Table of Contents

Volume 8, Issue 4

  April 2012

  Pages 439 - 713

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 Issue Catalogue (MARC XML)

EDITOR'S CORNER

Open Access Article

A human autophagy interaction network

Daniel J. Klionsky
Pages 439 - 441
http://dx.doi.org/10.4161/auto.19926
Abstract | Full Text | PDF

The original “Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes” has been well received and used by many researchers and authors. I consider these to be very important guidelines that require a consensus among the researchers in the field, because they are used by authors to defend against inappropriate reviewers’ comments, and by reviewers to point out to editors the flaws in research papers. Accordingly, I decided it was time to revise and update the guidelines. After all, the field has expanded substantially, as has the range of model systems being used to analyze autophagy. As a result, the list of authors has similarly increased. In addition, this version of the guidelines is not limited to higher eukaryotes nor to macroautophagy. Here, I explain the approach used to invite authors to participate in the revised guidelines, and briefly demonstrate one aspect of their utility.




AUTOPHAGIC FLUX

Open Access Article

Highlights from this issue

Andriy Nemchenko
Pages 442 - 444
http://dx.doi.org/10.4161/auto.20287
Full Text | PDF




REVIEW

Open Access Article

Guidelines for the use and interpretation of assays for monitoring autophagy

Daniel J. Klionsky and 1269 others
Pages 445 - 544
http://dx.doi.org/10.4161/auto.19496
Abstract | Full Text | PDF

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.




BASIC BRIEF REPORT

Open Access Article

Autophagy: A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

Ricky H. Bhogal, Christopher J. Weston, Stuart M. Curbishley, David H. Adams and Simon C. Afford
Pages 545 - 558
http://dx.doi.org/10.4161/auto.19012
Abstract | Full Text | PDF

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.




BASIC RESEARCH PAPERS

Open Access Article

Cucurbitacin induces autophagy through mitochondrial ROS production which counteracts to limit caspase-dependent apoptosis

Tiejun Zhang, Yuwen Li, Kyeong Ah Park, Hee Sun Byun, Minho Won, Juhee Jeon, Yoonjung Lee, Jeong Ho Seok, Seung-Won Choi, Sang-Hee Lee, Jin Man Kim, Ji Hoon Lee, Chang Gue Son, Zee-Won Lee, Han-Ming Shen and Gang Min Hur
Pages 559 - 576
http://dx.doi.org/10.4161/auto.18867
Abstract | Full Text | PDF

Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.

Open Access Article

Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

Satoru Kobayashi, Xianmin Xu, Kai Chen and Qiangrong Liang
Pages 577 - 592
http://dx.doi.org/10.4161/auto.18980
Abstract | Full Text | PDF

Hyperglycemia is linked to increased heart failure among diabetic patients. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Autophagy is a cellular degradation pathway that plays important roles in cellular homeostasis. Autophagic activity is altered in the diabetic heart, but its functional role has been unclear. In this study, we determined if mimicking hyperglycemia in cultured cardiomyocytes from neonatal rats and adult mice could affect autophagic activity and myocyte viability. High glucose (17 or 30 mM) reduced autophagic flux compared with normal glucose (5.5 mM) as indicated by the difference in protein levels of LC3-II (microtubule-associated protein 1 light chain 3 form II) or the changes of punctate fluorescence patterns of GFP-LC3 and mRFP-LC3 in the absence and presence of the lysosomal inhibitor bafilomycin A₁. Unexpectedly, the inhibited autophagy turned out to be an adaptive response that functioned to limit high glucose cardiotoxicity. Indeed, suppression of autophagy by 3-methyladenine or short hairpin RNA-mediated silencing of the Becn1 or Atg7 gene attenuated high glucose-induced cardiomyocyte death. Conversely, upregulation of autophagy with rapamycin or overexpression of Becn1 or Atg7 predisposed cardiomyocytes to high glucose toxicity. Mechanistically, the high glucose-induced inhibition of autophagy was mediated at least partly by increased mTOR signaling that likely inactivated ULK1 through phosphorylation at serine 467. Together, these findings demonstrate that high glucose inhibits autophagy, which is a beneficial adaptive response that protects cardiomyocytes against high glucose toxicity. Future studies are warranted to determine if autophagy plays a similar role in diabetic heart in vivo.

Open Access Article

Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts

Rui Guo, Nan Hu, Machender R. Kandadi and Jun Ren
Pages 593 - 608
http://dx.doi.org/10.4161/auto.18997
Abstract | Full Text | PDF

Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A₁, E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

Open Access Article

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo

Erin E. Congdon, Jessica W. Wu, Natura Myeku, Yvette H. Figueroa, Mathieu Herman, Paul S. Marinec, Jason E. Gestwicki, Chad A. Dickey, W. Haung Yu and Karen E. Duff
Pages 609 - 622
http://dx.doi.org/10.4161/auto.19048
Abstract | Full Text | PDF

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.

Open Access Article

PP2A regulates autophagy in two alternative ways in Drosophila

Ágnes Bánréti, Tamás Lukácsovich, György Csikós, Miklós Erdélyi and Miklós Sass
Pages 623 - 636
http://dx.doi.org/10.4161/auto.19081
Abstract | Full Text | PDF

Protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex, consisting of A, B and C subunits. The catalytic subunit PP2A-C (microtubule star/mts) binds to the C-terminal part of the scaffold protein PP2A-A (PP2A-29B). In Drosophila, there are three different forms of B subunits (widerborst/wdb, twins/tws and PP2A-B'), which determine the subcellular localization and substrate specificity of the holoenzyme. Previous studies demonstrated that PP2A is involved in the control of TOR-dependent autophagy both in yeast and mammals. Furthermore, in Drosophila, wdb genetically interacts with the PtdIns3K/PTEN/Akt signaling cascade, which is a main upstream regulatory system of dTOR. Here we demonstrate that in Drosophila, two different PP2A complexes (containing B' or wdb subunit) play essential roles in the regulation of starvation-induced autophagy. The PP2A-A/wdb/C complex acts upstream of dTOR, whereas the PP2A-A/B'/C complex functions as a target of dTOR and may regulate the elongation of autophagosomes and their subsequent fusion with lysosomes. We also identified three Drosophila Atg orthologs (Atg14, Atg17 and Atg101), which represent potential targets of the PP2A-A/B'/C complex during autophagy.

Open Access Article

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Guillaume Robert, Cecile Gastaldi, Alexandre Puissant, Amine Hamouda, Arnaud Jacquel, Maeva Dufies, Nathalie Belhacene, Pascal Colosetti, John C. Reed, Patrick Auberger and Fréderic Luciano
Pages 637 - 649
http://dx.doi.org/10.4161/auto.19084
Abstract | Full Text | PDF

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X_L bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.

Open Access Article

Interleukin-6: A bone marrow stromal cell paracrine signal that induces neuroendocrine differentiation and modulates autophagy in bone metastatic PCa cells

Nikki A. Delk and Mary C. Farach-Carson
Pages 650 - 663
http://dx.doi.org/10.4161/auto.19226
Abstract | Full Text | PDF

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.

Open Access Article

A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress

Virginie M.S. Betin, Thomas D.B. MacVicar, Stephen F. Parsons, David J. Anstee and Jon D. Lane
Pages 664 - 676
http://dx.doi.org/10.4161/auto.19227
Abstract | Full Text | PDF

The Atg4 cysteine proteases play crucial roles in the processing of Atg8 proteins during autophagy, but their regulation during cellular stress and differentiation remains poorly understood. We have found that two Atg4 family members—Atg4C and Atg4D—contain cryptic mitochondrial targeting sequences immediately downstream of their canonical (DEVD) caspase cleavage sites. Consequently, caspase-cleaved Atg4D (ΔN63 Atg4D) localizes to the mitochondrial matrix when expressed in mammalian cells, where it undergoes further processing to a ~42 kDa mitochondrial form. Interestingly, caspase cleavage is not needed for Atg4D mitochondrial import, because ~42 kDa mitochondrial Atg4D is observed in cells treated with caspase inhibitors and in cells expressing caspase-resistant Atg4D (DEVA⁶³). Using HeLa cell lines stably expressing ΔN63 Atg4D, we showed that mitochondrial Atg4D sensitizes cells to cell death in the presence of the mitochondrial uncoupler, CCCP, and that mitochondrial cristae are less extensive in these cells. We further showed that the organization of mitochondrial cristae is altered during the mitochondrial clearance phase in differentiating primary human erythroblasts stably expressing ΔN63 Atg4D, and that these cells have elevated levels of mitochondrial reactive oxygen species (ROS) during late stages of erythropoiesis. Together these data suggest that the import of Atg4D during cellular stress and differentiation may play important roles in the regulation of mitochondrial physiology, ROS, mitophagy and cell viability.




TOOLBOX

Open Access Article

A novel method for autophagy detection in primary cells: Impaired levels of macroautophagy in immunosenescent T cells

Kanchan Phadwal, Javier Alegre-Abarrategui, Alexander Scarth Watson, Luke Pike, Selvakumar Anbalagan, Ester M. Hammond, Richard Wade-Martins, Andrew McMichael, Paul Klenerman and Anna Katharina Simon
Pages 677 - 689
http://dx.doi.org/10.4161/auto.18935
Abstract | Full Text | PDF

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8⁺ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.




AUTOPHAGIC PUNCTA

Open Access Article

The fibroblast growth factor signaling axis controls cardiac stem cell differentiation through regulating autophagy

Jue Zhang, Junchen Liu, Leyuan Liu, Wallace L. McKeehan and Jue Zhang
Pages 690 - 691
http://dx.doi.org/10.4161/auto.19290
Abstract | Full Text | PDF

The fibroblast growth factor (FGF) signaling axis plays important roles in heart development. Yet, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. Using genetically engineered mouse and in vitro cultured embryoid body (EB) models, we demonstrate that FGF signaling suppresses premature differentiation of heart progenitor cells, as well as autophagy in outflow tract (OFT) myocardiac cells. The FGF also promotes mesoderm differentiation in embryonic stem cells (ESCs) but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, inhibition of FGF signaling increases myocardial differentiation and autophagy in both ex vivo cultured embryos and EBs, whereas activation of autophagy promotes myocardial differentiation. Thus, a link between FGF signals preventing premature differentiation of heart progenitor cells and suppression of autophagy has been established. These findings provide the first evidence that autophagy plays a role in heart progenitor differentiation, and suggest a new venue to regulate stem/progenitor cell differentiation.

Open Access Article

LRRK2 as a modulator of lysosomal calcium homeostasis with downstream effects on autophagy

Patricia Gómez-Suaga and Sabine Hilfiker
Pages 692 - 693
http://dx.doi.org/10.4161/auto.19305
Abstract | Full Text | PDF

Alterations in autophagy are thought to underlie various neurodegenerative diseases including Parkinson disease (PD). Previous studies have indicated that the PD gene leucine rich repeat kinase 2 (LRRK2) is involved in this process, but its mechanism of action has remained unknown. Our recent work describes how LRRK2 acts through calcium-mediated events originating from acidic stores to regulate autophagy and cell survival, which may give rise to novel therapeutic strategies.

Open Access Article

ATG5, autophagy and lung function in asthma

Audrey Poon, David Eidelman, Catherine Laprise and Qutayba Hamid
Pages 694 - 695
http://dx.doi.org/10.4161/auto.19315
Abstract | Full Text | PDF

Reactive oxidative species (ROS) are essential in cellular survival; however, excessive production and chronic exposure to ROS pose serious health threats. Excessive production of ROS is thought to play a pivotal role in the pathogenesis of asthma, where exhaled levels of ROS have been found to positively correlate with disease severity. Autophagy is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage, and presents itself as a good candidate pathway for investigation in asthma pathogenesis. Given the role of oxidative stress in the pathogenesis of asthma and disease severity, we hypothesized that autophagy is associated with asthma pathogenesis, and sought to detect its presence using both genetic and histological approaches. We found variant rs12212740, an intronic SNP of ATG5, to be associated with asthma and forced expiratory volume in 1 second (FEV₁) percent predicted in the French Canadian population and with FEV₁ in an American Caucasian cohort. Furthermore, double-membrane autophagosomes were more easily detected in fibroblast and epithelial cells from a bronchial biopsy tissue of a moderately severe asthma patient compared with corresponding cells of a healthy subject. Asthma is associated with a cytokine milieu [e.g., interleukin (IL)-13] that promotes transforming growth factor-β1 (TGFβ1) affiliated airway remodeling, and agonistic relationships existed among these cytokines and ROS. Hence, autophagy may be a cellular mechanism that promotes TGFβ1 airway remodeling and loss of lung function in asthma.

Open Access Article

MTOR regulates autophagic flux in the glomerulus

Davide P. Cinà, Tuncer Onay, Aarti Paltoo, Chengjin Li, Yoshiro Maezawa, Javier De Arteaga, Andrea Jurisicova and Susan E. Quaggin
Pages 696 - 698
http://dx.doi.org/10.4161/auto.19386
Abstract | Full Text | PDF

Sirolimus (rapamycin), an inhibitor of the mechanistic target of rapamycin (MTOR), was originally proposed as an immunosuppressant to prevent rejection of solid organ transplants. There were expectations that MTOR inhibitors would replace nephrotoxic calcineurin inhibitors (CNIs). Despite its potential advantages, evidence that sirolimus causes de novo or worsening proteinuria is unequivocal. Given the well-recognized proteinuric effect of MTOR inhibitors, we were interested in understanding its role in maintaining the glomerular filtration barrier. To investigate this in vivo, we developed a mouse model with a podocyte selective deletion of the Mtor gene (Mtor pod-KO).

Open Access Article

Mitophagy in cells with mtDNA mutations: Being sick is not enough

Rosa L.A. de Vries, Robert W. Gilkerson, Serge Przedborski and Eric A. Schon
Pages 699 - 700
http://dx.doi.org/10.4161/auto.19470
Abstract | Full Text | PDF

Despite the emergence of autophagy as a key process for mitochondrial quality control, the existence and persistence of pathogenic mtDNA mutations in human disease suggests that the degradation of dysfunctional mitochondria does not occur widely in vivo. During macroautophagy, a double-membraned cup-shaped structure engulfs cytosolic content. This autophagic vesicle then fuses with lysosomes, allowing hydrolytic enzymes to degrade the contents. Mitochondrial autophagy, or mitophagy, is thought to degrade damaged or nonfunctioning mitochondria specifically. The Parkinson disease-related proteins PINK1 (a mitochondrially localized kinase) and PARK2 (PARKIN, a cytosolically-localized E3 ubiquitin ligase) are essential for targeting mitochondria for mitophagy. Upon chemical uncoupling of the mitochondrial transmembrane potential (Δψ_m), PINK1 located in the mitochondrial outer membrane recruits PARK2 from the cytosol to the mitochondria, followed by delivery of the organelle to the autophagic machinery for degradation.

Open Access Article

Malin knockout mice support a primary role of autophagy in the pathogenesis of Lafora disease

Erwin Knecht, Olga Criado-García, Carmen Aguado, Javier Gayarre, Lara Duran-Trio, Ana M. Garcia-Cabrero, Santiago Vernia, Beatriz San Millán, Miguel Heredia, Carlos Romá-Mateo, Silvana Mouron, Lucía Juana-López, Mercedes Domínguez, Carmen Navarro, Jose M. Serratosa, Marina Sanchez, Pascual Sanz, Paola Bovolenta and Santiago Rodríguez de Córdoba
Pages 701 - 703
http://dx.doi.org/10.4161/auto.19522
Abstract | Full Text | PDF

Lafora disease (LD), a fatal neurodegenerative disorder characterized by intracellular inclusions called Lafora bodies (LBs), is caused by recessive loss-of-function mutations in the genes encoding either laforin or malin. Previous studies suggested a role of these proteins in regulating glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. Here we review our recent finding that dysfunction of autophagy is a common feature of both laforin- and malin-deficient mice, preceding other pathological manifestations. We propose that autophagy plays a primary role in LD pathogenesis and is a potential target for its treatment.

Open Access Article

Hypoxia-induced autophagy: A new player in cancer immunotherapy?

Muhammad Zaeem Noman, Bassam Janji, Guy Berchem, Fathia Mami-Chouaib and Salem Chouaib
Pages 704 - 706
http://dx.doi.org/10.4161/auto.19572
Abstract | Full Text | PDF

A major challenge in formulating an effective immunotherapy is to overcome the mechanisms of tumor escape from immunosurveillance. We showed that hypoxia-induced autophagy impairs cytotoxic T-lymphocyte (CTL)-mediated tumor cell lysis by regulating phospho-STAT3 in target cells. Autophagy inhibition in hypoxic cells decreases phospho-STAT3 and restores CTL-mediated tumor cell killing by a mechanism involving the ubiquitin proteasome system and SQSTM1/p62. Simultaneously boosting the CTL-response, using a TRP-peptide vaccination strategy, and targeting autophagy in hypoxic tumors, improves the efficacy of cancer vaccines and promotes tumor regression in vivo. Overall, in addition to its immunosuppressive effect, the hypoxic microenvironment also contributes to immunoresistance and can be detrimental to antitumor effector cell functions.

Open Access Article

Mammalian PIK3C3/VPS34: The key to autophagic processing in liver and heart

Nadia Jaber, Zhixun Dou, Richard Z. Lin, Jianhua Zhang and Wei-Xing Zong
Pages 707 - 708
http://dx.doi.org/10.4161/auto.19627
Abstract | Full Text | PDF

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.

Open Access Article

Targeting autophagy for drug-induced hepatotoxicity

Hong-Min Ni, Hartmut Jaeschke and Wen-Xing Ding
Pages 709 - 710
http://dx.doi.org/10.4161/auto.19659
Abstract | Full Text | PDF

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.

Open Access Article

Autophagy, neuron-specific degradation and neurodegeneration

Dong Wang and P. Robin Hiesinger
Pages 711 - 713
http://dx.doi.org/10.4161/auto.19660
Abstract | Full Text | PDF

Degradation of membrane compartments, organelles and other debris through macroautophagy (hereafter referred to as autophagy) is thought to occur in most, maybe all, cells. We recently reported the discovery of a neuron-specific endomembrane degradation mechanism that depends on the vesicle SNARE neuronal Synaptobrevin (n-Syb) and the vesicle ATPase component V100 (the V₀a1 subunit). Loss of n-Syb causes degeneration of adult photoreceptor neurons in Drosophila, reminiscent of adult-onset degeneration in neurons with defective autophagy. Here we explore the potential importance of this newly discovered neuron-specific degradation mechanism in comparison with ubiquitous autophagy machinery for adult-onset neurodegeneration.