Blockage Of Autophagic Flux Research Articles

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations.

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy.

CPKCγ-Modulated Sequential Reactivation of mTOR Inhibited Autophagic Flux in Neurons Exposed to Oxygen Glucose Deprivation/Reperfusion.

We have reported that conventional protein kinase Cγ (cPKCγ)-modulated neuron-specific autophagy improved the neurological outcome of mice following ischemic stroke through the Akt-mechanistic target of rapamycin (mTOR) pathway. However, its detailed molecular mechanism remains unclear. In this study, primary cortical neurons from postnatal one-day-old C57BL/6J cPKCγ wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice suffering oxygen glucose deprivation/reperfusion (OGD/R) were used to simulate ischemia/reperfusion injury in vitro. A block of autophagic flux was observed in cPKCγ+/+ neurons under OGD/R exposure, characterized by accumulation of p62. Immunofluorescent results showed a decrease in colocalization between LC3 and Atg14 or Stx17 in cPKCγ+/+ neurons when compared with cPKCγ−/− neurons after OGD/R. However, the colocalization between LC3 and Lamp2 was barely decreased, indicating the presence of autolysosomes. The larger lysotracker-positive structures were also significantly increased. These results suggest that cPKCγ-induced inhibition of autophagy occurred at the stages of autophagosome formation, Stx17 anchoring, and the degradation of autolysosomes in particular. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2481 was dependent on the site of Ser 2448, which may have blocked autophagic flux. cPKCγ-modulated sequential reactivation of mTOR inhibited autophagic flux in neurons exposed to OGD/R, which may provide endogenous interventional strategies for stroke, especially ischemia/reperfusion injury.

Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib.

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

Lysosome-dependent cell death and deregulated autophagy induced by amine-modified polystyrene nanoparticles.

Nanoparticles (NPs) typically accumulate in lysosomes. However, their impact on lysosomal function, as well as autophagy, a lysosomal degradative pathway, is still not well known. We have previously reported in the 1321N1 cell line that amine-modified polystyrene (NH2-PS) NPs induce apoptosis through damage initiated in the lysosomes leading ultimately to release of lysosomal content in the cytosol, followed by apoptosis. Here, by using a combination of biochemical and cell biological approaches, we have characterized in a mouse embryonic fibroblast cell line that the lysosomal alterations induced by NH2-PS NPs is progressive, initiating from mild lysosomal membrane permeabilization (LMP), to expansion of lysosomal volume and intensive LMP before the summit of cell death. Though the cells initially seem to induce autophagy as a surviving mechanism, the damage of NH2-PS NPs to lysosomes probably results in lysosomal dysfunctions, leading to blockage of autophagic flux at the level of lysosomes and the eventual cell death.

Resveratrol protects muscle cells against palmitate-induced cellular senescence and insulin resistance through ameliorating autophagic flux

Skeletal muscle, a highly metabolic tissue, is particularly vulnerable to increased levels of saturated free fatty acids (FFAs). The role of autophagy in saturated FFAs-induced cellular senescence and insulin resistance in skeletal muscle remains unclear. Therefore, the present study was aimed to explore autophagic flux in cellular senescence and insulin resistance induced by palmitate in muscle cells, and whether resveratrol limited these responses. Our results showed that palmitate induced cellular senescence in both myoblasts and myotubes. In addition, palmitate delayed differentiation in myoblasts and inhibited expression of insulin-stimulated p-AKTSer473 in myotubes. The accumulations of autophagosome assessed by tandem fluorescent-tagged LC3 demonstrated that autophagic flux was impaired in both palmitate-treated myoblasts and myotubes. Resveratrol protected muscle cells from palmitate-induced cellular senescence, apoptosis during differentiation, and insulin resistance via ameliorating autophagic flux. The direct influence of autophagic flux on development of cellular senescence and insulin resistance was confirmed by blockage of autophagic flux with chloroquine. In conclusion, impairment of autophagic flux is crucial for palmitate-induced cellular senescence and insulin resistance in muscle cells. Restoring autophagic flux by resveratrol could be a promising approach to prevent cellular senescence and ameliorate insulin resistance in muscle.

The cytotoxicity of coxsackievirus B3 is associated with a blockage of autophagic flux mediated by reduced syntaxin 17 expression

Coxsackievirus B3 (CVB3) is an important human pathogen linked to cardiac arrhythmias and acute heart failure. CVB3 infection has been reported to induce the formation of autophagosomes that support the viral replication in host cells. Interestingly, our study shows that the accumulation of autophagosomes during CVB3 infection is caused by a blockage of autophagosome–lysosome fusion rather than the induction of autophagosome biogenesis. Moreover, CVB3 decreases the transcription and translation of syntaxin 17 (STX17), a SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein involved in autophagosome–lysosome fusion. Overexpression of STX17 restored the autophagic flux, alleviated the virus-induced lysosomal dysfunction, and decreased the apoptosis induced by CVB3 infection in HeLa cells. Taken together, our results suggest that CVB3 infection impairs the autophagic flux by blocking autophagosome–lysosome fusion. These findings thus point to potential new therapeutic strategies targeting STX17 or autophagosome–lysosome fusion for treating CVB3-associated diseases.

Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking

Background/Aims: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. Results: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. Conclusion: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

Nanodiamond-based non-canonical autophagy inhibitor synergistically induces cell death in oxygen-deprived tumors

Blockage of autophagic flux by nanodiamonds induces apoptosis in hypoxic tumor cells with minimal toxicity to normal tissues and enhances the effects of anti-angiogenic therapy.

Autophagic flux response and glioblastoma sensitivity to radiation

Objective:Glioblastoma is the most common primary brain tumor in adults and one of the most lethal human tumors. It constitutes a unique non-metastasizing human tumor model with high resistance to radiotherapy and chemotherapy. The current study investigates the association between autophagic flux and glioblastoma cell resistance.Methods:The expression kinetics of autophagy- and lysosome-related proteins following exposure of two glioblastoma cell lines (T98 and U87) to clinically relevant radiation doses was examined. Then, the response of cells resistant to radiotherapy and chemotherapy was investigated after silencing of LC3A, LC3B, and TFEB genes in vitro and in vivo. Results:Following irradiation with 4 Gy, the relatively radioresistant T98 cells exhibited enhanced autophagic flux. The more radiosensitive U87 cell line suffered a blockage of autophagic flux. Silencing of LC3A, LC3B, and TFEB genes in vitro, significantly sensitized cells to radiotherapy and temozolomide (U87: P < 0.01 and < 0.05, respectively; T98: P < 0.01 and < 0.01, respectively). Silencing of the LC3A gene sensitized mouse xenografts to radiation. Conclusions:Autophagy in cancer cells may be a key factor of radio-resistance and chemo-resistance in glioblastoma cells. Blocking autophagy may improve the efficacy of radiochemotherapy for glioblastoma patients.

LncRNA UCA1 attenuates autophagy-dependent cell death through blocking autophagic flux under arsenic stress

Arsenic (As) is a naturally toxin which exists ubiquitously in foods and various environment media, incurring diverse toxicities and health problems. Previous studies have shown that oxidative stress, genotoxic damage and pro-apoptotic pathways are ascribed to As-associated detrimental effects. Meanwhile, epigenetic regulations (such as miRNAs and histone modifications) were also reported to contribute to As-induced adverse effects. Nonetheless, whether long non-coding RNAs (LncRNAs) are indispensable for the regulation of As-induced biological outcomes are nearly unknown. In this study, we identified that a lncRNA UCA1 was markedly induced by As treatment in human hepatocytes. Functional assessments revealed that UCA1 played a critical role in protecting hepatocytes from As-induced autophagy inhibition. Furthermore, through RNA-seq assay, oxidative stress induced growth inhibitor 1 (OSGIN1) was uncovered to be the most responsive target downstream of UCA1, and miR-184 acted as an intermediate for the regulation of UCA1 on the level of OSGIN1 through a competing endogenous RNAs (ceRNAs) mechanism. Further mechanistic investigations demonstrated that UCA1/OSGIN1 signaling contributed to As-induced autophagic flux blockage through activating mTOR/p70S6 K cascade, resulting in compromised cell death. Collectively, our study deciphered a lncRNA-dictated molecular mechanism responsible for As toxicity: UCA1 leads a protective role against As-induced cell death through blocking autophagic flux.

Maduramicin induces apoptosis and necrosis, and blocks autophagic flux in myocardial H9c2 cells.

Maduramicin, a polyether ionophore antibiotic, is widely used as an anticoccidial agent in the poultry industry. It has been reported that maduramicin may cause heart and skeletal muscle cell damage, resulting in heart failure, skeletal muscle degeneration and even death in animals and humans, if improperly used. However, the molecular mechanism behind its capability to cause death of cardiac cells is not known. Here, we show that maduramicin induced apoptosis and necrosis in rat myocardial cells (H9c2). Maduramicin did not apparently upregulate the expression of pro-apoptotic proteins (e.g., BAD, BAK and BAX) or downregulate the expression of anti-apoptotic proteins (e.g. Bcl-2, Bcl-xL, Mcl-1 and survivin). Interestingly, maduramicin increased the expression of DR4 and TRAIL, activating caspases 8/3 and triggering cleavage of poly ADP ribose polymerase (PARP). In addition, maduramicin induced nuclear translocation of apoptosis inducing factor. Furthermore, maduramicin blocked autophagic flux, as evidenced by inducing accumulation of both LC3-II and p62/SQSTM1. Taken together, the above results suggest that maduramicin executes its toxicity in the myocardial cells at least by inducing caspase-dependent cell death through TRAIL/DR4-mediated extrinsic pathway and caspase-independent cell death by inducing apoptosis inducing factor nuclear translocation and blocking autophagic flux. Our findings provide a new insight into the molecular mechanism of maduramicin's toxicity in myocardial cells.

Is trehalose an autophagic inducer? Unraveling the roles of non-reducing disaccharides on autophagic flux and alpha-synuclein aggregation

Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

Chemical chaperone 4-phenyl butyric acid (4-PBA) reduces hepatocellular lipid accumulation and lipotoxicity through induction of autophagy

Defective autophagy has been linked to lipotoxicity in several cellular models. We aimed to investigate autophagy in lipid-stimulated hepatoma (Huh7) cells and tested whether 4-phenyl butyric acid (4-PBA), a chemical chaperone, has a beneficial role in hepatic fat accumulation and lipotoxicity. We report that long-term (24 h) exposure of hepatocytes to palmitate block autophagic flux that leads to lipid accumulation and cell death. Western blotting analysis showed increased accumulation of SQSTM1/p62, and decreased expression of Beclin1 and Atg7 in palmitate-treated cells. Autophagy inhibition by 3-methyladenine (3-MA) in palmitate-treated cells neither increased SQSTMI/p62 accumulation nor cell death, thus suggesting complete blockade of autophagy by palmitate. 4-PBA reduced lipid accumulation and cell death that were associated with restoration of autophagy. siRNA-mediated knockdown of Atg7 and presence of autophagy inhibitors, 3-MA and chloroquine, resulted in the decrease in lipid-lowering effect of 4-PBA, suggesting that 4-PBA mediates its lipid-lowering effect via autophagy. Apoptotic parameters, including altered Bcl2:Bax ratio and PARP1 cleavage induced by palmitate, were improved by 4-PBA. Our results indicate that palmitate impairs autophagy and increases lipid accumulation in Huh7 cells, whereas 4-PBA plays a protective role in lipid accumulation and lipotoxicity through activation of autophagy.

Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K, AMPK and MAPK pathways

Crotonaldehyde is an ubiquitous hazardous pollutant in the environment which can be produced naturally, artificially and endogenously. Acute exposure of crotonaldehyde was reported to induce severe lung injury in humans and experimental animals. However, the exact toxicity mechanisms of crotonaldehyde in organisms have not been fully explored. In the present study, we explored the role autophagy played in the cytotoxicity induced by crotonaldehyde in human bronchial epithelial cells (BEAS-2B), and the pathways that mediated autophagy, including the phosphatidylinositol 3-kinase (PI3K) pathway, the AMP-activated protein kinase (AMPK) pathway and the mitogen-activated protein kinase (MAPK) pathways, were examined and validated. We found that crotonaldehyde induced cytotoxicity and autophagy simultaneously in BEAS-2B cells, and blockage of autophagic flux significantly elevated the viability of BEAS-2B exposed to high concentrations of crotonaldehyde. Crotonaldehyde down-regulated the activity of PI3K pathway, and elevated the activities of AMPK and MAPK pathways. Pretreatment of specific agonist or antagonist of these pathways could inhibit autophagy and partly improve the viability. These results suggested that acute exposure of crotonaldehyde induced cell death mediated by autophagy, which might be helpful to elucidate the toxicity mechanisms of crotonaldehyde and contribute to environmental and human health risk assessment.

The helminth product, ES-62 modulates dendritic cell responses by inducing the selective autophagolysosomal degradation of TLR-transducers, as exemplified by PKCδ.

We have previously shown that ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the parasitic filarial nematode Acanthocheilonema viteae targets dendritic cell (DC) responses, specifically by suppressing TLR4 signalling to inhibit Th1/Th17-driven inflammation. We have now investigated the molecular mechanisms underpinning such immunomodulation and show here that ES-62-mediated downregulation of protein kinase C-δ (PKC-δ), a TLR4-associated signalling mediator required for full activation of LPS-driven pro-inflammatory responses, is associated with induction of a low level of autophagic flux, as evidenced by upregulation and trafficking of p62 and LC3 and their consequent autophagolysosomal degradation. By contrast, the classical TLR4 ligand LPS, strongly upregulates p62 and LC3 expression but under such canonical TLR4 signalling this upregulation appears to reflect a block in autophagic flux, with these elements predominantly degraded in a proteasomal manner. These data are consistent with autophagic flux acting to homeostatically suppress proinflammatory DC responses and indeed, blocking of PKC-δ degradation by the autophagolysosomal inhibitors, E64d plus pepstatin A, results in abrogation of the ES-62-mediated suppression of LPS-driven release of IL-6, IL-12p70 and TNF-α by DCs. Thus, by harnessing this homeostatic regulatory mechanism, ES-62 can protect against aberrant inflammation, either to promote parasite survival or serendipitously, exhibit therapeutic potential in inflammatory disease.

The reverse-mode NCX1 activity inhibitor KB-R7943 promotes prostate cancer cell death by activating the JNK pathway and blocking autophagic flux.

We explored the effects of KB-R7943, an inhibitor of reverse-mode NCX1 activity, in prostate cancer (PCa). NCX1 was overexpressed in PCa tissues and cell lines, and higher NCX1 levels were associated higher PCa grades. At concentrations greater than 10 μM, KB-R7943 dose-dependently decreased PC3 and LNCaP cell viability. KB-R7943 also increased cell cycle G1/S phase arrest and induced apoptosis in PC3 cells. KB-R7943 increased autophagosome accumulation in PCa cells as indicated by increases in LC3-II levels and eGFP-LC3 puncta. Combined treatment with chloroquine (CQ) and KB-R7943 decreased P62 and increased LC3-II protein levels in PC3 cells, indicating that KB-R7943 blocked autophagic flux. KB-R7943 induced autophagosome accumulation mainly by downregulating the PI3K/AKT/m-TOR pathway and upregulating the JNK pathway. In xenograft experiments, KB-R7943 inhibited tumor growth. Combined treatment with KB-R7943 and an autophagy inhibitor inhibited growth and increased apoptosis. These results indicate that KB-R7943 promotes cell death in PCa by activating the JNK signaling pathway and blocking autophagic flux.

Chemical characterization and cytotoxic potential of an ellagitannin-enriched fraction from Fragaria vesca leaves

The hepatocellular carcinoma, a primary malignancy of the liver, has a very poor prognosis and a lower survival rate. Moreover, the inefficacy of conventional therapies emphasizes the importance of discovering new bioactive compounds. Several studies clearly state that plant-derived polyphenols, namely ellagitannins, have several health benefits. Fragaria vesca leaves contain high amounts of polyphenols, being especially rich in ellagitannins. Therefore, this study aimed to characterize an ellagitannin-enriched fraction (EEF) from F. vesca leaves and to unveil the anticancer potential of this fraction on human hepatocellular carcinoma cells.The analysis of EEF by HPLC-PDA-ESI/MSn allowed the detection of 12 ellagitannins. The cell viability of both EEF and crude extract was determined after 24h of cells treatment and the half-maximal inhibitory concentration (IC50) was evaluated. The IC50 of the EEF (113μg/mL) was about 6 times lower than the IC50 of the crude extract (690μg/mL). Furthermore, EEF induced cell cycle arrest at G2/M checkpoint and decreased cell proliferation in a dose-dependent way. This fraction also induced an accumulation of LC3-II protein through blockage of autophagic flux, and inhibited chymotrypsin-like activity of 26S proteasome. These results showed, for the first time, that EEF from F. vesca leaves inhibits both, autophagic and ubiquitin-proteasome system pathways, two main intracellular protein degradation systems that are targets for anticancer therapies. Additionally, a proteomic analysis allowed the identification of 914 proteins, among which 133 were modulated after cells treatment with EEF, most of them related to metabolic pathways. Overall, this study shows that the EEF from F. vesca leaves decreased cell proliferation, inhibited the proteolytic mechanisms and modulated the metabolic pathways of the cell. Additionally this study points out F. vesca as a source of valuable molecules with anticancer potential, suggesting that ellagitannins, the polyphenols identified in this fraction, could be useful in the development of new fine-tuned therapeutic strategies against carcinogenesis.

The autophagic response to polystyrene nanoparticles is mediated by transcription factor EB and depends on surface charge.

BackgroundA number of engineered nanoparticles induce autophagy, the main catabolic pathway that regulates bulk degradation of cytoplasmic material by the lysosomes. Depending on the specific physico-chemical properties of the nanomaterial, however, nanoparticle-induced autophagy may have different effects on cell physiology, ranging from enhanced autophagic degradation to blockage of autophagic flux. To investigate the molecular mechanisms underlying the impact of nanoparticle charge on the nature of the autophagic response, we tested polystyrene nanoparticles (50 nm) with neutral, anionic, and cationic surface charges.ResultsWe found all polystyrene nanoparticles investigated in this study to activate autophagy. We showed that internalization of polystyrene nanoparticles results in activation of the transcription factor EB, a master regulator of autophagy and lysosome biogenesis. Autophagic clearance, however, was observed to depend specifically on the charge of the nanoparticles. Particularly, we found that the autophagic response to polystyrene nanoparticles presenting a neutral or anionic surface involves enhanced clearance of autophagic cargo. Cell exposure to polystyrene nanoparticles presenting a cationic surface, on the other hand, results in transcriptional upregulation of the pathway, but also causes lysosomal dysfunction, ultimately resulting in blockage of autophagic flux.ConclusionsThis study furthers our understanding of the molecular mechanisms that regulate the autophagic response to nanoparticles, thus contributing essential design criteria for engineering benign nanomaterials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0149-6) contains supplementary material, which is available to authorized users.

The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1–transfected Chinese hamster ovary cells

Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-β (Aβ) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate–conjugated Aβ1–42 was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aβ oligomers.

Altered TFEB-mediated lysosomal biogenesis in Gaucher disease iPSC-derived neuronal cells.

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). The severe forms of GD are associated with neurodegeneration with either rapid (Type 2) or slow progression (Type 3). Although the neurodegenerative process in GD has been linked to lysosomal dysfunction, the mechanisms involved are largely unknown. To identify the lysosomal alterations in GD neurons and uncover the mechanisms involved, we used induced pluripotent stem cells (iPSCs) derived from patients with GD. In GD iPSC-derived neuronal cells (iPSC-NCs), GBA1 mutations caused widespread lysosomal depletion, and a block in autophagic flux due to defective lysosomal clearance of autophagosomes. Autophagy induction by rapamycin treatment in GD iPSC-NCs led to cell death. Further analysis showed that in GD iPSC-NCs, expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes, and lysosomal gene expression, were significantly downregulated. There was also reduced stability of the TFEB protein and altered lysosomal protein biosynthesis. Treatment of mutant iPSC-NCs with recombinant GCase (rGCase) reverted the lysosomal depletion and autophagy block. The effect of rGCase on restoring lysosomal numbers in mutant cells was enhanced in the presence of overexpressed TFEB, but TFEB overexpression alone did not reverse the lysosomal depletion phenotype. Our results suggest that GBA1 mutations interfere with TFEB-mediated lysosomal biogenesis, and that the action of GCase in maintaining a functioning pool of lysosomes is exerted in part through TFEB. The lysosomal alterations described here are likely to be a major determinant in GBA1-associated neurodegeneration.