (Fig. 5A and Table 1). The brain PK studies revealed that IBC crossed the blood-brain barrier with low penetration and showed a C_max value of 34 ± 04 ng/g of brain tissue (Fig. 5B and Table 2). We determined the brain-to-plasma ratio of 0.12 to 0.16. Therefore, to observe the effect of IBC on AD pathology, we decided to treat the six months old 5XFAD mice at two different doses that are 25 mg/kg and 50 mg/kg, for a period of two months (The complete study plan is given in Figure S4).

Table 1

Plasma pharmacokinetic studies of IBC
Parameters	IBC (30mg/kg)
C_max (ng/mL)	208 ± 46
T_max (h)	0.6 ± 0.1
T_1/2 (h)	5.6 ± 1.5
AUC_0 − t (ng.h/mL)	548 ± 59
AUC_0−∞ (ng.h/mL)	637 ± 74
V_d (L/Kg)	355 ± 81
Cl (L/h/Kg)	50 ± 7

Table 2

Brain pharmacokinetic studies of IBC.
Parameters	IBC (30mg/kg)
C_max (ng/g)	34 ± 4
T_max (h)	0.6 ± 0.1
T_1/2 (h)	1.8 ± 0.4
AUC_0 − t (ng.h/g)	72 ± 7
AUC_0−∞ (ng.h/g)	79 ± 7

After six weeks of dosing, the animals were trained for five days each for the radial arm maze and rota rod tests. We generated the baseline behavior data by comparing the age-matched wild-type C57BL/6J mice group with the 5XFAD control group and found a significant deviation in all the parameters tested during behavior analysis. In a radial arm maze test, the animals treated with IBC at both the doses 25 and 50 mg/kg took significantly less time (16.58 and 14.36 s, respectively) to enter the baited arm in comparison to the control group, which took an average of 32.3 s (Fig. 5C). Furthermore, the animals treated with IBC spent a significantly longer time in the baited arm than the control group (Fig. 5D). The treated animals also traveled significantly less distance to find the baited arm and showed less number of entries into the non-baited arm (Fig. 5E and 5F). These data clearly indicated improvement in spatial memory of 5XFAD mice treated with IBC. We also tested the exploratory behavior of mice. The 5XFAD mice displayed a significant reduction in their desire to explore the surrounding environment, which represents the behavior associated with AD. However, the animals treated with IBC showed improved exploratory behavior, which was evident from less time spent in the center, more time spent in the corners and were in mobile state for a longer time at higher speed along with greater distance traveled as compared to the 5XFAD control group (Fig. 5H, 5I, 5J, 5K and 5L). The track plots of the mice in the radial arm maze test and open field test, which were drawn using AnyMaze software, are shown in Figs. 5G and 5M, respectively. The treatment of 5XFAD mice with IBC also improved their neuro-muscular coordination and stamina evaluated by rota rod test. The animals treated with IBC were able to stay on moving rods for a significantly longer time (92 s at 25 mg/kg and 118 s at 50 mg/kg) in comparison to the 5XFAD control group, which could stay on rods for about 63 s only (Fig. 5N).

Improvement in the memory behavior in 5XFAD mice is caused by IBC-mediated clearance of Aβ ₄₂ and reduction of neuroinflammation

The improved memory behavior of 5XFAD mice after 2 months of treatment with IBC led us to explore the molecular changes in the brains of these mice. Therefore, we analyzed the effect of IBC on the deposition of Aβ, which is a key pathological feature of AD responsible for cognitive impairment. We evaluated the Aβ levels in the hippocampi of the 5XFAD mice by immunohistochemical (IHC) analysis. We observed a huge load of Aβ plaque (Average number of plaques = 55) in the 5XFAD control mice group. The counting of Aβ plaques, using the cell pathfinder software, revealed that the average number of plaques were reduced to 21 and 15 in the mice that were treated with IBC at 25 and 50 mg/kg, respectively (Fig. 6A and B and Supplementary Figure S5A). This was further validated by the fluorescence analysis of samples, which displayed a highly significant decline in the treated groups (Fig. 6A and C). Further, the analysis through ELISA also exhibited reduced levels of Aβ₄₂ in the blood plasma of mice treated with IBC compared to the untreated control group, indicating the decline in the Aβ load in the brains of these mice (Fig. 6D).

Deposition of Aβ₄₂ is strongly linked with increased neuroinflammation, which is reflected in the form of reactive astrogliosis and activation of NLRP3 inflammasome. Thus, we intended to know if the IBC meditated decline in Aβ₄₂ has reduced brain inflammation. The IHC analysis of the hippocampus for GFAP showed a large number of astrocytes expressing high levels of GFAP in the control group. However, in the mice treated with 25 and 50 mg/kg of IBC, the expression of GFAP was significantly reduced (Fig. 6E, 6F and S5B). Also, Aβ, being one of the damage-associated molecular patterns (DAMP) known to activate NLRP3 inflammasome, we checked if the dwindling load of Aβ after treatment with IBC could reduce the activation of NLRP3 inflammasome. For that, we analyzed the levels of IL-1β in the cortices in the brain of these mice using ELISA. The data indicated a highly significant decline in the levels of IL-1β in the 5XFAD mice treated with IBC at both doses 25 and 50 mg/kg, compared to the control group (Fig. 6G).

IBC induced autophagy and inhibited NLRP3 inflammasome in the hippocampus of 5XFAD mice

After confirming the clearance of Aβ deposits and alleviation of inflammation from the brain of 5XFAD mice, we analyzed the expression of some of the proteins that are directly involved in the autophagic pathway. The western blot analysis clearly showed increased expression of pAMPK and ATG7, whereas the expression of SQSTM1 was significantly decreased in the mice treated with IBC at 25 and 50 mg/kg (Fig. 7A). Further, the animals treated with IBC also expressed reduced levels of NLRP3 inflammasome proteins. The western blot analysis showed significant abrogation of CASP1 and ASC in mice treated with IBC. This was further reflected in the reduced cleavage of IL-1β in these mice (Fig. 7B). The treatment of mice with IBC also inhibited the level of the reactive phenotype of astrocytes and microglial cells, evidenced by decreased expression of GFAP and IBA1 (Fig. 7B).

The complex pathology of AD has led to the failure of multiple drugs in clinical trials. The failure rate (99.6%) of anti-AD drugs in clinical trials is the highest among all the diseases (Cummings et al., 2014; Cummings et al., 2017). Though, two anti- Aβ biologicals viz. aducanumab and lecanemab-irmb have recently been approved by the US FDA for treating AD (Sevigny et al., 2016; Cummings, 2023; van Dyck et al., 2023). However, the efficacy of these biologicals to provide long-term clinical benefits to AD patients will be decided in the future. The clinical trial data analysis shows that most drugs that failed against AD were based on a single target. This study was designed to employ the idea that hitting more than one AD target, that are, Aβ and NLRP3 inflammasome-mediated neuroinflammation, could effectively ameliorate AD pathology. To implicate this concept, we chose an experimental compound, IBC, which induced AMPK-mediated autophagy in primary astrocytes. We found that IBC caused the induction of CAMKK2 to activate AMPK and its downstream targets, which ultimately led to the completion of autophagy, as confirmed through the increased autophagy flux. Despite being present in large numbers in the brain and their recently emerging role in neurodegeneration, astrocytes have not been explored much as a target in AD drug discovery. Astrocytes regulate synaptic transmissions, protect and support neurons, and manage toxins infiltration along with microglia, etc. (Sofroniew and Vinters, 2010). Particularly in AD, the early physiological dysfunction of astrocytes to clear Aβ debris and their pathological involvement in neuroinflammation plays a crucial role in cognitive impairments (Garwood et al., 2017; Gonzalez-Reyes et al., 2017).

Autophagy is the major cellular pathway that is known to modulate the activation of the NLPR3 inflammasome (Shi et al., 2012; Houtman et al., 2019). It clears the pro-form of proteins from the cytoplasm and manages the clearance of NLRP3 protein ligands like ROS-producing dysfunctional mitochondria (Harris et al., 2011; Rodgers et al., 2014). In the AD brain, due to the continuous death of cells, the release of free ATP becomes a source for activation of NLRP3 inflammasome (Guo et al., 2020; Huang et al., 2021). Additionally, Aβ is another DAMP responsible for chronic NLRP3 inflammasome activity and release of IL-1β in AD (Luciunaite et al., 2020). IBC, through autophagy induction, limited the ATP-mediated NLRP3 inflammasome activation by inhibiting ASC oligomerization and speck formation. The involvement of autophagy in anti-NLRP3 inflammasome was confirmed when we inhibited the autophagy at two different steps. The inhibition of autophagy at the initial stage by using siAMPK and at the final stage by bafilomycin A₁ led to a reversal of the anti-NLRP3 inflammasome activity of IBC. Further, IBC displayed the auxiliary advantage of clearing Aβ deposits in the primary astrocytes through autophagy induction. Thus, IBC acted on two of the essential DAMPs in the brain responsible for NLRP3 inflammasome-mediated neuroinflammation.

After confirming the in vitro activity of IBC against Aβ clearance and NLRP3 inflammasome, the two important targets in AD, we validated its efficacy in the 5XFAD transgenic mouse model of the disease. Before that, we confirmed the IBC’s penetration into the brain through PK studies in C57BL/6J mice, a genetic background for transgenic 5XFAD mice. Due to relatively low brain availability, we planned to treat the 5XFAD mice at two different doses of IBC (25 and 50 mg/kg, p.o.) for two months. AD pathology progresses with time, and some of the latest antibodies-based clinical trials targeting Aβ have indicated that starting treatment at an early stage of AD may be helpful in better clinical outcomes (Yiannopoulou et al., 2019; Habashi et al., 2022). Therefore, we began the treatment of 5XFAD mice at six months of age, which is a relatively early stage of the disease (Forner et al., 2021).

The analysis of all the parameters related to cognitive behavior clearly indicated the impairment in 5XFAD control mice compared to wild-type C57BL6/J mice. However, the IBC treated groups showed significant improvement in spatial memory. Along with spatial memory, decline in other behavior parameters is a common feature of AD (Albert, 1996; Webster et al., 2014; Pottorf et al., 2022). Therefore, we also analyzed the exploratory behavior of mice after treatment with IBC. The 5XFAD mice displayed deficits in the parameters related to exploratory behavior and reduced neuromuscular coordination, which indicated deterioration of brain function. The mice treated with IBC displayed a reversal of functional decline in the brain at both the doses used for treatment. Intriguingly, the mice treated with IBC performed better than the wild-type mice in most of the evaluated parameters. This observation could be attributed to improved autophagic homeostasis and reduced NLRP3-mediated chronic inflammation, which helped the treated mice perform better.

We further confirmed whether the improvement in behavior parameters is related to the effect of IBC on the clearance of Aβ and reduction of inflammation through autophagy, as observed in the in vitro studies. Several recent studies have strongly linked the decline of autophagy as one of the reasons for the deposition of Aβ in the AD brain (Luo et al., 2020; Wani et al., 2021; Lee et al., 2022). Moreover, a complex cascade of events follows the deposition of Aβ, which involves the release of inflammatory cytokines by microglia and astrocytes, leading to the death of neurons (Kitazawa et al., 2004; Wu et al., 2013; Singh, 2022). Therefore, we analyzed the hippocampus of mice treated with IBC for Aβ deposits. The data confirmed that autophagy induced by IBC effectively cleared Aβ₄₂ plaques from the hippocampus at 25 and 50 mg/kg doses. Furthermore, the plasma levels of Aβ₄₂ were also significantly reduced, thereby confirming the effect of IBC on its clearance from the brain. The analysis of autophagic proteins like AMPK, ATG7, and SQSTM1, which we earlier observed in astrocytes, were also regulated in the hippocampus of treated mice. These data further emphasized the mechanism of action of IBC for clearance of Aβ from the brain. Apart from the deposition of Aβ, neuroinflammation is one of the critical hallmarks of AD. The release of pro-inflammatory cytokine IL-1β due to chronic activation of NLRP3 inflammasome by Aβ, TAU, and ATP creates an inflammatory microenvironment in the brain, contributing further to neurodegeneration (Akiyama et al., 2000; Heneka et al., 2018; Ising et al., 2019). In our study, IBC treatment mitigated the neuroinflammation in 5XFAD mice, which was confirmed by reduced GFAP and IL- 1β expression in the hippocampus and cortex, respectively. Furthermore, the expression levels of ASC and CASP1, the essential proteins of NLRP3 inflammasome complex, were significantly reduced in the mice treated with IBC, suggesting the involvement of autophagy in the inhibition of NLRP3 inflammasome in the hippocampus of mice treated with IBC.

In summary, we could clear Aβ through pharmacological induction of autophagy by IBC from astrocytes and hippocampus of 5XFAD mice. IBC, via its ability to induce autophagy, also acted as a potent inhibitor of NLRP3 inflammasome. Our data suggests that simultaneous targeting of two critical hallmarks of AD, that is, the deposition of Aβ and neuroinflammation, can effectively ameliorate complex AD pathology. In this study, we used IBC as an example to demonstrate the efficacy of this strategic approach for managing AD, which can be employed in future for developing new therapeutics against the disease.

Aβ: amyloid-beta

AD: Alzheimer disease

AMPK: 5’ adenine monophosphate-activated protein kinase

ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain

ATG: autophagy related

ATP: 5’ adenosine triphosphate

BCL2: B-cell lymphoma

BECN1: beclin 1, autophagy related

CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2

CASP1: caspase-1

DAMP: damage-associated molecular patterns

DAPI: 4,6-diaminido-2-phenylindole

GFAP: glial fibrillary acidic protein

IBA1: ionized calcium-binding adapter molecule 1

IBC-isobavachalcone

IHC: immunohistochemistry

IL-18: interleukin-18

IL-1β: interleukin-1β

LAMP-1: lysosomal-associated membrane protein 1

LC3: light chain 3

LKB1: liver kinase B1

LPS: lipopolysaccharide

MTOR: mechanistic target of rapamycin kinase

NeuN: neuronal nuclear protein

NFT: neurofibrillary tangles

NLRP3: NOD-like receptor family pyrin domain containing 3

SQSTM1: Sequestosome 1

ULK-1: unc-51-like kinase 1

Competing interests

The authors declare that they have no competing interests.

Funding

The funding for this project was provided by CSIR-Indian Institute of Integrative Medicine through the project MLP-6002 (WP-4).

Author Contribution

D.K. did most of the in vitro experiments along with some in vivo experiments. P.K. and Ankita S. performed ELISA experiments. K.S. performed animal behavior studies. Alpa S. and S.D.S. isolated and characterized the compound IBC. S.M.A. performed confocal microscope studies. P.W. and U.N. did pharmacokinetics studies. P.R. helped in genotyping of 5XFAD mice. Z.A. helped in designing the study. D.K. and A.K. designed the study, analyzed the data and wrote the manuscript. All authors reviewed the manuscript.

Akiyama H et al (2000) Inflammation and Alzheimer's disease. Neurobiol Aging 21:383–421
Al-Ghraiybah NF, Wang J, Alkhalifa AE, Roberts AB, Raj R, Yang E, Kaddoumi A (2022) Glial Cell-Mediated Neuroinflammation in Alzheimer's Disease. International journal of molecular sciences 23
Albert MS (1996) Cognitive and neurobiologic markers of early Alzheimer disease. Proc Natl Acad Sci USA 93:13547–13551
Cummings J (2023) Anti-Amyloid Monoclonal Antibodies are Transformative Treatments that Redefine Alzheimer's Disease Therapeutics. Drugs 83:569–576
Cummings J, Lee G, Mortsdorf T, Ritter A, Zhong K (2017) Alzheimer's disease drug development pipeline: 2017. Alzheimer's Dement 3:367–384
Cummings JL, Morstorf T, Zhong K (2014) Alzheimer's disease drug-development pipeline: few candidates, frequent failures. Alzheimers Res Ther 6:37
Forner S et al (2021) Systematic phenotyping and characterization of the 5xFAD mouse model of Alzheimer's disease. Sci data 8:270
Garwood CJ, Ratcliffe LE, Simpson JE, Heath PR, Ince PG, Wharton SB (2017) Review: Astrocytes in Alzheimer's disease and other age-associated dementias: a supporting player with a central role. Neuropathol Appl Neurobiol 43:281–298
Gonzalez-Reyes RE, Nava-Mesa MO, Vargas-Sanchez K, Ariza-Salamanca D, Mora-Munoz L (2017) Involvement of Astrocytes in Alzheimer's Disease from a Neuroinflammatory and Oxidative Stress Perspective. Front Mol Neurosci 10:427
Guo T, Zhang D, Zeng Y, Huang TY, Xu H, Zhao Y (2020) Molecular and cellular mechanisms underlying the pathogenesis of Alzheimer's disease. Mol neurodegeneration 15:40
Habashi M, Vutla S, Tripathi K, Senapati S, Chauhan PS, Haviv-Chesner A, Richman M, Mohand SA, Dumulon-Perreault V, Mulamreddy R, Okun E, Chill JH, Guerin B, Lubell WD, Rahimipour S (2022) Early diagnosis and treatment of Alzheimer's disease by targeting toxic soluble Abeta oligomers. Proc Natl Acad Sci USA 119:e2210766119
Harris J, Hartman M, Roche C, Zeng SG, O'Shea A, Sharp FA, Lambe EM, Creagh EM, Golenbock DT, Tschopp J, Kornfeld H, Fitzgerald KA, Lavelle EC (2011) Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation. J Biol Chem 286:9587–9597
Heneka MT, McManus RM, Latz E (2018) Inflammasome signalling in brain function and neurodegenerative disease. Nat Rev Neurosci 19:610–621
Houtman J, Freitag K, Gimber N, Schmoranzer J, Heppner FL, Jendrach M (2019) Beclin1-driven autophagy modulates the inflammatory response of microglia via NLRP3. EMBO J 38
Huang Y, Xu W, Zhou R (2021) NLRP3 inflammasome activation and cell death. Cell Mol Immunol 18:2114–2127
Ising C et al (2019) NLRP3 inflammasome activation drives tau pathology. Nature 575:669–673
Kelley N, Jeltema D, Duan Y, He Y (2019) The NLRP3 Inflammasome: An Overview of Mechanisms of Activation and Regulation. Int J Mol Sci 20
Kitazawa M, Yamasaki TR, LaFerla FM (2004) Microglia as a potential bridge between the amyloid beta-peptide and tau. Ann N Y Acad Sci 1035:85–103
Kumar A, Sidhu J, Goyal A, Tsao JW (2023) Alzheimer Disease. In: StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Jaskirat Sidhu declares no relevant financial relationships with ineligible companies. Disclosure: Amandeep Goyal declares no relevant financial relationships with ineligible companies. Disclosure: Jack Tsao declares no relevant financial relationships with ineligible companies
Lee JH, Yang DS, Goulbourne CN, Im E, Stavrides P, Pensalfini A, Chan H, Bouchet-Marquis C, Bleiwas C, Berg MJ, Huo C, Peddy J, Pawlik M, Levy E, Rao M, Staufenbiel M, Nixon RA (2022) Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Abeta in neurons, yielding senile plaques. Nat Neurosci 25:688–701
Liddelow SA et al (2017) Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541:481–487
Luciunaite A, McManus RM, Jankunec M, Racz I, Dansokho C, Dalgediene I, Schwartz S, Brosseron F, Heneka MT (2020) Soluble Abeta oligomers and protofibrils induce NLRP3 inflammasome activation in microglia. J Neurochem 155:650–661
Luo R, Su LY, Li G, Yang J, Liu Q, Yang LX, Zhang DF, Zhou H, Xu M, Fan Y, Li J, Yao YG (2020) Activation of PPARA-mediated autophagy reduces Alzheimer disease-like pathology and cognitive decline in a murine model. Autophagy 16:52–69
Mishra SR, Mahapatra KK, Behera BP, Patra S, Bhol CS, Panigrahi DP, Praharaj PP, Singh A, Patil S, Dhiman R, Bhutia SK (2021) Mitochondrial dysfunction as a driver of NLRP3 inflammasome activation and its modulation through mitophagy for potential therapeutics. Int J Biochem Cell Biol 136:106013
Pottorf TS, Nocera JR, Eicholtz SP, Kesar TM (2022) Locomotor Adaptation Deficits in Older Individuals With Cognitive Impairments: A Pilot Study. Front Neurol 13:800338
Rajmohan R, Reddy PH (2017) Amyloid-Beta and Phosphorylated Tau Accumulations Cause Abnormalities at Synapses of Alzheimer's disease Neurons. J Alzheimer's disease: JAD 57:975–999
Rodgers MA, Bowman JW, Liang Q, Jung JU (2014) Regulation where autophagy intersects the inflammasome. Antioxid Redox Signal 20:495–506
Saresella M, La Rosa F, Piancone F, Zoppis M, Marventano I, Calabrese E, Rainone V, Nemni R, Mancuso R, Clerici M (2016) The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer's disease. Mol neurodegeneration 11:23
Sevigny J et al (2016) The antibody aducanumab reduces Abeta plaques in Alzheimer's disease. Nature 537:50–56
Shi CS, Shenderov K, Huang NN, Kabat J, Abu-Asab M, Fitzgerald KA, Sher A, Kehrl JH (2012) Activation of autophagy by inflammatory signals limits IL-1beta production by targeting ubiquitinated inflammasomes for destruction. Nat Immunol 13:255–263
Singh D (2022) Astrocytic and microglial cells as the modulators of neuroinflammation in Alzheimer's disease. J Neuroinflamm 19:206
Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol 119:7–35
van Dyck CH, Swanson CJ, Aisen P, Bateman RJ, Chen C, Gee M, Kanekiyo M, Li D, Reyderman L, Cohen S, Froelich L, Katayama S, Sabbagh M, Vellas B, Watson D, Dhadda S, Irizarry M, Kramer LD, Iwatsubo T (2023) Lecanemab in Early Alzheimer's Disease. N Engl J Med 388:9–21
Van Zeller M, Dias D, Sebastiao AM, Valente CA (2021) NLRP3 Inflammasome: A Starring Role in Amyloid-beta- and Tau-Driven Pathological Events in Alzheimer's Disease. J Alzheimer's disease: JAD 83:939–961
Wang M, Lin L, Lu JJ, Chen X (2021) Pharmacological review of isobavachalcone, a naturally occurring chalcone. Pharmacol Res 165:105483
Wani A, Al Rihani SB, Sharma A, Weadick B, Govindarajan R, Khan SU, Sharma PR, Dogra A, Nandi U, Reddy CN, Bharate SS, Singh G, Bharate SB, Vishwakarma RA, Kaddoumi A, Kumar A (2021) Crocetin promotes clearance of amyloid-beta by inducing autophagy via the STK11/LKB1-mediated AMPK pathway. Autophagy 17:3813–3832
Webster SJ, Bachstetter AD, Nelson PT, Schmitt FA, Van Eldik LJ (2014) Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models. Front Genet 5:88
Wen M, Ding L, Zhang L, Zhang T, Teruyoshi Y, Wang Y, Xue C (2019) Eicosapentaenoic Acid-Enriched Phosphatidylcholine Mitigated Abeta1-42-Induced Neurotoxicity via Autophagy-Inflammasome Pathway. J Agric Food Chem 67:13767–13774
Wu Z, Sun L, Hashioka S, Yu S, Schwab C, Okada R, Hayashi Y, McGeer PL, Nakanishi H (2013) Differential pathways for interleukin-1beta production activated by chromogranin A and amyloid beta in microglia. Neurobiol Aging 34:2715–2725
Yiannopoulou KG, Anastasiou AI, Zachariou V, Pelidou SH (2019) Reasons for Failed Trials of Disease-Modifying Treatments for Alzheimer Disease and Their Contribution in Recent Research. Biomedicines 7

No competing interests reported.

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Isobavachalcone ameliorates Alzheimer disease pathology by autophagy-mediated clearance of amyloid beta and inhibition of NLRP3 inflammasome in 5XFAD mice

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Abstract

Figures

Introduction

Materials and Methods

Chemicals and Reagents

Primary astrocytes culture

NLRP3 inflammasome activation

Cytokines measurement by ELISA

ASC oligomer formation

Autophagy flux measurement

Immunoblotting

Confocal microscopy

Aβ clearance assay

Animals and Ethical Clearance

Pharmacokinetic analysis

Dosing in 5XFAD mice

Behavioral Analysis

Immunohistochemistry

Aβ estimation in plasma

Detection of IL-1β in mice cortex

Western blot analysis of proteins in mice hippocampi

Statistical Analysis

Results

IBC induced autophagy and inhibited NLRP3 inflammasome in primary astrocytes

IBC-induced autophagy by CAMKK2-mediated activation of AMPK pathway

IBC inhibited NLRP3 inflammasome through autophagy induction in primary astrocytes

Autophagy induced by IBC promotes the clearance of Aβ in primary astrocytes

IBC crossed the blood-brain barrier and improved the memory behavior in 5XFAD mice

IBC induced autophagy and inhibited NLRP3 inflammasome in the hippocampus of 5XFAD mice

Discussion

Conclusion

Abbreviations

Declarations

Competing interests

Funding

Author Contribution

References

Additional Declarations

Supplementary Files

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