Inhibition Of Autophagosome Formation Research Articles

SEC31a-ATG9a Interaction Mediates the Recruitment of COPII Vesicles for Autophagosome Formation.

Autophagy plays an important role in determining stem-cell differentiation. During the osteogenic differentiation of mesenchymal stem cells (MSCs), autophagosome formation is upregulated but the reason is unknown. A long-standing quest in the autophagy field is to find the membrane origin of autophagosomes. In this study, cytoplasmic coat protein complex II (COPII) vesicles, endoplasmic reticulum-derived vesicles responsible for the transport of storage proteins to the Golgi, are demonstrated to be a critical source of osteoblastic autophagosomal membrane. A significant correlation between the number of COPII vesicle and the autophagy level is identified in the rat bone tissues. Disruption of COPII vesicles restrained osteogenesis and decreased the number and size of autophagosomes. SEC31a (an outer coat protein of COPII vesicle) is found to be vital to regulate COPII vesicle-dependent autophagosome formation via interacting with ATG9a of autophagosomal seed vesicles. The interference of Sec31a inhibited autophagosome formation and osteogenesis in vitro and in vivo. These results identified a novel mechanism of autophagosome formation in osteogenic differentiation of stem cells and identified SEC31a as a critical protein that mediates the interplay between COPII and ATG9a vesicles. These findings broaden the understanding of the regulatory mechanism in the osteogenic differentiation of MSCs.

Dexmedetomidine alleviates CoCl2-induced hypoxic cellular damage in INS-1 cells by regulating autophagy.

Ischemia-reperfusion (I/R) injury is inevitable during the perioperative period. The pancreas is susceptible to I/R injury. Autophagy, a self-digestion process, is upregulated during I/R injury and strongly induced by hypoxia. This study aims to determine whether dexmedetomidine can decrease pancreatic β-cell damage by regulating autophagy under hypoxia. INS-1 rat insulinoma cells were cultured in dexmedetomidine before being exposed to cobalt chloride (CoCl2)-induced hypoxia. Cell viability and the expression of autophagy-related proteins (light chain 3B [LC3B]-II, p62, and ATGs) were assessed. The expression of apoptosis-related proteins (BCL-2 and P-BAD) were also evaluated. CoCl2-treated INS-1 cells were pretreated with the autophagosome formation inhibitor, 3-methyladenine (3-MA), to compare its effects with those of dexmedetomidine. Bafilomycin-A1 (Baf-A1) that inhibits autophagosome degradation was used to confirm the changes in autophagosome formation induced by dexmedetomidine. Dexmedetomidine attenuated the increased expression of autophagic proteins (LC3B-II, p62, and ATGs) and reversed the CoCl2-induced reduction in the proliferation of INS-1 cells after hypoxia. Dexmedetomidine also alleviated the decreased expression of the anti-apoptotic protein (BCL-2) and the increased expression of apoptotic protein (BAX). Dexmedetomidine reduces the activation of autophagy through inhibiting autophagosome formation, as confirmed by a decrease in LC3B-II/I ratio, a marker of autophagosome formation, in LC3B turnover assay combined with Baf-A1. Dexmedetomidine alleviates the degree of cellular damage in INS-1 cells against CoCl2-induced hypoxia by regulating autophagosome formation. These results provide a basis for further studies to confirm these effects in clinical practice.

Mangiferin prevents glucolipotoxicity-induced pancreatic beta-cell injury through modulation of autophagy via AMPK-mTOR signaling pathway

The aim of this study was to investigate the protective effects of Mangiferin (MG) on glucolipotoxicity-induced pancreatic beta-cell injury. In vivo administration of MG significantly reduced the level of blood glucose in high-fat diet (HFD)-fed mice. MG treatment inhibited beta-cell apoptosis in HFD-treated mice. In vitro, MG protected INS-1 cells against apoptosis and impairment of insulin secretion following High glucose/Palmitic acid (HG/PA) treatment. MG treatment enhanced autophagy flux which was blocked by HG/PA treatment. Inhibition of autophagosome formation by 3-Methyladenine or blockade of autolysosome by Chloroquine reversed the protective effects of MG on INS-1 cells. MG treatment increased AMPK phosphorylation and reduced mTOR activation in INS-1 cells. Administration of the AMPK blocker abrogated MG-induced autophagy, and similar results were observed in INS-1 cells after cotreatment with MG and mTOR activator. In conclusion, MG ameliorated pancreatic beta-cell injury induced by glucolipotoxicity through modulation of autophagy via the AMPK-mTOR pathway.

NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

Autophagy is closely related to the occurrence and development of human malignancies; however, the detailed mechanisms underlying autophagy in cervical cancer require further investigation. Previously, we found that the ectopic expression of NCAPH, a regulatory subunit of condensed protein complexes, significantly enhanced the proliferation of tumor cells; however, the underlying mechanisms were unclear. Here, we revealed that NCAPH is a novel autophagy-associated protein in cervical cancer that promotes cell proliferation by inhibiting autophagosome formation and reducing autophagy, with no effect on the cell cycle, apoptosis, or aging. Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in inflammation, autoimmunity and cancer, mainly via its E3 ubiquitin ligase activity. Mass spectrometry and immunoprecipitation assays showed that TRIM21 interacted with NCAPH and decreased the protein stability of NCAPH via ubiquitination at the K11 lysine residue. Structural domain mutation analysis revealed that TRIM21 combined with NCAPH through its PRY/SPRY and CC domains and accelerated the degradation of NCAPH through the RING domain. Furthermore, TRIM21 promoted autophagosome formation and reduced cell proliferation by inhibiting NCAPH expression and the downstream AKT/mTOR pathway in cervical cancer cells. Immunohistochemical staining revealed that the protein expression of TRIM21 was negatively correlated with that of NCAPH and positively correlated with that of beclin-1 in cervical cancer tissues. Therefore, we provide evidence for the role of the TRIM21-NCAPH axis in cervical cancer autophagy and proliferation and the involvement of the AKT/mTOR signaling pathway in this process. These results deepen our understanding of the carcinogenesis of cervical cancer, broaden the understanding of the molecular mechanisms of TRIM21 and NCAPH, and provide guidance for individualized treatment of cervical cancer in the future.

The mammalian actin elongation factor ENAH/MENA contributes to autophagosome formation via its actin regulatory function

ABSTRACT Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH’s colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH’s EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy. Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl’s balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin.

HMGB1 regulates autophagy of placental trophoblast through ERK signaling pathway†.

The purpose of this study is to investigate the role of high mobility group protein B1 (HMGB1) in placental development and fetal growth. We employed the Cre-loxP recombination system to establish a placenta-specific HMGB1 knockout mouse model. Breeding HMGB1flox/flox mice with Elf5-Cre mice facilitated the knockout, leveraging Elf5 expression in extra-embryonic ectoderm, ectoplacental cone, and trophoblast giant cells at 12.5days of embryonic development. The primary goal of this model was to elucidate the molecular mechanism of HMGB1 in placental development, assessing parameters such as placental weight, fetal weight, and bone development. Additionally, we utilized lentiviral interference and overexpression of HMGB1 in human trophoblast cells to further investigate HMGB1's functional role. Our findings indicate that the HMGB1flox/floxElf5cre/+ mouse displays fetal growth restriction, characterized by decreased placental and fetal weight and impaired bone development. The absence of HMGB1 inhibits autophagosome formation, impairs lysosomal degradation, and disrupts autophagic flux. Depletion of HMGB1 in human trophoblast cells also suppresses cell viability, proliferation, migration, and invasion by inhibiting the ERK signaling pathway. Overexpression of HMGB1 observed the opposite phenotypes. HMGB1 participates in the regulation of autophagy through the ERK signaling pathway and affects placental development.

Xie-Bai-San increases NSCLC cells sensitivity to gefitinib by inhibiting Beclin-1 mediated autophagosome formation

BackgroundAutophagy, a cellular process involving lysosomal self-digestion, plays a crucial role in recycling biomolecules and degrading dysfunctional proteins and damaged organelles. However, in non-small cell lung cancer (NSCLC), cancer cells can exploit autophagy to survive metabolic stress and develop resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which reduce treatment efficacies. Currently, most studies have found that late-stage autophagy inhibitors can hinder EGFR-TKIs resistance, while research on early-stage autophagy inhibitors is still limited. PurposeThis study investigates the mechanism via which the Xie-Bai-San (XBS) formula enhances NSCLC cell sensitivity to gefitinib, revealing the relationship between XBS-induced cell death and the inhibition of autophagosome formation. MethodsCell viability was assessed using CCK-8 and EdU assays, lentivirus transfection was utilized to generate PC9 cells harboring the PIK3CA E545K mutation (referred to as PC9-M), autophagic flux was monitored using mCherry-GFP-LC3 adenovirus. Protein expression and colocalization were observed through immunofluorescence staining. The interaction between Bcl-2 and Beclin-1 in PC9-GR and PC9-M cells was determined via co-immunoprecipitation (Co-IP) assay, cell apoptosis was assessed by flow cytometry and PI staining, and overall survival analysis of lung adenocarcinoma patients was conducted using the TCGA database. In vivo experiments included a patient-derived xenograft (PDX) model with EGFR and PIK3CA mutations and subcutaneous mice xenografts of NSCLC cell lines (PC9 and PC9-GR). In addition, autophagic vesicles in mouse tumor tissues were observed via transmission electron microscopy analysis. ResultsXBS effectively inhibits the proliferation of gefitinib-resistant NSCLC cells and induces apoptosis both in vitro and in vivo. Mechanistically, XBS suppresses gefitinib-induced autophagic flux by inhibiting autophagy through the upregulation of p-mTOR and Bcl-2 and downregulation of Beclin-1. Additionally, XBS enhances the interaction between Bcl-2 and Beclin-1, and the overexpression of Beclin-1 promotes NSCLC cell proliferation and counteracts XBS-induced cell death, while XBS demonstrates minimal impact on autophagosome-lysosome fusion or lysosome function. ConclusionThis study reveals a novel role for the XBS formula in impeding autophagy initiation and demonstrates its potential as a candidate drug to counteract autophagy-induced treatment resistance in NSCLC.

MET Oncogene Enhances Pro-Migratory Functions by Counteracting NMDAR2B Cleavage.

The involvement of the N-methyl-D-aspartate receptor (NMDAR), a glutamate-gated ion channel, in promoting the invasive growth of cancer cells is an area of ongoing investigation. Our previous findings revealed a physical interaction between NMDAR and MET, the hepatocyte growth factor (HGF) receptor. However, the molecular mechanisms underlying this NMDAR/MET interaction remain unclear. In this study, we demonstrate that the NMDAR2B subunit undergoes proteolytic processing, resulting in a low-molecular-weight form of 100 kDa. Interestingly, when the NMDAR2B and MET constructs were co-transfected, the full-size high-molecular-weight NMDAR2B form of 160 kDa was predominantly observed. The protection of NMDAR2B from cleavage was dependent on the kinase activity of MET. We provide the following evidence that MET opposes the autophagic lysosomal proteolysis of NMDAR2B: (i) MET decreased the protein levels of lysosomal cathepsins; (ii) treatment with either an inhibitor of autophagosome formation or the fusion of the autophagosome and lysosome elevated the proportion of the NMDAR2B protein's uncleaved form; (iii) a specific mTOR inhibitor hindered the anti-autophagic effect of MET. Finally, we demonstrate that MET coopts NMDAR2B to augment cell migration. This implies that MET harnesses the functionality of NMDAR2B to enhance the ability of cancer cells to migrate.

TGFβ1 Secretion in Megakaryocytes Is Autophagy-Dependent and Its Inhibition Ameliorates Myelofibrosis in Mice

Megakaryocytes (MKs) are large, polyploid platelet precursor cells that primarily reside within the bone marrow. Recent advances revealed that in addition to platelet production, MKs contribute to immunity and maintenance of bone marrow homeostasis, raising the question of whether these functions are hijacked in disease. One hallmark of primary and secondary myelofibrosis (MF), subsets of myeloproliferative neoplasms, is the accumulation of aberrant, immature MKs in the bone marrow. These release pro-fibrotic cytokines such as transforming growth factor β1 (TGFβ1) and interleukin 1β (IL1β) into the bone marrow, thus inducing fibrotic alterations that ultimately cause pancytopenia - the major cause of mortality in patients with MF. While current treatment strategies in MF aim to prevent MK proliferation and correct the differentiation defects, a direct targeting of cytokine release from MKs has not yet been explored. Recently, unconventional secretion of TGFβ1 and IL1β via secretory autophagy, a secretion pathway driven by the fusion of autophagosomes with the plasma membrane, has been shown to occur in other cell types. We therefore hypothesize that MKs may preferentially utilize secretory autophagy for cytokine release and that inhibiting autophagosome formation might therefore slow fibrosis progression in vivo. Previous studies suggested that TGFβ1 is stored within specialized α-granules in MKs. To test this, we cultured murine MKs and determined TGFβ1 co-localization with different α-granule proteins by super-resolution confocal microscopy. However, while we did not identify any correlation between TGFβ1 and α-granules, we found that TGFβ1 strongly co-localized with the autophagy marker light chain (LC) 3B, supporting a potential role for secretory autophagy. To investigate this, we disrupted secretory autophagy by inhibiting the GTPase RhoA and its downstream effector Rho kinase (ROCK), which were previously shown to promote autophagosome formation. Treatment of MKs with inhibitors of autophagy or RhoA/ROCK markedly reduced TGFβ1 secretion in vitro and led to intracellular accumulation of TGFβ1. Taken together, these data suggest that TGFβ1 is secreted by MKs via secretory autophagy. To examine whether secretory autophagy in MKs contributes to disease pathogenesis in vivo, we employed an MPL W515L-driven transplant model. Basal autophagy measured by LC3B conversion and RhoA protein levels were increased in platelets derived from mice with MPL W515L-driven MF. Conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system ( Mx1-Cre+; Atg5fl/fl mice) prevented leukocytosis, MK clustering and aberrant TGFβ1 secretion in the MPL W515L-driven transplant model, indicating that autophagy promotes the initiation of MF. Furthermore, MPL W515L-transplanted mice treated with a low-dose ROCK inhibitor (ROCKi) - several of which are FDA-approved - reduced MK clustering and fibrosis in spleens and femurs of treated mice (Figure 1a, b). To further verify our data, we utilized a second preclinical mouse model of MF - a knockout mouse lacking the inhibitory MK receptor G6b-B ( Mpig6b -/-), which exhibits thrombocytopenia, MK clustering and MF. In line with our previous findings, conditional deletion of Rhoa in MK lineage cells in Pf4-Cre+; Rhoafl/fl; Mpig6b -/- mice normalized TGFβ1 levels within the bone marrow and protected against the development of fibrosis in bone marrow and spleen. In ongoing work, ROCK inhibitors are further being tested in higher doses alone or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data suggest that TGFβ1 secretion is uncoupled from conventional granule secretion in MKs and is instead dependent on RhoA/autophagy signaling pathways. Targeting these pathways in aberrant MKs represents a new potential therapeutic target in the treatment of MF.

Abstract 17462: Angiotensin II-Mediated Suppression of Skeletal Muscle Autophagy via mTOR and FoxO3a Pathways

Background: Chronic skeletal muscle (SK.M) atrophy, cachexia, occurs in diseases such as congestive heart failure (CHF), which is associated with high levels of angiotensin II (Ang-II). We have shown that Ang-II infusion reproduces major aspects of CHF-induced SK.M atrophy and causes mitochondrial (MT) damage and dysfunction in SK.M. Ang-II also inhibits autophagy/mitophagy, and a combination of these effects likely causes accumulation of damaged MT, leading to SK.M atrophy. Hypothesis: Ang-II infusion suppresses autophagy in SK.M via activation of mTOR and inhibition of FoxO3a. Aim: To understand the molecular mechanisms whereby Ang-II inhibits autophagy/mitophagy in SK.M. Methods: 12 weeks old male FVB mice were infused with Ang-II (1.0 μg·kg -1 ·min -1 ) or saline via osmotic minipumps (ALZET 1007D) for 7 days (N=6). Expression of autophagic markers and FoxO3a target genes were analyzed in tibialis anterior muscles by western blotting (at d1 & d7) and qPCR (at d4), respectively. Colchicine treatment was included in the analyses to assess autophagic flux in vivo . For statistical analysis Two-way ANOVA and t-test were used. Results: In the presence of colchicine, LC3 lipidation was decreased in Ang-II infused mice, indicating that Ang-II inhibits autophagy at the autophagosome formation step. Furthermore, Ang-II increased p62 and PINK1 and decreased BNIP3, suggesting that Ang-II-mediated reduction of autophagy causes disruption of damaged MT clearance via mitophagy. Mechanistically, Ang-II likely inhibits autophagosome formation via two pathways, mTOR and FoxO3a: Ang-II increased phosphorylation of mTOR at Ser2448 (p<0.005) and its downstream target 4E-BP1 at Thr37/46 (p<0.0005). Lastly, Ang-II increased phosphorylation of FoxO3a at Thr32 (p<0.0005), associated with reduced mRNA expression of FoxO3a target genes, such as LC3B and BNIP3 (24% and 46% reductions, respectively). Conclusion: Ang-II inhibits autophagosome formation in SK.M via mTOR activation and FoxO3a inhibition. Suppression of autophagy via these pathways likely disrupts clearance of damaged MT, leading to SK.M atrophy. These results provide insights for future development of therapies for CHF-induced cachexia by targeting mTOR and FoxO3a pathways to restore autophagy.

Ethanol inhibits pancreatic acinar cell autophagy through upregulation of ATG4B, mediating pathological responses of alcoholic pancreatitis.

Excessive alcohol intake is a major risk factor for pancreatitis, sensitizing the exocrine pancreas to stressors by mechanisms that remain obscure. Impaired autophagy drives nonalcoholic pancreatitis, but the effects of ethanol (EtOH) and alcoholic pancreatitis on autophagy are poorly understood. Here, we find that ethanol reduces autophagosome formation in pancreatic acinar cells, both in a mouse model of alcoholic pancreatitis induced by a combination of EtOH diet and cerulein (a CCK ortholog) and in EtOH+CCK-treated acinar cells (ex vivo model). Ethanol treatments decreased pancreatic level of LC3-II, a key mediator of autophagosome formation. This was caused by ethanol-induced upregulation of ATG4B, a cysteine protease that, cell dependently, regulates the balance between cytosolic LC3-I and membrane-bound LC3-II. We show that ATG4B negatively regulates LC3-II in acinar cells subjected to EtOH treatments. Ethanol raised ATG4B level by inhibiting its degradation, enhanced ATG4B enzymatic activity, and strengthened its interaction with LC3-II. We also found an increase in ATG4B and impaired autophagy in a dissimilar, nonsecretagogue model of alcoholic pancreatitis induced by EtOH plus palmitoleic acid. Adenoviral ATG4B overexpression in acinar cells greatly reduced LC3-II and inhibited autophagy. Furthermore, it aggravated trypsinogen activation and necrosis, mimicking key responses of ex vivo alcoholic pancreatitis. Conversely, shRNA Atg4B knockdown enhanced autophagosome formation and alleviated ethanol-induced acinar cell damage. The results reveal a novel mechanism, whereby ethanol inhibits autophagosome formation and thus sensitizes pancreatitis, and a key role of ATG4B in ethanol's effects on autophagy. Enhancing pancreatic autophagy, particularly by downregulating ATG4B, could be beneficial in mitigating the severity of alcoholic pancreatitis.NEW & NOTEWORTHY Ethanol sensitizes mice and humans to pancreatitis, but the underlying mechanisms remain obscure. Autophagy is important for maintaining pancreatic acinar cell homeostasis, and its impairment drives pancreatitis. This study reveals a novel mechanism, whereby ethanol inhibits autophagosome formation through upregulating ATG4B, a key cysteine protease. ATG4B upregulation inhibits autophagy in acinar cells and aggravates pathological responses of experimental alcoholic pancreatitis. Enhancing pancreatic autophagy, particularly by down-regulating ATG4B, could be beneficial for treatment of alcoholic pancreatitis.

Inhibiting autophagy by miR-19a-3p/PTEN regulation protected retinal pigment epithelial cells from hyperglycemic damage

ObjectiveThe catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear. MethodsAn in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition. ResultsAutophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition. ConclusionsOur findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR.

Cardiomyocyte peroxisome proliferator-activated receptor α prevents septic cardiomyopathy via improving mitochondrial function.

Clinically, cardiac dysfunction is a key component of sepsis-induced multi-organ failure. Mitochondria are essential for cardiomyocyte homeostasis, as disruption of mitochondrial dynamics enhances mitophagy and apoptosis. However, therapies targeted to improve mitochondrial function in septic patients have not been explored. Transcriptomic data analysis revealed that the peroxisome proliferator-activated receptor (PPAR) signaling pathway in the heart was the most significantly decreased inthe cecal ligation puncture-treated mouse heart model, and PPARα was the most notably decreased among the three PPAR family members. Male Pparafl/fl (wild-type), cardiomyocyte-specific Ppara-deficient (PparaΔCM), and myeloid-specific Ppara-deficient (PparaΔMac) mice were injected intraperitoneally with lipopolysaccharide (LPS) to induce endotoxic cardiac dysfunction. PPARα signaling was decreased in LPS-treated wild-type mouse hearts. To determine the cell type in which PPARα signaling was suppressed, the cell type-specific Ppara-null mice were examined. Cardiomyocyte- but not myeloid-specific Ppara deficiency resulted in exacerbated LPS-induced cardiac dysfunction. Ppara disruption in cardiomyocytes augmented mitochondrial dysfunction, as revealed by damaged mitochondria, lowered ATP contents, decreased mitochondrial complex activities, and increased DRP1/MFN1 protein levels. RNA sequencing results further showed that cardiomyocyte Ppara deficiency potentiated the impairment of fatty acid metabolism in LPS-treated heart tissue. Disruption of mitochondrial dynamics resulted in increased mitophagy and mitochondrial-dependent apoptosis in Ppara△CM mice. Moreover, mitochondrial dysfunction caused an increase of reactive oxygen species, leading to increased IL-6/STAT3/NF-κB signaling. 3-Methyladenine (3-MA, an autophagosome formation inhibitor) alleviated cardiomyocyte Ppara disruption-induced mitochondrial dysfunction and cardiomyopathy. Finally, pre-treatment with the PPARα agonist WY14643 lowered mitochondrial dysfunction-induced cardiomyopathy in hearts from LPS-treated mice. Thus, cardiomyocyte but not myeloid PPARα protects against septic cardiomyopathy by improving fatty acid metabolism and mitochondrial dysfunction, thus highlighting that cardiomyocyte PPARα may be a therapeutic target for the treatment of cardiac disease.

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction

ABSTRACT Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy. Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

PACSIN2 as a modulator of autophagy and mercaptopurine cytotoxicity: mechanisms in lymphoid and intestinal cells.

PACSIN2 variants are associated with gastrointestinal effects of thiopurines and thiopurine methyltransferase activity through an uncharacterized mechanism that is postulated to involve autophagy. This study aims to clarify the role of PACSIN2 in autophagy and in thiopurine cytotoxicity in leukemic and intestinal models. Higher autophagy and lower PACSIN2 levels were observed in inflamed compared with non-inflamed colon biopsies of inflammatory bowel disease pediatric patients at diagnosis. PACSIN2 was identified as an inhibitor of autophagy, putatively through inhibition of autophagosome formation by a protein-protein interaction with LC3-II, mediated by a LIR motif. Moreover, PACSIN2 resulted a modulator of mercaptopurine-induced cytotoxicity in intestinal cells, suggesting that PACSIN2-regulated autophagy levels might influence thiopurine sensitivity. However, PACSIN2 modulates cellular thiopurine methyltransferase activity via mechanisms distinct from its modulation of autophagy.

Catalase-deficient mice induce aging faster through lysosomal dysfunction

BackgroundLysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice.MethodsWe performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group.ResultsWe found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice.ConclusionsThis study unveils the new role of catalase and its role in lysosomal function during aging.D3gySv46Ggy6LMJqdgNDW3Video abstract

Ginsenoside Re prevents 3-methyladenine-induced catagen phase acceleration by regulating Wnt/β-catenin signaling in human dermal papilla cells

BackgroundThe human hair follicle undergoes cyclic phases—anagen, catagen, and telogen—throughout its lifetime. This cyclic transition has been studied as a target for treating hair loss. Recently, correlation between the inhibition of autophagy and acceleration of the catagen phase in human hair follicles was investigated. However, the role of autophagy in human dermal papilla cells (hDPCs), which is involved in the development and growth of hair follicles, is not known. We hypothesized that acceleration of hair catagen phase upon inhibition of autophagy is due to the downregulation of Wnt/β-catenin signaling in hDPCs, and that components of Panax ginseng extract can increase the autophagic flux in hDPCs. MethodsWe generated an autophagy-inhibited condition using 3-methyladenine (3-MA), a specific autophagy inhibitor, and investigated the regulation of Wnt/β-catenin signaling using the luciferase reporter assay, qRT-PCR, and western blot analysis. In addition, cells were cotreated with ginsenoside Re and 3-MA and their roles in inhibiting autophagosome formation were investigated. ResultsWe found that the unstimulated anagen phase dermal papilla region expressed the autophagy marker, LC3. Transcription of Wnt-related genes and nuclear translocation of β-catenin were reduced after treatment of hDPCs with 3-MA. In addition, treatment with the combination of ginsenoside Re and 3-MA changed the Wnt activity and hair cycle by restoring autophagy. ConclusionsOur results suggest that autophagy inhibition in hDPCs accelerates the catagen phase by downregulating Wnt/β-catenin signaling. Furthermore, ginsenoside Re, which increased autophagy in hDPCs, could be useful for reducing hair loss caused by abnormal inhibition of autophagy.

Albendazole-induced autophagy blockade contributes to elevated apoptosis in cholangiocarcinoma cells through AMPK/mTOR activation

Albendazole (ABZ) is a broad-spectrum anti-parasitic drug that exhibits antitumor effects against several carcinomas. The effects of ABZ on cholangiocarcinoma (CCA) and its underlying mechanisms are still unclear. Our study aims to investigate the role of ABZ in inducing autophagy-mediated apoptosis of cholangiocarcinoma cells. The antitumor effects of ABZ were evaluated against CCA cells and HIBEC intrahepatic biliary epithelial cells. Furthermore, the apoptosis rates, and autophagy flux in RBE and FRH-0201 cells treated with ABZ were investigated. ABZ inhibited proliferation, induced cell death and apoptosis in CCA cells in vitro. In vivo, tumors from ABZ- treated BALB/c nude mice were significantly smaller than untreated mice. ABZ also induced the initiation of autophagy via AMPK/mTOR pathways, resulting in the formation of autophagosome. In addition, ABZ blocked autophagic flux by inhibiting the fusion of autophagosome-lysosome, which increased the apoptotic death of CCA cells. However, the apoptotic death of CCA cells induced by ABZ was reversed by 3-methyladenine (3-MA), an autophagosome formation inhibitor, but increased by chloroquine (CQ), an autophagosome-lysosome fusion inhibitor.Our work provides novel mechanisms for anti-tumor effects of ABZ on CCA, suggesting that ABZ may be used as a potent autophagy inhibitor in the treatment of CCA.

Isoliquiritigenin inhibits pancreatic cancer progression through blockade of p38 MAPK-regulated autophagy

BackgroundPancreatic cancer has been characterized by poor prognosis, early metastasis and dissatisfactory treatment outcome. The high basal level of autophagy in tumor cells leads to chemoresistance and tumor progression. Thus, it is imminent to explore novel effective chemotherapeutic adjuvants to increase patients’ survival rate. Isoliquiritigenin (ISL) is a bioactive flavonoid obtained from the Traditional Chinese herbal medicine Glycyrrhiza glabra, and it possesses a broad range of pharmacological effects. In this study, the anti-cancer effect of ISL in pancreatic cancer treatment and the underlying mechanism are investigated. MethodsMTT assay, colony formation and EdU analysis were performed to explore the growth inhibition of ISL on pancreatic cancer cells. Apoptosis were analyzed using TUNEL and flow cytometry. The formations of autophagosomes were analyzed by immunofluorescence microscopy and transmission electron microscopy. RFP-GFP-LC3B probe was applied to detect the autophagy flux. To assess the structural interaction of ISL with p38 protein, molecular docking assays were performed. The molecular mechanism was elucidated by using western immunoblotting. Subsequently, the inhibition of ISL on tumor growth was determined in vivo using pancreatic tumor mice model. ResultsISL inhibited pancreatic cancer cell growth and induced apoptosis, both in vitro and in vivo. ISL caused accumulation of autophagosome through blockade of late stage autophagic flux. Moreover, autophagy inducer rapamycin enhanced ISL-evoked cell growth inhibition and promoted apoptosis, while inhibition of autophagosome formation by siAtg5 attenuated ISL-induced apoptosis. It is remarkable that ISL synergistically sensitized the cytotoxic effect of gemcitabine and 5-fluorouracil on pancreatic cancer cells as both drugs induced autophagy. Molecular docking analysis has indicated that ISL acted by direct targeting of p38 MAPK, which was confirmed by ISL-induced phosphorylation of p38. The autophagy flux induced by p38 inhibitor SB203580 was blocked by ISL, with further increasing toxicity of ISL in pancreatic cancer cells. ConclusionThe results have revealed that ISL inhibited pancreatic cancer progression by blockade of autophagy through p38 MAPK signaling.

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling

BackgroundAutophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease (AD). This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.MethodsThe primary hippocampal neurons, N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy, which was analysed by Student's two-tailed t-test. The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1 (mTORC1) activity and the vacuolar H+-ATPase (v-ATPase) activity, respectively, which were analysed by One‐way ANOVA with post hoc tests. The Western blotting, co-immunoprecipitation and immunofluorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations, as analysed by Student's two-tailed t-test or One‐way ANOVA with post hoc tests. The autophagosome formation was detected by immunofluorescence staining and transmission electron microscopy. The amino acids (AA) levels were detected by high performance liquid chromatography (HPLC).ResultsWe observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits. Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1 (PRD-TIA1) and this association significantly increased the intercellular level of amino acids (Leucine, P = 0.0038; Glutamic acid, P = 0.0348; Alanine, P = 0.0037; Glycine, P = 0.0104), with concordant upregulation of mTORC1 activity [phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1), P < 0.0001; phosphorylated 70 kDa ribosomal protein S6 kinase 1 (p-p70S6K1), P = 0.0001, phosphorylated unc-51-like autophagy-activating kinase 1 (p-ULK1), P = 0.0015] and inhibition of autophagosome formation [microtubule-associated protein light chain 3 II (LC3 II), P = 0.0073; LC3 puncta, P < 0.0001]. As expected, this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation. Importantly, we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1, downregulating the endogenous TIA1 expression by shRNA, or downregulating tau protein level by a small proteolysis targeting chimera (PROTAC) could remarkably attenuate tau-induced autophagy impairment.ConclusionsOur findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway, and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treatment and that of related tauopathies.