Search tips
Search criteria

Results 1-25 (393)

Clipboard (0)
Year of Publication
Document Types
1.  [No title available] 
PMCID: PMC4294541  PMID: 19276647
2.  Analysis of a lung defect in autophagy-deficient mouse strains 
Autophagy  2013;10(1):45-56.
Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.
PMCID: PMC4028323  PMID: 24275123
Ulk1/2 DKO mice; Atg5 KO mice; perinatal mortality; glycogen; lung development
3.  The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death 
Autophagy  2013;9(12):2087-2102.
Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells.
PMCID: PMC3981748  PMID: 24113242
malignant melanoma; amodiaquine; chloroquine; autophagy; lysosome; cathepsin; E2F1; CDKN1A
4.  Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy 
Autophagy  2013;9(12):1983-1995.
Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.
PMCID: PMC4028342  PMID: 24013218
MTOR; autophagy; PIK3C3; ATG14; BECN1
5.  Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells 
Autophagy  2013;9(12):1996-2008.
How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.
PMCID: PMC4028343  PMID: 24145463
autophagy; oxidative stress; 4-hydroxynonenal; glycolysis; apoptosis; ATP; glutathione; Z-VAD; 3-MA
6.  Distinct functions of Ulk1 and Ulk2 in the regulation of lipid metabolism in adipocytes 
Autophagy  2013;9(12):2103-2114.
ULK1 (unc-51 like kinase 1) is a serine/threonine protein kinase that plays a key role in regulating the induction of autophagy. Recent studies using autophagy-defective mouse models, such as atg5- or atg7-deficient mice, revealed an important function of autophagy in adipocyte differentiation. Suppression of adipogenesis in autophagy-defective conditions has made it difficult to study the roles of autophagy in metabolism of differentiated adipocytes. In this study, we established autophagy defective-differentiated 3T3-L1 adipocytes, and investigated the roles of Ulk1 and its close homolog Ulk2 in lipid and glucose metabolism using the established adipocytes. Through knockdown approaches, we determined that Ulk1 and Ulk2 are important for basal and MTORC1 inhibition-induced autophagy, basal lipolysis, and mitochondrial respiration. However, unlike other autophagy genes (Atg5, Atg13, Rb1cc1/Fip200, and Becn1) Ulk1 was dispensable for adipogenesis without affecting the expression of CCAAT/enhancer binding protein α (CEBPA) and peroxisome proliferation-activated receptor gamma (PPARG). Ulk1 knockdown reduced fatty acid oxidation and enhanced fatty acid uptake, the metabolic changes that could contribute to adipogenesis, whereas Ulk2 knockdown had opposing effects. We also found that the expression levels of insulin receptor (INSR), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (SLC2A4/GLUT4) were increased in Ulk1-silenced adipocytes, which was accompanied by upregulation of insulin-stimulated glucose uptake. These results suggest that ULK1, albeit its important autophagic role, regulates lipid metabolism and glucose uptake in adipocytes distinctly from other autophagy proteins.
PMCID: PMC4028344  PMID: 24135897
ULK1; ULK2; mTORC1; adipogenesis; adipocytes; lipid metabolism
7.  Placental autophagy regulation by the BOK-MCL1 rheostat 
Autophagy  2013;9(12):2140-2153.
Autophagy is the catabolic degradation of cellular cytoplasmic constituents via the lysosomal pathway that physiologically elicits a primarily cytoprotective function, but can rapidly be upregulated in response to stressors thereby inducing cell death. We have reported that the balance between the BCL2 family proteins BOK and MCL1 regulates human trophoblast cell fate and its alteration toward cell death typifies preeclampsia. Here we demonstrate that BOK is a potent inducer of autophagy as shown by increased LC3B-II production, autophagosomal formation and lysosomal activation in HEK 293. In contrast, using JEG3 cells we showed that prosurvival MCL1 acts as a repressor of autophagy via an interaction with BECN1, which is abrogated by BOK. We found that MCL1-cleaved products, specifically MCL1c157, trigger autophagy while the splicing variant MCL1S has no effect. Treatment of JEG3 cells with nitric oxide donor SNP resulted in BOK-MCL1 rheostat dysregulation, favoring BOK accumulation, thereby inducing autophagy. Overexpression of MCL1 rescued oxidative stress-induced autophagy. Of clinical relevance, we report aberrant autophagy levels in the preeclamptic placenta due to impaired recruitment of BECN1 to MCL1. Our data provided the first evidence for a key role of the BOK-MCL1 system in regulating autophagy in the human placenta, whereby an adverse environment as seen in preeclampsia tilts the BOK-MCL1 balance toward the build-up of isoforms that triggers placental autophagy.
PMCID: PMC4028345  PMID: 24113155
placenta; MCL1; BOK; oxidative stress; autophagy; preeclampsia
8.  PINK1 is degraded through the N-end rule pathway 
Autophagy  2013;9(11):1758-1769.
PINK1, a mitochondrial serine/threonine kinase, is the product of a gene mutated in an autosomal recessive form of Parkinson disease. PINK1 is constitutively degraded by an unknown mechanism and stabilized selectively on damaged mitochondria where it can recruit the E3 ligase PARK2/PARKIN to induce mitophagy. Here, we show that, under steady-state conditions, endogenous PINK1 is constitutively and rapidly degraded by E3 ubiquitin ligases UBR1, UBR2 and UBR4 through the N-end rule pathway. Following precursor import into mitochondria, PINK1 is cleaved in the transmembrane segment by a mitochondrial intramembrane protease PARL generating an N-terminal destabilizing amino acid and then retrotranslocates from mitochondria to the cytosol for N-end recognition and proteasomal degradation. Thus, sequential actions of mitochondrial import, PARL-processing, retrotranslocation and recognition by N-end rule E3 enzymes for the ubiquitin proteosomal degradation defines the rapid PINK1 turnover. PINK1 steady-state elimination by the N-end rule identifies a novel organelle to cytoplasm turnover pathway that yields a mechanism to flag damaged mitochondria for autophagic elimination.
PMCID: PMC4028335  PMID: 24121706
mitochondrial import; PARKIN; ubiquitin; PARL; mitophagy
9.  Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy 
Autophagy  2013;9(11):1828-1836.
Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity.
PMCID: PMC4028336  PMID: 24025448
mitochondrial protease; mitophagy; starvation; vacuole; yeast
10.  Basal autophagy induction without AMP-activated protein kinase under low glucose conditions 
Autophagy  2009;5(8):1155-1165.
When ATP levels in a cell decrease, various homeostatic intracellular mechanisms initiate attempts to restore ATP levels. As a prominent energy sensor, AMP-activated protein kinase (AMPK) represents one molecular gauge that links energy levels to regulation of anabolic and catabolic processes to restore energy balance. Although pharmacological studies have suggested that an AMPK activator, AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) may link AMPK activation to autophagy, a process that can provide short-term energy within the cell, AICAR can have AMPK-independent effects. Therefore, using a genetic-based approach we investigated the role of AMPK in cellular energy balance. We demonstrate that genetically altered cells, mouse embryonic fibroblasts (MEFs), lacking functional AMPK, display altered energy balance under basal conditions and die prematurely under low glucose-serum starvation challenge. These AMPK mutant cells appear to be abnormally reliant on autophagy under low glucose basal conditions, and therefore cannot rely further on autophagy like wild-type cells during further energetic stress and instead undergo apoptosis. This data suggests that AMPK helps regulate basal energy levels under low glucose. Further, AMPK mutant cells show increased basal phosphorylation of p53 at serine 15, a residue phosphorylated under glucose deprivation. We propose that cells lacking AMPK function have altered p53 activity that may help sensitize these cells to apoptosis under energetic stress.
PMCID: PMC4203367  PMID: 19844161
AMPK; autophagy; apoptosis; p53; PTEN; LC3; ATP; AICAR
11.  PINK1-parkin-dependent mitophagy involves ubiquitination of mitofusins 1 and 2 
Autophagy  2011;7(2):243-245.
PMCID: PMC4196638  PMID: 21139416
mitochondria; mitofusin; mitophagy; neurodegeneration; Parkinson disease; parkin; PINK1
12.  How could Parkin-mediated ubiquitination of mitofusin promote mitophagy? 
Autophagy  2010;6(5):660-662.
PMCID: PMC4196639  PMID: 20484985
Parkinson disease; neurodegeneration; Parkin; PINK1; Mfn; mitochondrial dynamics; ubiquitination; Drosophila
13.  Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes 
Autophagy  2009;5(8):1099-1106.
In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serum-containing Waymouth’s medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1α mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.
PMCID: PMC4170191  PMID: 19783904
cyclosporin A; dedifferentiation; mitochondrial permeability transition; mitophagy; mtDNA; remodeling
14.  20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body 
Autophagy  2013;9(8):1172-1187.
Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.
PMCID: PMC3748190  PMID: 23674061
20-hydroxyecdysone; fat body; autophagy; Atg genes; Atg1; ATG8; transcriptional regulation; TORC1; Bombyx mori
15.  ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis 
Autophagy  2013;9(8):1188-1200.
The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.
PMCID: PMC3748191  PMID: 23782979
ATG4B; autophagin-1; autophagy; colitis; inflammation; intestinal homeostasis; cysteine peptidase; Paneth cell
16.  Drosophila Fip200 is an essential regulator of autophagy that attenuates both growth and aging 
Autophagy  2013;9(8):1201-1213.
Autophagy-related 1 (Atg1)/Unc-51-like protein kinases (ULKs) are evolutionarily conserved proteins that play critical physiological roles in controlling autophagy, cell growth and neurodevelopment. RB1-inducible coiled-coil 1 (RB1CC1), also known as PTK2/FAK family-interacting protein of 200 kDa (FIP200) is a recently discovered binding partner of ULK1. Here we isolated the Drosophila RB1CC1/FIP200 homolog (Fip200/CG1347) and showed that it mediates Atg1-induced autophagy as a genetically downstream component in diverse physiological contexts. Fip200 loss-of-function mutants experienced severe mobility loss associated with neuronal autophagy defects and neurodegeneration. The Fip200 mutants were also devoid of both developmental and starvation-induced autophagy in salivary gland and fat body, while having no defects in axonal transport and projection in developing neurons. Interestingly, moderate downregulation of Fip200 accelerated both developmental growth and aging, accompanied by target of rapamycin (Tor) signaling upregulation. These results suggest that Fip200 is a critical downstream component of Atg1 and specifically mediates Atg1’s autophagy-, aging- and growth-regulating functions.
PMCID: PMC3748192  PMID: 23819996
autophagy; neurodegeneration; Drosophila; aging; growth
17.  IκB kinase complex (IKK) triggers detachment-induced autophagy in mammary epithelial cells independently of the PI3K-AKT-MTORC1 pathway 
Autophagy  2013;9(8):1214-1227.
Adherent cells require proper integrin-mediated extracellular matrix (ECM) engagement for growth and survival; normal cells deprived of proper ECM contact undergo anoikis. At the same time, autophagy is induced as a survival pathway in both fibroblasts and epithelial cells upon ECM detachment. Here, we further define the intracellular signals that mediate detachment-induced autophagy and uncover an important role for the IκB kinase (IKK) complex in the induction of autophagy in mammary epithelial cells (MECs) deprived of ECM contact. Whereas the PI3K-AKT-MTORC1 pathway activation potently inhibits autophagy in ECM-detached fibroblasts, enforced activation of this pathway is not sufficient to suppress detachment-induced autophagy in MECs. Instead, inhibition of IKK, as well as its upstream regulator, MAP3K7/TAK1, significantly attenuates detachment-induced autophagy in MECs. Furthermore, function-blocking experiments corroborate that both IKK activation and autophagy induction result from decreased ITGA3-ITGB1 (α3β1 integrin) function. Finally, we demonstrate that pharmacological IKK inhibition enhances anoikis and accelerates luminal apoptosis during acinar morphogenesis in three-dimensional culture. Based on these results, we propose that the IKK complex functions as a key mediator of detachment-induced autophagy and anoikis resistance in epithelial cells.
PMCID: PMC3748193  PMID: 23778976
autophagy; anoikis; extracellular matrix; integrin; mammary epithelial cells
18.  β-adrenergic receptor-stimulated lipolysis requires the RAB7-mediated autolysosomal lipid degradation 
Autophagy  2013;9(8):1228-1243.
Hormone-stimulated lipolysis is a rapid way to mobilize fat from its storage depot for use in peripheral tissues. By convention, activation of cytosolic lipases via the β-adrenergic receptor (ADRB2)-cAMP signaling pathway is the only molecular mechanism considered to liberate fatty acids from triglycerides stored in lipid droplets (LDs) of cells. Herein, we provide evidence that, aside from the activation of cytosolic lipases, autophagy contributes to this hormone-stimulated lipolysis. The ADRB2-stimulated lipolysis was reduced after inhibition of early or late autophagy using either pharmacological inhibitors or shRNA-mediated autophagic gene knockdown. ADRB2 stimulation has caused a marked increase in the autophagy-targeted LDs for lysosomal degradation, which is dependent on the LD-associated RAB7 as evidenced by the use of both shRNA-mediated RAB7 knockdown and a dominant-negative RAB7 mutant. In addition, RAB7 is involved in unstimulated (basal) lipolysis, and mediates the enhanced basal lipolysis in PLIN1/perilipin 1 knockdown fat cells. In conclusion, our results showed a contribution of lipophagy to both basal and hormone-stimulated lipolysis and that RAB7 plays a pivotal role in the regulation of this autolysosome-mediated lipid degradation in fat cells.
PMCID: PMC3748194  PMID: 23708524
lipophagy; lipolysis; RAB7; autolysosome-mediated lipid degradation; 3T3-L1
19.  Reciprocal effects of rab7 deletion in activated and neglected T cells 
Autophagy  2013;9(7):1009-1023.
Mouse models lacking proteins essential for autophagosome formation have demonstrated that autophagy plays a critical role in T cell development and activation. To better understand the function of autophagy in quiescent and activated lymphocytes, we have generated a mouse deficient in rab7 selectively in T cells and compared the effects of blocking autophagy at an early (atg5−/−) or late (rab7−/−) stage on T cell biology. rab7−/− murine embryonic fibroblasts (MEFs) and T cells generated from these mice exhibit a profound block in autophagosome degradation and are as sensitive as atg5−/− cells to extracellular nutrient limitation. Rab7flox/floxCD4-Cre+ mice lacking the RAB7 protein in both CD4 and CD8 T cells had reduced numbers of peripheral T cells, but this defect was not as severe as in Atg5flox/floxCD4-Cre+ mice despite efficient rab7 deletion and inhibition of autophagic flux. This difference may stem from the reduced ROS generation and enhanced survival of rab7−/− T cells compared with wild-type and atg5−/− T cells in the absence of cytokine stimulation. rab7−/− and atg5−/− T cells exhibited similar defects in proliferation both following antibody-mediated T cell receptor (TCR) cross-linking and using a more physiologic activation protocol, allogeneic stimulation. Interestingly, autophagy was not required to provide building blocks for the upregulation of nutrient transporter proteins immediately following activation. Together, these studies suggest that autophagosome degradation is required for the survival of activated T cells, but that loss of rab7 is better tolerated in naïve T cells than the loss of atg5.
PMCID: PMC3722312  PMID: 23615463
RAB7; T cells; autophagy; lysosomal fusion; growth factor withdrawal; ATG5
20.  Live-cell imaging of Aspergillus nidulans autophagy 
Autophagy  2013;9(7):1024-1043.
We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This ‘autophagosome cycle’ gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabSRAB7)-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabSRAB7 or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabARAB5/RabBRAB5 (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE2-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabORAB1. TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabORAB1 localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabORAB1 are dissociable by mutation: rabOA136D hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.
PMCID: PMC3722313  PMID: 23722157
autophagosome; phagophore; intracellular traffic; nitrogen starvation; Rab; SNARE; Atg8
21.  Lumenal peroxisomal protein aggregates are removed by concerted fission and autophagy events 
Autophagy  2013;9(7):1044-1056.
We demonstrated that in the yeast Hansenula polymorpha peroxisome fission and degradation are coupled processes that are important to remove intra-organellar protein aggregates. Protein aggregates were formed in peroxisomes upon synthesis of a mutant catalase variant. We showed that the introduction of these aggregates in the peroxisomal lumen had physiological disadvantages as it affected growth and caused enhanced levels of reactive oxygen species. Formation of the protein aggregates was followed by asymmetric peroxisome fission to separate the aggregate from the mother organelle. Subsequently, these small, protein aggregate-containing organelles were degraded by autophagy. In line with this observation we showed that the degradation of the protein aggregates was strongly reduced in dnm1 and pex11 cells in which peroxisome fission is reduced. Moreover, this process was dependent on Atg1 and Atg11.
PMCID: PMC3722314  PMID: 23614977
yeast; peroxisome; protein aggregate; fission; autophagy
22.  Harnessing autophagy for cell fate control gene therapy 
Autophagy  2013;9(7):1069-1079.
We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides “cell-fate control” due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.
PMCID: PMC3722316  PMID: 23633667
autophagy; DTYMK/TMPK; rapamycin; cell-fate control; suicide gene
23.  Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity 
Autophagy  2013;9(7):1080-1093.
Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.
PMCID: PMC3722317  PMID: 23697944
autophagy; polyamines; Trypanosoma cruzi; spermidine; DFMO; autophagic response; LC3; ATG5
24.  Endosome-mediated autophagy 
Autophagy  2013;9(6):861-880.
Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4BC74A mutant and atg4b−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.
PMCID: PMC3672296  PMID: 23481895
MHC class II; dendritic cell; autophagy; LC3; electron tomography
25.  Autophagy facilitates organelle clearance during differentiation of human erythroblasts 
Autophagy  2013;9(6):881-893.
Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4BC74A; ATG4DC144A) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4BC74A-expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.
PMCID: PMC3672297  PMID: 23508006
ATG4B; ATG4D; erythropoiesis; electron microscopy; autophagosome; amphisome; mitochondrion

Results 1-25 (393)