Methamphetamine Increases Tubulo-Vesicular Areas While Dissipating Proteins from Vesicles Involved in Cell Clearance.

Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.

Bafilomycin 1A Affects p62/SQSTM1 Autophagy Marker Protein Level and Autophagosome Puncta Formation Oppositely under Various Inflammatory Conditions in Cultured Rat Microglial Cells.

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.

Phillyrin promotes autophagosome formation in A53T-αSyn-induced Parkinson's disease model via modulation of REEP1.

BACKGROUND: The preservation of autophagosome formation presents a promising strategy for tackling neurological disorders, such as Parkinson's disease (PD). Mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) serve not only as a focal point linked to various neurological disorders but also play a crucial role in supporting the biogenesis of autophagosomes. PURPOSE: This investigation aimed to elucidate the neuroprotective properties of phillyrin against PD and its underlying mechanisms in promoting autophagosome formation. METHODS: ER and mitochondria co-localization was assessed via fluorescent staining. Annexin V-fluorescein isothiocyanate (FITC) fluorescence was employed to quantify accessible cardiolipin (CL) on mitochondrial surfaces. The levels of CL within the MAM fraction of SH-SY5Y cells were evaluated using a CL probe assay kit. Monodansylcadaverine staining was utilized to detect autophagosome formation in SH-SY5Y cells. In an A53T-alpha-synuclein (αSyn)-induced PD mouse model, the anti-PD properties of phillyrin were assessed using open field, pole climbing, and rotarod tests, as well as immunohistochemistry staining of TH+ neurons in the brain sections. RESULTS: In A53T-αSyn-treated SH-SY5Y cells, phillyrin facilitated autophagosome formation by suppressing CL externalization and restoring MAM integrity. Phillyrin enhanced the localization of receptor expression-enhancing protein 1 (REEP1) within MAM and mitochondria, bolstering MAM formation. Increased REEP1 levels in mitochondria, attributed to phillyrin, enhanced the interaction between REEP1 and NDPK-D, thereby reducing CL externalization. Furthermore, phillyrin exhibited a dose-dependent enhancement of motor function in mice, accompanied by an increase in the abundance of dopaminergic neurons within the substantia nigra. CONCLUSIONS: These findings illuminate phillyrin's ability to enhance MAM formation through upregulation of REEP1 expression within MAM, while concurrently attenuating CL externalization via the REEP1-NDPK-D interaction. These mechanisms bolster autophagosome biogenesis, offering resilience against A53T-αSyn-induced PD. Thus, our study advances the understanding of phillyrin's complex mechanisms and underscores its potential as a therapeutic approach for PD, opening new avenues in natural product pharmacology.

Aloperine Suppresses Cancer Progression by Interacting with VPS4A to Inhibit Autophagosome-lysosome Fusion in NSCLC.

Aloperine (ALO), a quinolizidine-type alkaloid isolated from a natural Chinese herb, has shown promising antitumor effects. Nevertheless, its common mechanism of action and specific target remain elusive. Here, it is demonstrated that ALO inhibits the proliferation and migration of non-small cell lung cancer cell lines in vitro and the tumor development in several mouse tumor models in vivo. Mechanistically, ALO inhibits the fusion of autophagosomes with lysosomes and the autophagic flux, leading to the accumulation of sequestosome-1 (SQSTM1) and production of reactive oxygen species (ROS), thereby inducing tumor cell apoptosis and preventing tumor growth. Knockdown of SQSTM1 in cells inhibits ROS production and reverses ALO-induced cell apoptosis. Furthermore, VPS4A is identified as a direct target of ALO, and the amino acids F153 and D263 of VPS4A are confirmed as the binding sites for ALO. Knockout of VPS4A in H1299 cells demonstrates a similar biological effect as ALO treatment. Additionally, ALO enhances the efficacy of the anti-PD-L1/TGF-ß bispecific antibody in inhibiting LLC-derived subcutaneous tumor models. Thus, ALO is first identified as a novel late-stage autophagy inhibitor that triggers tumor cell death by targeting VPS4A.

Long Noncoding RNA NR_030777 Alleviates Cobalt Nanoparticles-Induced Neurodegenerative Damage by Promoting Autophagosome-Lysosome Fusion.

Potential exposure to cobalt nanoparticles (CoNPs) occurs in various fields, including hard alloy industrial production, the increasing use of new energy lithium-ion batteries, and millions of patients with metal-on-metal joint prostheses. Evidence from human, animal, and in vitro experiments suggests a close relationship between CoNPs and neurotoxicity. However, a systematic assessment of central nervous system (CNS) impairment due to CoNPs exposure and the underlying molecular mechanisms is lacking. In this study, we found that CoNPs induced neurodegenerative damage both in vivo and in vitro, including cognitive impairment, ß-amyloid deposition and Tau hyperphosphorylation. CoNPs promoted the formation of autophagosomes and impeding autophagosomal-lysosomal fusion in vivo and in vitro, leading to toxic protein accumulation. Moreover, CoNPs exposure reduced the level of transcription factor EB (TFEB) and the abundance of lysosome, causing a blockage in autophagosomal-lysosomal fusion. Interestingly, overexpression of long noncoding RNA NR_030777 mitigated CoNPs-induced neurodegenerative damage in both in vivo and in vitro models. Fluorescence in situ hybridization assay revealed that NR_030777 directly binds and stabilizes TFEB mRNA, alleviating the blockage of autophagosomal-lysosomal fusion and ultimately restoring neurodegeneration induced by CoNPs in vivo and in vitro. In summary, our study demonstrates that autophagic dysfunction is the main toxic mechanism of neurodegeneration upon CoNPs exposure and NR_030777 plays a crucial role in CoNPs-induced autophagic dysfunction. Additionally, the proposed adverse outcome pathway contributes to a better understanding of CNS toxicity assessment of CoNPs.

FUNDC1 alleviates doxorubicin-induced cardiotoxicity by restoring mitochondrial-endoplasmic reticulum contacts and blocked autophagic flux.

Rationale: Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. Methods: We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model in vivo. Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson's trichrome staining, respectively. Results: DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. Conclusions: In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.

Altered autophagic flux in GNE mutant cells of Indian origin: Potential drug target for GNE myopathy.

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of ß-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.

Novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease.

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

Alpha-Hederin induces incomplete autophagic injury in non-small cell lung cancer by interfering with the lysosomal acidification.

Lung cancer is the most common oncological disease worldwide, with non-small cell lung cancer accounting for approximately 85% of lung cancer cases. α-Hederin is a monodesmosidic triterpenoid saponin isolated from the leaves of Hedera helix L. or Nigella sativa and has been extensively studied for its antitumor activity against a variety of tumor cells. It has been suggested that α-Hederin is a potential regulator of autophagy and has high promise for application. However, the specific mechanism and characteristics of α-Hederin in regulating autophagy are not well understood. In this study, we confirmed the potential of α-Hederin application in lung cancer treatment and comprehensively explored the mechanism and characteristics of α-Hederin in regulating autophagy in lung cancer cells. Our results suggest that α-Hederin is an incomplete autophagy inducer that targets mTOR to activate the classical autophagic pathway, inhibits lysosomal acidification without significantly affecting the processes of autophagosome transport, lysosome biogenesis, autophagosome and lysosome fusion, and finally leads to impaired autophagic flux and triggers autophagic damage in NSCLC.

Targeted degradation of NDUFS1 by agrimol B promotes mitochondrial ROS accumulation and cytotoxic autophagy arrest in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a potential anticancer drug. Our recent report showed a favorable anticancer effect of agrimol B in HCC, however, the mechanism of action remains unclear. Here, we found agrimol B inhibits the growth and proliferation of HCC cells in vitro as well as in an HCC patient-derived xenograft (PDX) model. Notably, agrimol B drives autophagy initiation and blocks autophagosome-lysosome fusion, resulting in autophagosome accumulation and autophagy arrest in HCC cells. Mechanistically, agrimol B downregulates the protein level of NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) through caspase 3-mediated degradation, leading to mitochondrial reactive oxygen species (mROS) accumulation and autophagy arrest. NDUFS1 overexpression partially restores mROS overproduction, autophagosome accumulation, and growth inhibition induced by agrimol B, suggesting a cytotoxic role of agrimol B-induced autophagy arrest in HCC cells. Notably, agrimol B significantly enhances the sensitivity of HCC cells to sorafenib in vitro and in vivo. In conclusion, our study uncovers the anticancer mechanism of agrimol B in HCC involving the regulation of oxidative stress and autophagy, and suggests agrimol B as a potential therapeutic drug for HCC treatment.

Hernandezine promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation.

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum, is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her's dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies.

Tanreqing injection inhibits influenza virus replication by promoting the fusion of autophagosomes with lysosomes: An integrated pharmacological study.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanreqing injection (TRQ) is widely used, traditional Chinese medicine (TCM) injection used in China to treat respiratory infections. Modern pharmacological studies have confirmed that TRQ can protect against influenza viruses. However, the mechanism by which TRQ inhibits influenza viruses remains unclear. AIM OF THE STUDY: To explore the therapeutic effects and possible mechanisms of TRQ inhibition by the influenza virus. MATERIALS AND METHODS: Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to determine the chemical composition of TRQ. Isobaric tags for relative and absolute quantification (iTRAQ) were used to define differential proteins related to TRQ inhibition of viruses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation. For experimental validation, we established an in vitro model of the influenza virus infection by infecting A549 cells with the virus. The detection of the signaling pathway was carried out through qPCR, western blotting,and immunofluorescence. RESULTS: Fifty one components were identified using UPLC/Q-TOF MS. We confirmed the inhibitory effect of TRQ on influenza virus replication in vitro. Ninety nine differentially expressed proteins related to the inhibitory effect of TRQ were identified using iTRAQ. KEGG functional enrichment analysis showed that the TRQ may inhibit influenza virus replication by affecting autophagy. Through network analysis, 29 targets were selected as major targets, and three key targets, HSPA5, PARP1, and GAPDH, may be the TRQ targets affecting autophagy. In vitro experiments showed that TRQ inhibits influenza virus replication by interfering with the expression and localization of STX17 and VAMP8 proteins, thereby promoting the fusion of autophagosomes with lysosomes. CONCLUSION: TRQ inhibits influenza virus replication by promoting the fusion of autophagosomes with lysosomes. We additionally established potential gene and protein targets which are affected by TRQ. Therefore, our findings provide new therapeutic targets and a foundation further studies on influenza treatment with TRQ.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

The SAR and action mechanisms of autophagy inhibitors that eliminate drug resistance.

Autophagy is an essential homeostatic and catabolic process crucial for the degradation or recycling of proteins and cellular components. Drug resistance has been demonstrated to be closely implicated in increased autophagy. Autophagy inhibition to reverse drug resistance involves in the five stages of autophagy, including phagophore initiation, vesicle nucleation, vesicle elongation, vesicle fusion and cargo degradation. Herein, emphases were placed on discussions on the targets responsible for the upstream phagophore initiation and nucleation of autophagosome, as well as the ones mediating the downstream autophagosome and lysosome fusion and cargo degradation. The structure-activity relationships (SARs) and action mechanisms of the corresponding target-based small molecule autophagy inhibitors were analyzed and delineated. This review will provide a promising guidance for the design and optimization of drug-like scaffolds in the discovery of autophagy inhibitors able to eliminate drug resistance.

Kansl1 haploinsufficiency impairs autophagosome-lysosome fusion and links autophagic dysfunction with Koolen-de Vries syndrome in mice.

Koolen-de Vries syndrome (KdVS) is a rare disorder caused by haploinsufficiency of KAT8 regulatory NSL complex subunit 1 (KANSL1), which is characterized by intellectual disability, heart failure, hypotonia, and congenital malformations. To date, no effective treatment has been found for KdVS, largely due to its unknown pathogenesis. Using siRNA screening, we identified KANSL1 as an essential gene for autophagy. Mechanistic study shows that KANSL1 modulates autophagosome-lysosome fusion for cargo degradation via transcriptional regulation of autophagosomal gene, STX17. Kansl1+/- mice exhibit impairment in the autophagic clearance of damaged mitochondria and accumulation of reactive oxygen species, thereby resulting in defective neuronal and cardiac functions. Moreover, we discovered that the FDA-approved drug 13-cis retinoic acid can reverse these mitophagic defects and neurobehavioral abnormalities in Kansl1+/- mice by promoting autophagosome-lysosome fusion. Hence, these findings demonstrate a critical role for KANSL1 in autophagy and indicate a potentially viable therapeutic strategy for KdVS.

Novel and conventional inhibitors of canonical autophagy differently affect LC3-associated phagocytosis.

In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases.

Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells.

Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome-lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.

Interleukin 10 inhibits oxidative stress-induced autophagosome formation in hepatic stellate cells by activating the mTOR-STAT3 pathway.

Autophagy is involved in the activation of hepatic stellate cells (HSCs) and liver fibrosis. Previous studies have shown that interleukin 10 (IL-10) has a marked therapeutic effect against liver fibrosis. However, few studies have evaluated the effect of IL-10 on autophagy in HSCs and fibrotic livers. The aim of this study was to assess the effect of IL-10 on the autophagy of HSCs in vitro and in vivo and then to explore the underlying pathway. In vitro, The results revealed that IL-10 had inhibitory effects on hydrogen peroxide (H2O2)-induced autophagy, as evidenced by the decreased LC3II/I ratio and Beclin1 expression, increased p62 expression, reduced numbers of autophagosomes, and blocked autophagy initiation in HSCs. Mechanistically, IL-10 significantly promoted the phosphorylation of the signal transducer and activator of transcription 3(STAT3) and mammalian target of rapamycin (mTOR), leading to the activation of STAT3 and mTOR, which in turn inhibited autophagy. In vivo, the increased expression of IL-10 in fibrotic livers inhibited significantly liver fibrosis and decreased the autophagic activity in fibrotic livers and HSCs. Overall, our results indicate that IL-10 suppressed H2O2-induced autophagy in HSCs by activating the STAT3-mTOR signaling pathway. Present study provides a new theoretical basis for the anti-fibrotic effects of IL-10.

1α,25(OH)2D3(VD3) promotes Raddeanin A-induced anti-proliferative effects on HeLa cell apoptosis and autophagy through negative regulation of HPV18E6-E7/PD-L1/VDR axis.

Raddeanin A (RA) has indicated suppressive effects on various human tumor cells, and insufficient vitamin D was associated with human papillomavirus (HPV) persistence and gynecological tumors. However, combined effects of RA and vitamin D on HPV-positive cells remain elusive. Herein, we aimed to investigate the combined effects of RA and 1ɑ,25(OH)2D3 (VD3) on cellular viability and modulation of HPV18E6/E7, programmed cell death 1 ligand (PD-L1) and vitamin D receptor (VDR) expression in HeLa cells in vitro. HeLa cells were treated with RA alone or VD3 combined with RA. Cell viability was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and apoptosis was detected by flow cytometry. Real-time PCR (qRT-PCR) and Western blot were used to determine the gene/protein expression levels. The autophagosomes were observed by Transmission electron microscopy (TEM). The result showed that cell viability was inhibited by RA, and apoptosis in HeLa cells treated with RA was elevated accordingly. The expression of Bax, Cleaved-caspase-3, Cleaved-caspase-9 and Cleaved-PARP increased, and Bcl-2 decreased. The autophagy was induced by RA, as evidenced by elevated autophagosomes and the increased LC3-II/I ratio and Beclin-1. The expression of HPV18E6/E7, PD-L1 and VDR was reduced by RA. Moreover, RA combined with VD3 had a stronger effect on HeLa cells than RA alone. In conclusion, RA inhibits HeLa proliferation and induces apoptosis and autophagy via suppressing HPV18E6/E7, PD-L1 and VDR, and VD3 showed reinforced effects of RA on HeLa cells. Therefore, combined usage of VD3 with RA might be a potential novel immunotherapy strategy for HPV-related diseases.

Robust LC3B lipidation analysis by precisely adjusting autophagic flux.

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.