Loss of Pten,a tumor suppressor,causes the strong inhibition of autophagy without affecting LC3 lipidation

1Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [14C]-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microsopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.     

Loss of Pten, a tumor suppressor, causes the strong inhibition of autophagy without affecting LC3 lipidation

(1)Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [(14)C-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microscopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.     

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms: PERSISTENT INDUCTION OF AUTOPHAGOSOME SYNTHESIS AND IMPAIRMENT OF LYSOSOMAL INTEGRITY

Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.     

Aurantiamide acetate suppresses the growth of malignant gliomas in vitro and in vivo by inhibiting autophagic flux

We aim to investigate the effect of aurantiamide acetate isolated from the aerial parts of Clematis terniflora DC against gliomas. Human malignant glioma U87 and U251 cells were incubated with different concentrations (0–100 μM) of aurantiamide acetate. Aurantiamide acetate greatly decreased the cell viability in a dose‐ and time‐dependent manner. It induced moderate mitochondrial fragmentation and the loss of mitochondrial membrane potential. No significant difference was found in the alternation of other intracellular organelles, although F‐actin structure was slightly disturbed. Apparent ultrastructure alternation with increased autophagosome and autolysosome accumulation was observed in aurantiamide acetate‐treated cells. The expression of LC3‐II was greatly up‐regulated in cells exposed to aurantiamide acetate (P < 0.05 compared with control). The cytoplasmic accumulation of autophagosomes and autolysosomes induced by aurantiamide acetate treatment was confirmed by fluorescent reporter protein labelling. Administration of chloroquine (CQ), which inhibits the fusion step of autophagosomes, further increased the accumulation of autophagosomes in the cytoplasm of U87 cells. Autophagy inhibition by 3‐methyladenine, Bafilomycin A1 or CQ had no influence on aurantiamide acetate‐induced cytotoxicity, whereas autophagy stimulator rapamycin significantly suppressed aurantiamide acetate‐induced cell death. The anti‐tumour effects of aurantiamide acetate were further evaluated in tumour‐bearing nude mice. Intratumoural injection of aurantiamide acetate obviously suppressed tumour growth, and increased number of autophagic vacuoles was observed in tumour tissues of animals receiving aurantiamide acetate. Our findings suggest that aurantiamide acetate may suppress the growth of malignant gliomas by blocking autophagic flux.     

Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans

Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human beta-amyloid peptide (Abeta) in order to examine the mechanism(s) of Abeta-toxicity. In this model, Abeta expression causes autophagosome accumulation, thereby mimicking a pathology found in brains of Alzheimer's disease patients. Furthermore, we demonstrate that decreased insulin-receptor signaling [using the daf-2(e1370) mutation] suppresses Abeta-induced paralysis by a mechanism that requires autophagy. Surprisingly, the daf-2 mutation also decreases Abeta-induced autophagosome accumulation. These observations can be explained by a model in which decreased insulin-receptor signaling promotes the maturation of autophagosomes into degradative autolysosomes, whereas Abeta impairs this process. Consistent with this model, we find that RNAi-mediated knock-down of lysosomal components results in enhanced Abeta-toxicity and autophagosome accumulation. Also, Abeta; daf-2(e1370) nematodes contain more lysosomes than either Abeta or control strains. Finally, we demonstrate that decreased insulin-receptor signaling promotes the autophagic degradation of Abeta.     

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.     

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.     

Purification of autophagosomes from rat hepatocytes

To facilitate the purification of rat liver autophagosomes, isolated rat hepatocytes are first incubated for 2 h at 37°C with vinblastine, which induces autophagosome accumulation by blocking the fusion of these organelles with endosomes and lysosomes. The hepatocytes are then electrodisrupted and homogenized, and the various cellular organelles sequentially removed by subcellular fractionation. A brief incubation of the homogenate with the cathepsin C substrate, glycyl-phenylalanine-naphthylamide (GPN), causes rapid osmotic disruption of the lysosomes due to intralysosomal accumulation of GPN cleavage products. Nuclei are removed by differential centrifugation, and the postnuclear supernatant subsequently fractionated on a two-step Nycodenz density gradient. Autophagosomes are recovered in an intermediate density fraction, free from cytosol and mitochondria. The autophagosomes are finally separated from the membranes and vesicles of the endoplasmic reticulum, Golgi, endosomes, etc. by sieving through a density gradient of colloidal silica particles (Percoll). The final preparation contains about 95% autophagosomes and 5% amphisomes according to morphological and biochemical criteria.     

Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.     

Decreased Insulin-Receptor Signaling Promotes the Autophagic Degradation of β-Amyloid Peptide in C. elegans

Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human β-amyloid peptide (Aβ) in order to examine the mechanism(s) of Aβ-toxicity. In this model, Aβ expression causes autophagosome accumulation, thereby mimicking a pathology found in brains of Alzheimer’s disease patients. Furthermore, we demonstrate that decreased insulin-receptor signaling [using the daf-2(e1370) mutation] suppresses Aβ-induced paralysis by a mechanism that requires autophagy. Surprisingly, the daf-2 mutation also decreases Aβ-induced autophagosome accumulation. These observations can be explained by a model in which decreased insulin-receptor signaling promotes the maturation of autophagosomes into degradative autolysosomes, whereas Aβ impairs this process. Consistent with this model, we find that RNAi-mediated knock-down of lysosomal components results in enhanced Aβ-toxicity and autophagosome accumulation. Also, Aβ; daf-2(e1370) nematodes contain more lysosomes than either Aβ or control strains. Finally, we demonstrate that decreased insulin-receptor signaling promotes the autophagic degradation of Aβ.     

Patulin induces pro-survival functions via autophagy inhibition and p62 accumulation

Patulin (PAT) is one of the most common mycotoxins found in moldy fruits. Skin contact is one of the most likely exposure routes of PAT. Investigation of dermal toxicity of PAT is clearly needed and has been highlighted by WHO. In the present study, using human keratinocyte HaCaT cells as a model, we found that treatment with PAT caused an increased autophagosome accumulation. Measurements of autophagic flux demonstrated that the accumulation of autophagosomes by PAT was not directly due to enhanced autophagosome formation but due to inhibition of autophagosome degradation. Reductions in the activities of the lysosomal enzymes cathepsin B and cathepsin D by PAT might contribute to this inhibitory effect. Consistent with this, inhibition of autophagosome degradation by PAT resulted in accumulation of p62 that functioned as a pro-survival signal. The pro-survival function of p62 was found to be attributed to reactive oxygen species-mediated cytoprotective endoplasmic reticulum (ER) stress response. ER stress exerted cytoprotective effect via extracellular signal-regulated kinase1/2-dependent B-cell CLL/lymphoma 2-associated agonist of cell death inhibitory phosphorylation. Given the critical role of autophagy and its substrate p62 in carcinogenesis, our findings may have important implications in PAT-induced skin carcinogenesis.     

Energy dependence of autophagic protein degradation in isolated rat hepatocytes

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.     

Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease

Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM), Paget''s disease of the bone, and frontotemporal dementia (IBMPFD). Patient muscle has degenerating fibers, rimmed vacuoles (RVs), and sarcoplasmic inclusions containing ubiquitin and TDP-43 (TARDNA-binding protein 43). In this study, we find that IBMPFD muscle also accumulates autophagosome-associated proteins, Map1-LC3 (LC3), and p62/sequestosome, which localize to RVs. To test whether VCP participates in autophagy, we silenced VCP or expressed adenosine triphosphatase–inactive VCP. Under basal conditions, loss of VCP activity results in autophagosome accumulation. After autophagic induction, these autophagosomes fail to mature into autolysosomes and degrade LC3. Similarly, IBMPFD mutant VCP expression in cells and animals leads to the accumulation of nondegradative autophagosomes that coalesce at RVs and fail to degrade aggregated proteins. Interestingly, TDP-43 accumulates in the cytosol upon autophagic inhibition, similar to that seen after IBMPFD mutant expression. These data implicate VCP in autophagy and suggest that impaired autophagy explains the pathology seen in IBMPFD muscle, including TDP-43 accumulation.     

How Positive-Strand RNA Viruses Benefit from Autophagosome Maturation

The autophagic degradation pathway is a powerful tool in the host cell arsenal against cytosolic pathogens. Contents trapped inside cytosolic vesicles, termed autophagosomes, are delivered to the lysosome for degradation. In spite of the degradative nature of the pathway, some pathogens are able to subvert autophagy for their benefit. In many cases, these pathogens have developed strategies to induce the autophagic signaling pathway while inhibiting the associated degradation activity. One surprising finding from recent literature is that some viruses do not impede degradation but instead promote the generation of degradative autolysosomes, which are the endpoint compartments of autophagy. Dengue virus, poliovirus, and hepatitis C virus, all positive-strand RNA viruses, utilize the maturation of autophagosomes into acidic and ultimately degradative compartments to promote their replication. While the benefits that each virus reaps from autophagosome maturation are unique, the parallels between the viruses indicate a complex relationship between cytosolic viruses and host cell degradation vesicles.