Autophagy

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

Biallelic variants in genes regulating endosomal, lysosomal and autophagy pathways are increasingly implicated in severe neurodevelopmental disorders, yet the contribution of the Rab7 effector WDR91 to human disease remains incompletely defined. We report a child with a severe neurodevelopmental disorder characterized by progressive microcephaly, microlissencephaly, corpus callosum hypoplasia, and early-onset epilepsy, harboring compound heterozygous WDR91 variants: a truncating variant (p.Gln215*) and a missense variant (p.Tyr15Asn). Functional analyses show that p.Gln215* abolishes WDR91 expression, whereas p.Tyr15Asn reduces protein abundance through increased degradation. Reduced WDR91 expression was confirmed in primary patient-derived cells. In silico analyses suggest that p.Tyr15Asn induces a localized change within an N-terminal degron-containing region, potentially affecting protein stability. In cellular models, both variants impair early-to-late endosomal maturation and alter WDR91 localization to Rab7-positive compartments. WDR91 deficiency is further associated with transcriptional and functional evidence of autophagy dysregulation. While the p.Tyr15Asn variant partially restores autophagic flux under overexpression conditions, patient-derived cells display impaired autophagic turnover, consistent with a context-dependent functional and partial loss of function effect of this variant. Together, these findings provide functional evidence supporting the pathogenicity of WDR91 variants and implicate combined defects in endosomal maturation and autophagy in WDR91-related neurodevelopmental disease.

Mitochondria engage in extensive communication with other organelles through membrane contacts. Perturbed mitochondria-organelle interactions are indicated in a variety of neurodegenerative diseases, but the underlying mechanisms remain poorly understood. Here, we report a new class of mitochondria-organelle communication: autophagosome/autophagic vacuole (AV)-mitochondria (Mito) contact, which exhibits hyper-tethering in tauopathy neurons, consequently hampering AV retrograde transport. Such defects are attributed to accelerated turnover of the contact release factor TBC1D15, triggered by mitochondrial bioenergetic deficit-induced hyperactivity of the AMP-activated protein kinase (AMPK). Increasing TBC1D15 levels or repressing AMPK activity normalizes AV-Mito contact release and restores retrograde transport of AVs, thereby increasing autophagic cargo clearance and reducing tau burden in tauopathy axons. Furthermore, overexpression of TBC1D15 enhances autophagic clearance and attenuates tau pathology, alleviating neurodegeneration and cognitive dysfunction in tauopathy mice. Taken together, our study provides new insights into AV-Mito contact dysregulation in tauopathy-related autophagy failure, laying the groundwork for the development of potential therapeutics to combat tauopathy diseases.

The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features as well as surrounding and attached vesicles suggest that the enlarged rims are a result of cytosolic vesicles fusing with the growing phagophore. High-resolution imaging in this study shows that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

In mammals, most of the iron is found in the heme of red blood cells (RBCs), which must be recycled to support erythropoiesis in the bone marrow. Splenic red pulp macrophages (RPMs) play a crucial role in this process by phagocytosing senescent RBCs, metabolizing the heme and releasing iron back into the blood. Free cytoplasmic iron generates toxic reactive oxygen species, yet iron-specific adaptations of RPMs are not well documented. We previously reported that autophagy prevents ferroptosis in Langerhans cells, a cutaneous phagocyte subset. Thus, we hypothesized that autophagy may be important for the regulation of RPM metabolism and their maintenance of systemic iron homeostasis. To study this, we used Atg5flox/flox and Cd169cre mouse models to delete ATG5 in CD169+ macrophages, including RPMs. Atg5-deficient RPMs were decreased in number, and the remaining ones showed increased generation of toxic lipid peroxides. Spleens of Atg5{Delta}Cd169 mice were enlarged and contained more RBCs. Finally, autophagy impairment in RPMs exacerbated RBC loss in a model of phenylhydrazine-induced anemia. Our findings exemplify how dysregulation of macrophage metabolism alters their function and can disrupt tissue homeostasis upon challenge.

Macroautophagy is a conserved intracellular catabolic process in eukaryotes that participates in chloroplast degradation, through the selective breakdown of chloroplast components. Selective autophagy of membrane-bound organelles typically requires receptors that bridge organelle membranes and pre-autophagosomal structures. Here we identify OEP24.1 as a new receptor in the selective chloroplast piecemeal autophagy, supporting the degradation of stromal proteins. We found that the {beta}-barrel protein OEP24.1 is located at the outer membrane of plastid envelopes and on bodies budding off plastids into the cytosol and containing stroma proteins. OEP24.1 interacts physically with ATG8 autophagy proteins in a UIM dependent manner. OEP24.1-GFP and RFP-ATG8 colocalize with in mobile autophagosome-like puncta in the cytosol and in autophagic bodies within the vacuole. Delivery of OEP24.1 to vacuole lumen is dependent on active autophagy. OEP24.1 controls carbon allocation at the whole plant level, carbon concentrations in flowering stems and xylem composition. These phenotypes can be explained by the role of OEP24.1 in metabolite diffusion across the chloroplast envelope, and by its involvement in the facilitation of chloroplast quality control through piecemeal autophagy.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

The establishment of aphid-plant interaction involves the secretion of a salivary MIF protein. Morphological analyses revealed that aphid MpMIF1 prevents plant cell death, protects organelles from stress, and may promote plant cellular recovery. Co-expression of aphid MpMIF1 and the cell death inducer Npp1 revealed that MpMIF1 modulates autophagy-related genes ATG7/BECLIN1, impair plant senescence regulator ATAF1 and regulate apoptosis-like via Caspase-3-like activity. This effect on multiple-cell death pathways helps to maintain cellular homeostasis during aphid infection. Investigations on DNA Damage Response (DDR) signaling pathways demonstrated that aphid MpMIF1 reduces {gamma}H2A.X phosphorylation, maintains activity of the DNA repair protein RAD51 and stabilizes cell cycle checkpoint expression WEE1 under genotoxic stress. Therefore, MpMIF1 actively participates to the maintenance of a functional DDR. Finally, we showed that aphid MpMIF1 physically interacts with SOG1, a functional analog of animal p53 and central regulator of DDR, cell cycle arrest and programmed cell death in plants. These findings establish MpMIF1 as a key regulator of plant cell death during aphid-plant interactions and highlight its potential as a biotechnological tool for protecting major crops against aphid infection.

Alzheimers disease (AD) is the most prevalent form of dementia, characterized by progressive memory loss, cognitive decline, and emotional dysregulation. Adult hippocampal neurogenesis (AHN) critically contributes to cognition and mood but undergoes precipitous decline during AD progression. Here, we investigated whether enhancing AHN through genetic expansion of endogenous neural stem cells (NSC) ameliorates AD-related phenotypes. Using lentiviral overexpression of the cell cycle regulators Cdk4 and CyclinD1 in the dentate gyrus of 3xTg-AD mouse, we show that enhancing AHN partially rescues hippocampal-specific cognitive functions, namely: spatial navigation and exploratory behavior. These findings show that endogenous NSC can be exploited to ameliorate hippocampal cognitive functions in AD, providing additional evidence for exploiting AHN as a promising therapeutic target for neurodegenerative disease.

The loss of an organelle represents an extreme case of reductive evolution. Only a few plastid loss events have been documented and they seem to be particularly rare in free-living ancestrally photosynthetic taxa. We isolated a new phagotrophic protist, Ciliophrys sp. Baltic, representing a poorly studied non-photosynthetic lineage in the stramenopile algal class Dictyochophyceae. Genomic and transcriptomic data obtained from this organism enabled us to demonstrate that it lacks a plastid genome and nuclear genes critical for plastid biogenesis. The plastid organelle is thus most likely secondarily missing from Ciliophrys sp. Baltic, although a few enzymes originally located in the plastid persist with a changed localization. Unexpectedly, Ciliophrys sp. Baltic harbors a bacterial endosymbiont, Candidatus Penulousia baltica sp. nov. (Rickettsiales). Analysis of its complete genome sequence uncovered unique aspects of its metabolism not previously reported in other Rickettsiales. The bacterium exploits its host metabolically and presumably manipulates it with an arsenal of putative effector proteins. However, we also found that it may help offset some of the biosynthetic deficiencies of its host stemming from the plastid loss by providing haem and a lysine precursor. Altogether, our study establishes a new single-cell model system for studying plastid loss and endosymbiont acquisition.

Asthma is a chronic respiratory illness that causes mild to severe inflammation throughout narrowed airways. During allergic airways inflammation, autophagy prevents extensive lung tissue impairment while inducing a protective anti-pathogen response and macrophages in the lung to maintain homeostasis. Previous studies of autophagy genes and asthma have shown an association with variants in ATG5, but a comprehensive analysis of autophagy related genes and asthma has not been performed. Here we utilize summary statistic data generated from a two-stage genome-wide association study (GWAS) of asthma in the UK Biobank. We examined variants in 21 autophagy related genes and found statistically significant associations for 28 variants in two genes in the discovery dataset and nominally significant replication for 16 of these variants, all annotated to ATG4B. This is the first evidence of an association with variants in ATG4B with asthma which provides a novel potential for future drug development.

SummaryExtracellular vesicles (EVs) are bilayer vesicles that carry a diverse cargo of molecules, such as nucleic acids, proteins and metabolites. These EVs can be transported throughout the organism to specific recipient tissues. For this reason, EVs have been recognized as pivotal mediators of cell-to-cell communication (CCC). Importantly, alterations in EV-mediated communication have been linked to pathological processes, further highlighting their biological relevance. However, the in silico exploration of the functional effects of EV cargo in recipient tissues remains limited due to the lack of dedicated tools that can be applied to EV omics datasets. Most current bioinformatics tools for assessing CCC rely on ligand-mediated communication and therefore cannot be used to explore EV-mediated communication. To address this gap, we developed EV-Net, a bioinformatics tool designed to explore the effects of EV cargo on recipient tissues. EV-Net was built by adapting NicheNet, a CCC bioinformatics tool that relies on ligand-receptor mediated communication, for the analysis of EVs proteomics and RNA-seq data. The EV-Net framework enables the identification and prioritization of EV cargo molecules with high regulatory potential in a recipient tissue of interest. This prioritization facilitates the systematic translation of EV cargo profiles into testable biological hypotheses. Availability and documentationThe source code of EV-Net is stored in GitHub https://github.com/torrejoNia/EV-Net alongside instructions on how to install it. Comprehensive tutorials and additional documentation are available at https://torrejonia.github.io/EV-Net/. The datasets used in the use cases were deposited in Zenodo. The corresponding Zenodo links are provided in the tutorials for each use case. This software is distributed under a GLP3 licence.

Cellular senescence exerts powerful non-cell autonomous effects through the senescencelzlassociated secretory phenotype (SASP). This SASP comprises soluble factors and extracellular vesicles (EVs). Although soluble SASP components can induce senescence in neigbouring cells, the specific contribution of EVs to paracrine senescence is poorly defined. Here, we show that EVs released by senescent tumor cells are necessary and sufficient to propagate senescence. Conditioned media from bleomycinlzlinduced senescent A549 cells triggered a permanent growth arrest with morphological changes and upregulation of senescence markers in recipient tumor cells. Pharmacological inhibition of EV biogenesis using GW4869 or genetic downregulation of the EV secretion mediator RAB27A markedly attenuates paracrine senescence without affecting soluble SASP factor secretion or the senescent state of producer cells. Proteomic characterization reveals that senescent EVs exhibit a distinct molecular signature enriched for extracellular components and processes related to wound healing and hemostasis. Importantly, purified senescent EVs, devoid of soluble SASP factors, fully recapitulated paracrine senescence induction. These findings identify senescent EVs as key autonomous SASP effectors and highlight vesicular pathways as potential therapeutic targets in cancer and therapylzlinduced senescence.

Altered iron homeostasis has long been implicated in Parkinson's Disease (PD), although the mechanisms have not been clear. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of a homozygous mutant LRRK2G2019S on iron homeostasis within the RAW macrophage cell line with high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2G2019S mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. These conditions lead to an accumulation of phosphorylated Rab8 at the plasma membrane, which is selectively inhibited by LRRK type II kinase inhibitors. Iron overload then leads to increased oxidative stress and ferroptotic cell death. These data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD.

Aging causes a progressive loss of tissue homeostasis, with stem cell exhaustion as a major hallmark. Age-associated decline in organ function is widely perceived as emanating from progressive accumulation of cellular damage in adult tissues. However, whether aging trajectories are established early on during development remains an open question. Here, we demonstrate that genetic modulation of cellular aging pathways in larval adult midgut progenitors (AMPs), which serve as the precursors of adult intestinal stem cells and differentiated epithelial cells, dictates the long-term trajectory of intestinal aging in Drosophila. Accelerated cellular aging by genetic perturbation employing Toll or Imd pathway overactivation or elevation of reactive oxygen species (ROS) using ND42 (mitochondrial complex I) knockdown in the AMPs results in aberrant progenitor proliferation, skewed lineage allocation, epithelial barrier dysfunction, and genomic instability. These alterations are accompanied by marked destabilization of AMP islet architecture and widespread changes in age-related molecular signatures, as revealed by bulk transcriptomic analysis. In contrast, decelerated cellular aging mediated by Foxo or Atg8a overexpression results in a decrease in enteroendocrine population and the intestinal barrier remained unaffected. Intriguingly, early-life activation of immune and oxidative stress signaling manifested later in the adult gut as elevated enteroendocrine differentiation, highlighting lasting effects on intestinal regenerative capacity and lineage balance. Together, our findings demonstrate that cellular aging is tightly regulated early on in development and its perturbation can cause developmental disruption hampering adult gut homeostasis, establishing AMPs as key developmental determinants that regulate the trajectory of intestinal aging in Drosophila.

Frailty is a prevalent geriatric syndrome, and the shortage of objective biomarkers restricts its early diagnosis and intervention. This study aimed to identify robust molecular signatures and diagnostic markers for frailty using bioinformatics analyses of multiple independent datasets. Two transcriptome datasets (GSE144304, n=80; GSE287726, n=70) were obtained from the GEO database. We performed differential gene expression analysis, GO, KEGG and GSEA enrichment, and machine learning (70% training / 30% validation) to screen and validate core biomarkers. Numerous shared differentially expressed genes were identified. Vitamin D metabolism, ABC transporter, and inflammatory/immune pathways were consistently enriched and confirmed by GSEA. Machine learning models based on these signatures showed favorable diagnostic performance. Our study demonstrates that vitamin D metabolic disorders and chronic inflammation are core molecular features of frailty. The identified biomarkers provide new strategies for basic research, early clinical diagnosis, and therapeutic target development for frailty.

The unfolded protein response (UPR) helps reinstate cellular proteostasis upon an accumulation of misfolded proteins in the endoplasmic reticulum (ER), in part through ER-associated degradation (ERAD). Ube2j2 is an ER-localized E2 ubiquitin-conjugating enzyme that participates in ERAD. We used mass spectrometry analysis of cultured U2OS cells to investigate how the loss of Ube2j2 affects the cellular proteome in response to tunicamycin-induced ER stress. We constructed a network of twelve statistically distinct modules of protein abundance profiles across conditions. We describe the Gene Ontology annotations for each module along with the "hub gene" proteins whose abundance levels most closely adhere to each modules protein abundance profile. Our analysis identifies known Ube2j2-associated pathways (e.g., the UPR and ERAD) and cellular functions that were previously unassociated with Ube2j2 (e.g., RNA metabolism, ER-Golgi transport, and cell-cycle progression). These data are available via ProteomeXchange with identifier PXD076153 and provide avenues for further investigation into the cellular functions of Ube2j2 under basal and ER-stressed conditions.

Parkinsons disease (PD) exhibits well-established sex differences in prevalence and clinical phenotypes, yet the underlying molecular mechanisms remain largely elusive. Here, we conducted a comprehensive sex-stratified multi-omic integration to identify sex-specific causal proteins and biological pathways in PD. We performed gene-based association analysis, transcriptome-wide association studies (TWAS), and proteome-wide Mendelian randomization (PWMR) with colocalization analysis using GWAS summary statistics from the International PD Genetics Consortium (IPDGC; 12,054 male cases/11,999 controls; 7,384 female cases/12,389 controls) for sex-stratified analyses and Global Parkinsons Genetics Program (GP2; 34,933 cases/31,009 controls) for sex-combined analyses. Prioritized candidates were further evaluated through MR with brain expression quantitative trait loci (eQTLs) from MetaBrain and differential protein abundance analysis using the Global Neurodegeneration Proteomics Consortium (GNPC; 704 PD cases/5,629 controls in plasma; 78 cases/1,411 controls in cerebrospinal fluid). Additionally, pathway enrichment analysis was performed for prioritized molecules. Integration across three analytical layers prioritized 102 molecular candidates across 31 unique loci, significant from multiple analyses. Of these, eleven genes reached significance across all three layers, including SNCA, MAPT, and CTSB significant in both sexes; CD160, GPNMB, and LRRC37A2 as male-predominant; STX4 and PRSS53 as female-predominant; and BST1, SCARB2, and LGALS3 significant only in sex-combined analysis. In males, CD160 emerged as a novel candidate with convergent evidence across all three analyses and colocalization, while L3MBTL2 was identified as a novel risk gene from gene-based association and TWAS analyses. In females, STX4 and PRSS53 at the 16p11.2 locus showed female-predominant associations. Pathway enrichment analysis revealed innate immune and SUMOylation pathways in males, with CD160 and L3MBTL2 as key contributors respectively, contrasting with WDR5-mediated chromatin remodeling in females. Brain eQTL-based MR confirmed significant associations for 69 of 86 testable candidates (80.2%) in at least one tissue. Protein abundance analysis confirmed sex-specific patterns, and several candidates showed discordant directions between genetically predicted causal effects and observed protein abundance -- including male-specific plasma elevation of CD160 and female-specific patterns for STX4 -- underscoring the distinction between causal risk mechanisms and disease-state molecular changes. These findings demonstrate that PD is a molecularly heterogeneous disorder with sexually dimorphic pathogenic drivers. While shared axes such as lysosomal dysfunction and vesicle trafficking disruption exist, the divergence into male-specific immune dysregulation and female-specific chromatin remodeling suggests that the primary triggers of neurodegeneration differ by sex. Our results underscore the necessity of sex-stratified approaches in biomarker discovery and the development of precision therapeutic strategies for PD.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.