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1.  Sustained Autophagy Contributes to Measles Virus Infectivity 
PLoS Pathogens  2013;9(9):e1003599.
The interplay between autophagy and intracellular pathogens is intricate as autophagy is an essential cellular response to fight against infections, whereas numerous microbes have developed strategies to escape this process or even exploit it to their own benefit. The fine tuned timing and/or selective molecular pathways involved in the induction of autophagy upon infections could be the cornerstone allowing cells to either control intracellular pathogens, or be invaded by them. We report here that measles virus infection induces successive autophagy signallings in permissive cells, via distinct and uncoupled molecular pathways. Immediately upon infection, attenuated measles virus induces a first transient wave of autophagy, via a pathway involving its cellular receptor CD46 and the scaffold protein GOPC. Soon after infection, a new autophagy signalling is initiated which requires viral replication and the expression of the non-structural measles virus protein C. Strikingly, this second autophagy signalling can be sustained overtime within infected cells, independently of the expression of C, but via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Whereas this sustained autophagy signalling leads to the autophagy degradation of cellular contents, viral proteins escape from degradation. Furthermore, this autophagy flux is ultimately exploited by measles virus to limit the death of infected cells and to improve viral particle formation. Whereas CD150 dependent virulent strains of measles virus are unable to induce the early CD46/GOPC dependent autophagy wave, they induce and exploit the late and sustained autophagy. Overall, our work describes distinct molecular pathways for an induction of self-beneficial sustained autophagy by measles virus.
Author Summary
Autophagy is an evolutionarily conserved lysosomal dependent degradative pathway for recycling of long-lived proteins and damaged organelles. Autophagy is also an essential cellular response to fight infection by destroying infectious pathogens trapped within autophagosomes and plays a key role in the induction of both innate and adaptive immune responses. Numerous viruses have evolved strategies to counteract autophagy in order to escape from degradation or/and to inhibit immune signals. The kinetic and molecular pathways involved in the induction of autophagy upon infections might determine if cells would be able to control pathogens or would be invaded by them. We showed that measles virus (MeV) infection induces successive autophagy signallings in cells via distinct molecular pathways. A first autophagy wave is induced by the engagement of the MeV cellular receptor CD46 and the scaffold protein GOPC. A second wave is initiated after viral replication by the expression of the non-structural MeV protein C and is sustained overtime within infected cells thanks to the formation of syncytia. This sustained autophagy is exploited by MeV to limit the death of infected cells and to improve viral particle formation. We describe new molecular pathways by which MeV hijacks autophagy to promote its infectivity.
doi:10.1371/journal.ppat.1003599
PMCID: PMC3784470  PMID: 24086130
2.  IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network 
PLoS Pathogens  2011;7(12):e1002422.
Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.
Author Summary
Autophagy is a highly regulated cellular degradative pathway for recycling of long-lived proteins and damaged organelles. Autophagy is also used by host cells as a defense mechanism against intracellular pathogens. Autophagy can degrade pathogens or pathogen-derived molecules trapped within specialized vesicles named autophagosomes. Viruses and viral proteins are not an exception. However, since autophagy is a conserved pathway, viruses were submitted to an evolutionary pressure that led to the selection of molecular strategies which avoid or subvert this process to promote viral replication. Nevertheless the molecular details of viral interaction with autophagy remain largely unknown. We determined the ability of 83 proteins of several families of RNA viruses (including Hepatitis C virus (HCV), human immunodeficiency virus 1 (HIV-1), Measles virus (MeV) and influenza A virus) to interact with 44 human proteins known to regulate autophagy and found that autophagy is highly targeted by RNA viruses. Strikingly, immunity-associated GTPase family M (IRGM), known for its role in autophagy against bacteria, is the most targeted autophagy protein. Its absence is detrimental for HCV, HIV-1 and MeV production. Therefore, our data show that different RNA viruses families use similar strategies to fine tune autophagy to their own benefit.
doi:10.1371/journal.ppat.1002422
PMCID: PMC3234227  PMID: 22174682
3.  A Role for Autophagy in the Extension of Lifespan by Dietary Restriction in C. elegans 
PLoS Genetics  2008;4(2):e24.
In many organisms, dietary restriction appears to extend lifespan, at least in part, by down-regulating the nutrient-sensor TOR (Target Of Rapamycin). TOR inhibition elicits autophagy, the large-scale recycling of cytoplasmic macromolecules and organelles. In this study, we asked whether autophagy might contribute to the lifespan extension induced by dietary restriction in C. elegans. We find that dietary restriction and TOR inhibition produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan. The longevity response to dietary restriction in C. elegans requires the PHA-4 transcription factor. We find that the autophagic response to dietary restriction also requires PHA-4 activity, indicating that autophagy is a transcriptionally regulated response to food limitation. In spite of the rejuvenating effect that autophagy is predicted to have on cells, our findings suggest that autophagy is not sufficient to extend lifespan. Long-lived daf-2 insulin/IGF-1 receptor mutants require both autophagy and the transcription factor DAF-16/FOXO for their longevity, but we find that autophagy takes place in the absence of DAF-16. Perhaps autophagy is not sufficient for lifespan extension because although it provides raw material for new macromolecular synthesis, DAF-16/FOXO must program the cells to recycle this raw material into cell-protective longevity proteins.
Author Summary
Dietary restriction (limited food intake) increases lifespan in many organisms. However, the cellular processes underlying this fascinating phenomenon are still poorly understood. When an animal is starved, it degrades and recycles its organelles and other cellular components in a process called autophagy (literally “self-eating”). Here, we have asked whether autophagy also occurs in response to dietary restriction, using the roundworm C. elegans for our studies. We find that autophagy does take place when food intake is limited. Moreover, inhibiting genes required for autophagy has little effect on well-fed animals but prevents food limitation from extending lifespan. This autophagy requires PHA-4/FOXA, a life-extension protein that regulates gene expression, suggesting that changes in gene expression are required for dietary restriction to stimulate autophagy. Because autophagy seems like such a rejuvenating process, it might seem to be sufficient to increase longevity. However, we find that, in long-lived hormone-pathway mutants, both autophagy and DAF-16/FOXO, another protein that controls gene expression, are required for longevity. We propose that autophagy frees up new resources for the cell, but that transcription factors like the DAF-16/FOXO protein must channel this raw material into new cell-protective proteins in order for lifespan to be increased.
doi:10.1371/journal.pgen.0040024
PMCID: PMC2242811  PMID: 18282106
4.  Myc-Driven Overgrowth Requires Unfolded Protein Response-Mediated Induction of Autophagy and Antioxidant Responses in Drosophila melanogaster 
PLoS Genetics  2013;9(8):e1003664.
Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.
Author Summary
The evolutionarily conserved transcription factor Myc promotes protein synthesis, cell growth and cancer progression through incompletely understood mechanisms. In this work, we show that forced expression of Myc induces the accumulation of abnormal proteins leading to unfolded protein responses (UPR), presumably by overloading the protein synthetic capacity of cells in Drosophila. UPR then results in autophagy-mediated breakdown and recycling of cytoplasmic material, and at the same time, to activation of antioxidant responses in these cells. Blocking the UPR stress signaling, autophagy and antioxidant pathways genetically, or by feeding larvae an autophagy-inhibiting drug, prevents overgrowth of Myc-expressing cells, but these treatments do not affect the growth of control cells in the same tissues. These results, together with recent reports in mammalian cancer models, suggest that drugs targeting UPR, autophagy and antioxidant responses may specifically inhibit cancer cell proliferation driven by oncogenic Myc.
doi:10.1371/journal.pgen.1003664
PMCID: PMC3738540  PMID: 23950728
5.  Regulators of Autophagosome Formation in Drosophila Muscles 
PLoS Genetics  2015;11(2):e1005006.
Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.
Author Summary
Since the identification of the core autophagy genes in yeast, tissue culture cell lines have been the primary tool to evaluate the role and regulation of autophagy in higher organisms. However, since autophagy is a tissue-specific, context dependent process, stable cell lines can only give a limited view of the autophagic process. Here, we focus on the role of putative autophagy regulators in the specific context of the skeletal muscle, which has one of the most robust autophagy responses in mammals. We describe a fruitfly model of autophagy for skeletal muscles that integrates rapid genetic screening in primary cultured cells with robust in vivo validation in the larval muscle. We screened a set of genes previously linked to the autophagy pathway in humans, and identified both positive and negative regulators of autophagy. Our observation that genes involved in sugar metabolism impact muscle autophagy has important implications for both skeletal and cardiac myopathies associated with aberrant sugar storage. Surprisingly, we found that the proteasome is required to maintain autophagy in the muscle, suggesting that therapeutic treatments aiming to induce autophagy by proteasome inhibition must be carefully calibrated to ensure that the opposite phenotype does not occur.
doi:10.1371/journal.pgen.1005006
PMCID: PMC4334200  PMID: 25692684
6.  Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors 
PLoS Genetics  2013;9(8):e1003715.
Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.
Author Summary
Autophagy is a eukaryotic cellular process that transports cytoplasmic contents into lysosomes/vacuoles for degradation. It has been linked to multiple human diseases, including cancer and neurodegenerative disorders. The molecular machinery of autophagy was first identified and has been best characterized in the budding yeast Saccharomyces cerevisiae, but little is known about the autophagy machinery in another important unicellular model organism, the fission yeast Schizosaccharomyces pombe. In this study, we performed an unbiased and comprehensive screening of the fission yeast autophagy genes by profiling the mating phenotypes of nearly 3000 deletion strains. Following up on the screening results, we systematically characterized both previously known and newly identified fission yeast autophagy factors by examining their localization and the phenotype of their mutants. Our analysis increased the number of experimentally defined fission yeast autophagy factors from 14 to 23, including two novel factors that act in ways different from all previously known autophagy proteins. Together, our data reveal unexpected evolutionary divergence of autophagy mechanisms and establish a new model system for unraveling the molecular details of the autophagy process.
doi:10.1371/journal.pgen.1003715
PMCID: PMC3738441  PMID: 23950735
7.  Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates 
PLoS Genetics  2015;11(2):e1004987.
Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease.
Author Summary
Accumulation of misfolded proteins deposited in the form of inclusion bodies is a common pathological hallmark for many human genetic diseases, particularly for the neurodegenerative disorders. The aggregation of the disease related proteins suggests a failure of the cellular machineries that maintain the protein homeostasis or proteostasis. The cellular clearance pathways, e.g. autophagy-lysosomal pathway, may not be of high efficiency in the face of rapid formation of misfolded protein aggregates. Thus, understanding of intrinsic mechanism whereby autophagy offers protection to cells by removing toxic protein aggregates is important. Here we report that a signaling transduction event that chemically modifies autophagy receptor protein p62/SQSTM1 regulates the receptor’s binding affinity to small molecule called ubiquitin(essential for marking the protein for degradation), as well as the selective degradation of targeted proteins. Furthermore, we find that expression of Huntington’s disease (HD) associated protein aggregates (containing polyglutamine or polyQ expansion) triggers the same modification of p62, which is dependent on the length of the polyQ expansion, suggesting a protective response of the cell by activating autophagy toward degradation of toxic aggregates. The modification of p62 also occurs in HD model brains in an age-dependent manner. Our study sheds light on the regulation of selective autophagy and provides a rationale for targeting p62 modification to treat aggregate diseases including HD.
doi:10.1371/journal.pgen.1004987
PMCID: PMC4344198  PMID: 25723488
8.  Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans 
PLoS Medicine  2014;11(11):e1001755.
Theo Rein and colleagues examine the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.
Please see later in the article for the Editors' Summary
Background
FK506 binding protein 51 (FKBP51) is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.
Methods and Findings
Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO) mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR) treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways.
Importantly, the clinical antidepressant response of patients with depression (n = 51) could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r = 0.569, p = 0.004; Beclin1/fluoxetine: r = 0.454, p = 0.026; pAkt/amitriptyline: r = −0.416, p = 0.006; pAkt/PAR: r = −0.355, p = 0.021; LC3B-II/PAR: r = 0.453, p = 0.02), as well as by the lymphocytic expression levels of FKBP51 (r = 0.631, p<0.0001), pAkt (r = −0.515, p = 0.003), and Beclin1 (r = 0.521, p = 0.002) at admission. Limitations of the study include the use of male mice only and the relatively low number of patients for protein analyses.
Conclusions
To our knowledge, these findings provide the first evidence for the molecular mechanism of FKBP51 in priming autophagic pathways; this process is linked to the potency of at least some antidepressants. These newly discovered functions of FKBP51 also provide novel predictive markers for treatment outcome, consistent with physiological and potential clinical relevance.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Everyone feels miserable sometimes, but about one in six people will have an episode of clinical depression during their lifetime. For people who are clinically depressed, overwhelming feelings of sadness, anxiety, and hopelessness can last for months or years. Affected individuals lose interest in activities they used to enjoy, they sometimes have physical symptoms such as disturbed sleep, and they may contemplate suicide. Clinicians diagnose depression and determine its severity using questionnaires (“depression rating scales”) that explore the patient's feelings and symptoms. Mild depression is often treated with talking therapies (psychotherapy) such as cognitive behavioral therapy, which helps people change negative ways of thinking. For more severe depression, patients are also usually prescribed an antidepressant, most commonly a “selective serotonin reuptake inhibitor” such as paroxetine or a tricyclic antidepressant such as amitriptyline.
Why Was This Study Done?
Unfortunately, antidepressants don't work for more than half of patients. Moreover, because it is unclear how antidepressants work, it is not possible to predict which patients will respond to which antidepressants. Thus, matching patient to drug can be a lengthy, sometimes unsuccessful, process. Here, the researchers use several approaches to test the hypothesis that a protein called FK506 binding protein 51 (FKBP51) is involved in the actions of antidepressants and to investigate whether the ability of both FKBP51 and antidepressants to regulate a process called autophagy underlies the impact of FKBP51 on antidepressant responses. FKBP51 is a regulator of stress physiology, which is connected to the development and treatment of depression; genetic studies have suggested a link between FKBP51 expression and the antidepressant response rate. Some antidepressants are known to alter the initial steps in the autophagy pathway, a multistep process that maintains the integrity of cells through regulated degradation and recycling of cellular components; however, the potential synergistic role of FKBP51 and antidepressants in regulating pathways of autophagy are unknown.
What Did the Researchers Do and Find?
The researchers first treated wild-type mice and FKBP51 knockout mice (genetically altered animals that make no FKBP51) with an acute dose of antidepressant and compared their behavior in a forced swim test, an assay that measures the action of antidepressants in mice by determining how long the mice struggle or float inertly when placed in deep water. As expected, acute antidepressant treatment increased the time that wild-type mice spent struggling. However, this effect of antidepressant treatment was greatly attenuated in the FKBP51 knockout mice. Moreover, the levels of several autophagy markers increased in the brains of wild-type mice following antidepressant treatment but not in the brains of FKBP51 knockout mice. Next, using “chronic social defeat stress” to model the “endophenotype” of depression (a combination of physiological, hormonal, and behavioral traits seen in people with depression) in mice, the researchers showed that the stress response and the effect of chronic antidepressants on behavior and on autophagic markers all depend on FKBP51. Using cell-based assays, the researchers showed that antidepressants and FKBP51 had synergistic (interactive) effects on the autophagic pathway and that, in human blood cells, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways. Finally, the researchers report that the clinical response to antidepressant treatment in 51 patients with depression was associated with the response of autophagic markers in their peripheral blood lymphocytes to antidepressant treatment in test tubes, and that the expression levels of FKBP51 and autophagy markers in patient lymphocytes at admission were associated with subsequent clinical responses to antidepressants.
What Do These Findings Mean?
These findings suggest that the protein FKBP51 is required for the effects of both acute and chronic treatment with some antidepressants on behavior and on autophagic pathways in mice. These findings also reveal an association between antidepressant treatment responses in patients and both the expression levels of FKBP51 and autophagy markers in lymphocytes at admission and the response of autophagic markers to antidepressant treatment in patient lymphocytes. The accuracy of these findings is limited by the small number of clinical samples available for analysis, by the use of only male mice in the animal experiments, and by the inability of animal models of depression to fully replicate the human condition. Nevertheless, these findings identify the early stages of autophagy as potential targets for the development of new antidepressants and identify several potential biomarkers that might, after further clinical validation, help clinicians predict antidepressant efficacy in patients with depression.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001755.
The US National Institute of Mental Health provides information on all aspects of depression (in English and Spanish), including information on antidepressants
The UK National Health Service Choices website provides detailed information about depression and about antidepressants; it also provides personal stories about depression
The UK charity Mind provides information on depression, including some personal stories about depression
More personal stories about depression are available from healthtalk.org
MedlinePlus provides links to other resources about depression
Wikipedia has a page on autophagy (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The patients included in this study were all enrolled in the Munich Antidepressant Response Signature project, which aims to identify gene variants and biomarkers that predict treatment outcomes with antidepressants
doi:10.1371/journal.pmed.1001755
PMCID: PMC4227651  PMID: 25386878
9.  Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum 
PLoS ONE  2014;9(11):e113220.
Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.
doi:10.1371/journal.pone.0113220
PMCID: PMC4245143  PMID: 25426852
10.  Prohibitin 1 Modulates Mitochondrial Stress-Related Autophagy in Human Colonic Epithelial Cells 
PLoS ONE  2012;7(2):e31231.
Introduction
Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.
Methods
We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.
Results
TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.
Conclusions
Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.
doi:10.1371/journal.pone.0031231
PMCID: PMC3281932  PMID: 22363587
11.  Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat in CD4+ T Lymphocytes 
Journal of Virology  2014;89(1):615-625.
ABSTRACT
Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4+ T lymphocytes, a mechanism likely contributing to the loss of CD4+ T cells. In contrast, in productively infected CD4+ T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4+ T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread.
IMPORTANCE Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4+ T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.
doi:10.1128/JVI.02174-14
PMCID: PMC4301118  PMID: 25339774
12.  Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway 
Journal of Virology  2014;88(20):12133-12145.
ABSTRACT
Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.
IMPORTANCE
Autophagy is a cellular process that degrades and recycles nutrients under stress conditions to promote cell survival. Although autophagy commonly serves as a defense mechanism against viral infection, many viruses exploit this mechanism to promote their replication. This study finds that a transcription factor that is encoded by Epstein-Barr virus (EBV), Rta, activates autophagy, and the inhibition of autophagy reduces the ability of the virus to express viral lytic proteins and to generate progeny. Unlike other virus-encoded proteins that modulate autophagy by interacting with proteins that are involved in the autophagic pathway, Rta activates the transcription of the autophagy-related genes via the ERK pathway. The results of this study reveal how the virus manipulates autophagy to promote its lytic development.
doi:10.1128/JVI.02033-14
PMCID: PMC4178756  PMID: 25122800
13.  NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses 
PLoS Genetics  2013;9(1):e1003196.
Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.
Author Summary
Autophagy is an evolutionarily conserved process that sequestrates and delivers cytoplasmic macromolecules and organelles to the vacuoles or lysosomes for degradation. In plants, autophagy is involved in supplying internal nutrients during starvation and in promoting cell survival during senescence and during biotic and abiotic stresses. Arabidopsis NBR1 is a homolog of mammalian autophagy cargo adaptors P62 and NBR1. Disruption of Arabidopsis NBR1 caused increased sensitivity to a spectrum of abiotic stresses but had no significant effect on plant senescence, responses to carbon starvation, or resistance to a necrotrophic pathogen. NBR1 contains an ubiquitin-binding domain, and the compromised stress tolerance of autophagy mutants was associated with increased accumulation of NBR1 and ubiquitin-positive cellular protein aggregates in the insoluble protein fraction under stress conditions. Based on these results, we propose that NBR1 targets ubiquitinated protein aggregates most likely derived from denatured and otherwise damaged nonnative proteins for autophagic clearance under stress conditions.
doi:10.1371/journal.pgen.1003196
PMCID: PMC3547818  PMID: 23341779
14.  Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration 
PLoS Pathogens  2015;11(6):e1005018.
HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.
Author Summary
Under basal conditions, the mammalian target of rapamycin (MTOR) phosphorylates transcription factor EB (TFEB) resulting in its cytoplasmic retention. When MTOR is inhibited, TFEB is dephosphorylated and translocated to the nucleus where it increases autophagy and lysosomal gene expression. As human immunodeficiency virus type 1 (HIV) Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1), we investigated the role of Nef and TFEB in the modulation of autophagy during HIV infection of human macrophages. We found that upon exposure to HIV, macrophages elicited an autophagic response through a toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB. However, once HIV infection is established, phosphorylation and cytoplasmic sequestration of TFEB as well as autophagy revert to pre-infection levels. Moreover, this reversion is dependent upon the presence of HIV Nef. Collectively, the data suggests that the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. Once HIV establishes a productive infection, Nef binds BECN1 resulting in MTOR activation, TFEB phosphorylation and cytosolic sequestration and the inhibition of autophagy.
doi:10.1371/journal.ppat.1005018
PMCID: PMC4482621  PMID: 26115100
15.  Autophagy Attenuates Diabetic Glomerular Damage through Protection of Hyperglycemia-Induced Podocyte Injury 
PLoS ONE  2013;8(4):e60546.
Despite the recent attention focused on the important role of autophagy in maintaining podocyte homeostasis, little is known about the changes and mechanisms of autophagy in podocyte dysfunction under diabetic condition. In this study, we investigated the role of autophagy in podocyte biology and its involvement in the pathogenesis of diabetic nephropathy. Podocytes had a high basal level of autophagy. And basal autophagy inhibition either by 3-methyladenenine (3-MA) or by Beclin-1 siRNA was detrimental to its architectural structure. However, under diabetic condition in vivo and under high glucose conditions in vitro, high basal level of autophagy in podocytes became defective and defective autophagy facilitated the podocyte injury. Since the dynamics of endoplasmic reticulum(ER) seemed to play a vital role in regulating the autophagic flux, the results that Salubrinal/Tauroursodeoxycholic acid (TUDCA) could restore defective autophagy further indicated that the evolution of autophagy may be mediated by the changes of cytoprotective output in the ER stress. Finally, we demonstrated in vivo that the autophagy of podocyte was inhibited under diabetic status and TUDCA could improve defective autophagy. Taken together, these data suggested that autophagy might be interrupted due to the failure of ER cytoprotective capacity upon high glucose induced unmitigated stress, and the defective autophagy might accelerate the irreparable progression of diabetic nephropathy.
doi:10.1371/journal.pone.0060546
PMCID: PMC3623813  PMID: 23593240
16.  Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy 
PLoS Biology  2013;11(11):e1001708.
A novel Drosophila model system of chloroquine myopathy reveals how glycogen is targeted to the lysosome and what the significance of this process is for muscle cells.
Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8.
Author Summary
Lysosomes are organelles that work as a disposal system for the cell. It is known that lysosomes can degrade glycogen and that defects in this function trigger the accumulation of vesicles containing glycogen in animals that lead to vacuolar myopathies—diseases that result in muscle weakness. However, it remains unclear how and why glycogen is degraded through this system, and what significance it has for the pathology of such diseases. Here, we addressed these questions by establishing a fruitfly model system to study glycogen autophagy in skeletal muscles. By feeding the flies chloroquine (CQ), we induce a vacuolar myopathy associated with massive accumulation of glycogen-filled vesicles, and assay the role of autophagy and glycogen metabolic enzymes in this process. We show that CQ-induced glycogen autophagy is completely dependent on the core conserved autophagy genes and that this autophagy is triggered by nutrient deprivation in a Tor-dependent manner. Interestingly, while glycogen autophagy and enzymatic glycogen breakdown can compensate for each other, concurrent inhibition of both systems blocks glycogen breakdown. Finally, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of glycogen synthase—the main enzyme involved in converting glucose to glycogen—in regulating autophagy through its interaction with the autophagosome.
doi:10.1371/journal.pbio.1001708
PMCID: PMC3825659  PMID: 24265594
17.  Autophagy Pathway Is Required for IL-6 Induced Neuroendocrine Differentiation and Chemoresistance of Prostate Cancer LNCaP Cells 
PLoS ONE  2014;9(2):e88556.
Prostate cancer (PCa) cells undergoing neuroendocrine differentiation (NED) are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE) tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ) markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA), a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results reveal the potential of targeting autophagy as part of a combined therapeutic regime for NE tumors.
doi:10.1371/journal.pone.0088556
PMCID: PMC3925144  PMID: 24551118
18.  HIV-1 Inhibits Autophagy in Bystander Macrophage/Monocytic Cells through Src-Akt and STAT3 
PLoS ONE  2010;5(7):e11733.
Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, β-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1+ patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.
doi:10.1371/journal.pone.0011733
PMCID: PMC2908694  PMID: 20661303
19.  The Zebrafish as a New Model for the In Vivo Study of Shigella flexneri Interaction with Phagocytes and Bacterial Autophagy 
PLoS Pathogens  2013;9(9):e1003588.
Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.
Author Summary
Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, a complete understanding of the molecules and mechanisms restricting cytosolic bacteria has not been obtained, and the role of bacterial autophagy in vivo remains poorly understood. Shigella flexneri are human-adapted Escherichia coli that have gained the ability to invade the colonic mucosa, causing inflammation and diarrhea. The intracellular lifestyle of this pathogen has been well-studied in vitro, and Shigella has recently gained recognition as a paradigm of bacterial autophagy. We show that the zebrafish larva represents a valuable new host for the analysis of S. flexneri infection. Interactions between bacteria and host phagocytes can be imaged at high resolution in vivo, and the zebrafish model should prove useful for understanding the cell biology of Shigella infection. We use zebrafish larvae to investigate the role of bacterial autophagy in host defense, and observed that the perturbation of autophagy can adversely affect host survival in response to Shigella infection. Therefore, the zebrafish constitutes a valuable system to develop new strategies aimed at pathogen clearance by manipulation of anti-bacterial autophagy.
doi:10.1371/journal.ppat.1003588
PMCID: PMC3764221  PMID: 24039575
20.  Decreased Autophagy Contributes to Myocardial Dysfunction in Rats Subjected to Nonlethal Mechanical Trauma 
PLoS ONE  2013;8(8):e71400.
Autophagy is important in cells for removing damaged organelles, such as mitochondria. Insufficient autophagy plays a critical role in tissue injury and organ dysfunction under a variety of pathological conditions. However, the role of autophagy in nonlethal traumatic cardiac damage remains unclear. The aims of the present study were to investigate whether nonlethal mechanical trauma may result in the change of cardiomyocyte autophagy, and if so, to determine whether the changed myocardial autophagy may contribute to delayed cardiac dysfunction. Male adult rats were subjected to nonlethal traumatic injury, and cardiomyocyte autophagy, cardiac mitochondrial function, and cardiac function in isolated perfused hearts were detected. Direct mechanical traumatic injury was not observed in the heart within 24 h after trauma. However, cardiomyocyte autophagy gradually decreased and reached a minimal level 6 h after trauma. Cardiac mitochondrial dysfunction was observed by cardiac radionuclide imaging 6 h after trauma, and cardiac dysfunction was observed 24 h after trauma in the isolated perfused heart. These were reversed when autophagy was induced by administration of the autophagy inducer rapamycin 30 min before trauma. Our present study demonstrated for the first time that nonlethal traumatic injury caused decreased autophagy, and decreased autophagy may contribute to post-traumatic organ dysfunction. Though our study has some limitations, it strongly suggests that cardiac damage induced by nonlethal mechanical trauma can be detected by noninvasive radionuclide imaging, and induction of autophagy may be a novel strategy for reducing posttrauma multiple organ failure.
doi:10.1371/journal.pone.0071400
PMCID: PMC3747162  PMID: 23977036
21.  MicroRNA-155 Promotes Autophagy to Eliminate Intracellular Mycobacteria by Targeting Rheb 
PLoS Pathogens  2013;9(10):e1003697.
Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3′-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.
Author Summary
microRNA-155 (miR-155) plays an essential role in regulating the host immune response by post-transcriptionally repressing the expression of target genes. However, little is known regarding its activity in modulating autophagy, an important host defense mechanism against intracellular bacterial infection. Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects approximately one-third of the global population, and causes 1.5 million deaths annually. The present study explores a novel role of miR-155 in the host response against mycobacterial infection. Our data demonstrates that mycobacterial infection triggers the expression of miR-155, and the induction of miR-155 in turn activates autophagy by targeting Rheb, a negative regulator of autophagy. miR-155-promoted autophagy accelerates the maturation of the mycobacterial phagosome, thus decreasing the survival of intracellular mycobacteria in macrophages. These findings contribute to a better understanding of the host defense mechanisms against mycobacterial infection, providing useful information for development of potential therapeutic interventions against tuberculosis.
doi:10.1371/journal.ppat.1003697
PMCID: PMC3795043  PMID: 24130493
22.  Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress 
Oncotarget  2014;5(6):1526-1537.
The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ.
PMCID: PMC4039229  PMID: 24681637
autophagy; reactive oxygen species; mitoquinone; breast cancer
23.  Oxidative Damage and Autophagy in the Human Trabecular Meshwork as Related with Ageing 
PLoS ONE  2014;9(6):e98106.
Autophagy is an intracellular lysosomal degradation process induced under stress conditions. Autophagy also plays a major role in ocular patho-physiology. Molecular aging does occur in the trabecular meshwork, the main regulator of aqueous humor outflow, and trabecular meshwork senescence is accompanied by increased oxidative stress. However, the role of autophagy in trabecular meshwork patho-physiology has not yet been examined in vivo in human ocular tissues. The purpose of the herein presented study is to evaluate autophagy occurrence in ex-vivo collected human trabecular meshwork specimens and to evaluate the relationship between autophagy, oxidative stress, and aging in this tissue. Fresh trabecular meshwork specimens were collected from 28 healthy corneal donors devoid of ocular pathologies and oxidative DNA damage, and LC3 and p62 protein expression analyzed. In a subset of 10 subjects, further to trabecular meshwork proteins, the amounts of cathepesin L and ubiquitin was analyzed by antibody microarray in aqueous humor. Obtained results demonstrate that autophagy activation, measured by LC3II/I ratio, is related with. oxidative damage occurrence during aging in human trabecular meshwork. The expression of autophagy marker p62 was lower in subjects older than 60 years as compared to younger subjects. These findings reflect the occurrence of an agedependent increase in the autophagy as occurring in the trabecular meshwork. Furthermore, we showed that aging promotes trabecular-meshwork senescence due to increased oxidative stress paralleled by autophagy increase. Indeed, both oxidative DNA damage and autophagy were more abundant in subjects older than 60 years. These findings shed new light on the role of oxidative damage and autophagy during trabecular-meshwork aging.
doi:10.1371/journal.pone.0098106
PMCID: PMC4063984  PMID: 24945152
24.  Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells 
Background
Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels.
Methods and results
In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death.
Conclusion
Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.
doi:10.1186/1423-0127-19-64
PMCID: PMC3430578  PMID: 22788551
Insulin receptor substrate; Oxidative stress; Autophagy; Cell death; Cancer; Mammalian target of rapamycin; p70 ribosomal protein S6 kinase; Reactive oxygen species; Glucose oxidase
25.  Autophagy and Apoptosis Act as Partners to Induce Germ Cell Death after Heat Stress in Mice 
PLoS ONE  2012;7(7):e41412.
Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis.
doi:10.1371/journal.pone.0041412
PMCID: PMC3405141  PMID: 22848486

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