Autophagy regulates selective HMGB1 release in tumor cells that are destined to die

Macroautophagy (hereafter referred to as autophagy) can increase or decrease the amount of cell death in response to various stimuli. To test whether autophagy also controls the characteristics associated with dying cells, we studied tumor cell killing by epidermal growth factor receptor-targeted diphtheria toxin (DT-EGF). DT-EGF kills epithelial and glioblastoma tumor cells with similar efficiency but by different mechanisms that depend on whether the cells activate autophagy when treated with the drug. Dying cells in which autophagy is induced selectively release the immune modulator high-mobility group B1 (HMGB1) without causing lysis of the cell membrane and classical necrosis. Conversely, cells that are killed by DT-EGF where autophagy is blocked, activate caspases but retain HMGB1. These data suggest that it may be feasible to manipulate the immunogenicity of dying cells by increasing or decreasing autophagy.     

Neuronal autophagy: going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that "constitutive autophagy" in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.     

Proper autophagy is indispensable for angiogenesis during chick embryo development

People have known that autophagy plays a very important role in many physiological and pathological events. But the role of autophagy on embryonic angiogenesis still remains obscure. In this study, we demonstrated that Atg7, Atg8 and Beclin1 were expressed in the plexus vessels of angiogenesis at chick yolk sac membrane and chorioallantoic membrane. Interfering in autophagy with autophagy inducer or inhibitor could restrict the angiogenesis in vivo, which might be driven by the disorder of angiogenesis-related gene expressions, and also lead to embryonic hemorrhage, which was due to imperfection cell junctions in endothelial cells including abnormal expressions of tight junction, adheren junction and desmosome genes. Using HUVECs, we revealed that cell viability and migration ability changed with the alteration of cell autophagy exposed to RAPA or 3-MA. Interestingly, tube formation assay showed that HUVECs ability of tube formation altered with the change of Atg5, Atg7 and Atg8 manipulated by the transfection of their corresponding siRNA or plasmids. Moreover, the lost cell polarity labeled by F-actin and the absenced β-catenin in RAPA-treated and 3-MA-treated cell membrane implied intracellular cytoskeleton alteration was induced by the activation and depression of autophagy. Taken together, our current experimental data reveal that autophagy is really involved in regulating angiogenesis during embryo development.     

Neuronal macroautophagy: from development to degeneration

Macroautophagy, a lysosomal pathway responsible for the turnover of organelles and long-lived proteins, has been regarded mainly as an inducible process in neurons, which is mobilized in states of stress and injury. New studies show, however, that macroautophagy is also constitutively active in healthy neurons and is vital to cell survival. Neurons in the brain, unlike cells in the periphery, are protected from large-scale autophagy induction because they can use several different energy sources optimally, receive additional nutrients and neurotrophin support from glial cells, and benefit from hypothalamic regulation of peripheral nutrient supplies. Due to its exceptional efficiency, constitutive autophagy in healthy neurons proceeds in the absence of easily detectable autophagic vacuole intermediates. These intermediates can accumulate rapidly, however, when late steps in the autophagic process are blocked. Autophagic vacuoles also accumulate abnormally in affected neurons of several major neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, where they have been linked to various aspects of disease pathogenesis including neuronal cell death. The build-up of autophagic vacuoles in these neurological disorders and others may reflect either heightened autophagy induction, impairment in later digestive steps in the autophagy pathway, or both. Determining the basis for AV accumulation is critical for understanding the pathogenic significance of autophagy in a given pathologic state and for designing possible therapies based on modulating autophagy. In this review, we discuss the special features of autophagy regulation in the brain, its suspected roles in neurodevelopment and plasticity, and recent progress toward understanding how dysfunctional autophagy contributes to neurodegenerative disease.     

Non-canonical autophagy: an exception or an underestimated form of autophagy?

Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.     

EndoA/Endophilin-A creates docking stations for autophagic proteins at synapses

Synapses are very specialized compartments with high metabolic demand to maintain neurotransmission, an essential step for basic brain function. Neurons are post-mitotic and synapses need to stay functional over time—sometimes over decades. Given that synapses are often at a long distance from the cell body, they must use local mechanisms to regulate protein quality control. We show that macroautophagy/autophagy is one of these local processes and found that it is under strict control of the synapse-enriched protein EndoA/Endophilin-A, previously only implicated in endocytosis. Metabolic and neuronal stimulation induce synaptic autophagy and phosphorylation of EndoA by the Parkinson disease kinase Lrrk/LRRK2 is essential to promote the process. EndoA induces membrane curvature in vitro, and, mechanistically, phosphorylated EndoA creates curved membrane-protein docking sites that are capable of recruiting Atg3. Our work reveals a synapse-enriched branch of autophagy under the control of EndoA that may be deregulated in Parkinson disease.     

Non-canonical autophagy: An exception or an underestimated form of autophagy?

Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.     

TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.     

Autophagy modulates cell migration and β1 integrin membrane recycling

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3^?/? and atg5^?/? MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.     

Neuronal autophagy: Going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that “constitutive autophagy” in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.

Addendum to: Komatsu M, Wang QJ, Holstein GR, Friedrich Jr. VL, Iwata J, Kominami E, Chait BT, Tanaka K, Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 2007; 104:14489-94.     

Ultrastructure of autophagy in plant cells

Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.     

Autophagy paradox and ceramide

Sphingolipid molecules act as bioactive lipid messengers and exert their actions on the regulation of various cellular signaling pathways. Sphingolipids play essential roles in numerous cellular functions, including controlling cell inflammation, proliferation, death, migration, senescence, tumor metastasis and/or autophagy. Dysregulated sphingolipid metabolism has been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) “self-eating” is characterized by nonselective sequestering of cytosolic materials by an isolation membrane, which can be either protective or lethal for cells. Ceramide (Cer), a central molecule of sphingolipid metabolism, has been extensively implicated in the control of autophagy. The increasing evidence suggests that Cer is highly involved in mediating two opposing autophagic pathways, which regulate either cell survival or death, which is referred here as autophagy paradox. However, the underlying mechanism that regulates the autophagy paradox remains unclear. Therefore, this review focuses on recent studies with regard to the regulation of autophagy by Cer and elucidates the roles and mechanisms of action of Cer in controlling autophagy paradox. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Cellular Autophagy Machinery is not Required for Vaccinia Virus Replication and Maturation

The origin of the primary membrane of vaccinia virus, the double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane is of autophagy origin. Morphologic studies by electron microscopy showed no apparent difference in viral maturation in the autophagy deficient cell lines, the Atg5-/- mouse embryonic fibroblast (MEF) and the Beclin 1 -/- embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, quantitative assays indicated that the viruses replicate and maturate in the autophagy deficient cell lines as efficiently as they do in the corresponding isogenic wild type cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane likely does not originate from the autophagic membrane.     

c-myc induces autophagy in rat 3Y1 fibroblast cells

The proto-oncogene c-myc is a multifunctional gene that regulates cell division, cell growth, and apoptosis. Here we report a new function of c-myc: induction of autophagy. Autophagy is a bulk degradation system for intracellular proteins. Autophagy proceeds with characteristic morphologies, which begins with the formation of a double-membrane structure called the autophagosome surrounding a portion of the cytoplasm, after which its outer membrane then fuses with the lysosomal membrane to become an autolysosome. Autophagosomes and autolysosomes are generally called autophagic vacuoles. When c-Myc protein was overexpressed in rat 3Y1 fibroblasts or when the chimeric protein c-MycER was activated by estrogen, the number of autophagic vacuoles in cells increased significantly. The formation of autophagic vacuoles induced by c-Myc was completely blocked by a specific inhibitor of autophagosome formation, 3-methyladenine. A c-Myc mutant lacking Myc Box II induced neither apoptosis nor oncogenic transformation, but still stimulated autophagy. An inhibitor of caspases suppressed apoptosis but not autophagy. These results suggest that the autophagy caused by c-myc is not due to the apoptosis or tumorigenesis induced by c-myc. Taken together, our results suggest that the induction of autophagy is a novel function of c-myc.     

Targeting autophagy to fight hematopoietic malignancies

Macroautophagy, referred hereafter to as autophagy is an evolutionary conserved catabolic process for the degradation and recycling of macromolecules, bulk cytoplasm and dammaged organelles. Autophagy is activated under stress conditions induced by nutrient deprivation, hypoxia and drug treatments. Morphologically, autophagic cells are characterized by the accumulation of double membrane cytoplasmic vesicules called autophagosomes that surrounds cytoplasmic proteins and/or organelles. Autophagosomes next fuse with lysosomes to generate autolysosomes, the structures in which the retained constituents are digested before recycling into the cytoplasm. In this context, autophagy promotes cell survival under adverse conditions. In contrast, under certain circumstances autophagic cells may engage a specific mode of cell death called type II cell death or autophagic cell death (ACD). Considering the strategic positionnement of this process at the crossroads of cell death and survival, it is not surprising that defects in autophagy have been linked to a plethora of human diseases, including hematopoietic malignancies. Finally, autophagy induction is repressed by the mammalian target of rapamycin complex 1 (mTORC1) and favored by the adenosine-monophosphate activated-protein kinase (AMPK). In the present review, we focus on the functions of autophagy in normal and malignant hematopoiesis and discuss the opportunity to target the AMPK/mTOR pathways as a new therapeutic strategy to fight hematopoietic malignancies with a special emphasis on Chronic Myelogenous Leukemia (CML).     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.     

Vorinostat-induced autophagy switches from a death-promoting to a cytoprotective signal to drive acquired resistance

Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, de novo or acquired resistance to HDACi therapy is inevitable, and their molecular mechanisms are still unclear. To gain insight into HDACi resistance, we developed vorinostat-resistant clones from the hematological cell lines U937 and SUDHL6. Although cross-resistant to some but not all HDACi, the resistant cell lines exhibit dramatically increased sensitivity toward chloroquine, an inhibitor of autophagy. Consistent with this, resistant cells growing in vorinostat show increased autophagy. Inhibition of autophagy in vorinostat-resistant U937 cells by knockdown of Beclin-1 or Lamp-2 (lysosome-associated membrane protein 2) restores sensitivity to vorinostat. Interestingly, autophagy is also activated in parental U937 cells by de novo treatment with vorinostat. However, in contrast to the resistant cells, inhibition of autophagy decreases sensitivity to vorinostat. These results indicate that autophagy can switch from a proapoptotic signal to a prosurvival function driving acquired resistance. Moreover, inducers of autophagy (such as mammalian target of rapamycin inhibitors) synergize with vorinostat to induce cell death in parental cells, whereas the resistant cells remain insensitive. These data highlight the complexity of the design of combination strategies using modulators of autophagy and HDACi for the treatment of hematological malignancies.     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.