Autophagy-related Research Articles

Transcriptional Regulation by Pho23 Modulates the Frequency of Autophagosome Formation

Autophagy as a conserved lysosomal/vacuolar degradation and recycling pathway is important in normal development and physiology, and defects in this process are linked to many kinds of disease. Because too much or too little autophagy can be detrimental, the process must be tightly regulated both temporally and in magnitude. Two parameters that affect this regulation are the size and the number of autophagosomes; however, although we know that the amount of Atg8 affects the size of autophagosomes, the mechanism for regulating their number has not been elucidated. The transcriptional induction and repression of the autophagy-related (ATG) genes is one crucial aspect of autophagy regulation, but the transcriptional regulators that modulate autophagy are not well characterized. We detected increased expression levels of ATG genes, and elevated autophagy activity, in cells lacking the transcriptional regulator Pho23. Using transmission electron microscopy, we found that PHO23 null mutant cells contain significantly more autophagosomes than the wild-type. By RNA sequencing transcriptome profiling, we identified ATG9 as one of the key targets of Pho23, and our studies with strains expressing modulated levels of Atg9 show that the amount of this protein directly correlates with the frequency of autophagosome formation and the level of autophagy activity. Our results identified Pho23 as a master transcriptional repressor for autophagy that regulates the frequency of autophagosome formation through its negative regulation of ATG9.

Role and regulation of autophagy in heat stress responses of tomato plants.

As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.

Expression analysis of autophagy-related genes in the hard tick Haemaphysalis longicornis

Ticks are obligate hematophagous arthropods with unique life cycles characterized by relatively short feeding periods and long non-feeding periods. They ambush a suitable host animal while staying in a pasture without any food source for up to several months. To understand the molecular mechanisms underlying their exceptional viability, we focused on autophagy, a proteolysis system via the lysosomes that is induced by starvation in eukaryotes. We hypothesized that starved conditions facilitate autophagy during host-seeking periods in the life cycle of the tick. To date, homologues of five autophagy-related (ATG) genes, ATG3, ATG4, ATG6, ATG8, and ATG12, have been identified from the hard tick Haemaphysalis longicornis. We showed previously that the mRNA levels of H. longicornis ATG (HlATG) genes were higher during the non-feeding period than the feeding period in the nymphal to adult stages. In addition, the expressions of HlATG3, HlATG4, HlATG8 and HlATG12 were highest in the egg compared to the other developmental stages in the same tick. In the present study, we used real-time polymerase chain reaction to examine the expression profiles of HlATG genes in the embryonic stage, larval to nymphal stages, and in internal organs of female ticks. We found that the HlATG genes were expressed at the highest levels in developing eggs on day 0 after oviposition. The levels of HlATG4 and HlATG8 were higher during the non-feeding period than the feeding period in the larval to nymphal stages. In the adults, the unfed condition appeared to be associated with the increased expression of HlATG genes in the fat body and midgut, which are nutrient storage organs; however, the expression patterns of HlATG genes varied in other organs. These results suggest that an up-regulation of HlATG genes is not always induced in different organs of unfed female ticks. Taken together, our findings raise the new possibility that HlATG genes play distinct biological roles in eggs, unfed ticks, engorged ticks (metamorphosis), and in each organ.

Quality control of plant peroxisomes in organ specific manner via autophagy

Peroxisomes are essential organelles that are characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG)-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5-knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles for degradation by autophagy. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves and for proper plant development under natural growth conditions.

Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells.

Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts (MEFs) and HeLa cells deficient in various ATG proteins reveals that the emergence of the isolation membrane (phagophore) requires FIP200 (also known as RB1CC1), ATG9A and phosphatidylinositol (PtdIns) 3-kinase activity. By contrast, small premature isolation-membrane-like and autophagosome-like structures were generated in cells lacking VMP1 and both ATG2A and ATG2B, respectively. The isolation membranes could elongate in cells lacking ATG5, but did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62 (also known as SQSTM1) in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the site of autophagosome formation.

Detection of WIPI1 mRNA as an indicator of autophagosome formation

Autophagy is a cellular bulk degradation system for long-lived proteins and organelles that operates during nutrient starvation and is thus a type of recycling system. In recent years, a series of mammalian orthologs of yeast autophagy-related (ATG) genes have been identified; however, the importance of the transcriptional regulation of ATG genes underlying autophagosome formation is poorly understood. In this study, we identified several ATG genes, including the genes ULK1, MAP1LC3B, GABARAPL1, ATG13, WIPI1, and WDR45/WIPI4, with elevated mRNA levels in thapsigargin-, C2-ceramide-, and rapamycin-treated as well as amino acid-depleted HeLa cells except for MAP1LC3B mRNA in rapamycin-treated HeLa cells. Rapamycin had a weaker effect on the expressions of ATG genes. The increase in WIPI1 and MAP1LC3B mRNA was induced prior to the accumulation of the autophagy marker protein MAP1LC3 in the thapsigargin- and C2-ceramide-treated A549 cells. By counting the puncta marked with MAP1LC3B in HeLa cells treated with different autophagy inducers, we revealed that the time-dependent mRNA elevation of a specific set of ATG genes was similar to that of autophagosome accumulation. The transcriptional attenuation of WIPI1 mRNA using RNA interference inhibited the puncta number in thapsigargin-treated HeLa cells. Remarkably, increases in the abundance of WIPI1 mRNA were also manifested in thapsigargin- and C2-ceramide-treated human fibroblasts (WI-38 and TIG-1), human cancer cells (U-2 OS, Saos-2, and MCF7), and rodent fibroblasts (Rat-1). Taken together, these results suggest that the detection of WIPI1 mRNA is likely to be a convenient method of monitoring autophagosome formation in a wide range of cell types.

Amphisomes: out of the autophagosome shadow?

Amphisomes are intermediate organelles, formed during autophagy through the fusion between autophagosomes and endosomes. Complex multivesicular vacuoles that resemble amphisomes have been observed in various cell types, but whether they have cellular roles other than being a precursor structure is still enigmatic. While autophagy-related (ATG) proteins interact with the endocytic pathways in other processes different from autophagy, Patel and colleagues now report that these factors come together to generate amphisome-like compartments that regulate mucin secretion in goblet cells.

The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.

The autophagosome: origins unknown, biogenesis complex

Healthy cells use autophagy as a general 'housekeeping' mechanism and to survive stress, including stress induced by nutrient deprivation. Autophagy is initiated at the isolation membrane (originally termed the phagophore), and the coordinated action of ATG (autophagy-related) proteins results in the expansion of this membrane to form the autophagosome. Although the biogenesis of the isolation membrane and the autophagosome is complex and incompletely understood, insight has been gained into the molecular processes involved in initiating the isolation membrane, the source from which this originates (for example, it was recently proposed that the isolation membrane forms from the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM)) and the role of ATG proteins and the vesicular trafficking machinery in autophagosome formation.

Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site

Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1, and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.

Non-canonical Autophagy: Facts and Prospects

Macroautophagy is a catabolic process for cell components and terminates in the lysosomal compartment. Macroautophagy maintains the homeostasis of cells, tissues, and organisms, and plays important physiological roles during the development and aging processes. It also contributes to the innate and adaptive immune responses. The cytoprotective function of macroautophagy is compromised in diseases such as cancer, neurodegenerative disorders, and diabetes. The autophagosome, the central organelle in macroautophagy that sequesters cytoplasmic material, is formed by the hierarchical recruitment of 15 autophagy-related (ATG) proteins. However, alternatively to this canonical pathway, autophagosomes can also be formed by using only a subset of ATGs. Here, we will discuss recent discoveries concerning the molecular regulation of these non-canonical forms of macroautophagy and their potential role in the cellular response to stressful situations.

Hepatic autophagy after severe burn in response to endoplasmic reticulum stress

BackgroundPrevious studies showed that liver dysfunction develops soon after severe burn, and this is associated with activation of endoplasmic reticulum (ER) stress. Autophagy is a catabolic process to maintain cellular organelle balance; ER stress is associated with autophagy signaling cascades. We thus sought to determine whether autophagy signals were associated with damage in the liver after burn, and further whether burn-associated ER stress activates autophagy signals in hepatocytes. MethodsC57BL/6 male mice received a 25% total body surface area full-thickness scald burn, and liver was harvested at 24 h after burn. HepG2 cells were stimulated with an ER stress inducer thapsigargin (TG) for 24 h to mimic ER stress in vitro. Terminal deoxyuridine nick-end labeling staining was performed on histologic sections of liver. Autophagy was assessed by immunoblotting. Statistical analysis was performed using the Student t-test and significance was accepted at P < 0.05. ResultsTerminal deoxyuridine nick-end labeling positive–stained hepatocytes increased in burned animals with a significant elevation of caspase 3 activity (P < 0.05). Hepatic autophagy-related (ATG) protein 3, ATG5 and light chain (LC) 3B elevated significantly in burn animals as well (P < 0.05). Expression of Beclin-1, LC3A, and LC3B increased in HepG2 cells in response to TG, similar to the response seen in vivo. Cytosolic adenosine triphosphate dropped significantly, and adenosine monophosphate–activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were phosphorylated as well in response to TG (P < 0.05). ConclusionsER stress, which occurs in hepatocytes after severe injury, is associated with autophagy and liver damage after severe burn. In response to ER stress, activated autophagy is associated with adenosine monophosphate–activated protein kinase and mammalian target target of rapamycin pathway.

Abstract A45: Beclin1-independent autophagy promotes the survival of metastatic ovarian cancer cells

Abstract Epithelial ovarian cancer (EOC) metastasis proceeds by an intra-peritoneal route, involving detachment of cells from the primary tumor and dissemination throughout the peritoneal cavity as multicellular aggregates, or spheroids. Herein, we demonstrate that ascites-derived cells upregulate the process of macroautophagy (autophagy), a highly-conserved self-digestion mechanism that functions to mitigate cellular stresses. Both non-adherent culture of these cells to form in vitro spheroids as well as exposure to an allosteric AKT inhibitor stimulated this process, as evidenced by Western blot and immunofluorescence staining for the autophagy marker MAP1LC3B (LC3). Likewise, expression of an eGFP-labeled LC3 protein revealed a shift from diffuse to ‘punctate’ cytoplasmic localization, demonstrating the formation of intracellular autophagic vesicles (autophagosomes). Additionally, we performed transmission electron microscopy and autophagy flux assays, confirming that autophagic degradation proceeded to completion. To determine the role of autophagy upregulation, we attempted to block this process using siRNAs targeting critical autophagy-related (ATG) genes. Interestingly, we found that depletion of Beclin1/ATG6 had no effect on autophagy, despite its role as a canonical inducer of the process. Conversely, depletion of the autophagy regulators ATG5 &amp; ATG7 efficiently blocked LC3 processing and autophagosome formation. Likewise, autophagy was also blocked pharmacologically using the classical inhibitor Chloroquine and the novel agent Spautin-1 (the first targeted autophagy inhibitor to be published). Autophagy blockade by any of these mechanisms reduced EOC cell viability in both adherent and spheroid cultures, suggesting that the upregulation of this process serves to promote cell survival. Moreover, when autophagy blockade was combined with AKT inhibition, a synergistic reduction in cell survival was observed based on combination index analysis. Taken together, this work describes spheroid formation as a stimulus for a non-canonical, Beclin1-independent autophagy program, but also reveals autophagy to be a promoter of tumor cell survival during ovarian cancer metastasis, particularly in cells subjected to AKT/mTOR inhibition. In light of these findings, we propose that clinical autophagy inhibition could enhance the effectiveness of current or investigational ovarian cancer therapies. Citation Format: Rohann Correa, Yudith Ramos-Valdes, Teresa Peart, Elena Fazio, Monique Bertrand, Jacob McGee, Michel Préfontaine, Akira Sugimoto, Trevor G. Shepherd, Gabriel E. DiMattia. Beclin1-independent autophagy promotes the survival of metastatic ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A45.

The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil

Autophagy is a fundamental cellular process required for organelle degradation and removal of invasive pathogens. Autophagosome formation involves the recruitment of, and interaction between, multiple proteins produced from autophagy-related (ATG) genes. One of the key complexes in autophagosome formation is the ATG12-ATG5-ATG16L1 complex. ATG16L1 functions as a molecular scaffold mediating protein-protein interactions necessary for formation of the autophagosome in response to both classical and pathogen-related autophagy stimuli. The coiled-coil domain of the yeast ortholog, ATG16, exists as a homodimer both in solution and in the crystal form. The yeast and human orthologs show poor sequence identity. Here we have sought to determine the minimal boundaries of the human ATG16L1 coiled-coil domain and ascertain its oligomeric status in solution. Using a range of biochemical and biophysical techniques we show that the secondary structure of the human ATG16L1 coiled-coil has the expected helical composition and that the domain forms a homodimer in solution. We also observe extensive sequence conservation across vertebrates providing strong support for the crucial functional role of the ATG16L1 coiled-coil.

Mechanism of Ginsenoside Rh2 Inhibit Non-Small Cell Lung Cancer

To study function and relativity of autophagy in NSCLC treated with ginsenoside Rh2, and to explore some molecular mechanism that ginsenoside Rh2 induces A549 cell lines of NSCLC apoptosis. Methods: Immunofluorescence assays detects activity of caspase-8 / 9 and expression of autophagy-related molecules Beclin-1 and LC3. Results: Immunofluorescence assays shows that, 20μg/mL ginsenoside Rh2 acts 3h, fluorescent intensity that marks activity of caspase-8 and expression level of Beclin-1 significantly increases, it means that activity of caspase-8 significantly rises and expression level of Beclin-1 significantly increases. Fluorescent intensity that marks expression of LC3and activity of caspase-9 has increase but without significant change in 1-6h, it means expression of LC3 and activity of caspase-9 has no significant change. Conclusion: Ginsenoside Rh2 can adjust expression of membrane receptor pathway (caspase-8) and or autophagy related gene Beclin-1,then play the biological role of promoting apoptosis of tumor cell.

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34+ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.

Probing the Key Interactions between Human Atg5 and Atg16 Proteins: A Prospective Application of Molecular Modeling

Autophagy, a conserved catabolic process in eukaryotes, mediates the nutrient and energy homeostasis via the degradation of intracellular cytoplasmic materials and organelles by lysosomal machinery. It serves as a recycling mechanism for the rejuvenation of cell contents, which is known to be critical in various biological events, such as starvation response, neurodegeneration, immunity, inflammation, cancer and so on. Due to its complex nature, autophagy actually plays a dual role in relevant diseases. For example, as most anticancer drugs cause cellular stress, autophagy is often activated in cancer cells after drug treatment to promote cell survival under stress. Thus, both inducers and inhibitors of autophagy have been investigated for their potential therapeutic applications. Studies on the molecular mechanism of autophagy began with the identification of the autophagy-related (ATG) genes in yeast in the 1990s. So far, many protein factors have been found to participate in the formation of autophagosome. In particular, formation of the preautophagosomal structure (PAS) is essential for autophagy execution in yeast as well as mammals (Figure 1). Two ubiquitin-like protein conjugates are known to be involved in this process: Atg8–phosphatidylethanolamine (PE) and the Atg12–Atg5–Atg16 complex. Atg8–PE is equivalent to the LC3–phosphatidylethanolamine conjugate (LC3-II) in mammals. Atg8 was found to act as an important component of the scaffold during membrane expansion of PAS, and its amount in cell quantitatively relates to the PAS vesicle size. The Atg12–Atg5–Atg16 complex, which facilitates the lipidation of Atg8, is also believed to help direct Atg8–PE towards PAS. Mutations in the Atg12– Atg5–Atg16 complex lead to a decrease in Atg8–PE/LC3-II levels as well as a defective autophagosome. The studies mentioned above illustrate the importance of the Atg12–Atg5–Atg16 complex in autophagy. It is possible to develop effective autophagy inhibitors by targeting the protein–protein interactions (PPI) between Atg5–Atg16 and Atg5– Atg12. Atg12 is known to be covalently linked with Atg5 via a glycine residue at its C terminus. Mediated by the oligomerization of Atg16, the Atg12–Atg5 complex interacts noncova-

Autophagy protects from liver injury

Autophagy is understood as a mechanism by which subcellular debris are recycled and metabolic intermediates produced for maintaining cellular homeostasis. Moreover, autophagy can be induced under stress conditions with the goal of cell survival. On the other hand, autophagy also has a role in the cell death pathways, and the existence of a complex cross-talk between autophagy and apoptosis has recently emerged.1 For instance, key proteins originally thought to be apoptosis-specific inhibitors are also able to block autophagy,2 while some proteases involved in apoptosis block autophagy by cleaving autophagy-regulating proteins.3 In still other cases, the autophagy-related (ATG) proteins can be converted into a pro-apoptotic factor4 or can directly bind FAS-associated protein with death domain to support apoptosis.5 Unfortunately, all these findings currently fail to provide a consistent interpretation for the general function of autophagy in the apoptosis pathways. Nonetheless, an understanding of the molecular mechanisms regulating the interplay between autophagy and apoptosis promises to provide new hints on how to modulate the cell death pathways under pathological conditions such as cancer, neurodegenerative, or chronic inflammatory disorders. In this issue of the Journal, Amir et al.6 report the development of a mouse line exhibiting an inducible, hepatocyte-specific lack of the key ATG gene atg7. These mice have been challenged with &#x1d49f;-galactosamine (GalN) and lipopolysaccharide (LPS), both of which are stimuli commonly used to study tumor necrosis factor (TNF)-dependent liver injury. With the purpose of studying autophagy in TNF-induced cytotoxicity in the liver, these investigators found that autophagy protects the hepatocytes from TNF-dependent cell death. Mice with atg7-deficient hepatocytes showed a higher rate of hepatocytic apoptosis, liver injury, and mortality as compared with the wild-type mice following combined GalN and LPS treatment. Interestingly, these effects were not due to altered, LPS-induced cytokine production by macrophages in the liver, because TNF-α, IFN-γ, IL-6, and IL-1β levels were not altered as a consequence of the ATG7 deficiency. This suggests that the accelerated TNF-induced toxicity in such knockout mice resulted from an inhibition of autophagy. Furthermore, atg7−/− mice showed features of an accelerated extrinsic apoptotic pathway in liver cells, including caspase-8 activation, increased expression of truncated Bid, and cytochrome-c release, as well as caspase-3, caspase-7, and poly (ADP-ribose) polymerase cleavage. It had previously been shown that overactivation of c-Jun N-terminal kinase (JNK) can activate caspase-8, resulting in increased TNF-dependent cell death.7 Amir et al.6 observed increased phosphorylated JNK and its downstream target c-Jun in the atg7−/− mice upon GalN/LPS stimulation, indicating that autophagy protects TNF toxicity by blocking JNK activity (Figure 1). Figure 1 Autophagy overlaps with the extrinsic apoptosis pathway in liver cells. Deficient autophagy results in overactivation of JNK, which increases caspase-8 activation and allows the induction of TNF-dependent apoptosis. How autophagy regulates JNK activity ... Interestingly, this study also showed that GalN can inhibit LPS-induced autophagy by blocking the activation of adenosine monophosphate-activated protein kinase, which in turn positively regulates autophagy. Increased autophagy resulting from Beclin 1 overexpression prevented GalN/LPS-induced liver injury. This suggests that the toxicity caused by GalN is at least partially dependent on the inhibition of autophagy. Because autophagy is crucial for the survival of the GalN/LPS-sensitized hepatocytes, its inhibition leads to extra cell death and tissue injury in the knockout mice. Therefore, it seems likely that it is the autophagic suppression of apoptosis that helps the liver cells to survive by delaying the onset of death signals under toxic conditions. Hence, upregulating the autophagic pathway might be an attractive therapeutic strategy in several inflammatory liver diseases.

Autophagy in the liver

Autophagy in the liver

Autophagy Contributes to Leaf Starch Degradation

Transitory starch, a major photosynthetic product in the leaves of land plants, accumulates in chloroplasts during the day and is hydrolyzed to maltose and Glc at night to support respiration and metabolism. Previous studies in Arabidopsis thaliana indicated that the degradation of transitory starch only occurs in the chloroplasts. Here, we report that autophagy, a nonplastidial process, participates in leaf starch degradation. Excessive starch accumulation was observed in Nicotiana benthamiana seedlings treated with an autophagy inhibitor and in autophagy-related (ATG) gene-silenced N. benthamiana and in Arabidopsis atg mutants. Autophagic activity in the leaves responded to the dynamic starch contents during the night. Microscopy showed that a type of small starch granule-like structure (SSGL) was localized outside the chloroplast and was sequestered by autophagic bodies. Moreover, an increased number of SSGLs was observed during starch depletion, and disruption of autophagy reduced the number of vacuole-localized SSGLs. These data suggest that autophagy contributes to transitory starch degradation by sequestering SSGLs to the vacuole for their subsequent breakdown.