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Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.

Bafilomycin A1 (BAF A1) is a macrolide antibiotic that, in addition to its antibacterial activity, can induce apoptosis of cancer cells. Glycosylation plays an important role in the modification of antibiotics as it can improve their bioactivity. However, glycosylation of BAF A1 has not been previously reported. Bacillus licheniformis glycosyltransferase (GTs) Bl-YjiC and Bacillus subtilis GTs Bs-YjiC were expressed successfully in a heterologous manner. The glycosylation of BAF A1 with UDP-glucose, UDP-galactose or UDP-N-acetylglucosamine was catalyzed using the enzymes Bl-YjiC and Bs-YjiC. Our results demonstrated that Bl-YjiC can only utilize UDP-glucose as the donor, while Bs-YjiC can utilize all three glycosyl donors. The glycosylation site was demonstrated by MS/MS to be the hydroxyl group at the C21 position of BAF A1. The anti-proliferative effects of glucosyl BAF A1 (BAF-Glc) on HeLa cells indicate that this novel antibiotic is superior to BAF A1. The IC50 for BAF-Glc was determined to be 5.47 μM. Here, we report the production of glycosylated BAF A1 for the first time, and we show that the produced BAF-Glc exhibited better anticancer activity than BAF A1. This work provides theoretical and experimental support for the development of novel anticancer bafilomycins.

Cisplatin plays a key role in treating small cell lung cancer (SCLC); however, the rapid development of cisplatin resistance limits its treatment effect. The detailed mechanisms of cisplatin-resistance, particularly in SCLC, remain unclear. We analyzed the differentially expressed genes (DEGs) between cisplatin-resistant small cell lung cancer cell line H446/CDDP and its parental cell line H446, using the transcriptome sequencing technique. Gene ontology (GO) analysis and the subsequent tests demonstrated that the functions of protein ubiquitination and autophagy are more active in the H446/CDDP cells. Autophagy plays a protective role in the H446/CDDP cells by using the autophagy inhibitors, 3-methyladenine and bafilomycin A1. Moreover, antimalarial drugs that inhibit autophagy by increasing the pH of lysosomes can also enhance cisplatin-induced cell death.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Oxidative stress-induced DNA damage, the senescence-associated secretory phenotype (SASP), and impaired autophagy all are general features of senescent cells. However, the cross-talk among these events and processes is not fully understood. Here, using NIH3T3 cells exposed to hydrogen peroxide stress, we show that stress-induced DNA damage provokes the SASP largely via cytosolic chromatin fragment (CCF) formation, which activates a cascade comprising cGMP-AMP synthase (cGAS), stimulator of interferon genes protein (STING), NF-κB, and SASP, and that autolysosomal function inhibits this cascade. We found that CCFs accumulate in senescent cells with activated cGAS-STING-NF-κB signaling, promoting SASP and cellular senescence. We also present evidence that the persistent accumulation of CCFs in prematurely senescent cells is partially associated with a defect in DNA-degrading activity in autolysosomes and reduced abundance of activated DNase 2α. Intriguingly, we found that metformin- or rapamycin-induced activation of autophagy significantly lessened the size and levels of CCFs and repressed the activation of the cGAS-STING-NF-κB-SASP cascade and cellular senescence. These effects of autophagy activators indicated that autolysosomal function contributes to CCF clearance and SASP suppression, further supported by the fact that the lysosome inhibitor bafilomycin A1 blocked the role of autophagy-mediated CCF clearance and senescence repression.

Grass carp reovirus (GCRV), the genus Aquareovirus in family Reoviridae, is viewed as the most pathogenic aquareovirus. To understand the molecular mechanism of how aquareovirus initiates productive infection, the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots (QDs)-tracking in combination with biochemical approaches. We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH(4)Cl, chloroquine and bafilomycin A1 (Bafi). Confocal images indicated that GCRV particles could colocalize with Rab5, Rab7 and lysosomes in host cells. Further ultrastructural examination validated that viral particle was found in late endosomes. Moreover, disruption of microtubules with nocodazole clearly blocked GCRV entry, while no inhibitory effects were observed with cytochalasin D treated cells in viral infection, hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament. Notably, viral particles were observed to transport along microtubules by using QD-labeled GCRV. Altogether, our results suggest that GCRV can use endosomes and microtubules to initiate productive infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00178-1) contains supplementary material, which is available to authorized users.

Autophagy is a conserved cellular process involving intracellular membrane trafficking and degradation. Pathogens, including hepatitis C virus (HCV), often exploit this process to promote their own survival. The aim of this study was to determine the mechanism by which HCV increases steady-state autophagosome numbers while simultaneously inhibiting flux through the autophagic pathway. Using the lysosomal inhibitor bafilomycin A1, we showed that HCV-induced alterations in autophagy result from a blockage of autophagosome degradation rather than an increase in autophagosome generation. In HCV-infected cells, lysosome function was normal, but a tandem RFP–GFP–LC3 failed to reach the lysosome even under conditions that activate autophagy. Autophagosomes and lysosomes isolated from HCV-infected cells were able to fuse with each other normally in vitro, suggesting that the cellular fusion defect resulted from trafficking rather than an inability of vesicles to fuse. Arl8b is an Arf-like GTPase that specifically localizes to lysosomes and plays a role in autophagic flux through its effect on lysosomal positioning. At basal levels, Arl8b was primarily found in a perinuclear localization and co-localized with LC3-positive autophagosomes. HCV infection increased the level of Arl8b 3-fold and redistributed Arl8b to a more diffuse, peripheral pattern that failed to co-localize with LC3. Knockdown of Arl8b in HCV-infected cells restored autophagosome–lysosome fusion and autophagic flux to levels seen in control cells. Thus, HCV suppresses autophagic flux and increases the steady-state levels of autophagosomes by increasing the expression of Arl8b, which repositions lysosomes and prevents their fusion with autophagosomes.

Recent studies suggest that phytochemicals, as part of the food matrix, might alter the toxicity of nanoparticles (NPs); however, relatively few studies have investigated the impact of anthocyanidins on the toxicity of NPs to cells lining the gastrointestinal tract. Therefore, this study used cyanidin chloride (CC) as the model for anthocyanidins and investigated the effects of CC on the toxicity of ZnO or Ag NPs to Caco-2 cells. Exposure to ZnO but not Ag NPs significantly induced cytotoxicity. The presence of CC, but not its analog quercetin (Qu), modestly protected Caco-2 cells from ZnO NP exposure. However, the intracellular superoxide, Zn ions, or release of interleukin-8 after ZnO NP exposure were not significantly affected by the presence of CC. Rather, CC promoted the expression of autophagic genes ATG5, ATG7, and BECN1 as well as the ratio of LC3-II/I after exposure to ZnO NPs. Meanwhile, the presence of autophagic inhibitors (chloroquine, NH4Cl, bafilomycin A1) significantly promoted the cytotoxicity of ZnO NPs and inhibited the cytoprotective effects of CC. In conclusion, these data suggest that CC could modestly protect Caco-2 cells from ZnO NP exposure, probably through the induction of autophagy.

Objective To investigate how a high glucose environment influences the osteogenic ability of periodontal ligament stem cells (PDLSCs) and the function of autophagy in this process, we explored whether the osteogenic ability of PDLSCs could be protected by autophagy. Design PDLSC proliferation and osteogenesis were evaluated by CCK-8 and western blotting under gradient glucose conditions. The Autophagy RT2 Profiler PCR Array was used to screen autophagy-related mRNA expression during PDLSC osteoblastic differentiation on 5.5 mM + osteogenic induction (OI) medium or 25 mM + OI medium on day 3. Autophagy was regulated by an inducer (rapamycin) and inhibitor (bafilomycin) to investigate its protective effects on PDLSCs. A periodontal trauma model was established in diabetic rats to verify the effects of enhanced autophagy activity on PDLSCs. Results A high glucose concentration (25 mM) impeded PDLSC proliferation on day 1, and compared with the control condition, high glucose also decreased the osteogenic ability of PDLSCs. The Autophagy RT2 Profiler PCR Array showed obvious fluctuations in many autophagy-related genes, such as ULK1 (9.27), MTOR (3.15), MAP1LC3B (4.22), GABARAPL1 (7.09), ATG10 (6.5), AMPK14 (4.47), WIPI1 (3.29), and IGF1 (24.65). Compared with the control condition, an autophagy inducer or inhibitor markedly impaired or enhanced osteogenic differentiation in cells. The diabetic rat periodontal trauma model demonstrated that periodontium tissue partly recovered in the autophagy-enhanced cell injection diabetic rat group. Conclusions High glucose inhibited the activity of PDLSCs, and regulating autophagy protected cell function. Upregulating autophagy partially reversed the adverse effect of high glucose conditions on PDLSCs.

Molecular behavior under bilayer membrane environments is one of the important research topics concerning how organic molecules exert their biological activities when interacting with cellular membranes. However, chemistry-based approaches to this property have not been successful when compared with the structural biological strategy on ligand-receptor interactions. Here, we investigated the molecular behavior of the lipophilic ATPase inhibitor bafilomycin A1 and its derivatives under a lipid environment from a chemical point of view. Our results revealed significant differences in membrane affinity and dynamics among ligands having different inhibitory potencies, suggesting the specific contribution of ligand-membrane interactions to their biological activity.

The oxygen partial pressure generally increases when cancerous cells become part of the blood vessels. The study was to investigate the influence of oxygen partial pressure on the apoptosis of B16 melanoma cells. Our results demonstrated that both short-term and long-term hypoxia/reoxygenation (H/R) treatment increased stress-induced intracellular reactive oxygen species (ROS). H/R treatment also increased apoptosis and autophagy in B16 cells. N-acetylcysteine (NAC), a ROS scavenger, can reduce ROS and aid survival. However, Bafilomycin A1, an autophagy inhibitor, can accelerate cell death. Thus, our work revealed that ROS and autophagy play critical roles in cellular H/R.

Damage-associated molecular pattern molecules (DAMPs) are endogenous danger signals that alert the innate immune system and shape the inflammation response to cell death. However, the release and activity of DAMPs in ferroptosis, a recently identified form of regulated necrosis characterized by iron overload and lipid peroxidation, still remain poorly understood. Here, we demonstrate that HMGB1 is a DAMP released by ferroptotic cells in an autophagy-dependent manner. Both type I and II ferroptosis activators, including erastin, sorafenib, RSL3, and FIN56, induce HMGB1 release in cancer and noncancer cells. In contrast, genetic ablation (using ATG5-/- or ATG7-/- cells) or pharmacologic inhibition (the administration of bafilomycin A1 or chloroquine) of autophagy was found to block ferroptosis activator-induced HMGB1 release. Mechanically, autophagy-mediated HDAC inhibition promotes HMGB1 acetylation, resulting in HMGB1 release in ferroptosis. Moreover, AGER, but not TLR4, is required for HMGB1-mediated inflammation in macrophages in response to ferroptotic cells. These studies suggest that HMGB1 inhibition might have some potential therapeutic effects in ferroptosis-associated human disease.

We hypothesized that autophagy and associated lysosome function serve as a critical modulator during Nod-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome activation on proatherogenic stimuli. We first demonstrated that 7-ketocholesterol stimulated Nlrp3 inflammasome formation and activation as shown by increased colocalization of inflammasome components [Nlrp3 versus apoptosis associated speck-like protein (Asc) or caspase-1] and enhanced cleavage of caspase-1 into active caspase-1 to generate IL-1β in coronary artery smooth muscle cells. Deletion of the CD38 gene (CD38(−/−)) that regulates lysosome function and autophagic flux also led to Nlrp3 inflammasome formation and activation. In the presence of rapamycin, the effects of either 7-ketocholesterol treatment or CD38 gene deletion were abolished. The autophagy inhibitor spautin-1 and the lysosome function blocker bafilomycin A1 also enhanced Nlrp3 inflammasome formation and activation. In animal experiments, we found that increased colocalization of Nlrp3 versus Asc or caspase-1 enhanced IL-1β accumulation and caspase-1 activity in the coronary arterial wall of CD38(−/−) mice on the Western diet compared with CD38(+/+) mice. This increased colocalization was blocked by treatment with rapamycin but enhanced by chloroquine, a water-soluble blocker of autophagic flux. Morphologic examinations confirmed that the media of coronary arteries was significantly thicker in CD38(−/−) mice on the Western diet than CD38(+/+) mice. In conclusion, the deficiency of autophagic flux promotes Nlrp3 inflammasome formation and activation in coronary artery smooth muscle cells on proatherogenic stimulation, leading to medial thickening of the coronary arterial wall.

Schistosomiasis is a disease of global importance caused by parasitic flatworms, schistosomes, which cause pathogenicity through eggs laid by the female worm inside the host's blood vessels. Maintenance of cellular homeostasis is crucial for parasites, as for other organisms, and is quite likely important for schistosome reproduction and vitality. We hypothesize a role for autophagy in these processes, an evolutionarily conserved and essential cellular degradation pathway. Here, for the first known time, we shed light on the autophagy machinery and its involvement in pairing-dependent processes, vitality and reproduction of Schistosoma mansoni. We identified autophagy genes by in silico analyses and determined the influence of in vitro culture on the transcriptional expression in male and female worms using quantitative real-time PCR. Among the identified autophagy genes were Beclin, Ambra1, Vps34, DRAM, DAP1, and LC3B, of which some showed a sex-dependent expression. Specifically, the death-associated protein DAP1 was significantly more highly expressed in females compared with males, while for the damage-regulated autophagy modulator DRAM it was the opposite. Furthermore, in-vitro culture significantly changed the transcript expression level of DAP1 in female worms. Next, worms were treated with an autophagy inducer (rapamycin) or inhibitors (bafilomycin A1, wortmannin and spautin-1) to evaluate effects on autophagy protein expression, worm vitality, and reproduction. The conversion of the key autophagy protein LC3B, a marker for autophagic activity, was increased by rapamycin and blocked by bafilomycin. All inhibitors affected worm fitness, egg production, and negatively affected the morphology of gonads and intestine. In summary, autophagy genes in S. mansoni show an interesting sex-dependent expression pattern and manipulation of autophagy in S. mansoni by inhibitors induced detrimental effects, which encourages subsequent studies to identify antischistosomal targets within the autophagy machinery.

The role of autophagy in tongue squamous cell carcinoma (TSCC) cisplatin resistance is unclear. We aimed to identify a possible synergistic effect of autophagy inhibitors and cisplatin in TSCC cells and explore the underlying mechanism. Our results indicate that autophagic flux was high in TSCC cells; Autophagy inhibitor bafilomycin A1 increased cisplatin cytotoxicity in TSCC cells by inhibiting lysosomal uptake of platinum and enhancing intracellular platinum ion binding to DNA; Autophagy gene (Atg5) knockout in TSCC cells did not duplicate the above-mentioned sensitization of bafilomycin A1. Furthermore, we found that cisplatin resistance of TSCC cells was related to cisplatin inducing lysosome biogenesis in a TFEB-dependent manner, which was regulated by c-Abl. In summary, this is the first study to show that Bafilomycin A1 increases the sensitivity of TSCC cells to cisplatin by inhibiting lysosomal function but not autophagy. Lysosomes may be a potential target to increase cisplatin cytotoxicity toward TSCC cells.

Autophagy and apoptosis are two different biological processes that determine cell fates. We previously reported that autophagy inhibition and apoptosis induction are involved in lead(II)-induced cytotoxicity in primary rat proximal tubular (rPT) cells, but the interplay between them remains to be elucidated. Firstly, data showed that lead(II)-induced elevation of LC3-II protein levels can be significantly modulated by 3-methyladenine or rapamycin; moreover, protein levels of Autophagy-related protein 5 (Atg5) and Beclin-1 were markedly up-regulated by lead(II) treatment, demonstrating that lead(II) could promote the autophagosomes formation in rPT cells. Next, we applied three pharmacological agents and genetic method targeting the early stage of autophagy to validate that enhancement of autophagosomes formation can inhibit lead(II)-induced apoptotic cell death in rPT cells. Simultaneously, lead(II) inhibited the autophagic degradation of rPT cells, while the addition of autophagic degradation inhibitor bafilomycin A1 aggravated lead(II)-induced apoptotic death in rPT cells. Collectively, this study provided us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells, and the interplay between autophagy and apoptosis highlights a new sight into the mechanism of lead(II)-induced nephrotoxicity.

Precise regulation of vas deferens fluid pH is essential for sperm. However, the mechanisms underlying effect of testosterone on vas deferens fluid pH have never been identified, which could involve changes in expression and functional activity of vacoular (V)-ATPase. Methods Orchidectomized, adult male Sprague-Dawley rats were treated subcutaneously with 125 μg/kg/day and 250 μg/kg/day testosterone with or without flutamide (androgen receptor blocker) and finasteride (5α-reductase inhibitor) for seven (7) days. Following treatment completion, in vivo perfusion of vas deferens lumen was performed and changes in fluid secretion rate, pH and HCO3− content were measured with and without bafilomycin, a V-ATPase inhibitor. Rats were then sacrificed and vas deferens were harvested and subjected for V-ATPase A1 and B1/2 protein expression and distribution analysis by western blotting and immunohistochemistry, respectively. Results In sham-operated and testosterone-treated orchidectomized rats, higher fluid secretion rate, which was not antagonized by bafilomycin but lower HCO3− content and pH which were antagonized by bafilomycin were observed when compared to orchidectomized-only and orchidectomized, testosterone-treated rats receiving flutamide or finasteride, respectively. Bafilomycin had no effect on fluid secretion rate, HCO3− content and pH in orchidectomized and testosterone-treated orchidectomized rats receiving flutamide and finasteride. V-ATPase A1 and B1/2 proteins were expressed at high levels in vas deferens and were highly distributed at the apical membrane of luminal epithelium and in muscle layer of this organ, mainly in sham and testosterone-treated orchidectomized rats. Conclusions V-ATPase is involved in acidification of vas deferens fluid under testosterone influence.

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment.

The current research hot spot in the field of autophagic flux is to explain and alleviate disease from the perspective of autophagy. A highly sophisticated, sensitive, quantifiable and comprehensive method is required to accurately determine the dynamic process of autophagic flux. There are very few methods in neuroscience that specifically examine autophagic flux. Therefore, primary cortical neurons were divided into oxygen glucose deprivation/reperfusion (OGD/R) (group A) and OGD/R plus bafilomycin A1 (BafA1) (group B) groups. ① Transfection of the LC3 gene with the RFP-GFP tandem fluorescent label was performed. ② Direct quantification was performed using transmission electron microscopy (TEM). ③ Autophagy-related tools were used to detect the transformation of LC3I/II. ④ SQSTM1/P62 combined with the LC3 protein flip test was performed to comprehensively evaluate autophagic flux. Using method one, the ratio of autophagolysosomes to autophagosomes in group A was significantly increased based on fluorescence microscopy analysis. Using method two, the autophagy process in group A was more continuous and unobstructed based on TEM analysis, while only some partial processes were observed in group B, and the number of autophagosomes and autophagy lysosomes in group A was significantly greater more than that in group B. The LC3II/I ratio measured in method three was analysed in detail to explain the autophagic flux. The ratio of soluble p62 combined with the ratio of LC3II/I detected using method four reflected the activation of autophagy. In summary, each method has its own advantages, and different methods and indicators can be used to monitor different stages of autophagy. An understanding of these advantages and mastery of these methods, is a very promising strategy to systematically and objectively study central nervous system diseases, facilitate the rational use of drugs, and formulate effective treatment plans from the perspective of autophagy.

Summary Rhinovirus species C (RV-C) causes more severe asthma attacks than other rhinovirus species. However, the modulation of RV-C replication by drugs has not been well studied. Primary human nasal epithelial (HNE) cells cultured on filter membranes with air-liquid interface methods were infected with RV-C03, and the levels of RV-C03 RNA collected from the airway surface liquid (ASL) of HNE cells were measured with a SYBR Green assay. Pretreatment of HNE cells with the specific vacuolar H+-ATPase inhibitor bafilomycin A1 reduced the RV-C03 RNA levels in the ASL; inflammatory cytokines, including interleukin (IL)-1β, IL-6 and IL-8, in the supernatant; the mRNA expression of the RV-C receptor cadherin-related family member 3 (CDHR3) in the cells; and the number of acidic endosomes where RV-B RNA enters the cytoplasm. The levels of RV-C03 RNA in the ASL obtained from HNE cells with the CDHR3 rs6967,330 G/A genotype tended to be higher than those obtained from HNE cells with the G/G genotype. Pretreatment with the Na+/H+ exchanger inhibitor ethyl-isopropyl amiloride or either of the macrolides clarithromycin or EM900 also reduced RV-C03 RNA levels in the ASL and the number of acidic endosomes in HNE cells. In addition, significant levels of RV-A16, RV-B14 and RV-C25 RNA were detected in the ASL, and bafilomycin A1 also decreased the RV-C25 RNA levels. These findings suggest that bafilomycin A1 may reduce the release of RV-Cs and inflammatory cytokines from human airway epithelial cells. RV-Cs may be sensitive to drugs, including bafilomycin A1, that increase endosomal pH, and CDHR3 may mediate virus entry through receptor-mediated endocytosis in human airway epithelial cells.

Two novel 16-membered tetraene marcolides, bafilomycins N (1) and O (2), along with two known analogues, JBIR-100 (3) and bafilomycin K (4) were produced from isolate GIC10-1, a Streptomyces sp. strain that was originally cloned from bacterial communities associated with marine sponge Theonella sp. The structures of new bafilomycins 1 and 2 were established by using spectroscopic methods and comparing the spectroscopic data with those of known related metabolites. Compounds 1 and 2 were proven to be the first 14-methylbafilomycins to be isolated. Cytotoxicity of these compounds toward various cancer cell lines also is described.

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca2+ leak using synthetic Ca2+ indicators that report changes in both the cytoplasmic ([Ca2+]i) and the luminal ER ([Ca2+]ER) Ca2+ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca2+ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca2+]ER but did not decrease the ER Ca2+ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP3R and only Orai3 channel supported the 2-APB-induced ER Ca2+ leak, while Orai1 and Orai2 inhibited this type of ER Ca2+ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca2+ leak but did not modify the ER Ca2+ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca2+]i response after TG but only when the ER store had been overloaded with Ca2+ by eliminating the acidic internal Ca2+ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca2+ leak but forms an ER Ca2+ leak channel that is limiting the overloading with Ca2+ of the ER store.

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei. When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30–60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

Bafilomycins are an important subgroup of polyketides with diverse biological activities and possible applications as specific inhibitors of vacuolar H+-ATPase. However, the general toxicity and structural complexity of bafilomycins present formidable challenges to drug design via chemical modification, prompting interests in improving bafilomycin activities via biosynthetic approaches. Two bafilomycin biosynthetic gene clusters have been identified, but their post-polyketide synthase (PKS) tailoring steps for structural diversification and bioactivity improvement remain largely unknown. In this study, the post-PKS tailoring pathway from bafilomycin A1 (1)→C1 (2)→B1 (3) in the marine microorganism Streptomyces lohii was elucidated for the first time by in vivo gene inactivation and in vitro biochemical characterization. We found that fumarate is first adenylated by a novel fumarate adenylyltransferase Orf3. Then, the fumaryl transferase Orf2 is responsible for transferring the fumarate moiety from fumaryl-AMP to the 21-hydroxyl group of 1 to generate 2. Last, the ATP-dependent amide synthetase BafY catalyzes the condensation of 2 and 2-amino-3-hydroxycyclopent-2-enone (C5N) produced by the 5-aminolevulinic acid synthase BafZ and the acyl-CoA ligase BafX, giving rise to the final product 3. The elucidation of fumarate incorporation mechanism represents the first paradigm for biosynthesis of natural products containing the fumarate moiety. Moreover, the bafilomycin post-PKS tailoring pathway features an interesting cross-talk between primary and secondary metabolisms for natural product biosynthesis. Taken together, this work provides significant insights into bafilomycin biosynthesis to inform future pharmacological development of these compounds.

Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy., Highlights • Autophagy inhibition decreased mitochondrial bioenergetics in intact neurons. • Autophagy inhibition decreased mitochondrial complexes I, II or IV substrate linked respiration. • Autophagy inhibition increased mitochondrial DNA damage. • Autophagy inhibition decreased major components of the Krebs cycle. • Autophagy inhibition resulted in decreased citrate synthase activities., Graphical abstract fx1

We analyzed V-type H+-ATPase (VA) and Na+/K+-ATPase (NKA) along the caeca and midgut of third instar Drosophila larvae using immunohistochemistry and ATPase activity assays. Corresponding H+ and K+ fluxes were characterized using the Scanning Ion-Selective Electrode Technique (SIET), and the roles of transport ATPases in energizing ion transport across the larval gut were investigated by basal application of bafilomycin, a VA inhibitor, and ouabain, a NKA inhibitor. Addition of bafilomycin led to a decrease in H+ absorption along the caeca and midgut except at the copper cells and large flat cell zone of the middle midgut. H+ absorption was decreased by acetazolamide, consistent with carbonic anhydrase activity in all regions except at the large flat cell zone of the middle midgut. Bafilomycin or acetazolamide also led to decreased K+ absorption across the caeca and the anterior midgut. Our data show the dependence of K+ transport on H+ gradients established by the VA in the latter regions, consistent with the presence of a Cation-Proton Antiporter (CPA2) identified in other insect epithelia. Addition of ouabain led to the increase of K+ absorption along the anterior midgut and the large flat cell zone of the middle midgut, suggesting a role for the NKA in these regions. This study shows the importance of both ATPases in driving ion transport across the gut of larval Drosophila.

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle-exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica-induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle-induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA-074-Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle-induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1.

Autophagy is believed to play an important role in maintaining homeostasis in pancreatic β-cells during insulin resistance. This study investigated the role of autophagy in β-cell damage induced by cholesterol and its possible activation mechanism. Rat and mouse pancreatic β-cell lines INS-1 and βTC-6 were incubated with cholesterol alone or in combination with autophagy inhibitors E-64d/Pepstatin A or bafilomycin A1. DAPI staining, western blotting, transmission electron microscopy and immunofluorescence were conducted to assess the effects of autophagy inhibitors on cholesterol-induced apoptosis and autophagy activity. An increase in FITC-LC3 fluorescence dots, autophagic vacuoles and LC3-II protein indicated that autophagy was activated in cells treated with cholesterol. This was further confirmed by blocking the natural turnover processes in lysosomes and autolysosomes with autophagy inhibitors, suggesting enhanced autophagic activity rather than blockage of autophagy. Furthermore, inhibition of autophagy significantly augmented the activation of caspase 3 and the percentage of cholesterol-induced apoptotic nuclei. These results demonstrate that autophagy plays a protective role against cholesterol-induced apoptosis in pancreatic β-cells.

Rotaviruses infect mature enterocytes from small intestine, however most data about their cellular entry are from studies carried out in non-intestinal polarized or non-polarized cell lines. In this work the entry of porcine rotavirus YM strain into small intestinal cell line IPEC-J2 was studied. It was found that YM and the human rotavirus Wa strain infect preferentially from the basolateral cell surface. Cell infection from the apical and basolateral surfaces was dependent on the presence of cholesterol. The treatment with neuraminidase, sucrose, and bafilomycin suggests that there are differences in the receptor usage and entry mechanism of the virus from the apical and basolateral surface. While cell entry is more efficient from basolateral surface, the viruses egressed mainly from the apical cell side.

MG132 has been used as a proteasome inhibitor on Bombyx cells, but its physiological effects on autophagy still have not been elucidated. In this study, we find that the lipidated BmAtg8, BmAtg8-PE as an autophagosomal marker protein, is only localized to membranes. Then we established systems to monitor autophagic flux in Bombyx cells: Induction of autophagy reduces exogenous BmAtg8 and exogenous BmAtg8-PE, facilitates formation of autophagosomes indicated by green EGFP-BmAtg8 puncta after cotreatment by Rapamycin and Bafilomycin A1, and causes accumulation of free EGFP from EGFP-BmAtg8 cleavage in autolysosomes. Using these established systems, we find that exposure of MG132 inhibits both basal and Rapamycin-induced autophagy when polyubiquitinated proteins are accumulated markedly in Bombyx cells. Interestingly, we reveal that attenuation of autophagy in these cells is ascribed as distinct suppression of formation of autophagosomes after MG132 treatment.

Doxorubicin (Doxo) is one of the most effective anti-neoplastic agents but its cardiotoxicity has been an important clinical limitation. The major mechanism of Doxo-induced cardiotoxicity is associated to its oxidative capacity. However, other processes are also involved with significant consequences for the cardiomyocyte. In recent years, a number of studies have investigated the role of autophagy on Doxo-induced cardiotoxicity but to date it is not clear how Doxo alters that process and its consequence on cardiomyocytes viability. Here we investigated the effect of Doxo 1uM for 24h of stimulation on cultured neonatal rat cardiomyocytes. We showed that Doxo inhibits basal autophagy. This inhibition is due to both Akt/mTOR signaling pathway activation and Beclin 1 level decrease. To assess the role of autophagy on Doxo-induced cardiomyocyte death, we evaluated the effects 3-methyladenine (3-MA), bafilomycin A1 (BafA), siRNA Beclin 1 (siBeclin 1) and rapamycin (Rapa) on cell viability. Inhibition of autophagy with 3-MA, BafA and siBeclin 1 increased lactate dehydrogenase (LDH) release but, when autophagy was induced by Rapa, Doxo-induced cardiomyocyte death was decreased. These results suggest that Doxo inhibits basal autophagy and contributes to cardiomyocyte death. Activation of autophagy could be used as a strategy to protect the heart against Doxo toxicity.

Based on previously reported structure–activity relationships, an analogue of bafilomycin A1 with a site-specific fluorine label at the C2 position was designed and efficiently synthesized. The fluorinated compound exhibited potent vacuolar-type ATPase (V-ATPase) inhibitory activity, comparable to that of the natural product, representing the first example of highly bioactive analogues with a modified macrolactone core from the plecomacrolide family compounds.

Ambroxol (Ax) is a frequently prescribed drug used to facilitate mucociliary clearance, but its mode of action is yet poorly understood. Here we show by X-ray spectroscopy that Ax accumulates in lamellar bodies (LBs), the surfactant storing, secretory lysosomes of type II pneumocytes. Using lyso- and acidotropic substances in combination with fluorescence imaging we confirm that these vesicles belong to the class of acidic Ca2+ stores. Ax lead to a significant neutralization of LB pH, followed by intracellular Ca2+ release, and to a dose-dependent surfactant exocytosis. Ax-induced Ca2+ release was significantly reduced and slowed down by pretreatment of the cells with bafilomycin A1 (Baf A1), an inhibitor of the vesicular H+ ATPase. These results could be nearly reproduced with NH3/NH4+. The findings suggest that Ax accumulates within LBs and severely affects their H+ and Ca2+ homeostasis. This is further supported by an Ax-induced change of nanostructural assembly of surfactant layers. We conclude that Ax profoundly affects LBs presumably by disordering lipid bilayers and by acting as a weak base. The pH change triggers – at least in part – Ca2+ release from stores and secretion of surfactant from type II cells. This novel mechanism of Ax as a lysosomal secretagogue may also play a role for its recently discussed use for lysosomal storage and other degenerative diseases.

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na(+) and K(+) in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na(+), K(+), H(+)) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K(+) and Na(+) were secreted by the distal iliac plexus (DIP) and K(+) was reabsorbed in downstream regions. The fluid secretion rate decreased>50% after 25μM bafilomycin A1, 500μM amiloride or 50μM bumetanide was added to the bath. The concentration of K(+) in the secreted fluid did not change, whereas the concentration of Na(+) in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500μM cAMP or 1μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K(+) concentration in the secreted fluid. An increase in Na(+) concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K(+), whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K(+)/Na(+) ratio.

Although iron disequilibrium has been observed frequently in high-fat diet (HFD) related insulin resistance (IR) the exact mechanism still obscure. Herein, we explore the potential mechanism, focusing on hepatic ferritinophagy flow. Male C57/6J mice were administered with HFD or low-fat diet (LFD) for 10 weeks, and HepG2 cells were treated with palmitate (PA, 200 mM) for 24 h. HFD led to abnormal hepatic steatosis and decline p-AKT and p-GSK3β by 67.1% and 66.3%, respectively. Also, not only decreased iron level but increased endoplasmic reticulum stress (ERS) were observed in the liver of HFD mice and that both them impaired glucose uptake and reduced the expression of p-AKT. However, ferric ammonium citrate (FAC) supplementation improved hepatic IR, as well as ERS. What's more, HFD/PA depleted the labile iron pool (LIP), accumulated p62 and disturbed the expression of nuclear receptor coactivator 4 (NCOA4) and ferritin. While NCOA4 overexpression or rapamycin improved the ERS and impaired glucose uptake in PA incubated HepG2 cells, which was abolished by NCOA4 knockdown or bafilomycin A1. Taken together, these findings suggest that HFD could restrict ferritinophagy flux and interfere with iron metabolism, which resulting in hepatic IR via ERS.

Two bafilomycin derivatives with an unprecedented 6/5/5 tricyclic ring system (1 and 2), along with five known analogues (3–7), were identified from a marine Streptomyces sp. The structures of 1 and 2 were elucidated by analysis of HRESIMS, extensive NMR spectroscopic methods, GIAO DET 13C NMR calculations and comparison of the spectroscopic data with those of known related compounds. Compound 2 exhibited significant cytotoxicity against PC3 human prostate carcinoma cells with IC50 value of 4.99 ± 0.15 μM.

Two new macrolide metabolites of the hygrolidin family, catenulisporidins A and B (1 and 2), together with a known compound hygrolidin (3), were isolated from the culture broth of the rare actinobacterium Catenulispora sp. KCB13F192. Their structures were elucidated on the basis of HRESIMS spectrometric and NMR spectroscopic analyses. Catenulisporidins A and B are the first example of natural hygrolidin and bafilomycin derivatives featuring a modified macrolide ring, and catenulisporidin A possesses a tetrahydrofuran ring through an ether linkage between C-7 and C-10. In cell-based fluorescent imaging and immunoblot assays, the three compounds were shown to inhibit autophagic flux in HeLa cells.

Recently, stimuli-responsive gene carriers have been widely studied to overcome the extra- and intracellular barriers in cancer treatment. In this study, we modified polydopamine nanoparticles with low-molecular weight polyethylenimine (PEI1.8k) and polyethylene glycol-phenylboronic acid (PEG-PBA) to prepare pH-responsive gene carrier PDANP-PEI-rPEG. PBA and polydopamine could form pH-responsive boronate ester bonds. Non-responsive PDANP-PEI-nPEG and non-PEGylated PDANP-PEI were also studied as control. Both PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes remained stable in the pH environment of blood circulation or extracellular delivery (pH 7.4) owing to the PEG modification. And after being internalized into endosomes, the boronate ester bonds could be cleaved. The pH responsive ability of PDANP-PEI-rPEG might facilitate complexes dissociation and gene release inside cells. The transfection level of PDANP-PEI-rPEG/DNA complexes was about 100 times higher than that of PDANP-PEI-nPEG/DNA complexes with the same mass ratios. Moreover, after NIR light irradiation at the power density of 2.6 W/cm2 for 20 min, the good photothermal conversion ability of PDANP resulted in quick endosomal escape. The transfection level of PDANP-PEI-rPEG/DNA complexes doubled, even higher than that of lipofectamine 2000/DNA complexes. This was also confirmed by Bafilomycin A1 inhibition test and CLSM observation. In response to the acidic pH within cancer cells and the NIR light irradiation, the PDANP-PEI-rPEG carrier could overcome multiple obstacles in gene delivery, which was promising for further application in gene therapy.

Baicalein has been proved as a promising compound for non-alcoholic fatty liver disease (NAFLD); however, the molecular mechanisms underlying the progression of NAFLD and its intervention by baicalein remain largely obscure. Male C57BL/6J fed high-fat diet (HFD) and HepG2 cells stimulated with free fatty acid were treated with baicalein and various pharmacological reagents to explore the effect of signaling pathways involved in lysosomal acidification. Baicalein intake declined ALT and AST activities by 25.1% and 18.7%, respectively, compared with the HFD group. Moreover, baicalein markedly ameliorated the HFD-trigged lysosomal membrane permeabilization evidenced by declined cathepsin B release. Subsequent disorders, including cathepsin D maturation and mitochondrion membrane potential were also partially normalized by baicalein administration to HFD-fed mice. Meanwhile, an increase in V-ATPase V1 subunits expression in lysosomes, V-ATPase activity and the colocalization of cytosol V-ATPase V1 subunits and lysosomes was observed in baicalein-supplement mice. Bafilomycin A1 stimulation resulting in elevated lysosomal pH and triacylglycerol accumulation partially abolished the effect of baicalein. Furthermore, incubation with mTOR inhibitor rapamycin restored lysosomal pH and decreased cellular triacylglycerol content. Collectively, these findings demonstrate that hepatic lysosomal acidification is the main target of baicalein against NAFLD via maintaining lysosomal V-ATPase assembly through mTOR pathway.

A new 16-membered diene macrolide, bafilomycin M ( 1 ), was produced from a Streptomyces sp. GIC10-1, which was originally isolated from a marine sponge Theonella sp. The structure of 1 was established by spectroscopic methods and this compound was found to exhibit cytotoxicity toward K-562, HL-60, SUPT-1, and LNCaP tumor cells with IC 50 values 0.060, 0.011, 0.047, and 0.389 μg/mL, respectively.

Justicidin A (JA), a novel arylnaphthalide lignan, was previously found to reduce the growth of human hepatocellular carcinoma cells (HCC), Hep 3B cells, in NOD-SCID mice via induction of apoptosis. Here, using L1000 microarray profiling obtained from JA-treated HCC cells, the z-score signatures of JA exhibited positive connectivity to known autophagy inducers among 3122 compounds from the Library of Integrated Network-based Cellular Signatures database. JA-induced autophagy in HCC Hep 3B cells was confirmed by formation of acidic vesicular organelles and an increase in the expression of LC3-II and LAMP2a. JA-induced autophagic flux was demonstrated by accumulation of LC3-II and LAMP2a, as well as a decrease in colocalization of LC3 and LAMP2a puncta in the presence of autophagy inhibitor bafilomycin A1. Administration of bafilomycin A1 also demonstrated that JA-induced autophagy suppressed apoptosis. Activation of Ras/MEK/ERK pathway was observed. Following administration of apoptosis inhibitor zVAD, it was found that JA-induced apoptosis did not affect JA-induced autophagy. It is noteworthy that addition of JA promoted chemosensitivity of sorafenib, the only FDA-approved targeted therapy for advanced HCC. The results demonstrate the effectiveness of this novel strategy for rapid discovery of molecular actions of small molecules and suggest the applications of JA in HCC treatment.

Background: Astrocyte pH regulation is essential for brain function. Results: Bicarbonate-independent acid load recovery in whole-mount astrocytes was achieved primarily via V-ATPase, which also held astrocytes at a basic resting pH. Conclusion: V-ATPase is the major bicarbonate-independent H+ extruder in a whole-mount astrocyte population. Significance: High levels of V-ATPase expression will isolate astrocyte pH regulation from cytoplasm changes in other ion species., The mechanisms of HCO3−-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H+ pump by Cl− removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3−-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

Retinal pigment epithelium (RPE) plays a major role in the maintenance of photoreceptors, and degeneration of RPE results in the development of age-related macular degeneration (AMD). Accumulation of intracellular protein aggregates, increased oxidative stress, and chronic inflammation are all factors damaging the functionality of aged RPE cells. Here, we report that inhibition of proteasomal degradation with MG-132 and autophagy with bafilomycin A1 resulted in the release of IL-1β but not that of IL-18 in human ARPE-19 cells. NLRP3 receptor became upregulated, and caspase-1, the functional component of an inflammasome complex, was activated. In addition to accumulating intracellular protein aggregates, inhibition of degradation systems induced oxidative stress which was demonstrated by elevated amounts of intracellular 4-hydroxynonenal (HNE)-protein adducts. Along with IL-1β, exposure to MG-132 and bafilomycin A1 resulted in the secretion of IL-8. A low concentration (1pg/ml) of IL-1β was capable of triggering significant IL-8 production which also became attenuated by treatment with a specific caspase-1 inhibitor. These results suggest that decline in intracellular degradation systems results not only in increased amounts of intracellular protein aggregates and oxidative stress but also in the activation of NLRP3 inflammasomes, arisen as a result of elevated production of biologically active IL-1β.

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10-100nM) increases the vesicular pH in these cells. Archazolid (10nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000nM. Of interest, secretion of interleukin-1β was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.

Autophagy is important for a variety for virus life cycles. We sought to determine the role of autophagy in human BK polyomavirus (BKPyV) infection. The addition excess amino acids during viral infection reduced BKPyV infection. Perturbing autophagy levels using inhibitors, 3-MA, bafilomycin A1, and spautin-1, also reduced infection, while rapamycin treatment of host cells increased infection. siRNA knockdown of autophagy genes, ATG7 and Beclin-1, corresponded to a decrease in BKPyV infection. BKPyV infection not only correlated with autophagosome formation, but also virus particles localized to autophagy-specific compartments early in infection. These data support a novel role for autophagy in the promotion of BKPyV infection., Highlights • Amino acid supplementation decreases BKPyV infection. • Autophagy inhibitors, 3-MA, bafilomycin A, and spautin-1, decrease BKPyV infection, while rapamycin increases infection. • Inhibitors are most effective when added early in viral life cycle. • Knockdown of autophagy genes, Beclin-1 and ATG7, in host cells decreases BKPyV infection levels. • BKPyV localizes to LC3+ autophagosome 3 h post infection.

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and binding component (C2II). The activated binding component (C2IIa) forms heptamers and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the intracellular trafficking of C2 toxin. When MDCK cells were incubated with C2I and C2IIa at 37 °C, C2I colocalized with C2IIa in cytoplasmic vesicles at 5 min, and C2I then disappeared (15 min incubation and later), and C2IIa was observed in the vesicles. Internalized C2I and C2IIa were transported to early endosomes. Some of both components were returned to the plasma membrane through recycling endosomes, whereas the rest of C2IIa was transported to late endosomes and lysosomes for degradation. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of C2IIa in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted C2IIa's movement in the cytosol. These results indicated that an internalized C2I and C2IIa complex was delivered to early endosomes, and that subsequent delivery of C2I to the cytoplasm occurred in early endosomes. C2IIa was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation.

We previously developed a novel photosensitizer, chlorophyllin e4, and found that chlorophyllin e4 mediated-PDT could kill 5637 and T24 cells by inducing apoptotic cell death. Here, we further investigated the new mechanism of autophagy and determined its relevance to apoptosis in e4-PDT. We demonstrated that chlorophyllin e4 was located in both lysosome and mitochondria, and autophagy also occurred in bladder cancer cells upon e4-PDT. More importantly, autophagy played a pro-survival role, and its inhibition enhanced e4-PDT-associated apoptotic cell death because cells pretreated with the typical autophagy inhibitor either 3-methyladenine or Bafilomycin A1 exhibited much lower cell viability and higher apoptotic cell death. Thus, these data imply that the combination of PDT, when mediated by our new photosensitizer chlorophyllin e4, and an autophagy inhibitor might be a promising approach to the eliminationof non-muscle invasive bladder cancer.

Chronic obstructive pulmonary disease affects 64 million people and is currently the fourth leading cause of death worldwide. Chronic obstructive pulmonary disease includes both emphysema and chronic bronchitis, and in the case of chronic bronchitis represents an inflammatory response of the airways that is associated with mucus hypersecretion and obstruction of small airways. Recently, it has emerged that exposure to cigarette smoke (CS) leads to an inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, causing airway surface liquid dehydration, which may play a role in the development of chronic bronchitis. CS rapidly clears CFTR from the plasma membrane and causes it to be deposited into aggresome-like compartments. However, little is known about the mechanism(s) responsible for the internalization of CFTR following CS exposure. Our studies revealed that CS triggered a rise in cytoplasmic Ca(2+) that may have emanated from lysosomes. Furthermore, chelation of cytoplasmic Ca(2+), but not inhibition of protein kinases/phosphatases, prevented CS-induced CFTR internalization. The macrolide antibiotic bafilomycin A1 inhibited CS-induced Ca(2+) release and prevented CFTR clearance from the plasma membrane, further linking cytoplasmic Ca(2+) and CFTR internalization. We hypothesize that CS-induced Ca(2+) release prevents normal sorting/degradation of CFTR and causes internalized CFTR to reroute to aggresomes. Our data provide mechanistic insight into the potentially deleterious effects of CS on airway epithelia and outline a hitherto unrecognized signaling event triggered by CS that may affect the long term transition of the lung into a hyper-inflammatory/dehydrated environment.