Distinct requirements of Autophagy-related genes in programmed cell death

Although most programmed cell death (PCD) during animal development occurs by caspase-dependent apoptosis, autophagy-dependent cell death is also important in specific contexts. In previous studies, we established that PCD of the obsolete Drosophila larval midgut tissue is dependent on autophagy and can occur in the absence of the main components of the apoptotic pathway. As autophagy is primarily a survival mechanism in response to stress such as starvation, it is currently unclear if the regulation and mechanism of autophagy as a pro-death pathway is distinct to that as pro-survival. To establish the requirement of the components of the autophagy pathway during cell death, we examined the effect of systematically knocking down components of the autophagy machinery on autophagy induction and timing of midgut PCD. We found that there is a distinct requirement of the individual components of the autophagy pathway in a pro-death context. Furthermore, we show that TORC1 is upstream of autophagy induction in the midgut indicating that while the machinery may be distinct the activation may occur similarly in PCD and during starvation-induced autophagy signalling. Our data reveal that while autophagy initiation occurs similarly in different cellular contexts, there is a tissue/function-specific requirement for the components of the autophagic machinery.There is a fundamental requirement for multicellular organisms to remove excess, detrimental, obsolete and damaged cells by programmed cell death (PCD).^{1, 2} In the majority of cases caspase-dependent apoptosis is the principle pathway of PCD; however, there are other modes of cell death with important context-specific roles, such as autophagy.^{3, 4} Defects in autophagy have significant adverse consequences to normal cellular functions and contribute to the pathogenesis of numerous human diseases. This is particularly evident in cancer where depending on the context autophagy can have tumour-suppressing or -promoting roles. Given the number of clinical trials targeting autophagy in cancer therapy, it will be critically important to understand the context-specific regulation and functions of autophagy.⁵Autophagy is a highly conserved multi-step catabolic process characterised by the encapsulation of part of the cytoplasm inside a double-membrane vesicle called the autophagosome. Autophagosomes then fuse with lysosomes and the components are subsequently degraded by acidic lysosomal hydrolases.⁶ The process of autophagy can be functionally divided into four groups: (1) serine/threonine kinase Atg1 (ULK1 in mammals) complex and its regulators responsible for the induction of autophagy; (2) the class III phosphatidylinositol 3-kinase (PI3K) complex, which involves Atg6 and functions in the nucleation of the autophagosome; (3) the Atg8 and Atg12 conjugation systems, which involves several Autophagy-related (Atg) proteins essential for the expansion of autophagosome; and (4) Atg9 and its associated proteins including Atg2 and Atg18, which aids the recycling of lipid and proteins.⁷ In addition, several of the Atg proteins can function in multiple steps. For example, Atg1 interacts with proteins with different functions (e.g. Atg8, Atg18 and others), suggesting that it is not only required for initiation but also participates in the formation of autophagosomes.⁸ It is yet to be fully established if the context-specific functions of autophagy have distinct requirements for select components of the autophagy pathway.High levels of autophagy are induced in response to stress, such as nutrient deprivation, intracellular stress, high temperature, high culture density, hormones and growth factor deprivation.^{9, 10} The target of rapamycin (TOR) pathway is a central mediator in regulating the response to nutrients and growth signalling. TOR functions in two distinct complexes, with regulatory associated protein of TOR (Raptor) in TOR complex 1 (TORC1) or with rapamycin insensitive companion of TOR (Rictor) in TOR complex 2 (TORC2).^{11, 12, 13, 14, 15} Of these, TORC1 regulates autophagy; in nutrient-rich conditions, TORC1 activity inhibits the Atg1 complex preventing autophagy and cellular stress such as starvation leads to inactivation of TORC1 promoting a dramatic increase in autophagy. TORC2 can also negatively regulate autophagy via the FoxO3 complex in specific context.¹⁶Most direct in vivo evidence for a role of autophagy in cell death has emerged from studies in Drosophila.⁵ Developmentally regulated removal of the Drosophila larval midgut can occur in the absence of canonical apoptosis pathway, whereas inhibiting autophagy delays the process.^{17, 18} Also, inhibition of autophagy leads to delayed degradation of larval salivary glands in Drosophila.¹⁹ Genetic studies have shown that many of the Atg genes known to be involved in starvation-induced autophagy in the Drosophila fat body are also involved in autophagy-dependent degradation of salivary glands and midgut.^{5, 20, 21} However, systematic studies to test whether starvation-induced autophagy and autophagy required for PCD require identical components have not been carried out, and there are some observations suggesting that there may be distinctions. For example, in Atg7-null mutants autophagy is perturbed but the larval–adult midgut transition proceeds normally.²² In addition, a novel Atg7- and Atg3-independent autophagy pathway is required for cell size reduction during midgut removal.²³ Here we show that downregulation of TORC1 activity is required for induction of autophagy during midgut removal. Surprisingly, however, the requirement of part of the autophagy machinery during midgut degradation was found to be distinct to that which is required during autophagy induced by starvation. We report that Atg genes required for autophagy initiation, Atg8a and recycling are all essential for autophagy-dependent midgut removal, whereas other components of the elongation and nucleation steps are not essential.     

Autophagy supports survival and phototransduction protein levels in rod photoreceptors

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomes for degradation. Defective autophagy can contribute to the age-dependent accumulation of damaged proteins and organelles leading to altered cellular homeostasis and loss of function.^{1, 2, 3, 4, 5} The metabolic roles of autophagy can be classified into two types, basal and induced. In nutrient-rich conditions, autophagy is suppressed but still occurs at low levels (basal autophagy); however, when cells are subjected to stress (starvation, injury, hypoxia), autophagy is activated immediately (induced autophagy).⁶ Induced autophagy maintains the amino acid pool inside cells to adapt to starvation while constitutive autophagy has been shown to function as a cell-repair mechanism that is important for long-lived postmitotic cells.^{7, 8, 9, 10, 11} Defects in autophagy have been associated with neurodegenerative diseases,^{12, 13, 14, 15} diabetes,^{16, 17} lysosomal storage disease¹⁸ and the loss of vision.¹⁹ In addition to macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) have been described. Although little is known about microautophagy in mammalian cells, macroautophagy (hereafter autophagy) is a major pathway for bulk degradation of cytoplasmic components. CMA is a more selective pathway for degradation of cytosolic proteins that can compensate for the loss of macroautophagy.^{2, 20, 21, 22}Inherited retinal degenerative diseases such as retinitis pigmentosa or Leber''s congenital amaurosis are characterized by premature and progressive death of rod and cone photoreceptor cells.²³ These diseases are characterized by the loss of night vision due to the death of rods followed by the loss of cones leading to diminished visual acuity and a reduction in the quality of life for patients. Disease is typically associated with the production of harmful gene products that promote pathology by inhibiting critical pathways resulting in cell death.^{24, 25, 26} Strategies to prevent photoreceptor death during retinal degenerative disease such as gene replacement therapies or inhibition of cell death pathways have been undertaken with some success;^{27, 28, 29} however, effective treatments for these blinding disorders are lacking.Another strategy that could be used in conjunction with other therapies might be to enhance survival by stimulating autophagy. Augmenting autophagy would increase the supply of nutrients to stressed cells and accelerate removal of damaged proteins thereby prolonging cell survival beyond what would be possible by only preventing cell death. Although canonical^{22, 30, 31, 32, 33} and noncanonical autophagic mechanisms³⁴ have been detected in the eye, our knowledge of basic autophagy functions in this organ is still limited. In order to understand how autophagy maintains retinal homeostasis and function, we undertook studies to examine the consequences of deleting the essential autophagy gene Atg5 in rod photoreceptors.     

Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model

Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9.Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy characterized by the uncontrolled proliferation of immature myeloid cells within the bone marrow (BM), eventually suppressing normal hematopoiesis.¹ Recurrent chromosomal translocations frequently occur in AML, one of which involves the fusions of the KMT2A gene on chromosome 11 to a number of potential partners that are diagnosed as prognostically intermediate to poor.¹ Among these fusions, the MLL-AF9 fusion oncogene, resulting from the t(9;11)(p22;q23) translocation, is well studied owing to its robust phenotype in various mouse models of AML.^{2, 3, 4} It has been previously reported that BM transplantation of hematopoietic progenitors expressing exogenous MLL-AF9 leads to rapid in vivo transformation and progression of AML in a syngeneic, immunocompetent mouse model and recapitulates the poor chemotherapy response of t(9;11)(p22;q23) fusion human AML.^{2, 5}Autophagy is an evolutionarily conserved catabolic pathway by which cellular components are engulfed by double-membraned vesicles, called autophagosomes, and delivered to the lysosome for degradation and recycling. Autophagy is best characterized to be induced under stressful conditions, such as organelle damage or nutrient deprivation, and is followed by the elongation of the autophagosome membrane around its cargo. In Atg5-dependent autophagy, the conversion of LC3-I to LC3-II by lipidation is crucial for autophagosome membrane expansion, which is mediated by a series of ubiquitin-like conjugation systems.⁶ Within this pathway, the Atg5-Atg12-Atg16 complex acts as an E3-ubiquitin-ligase-like enzyme that specifically mediates the conjugation of LC3-I to phosphatidylethanolamine to form LC3-II, which inserts to the autophagosomal membrane. Autophagosome maturation is followed by fusion to lysosomes, at which time the inner compartment is degraded. The genetic ablation of Atg5 leads to a complete and highly selective inhibition of LC3-dependent autophagosome formation.^{6, 7}Autophagy is known to be implicated in cancer as both a tumor promoter and a tumor suppressor.⁸ The genetic ablation of autophagy in mouse hematopoietic stem cells (HSCs) has been shown to result in severe impairments to HSC maintenance.^{9, 10, 11, 12, 13} Autophagy dysregulation has also been implicated in AML,^{12, 13, 14} suggesting that targeting autophagy could be promising for AML treatment. As an expanding arsenal of pharmacological autophagy modulators are being developed,^{15, 16} it has become increasingly important to specifically determine whether autophagy has an important role in AML using a genetic mouse model. Therefore, we sought to dissect the role of autophagy through the in vivo homozygous deletion of Atg5 in MLL-AF9-driven murine AML. We discover in this study that Atg5 deletion during primary transplantation prolongs the survival of animals, whereas Atg5 deletion after secondary transplantation has no effect on animal survival, suggesting a role for autophagy in the initiation, but not maintenance, of AML in our model. We additionally assessed the effect of autophagy in chemotherapeutic response and found that Atg5 deletion in our MLL-AF9 model had no effect on the in vivo response to cytarabine and doxorubicin combination therapy, suggesting that autophagy does not significantly contribute to chemotherapy response in this model.     

Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice

Primary ovarian insufficiency (POI) is a common cause of infertility in around 1–2% of women aged <40 years. However, the mechanisms that cause POI are still poorly understood. Here we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to subfertility in female mice. The subfertility of Atg7 deletion females was caused by severe ovarian follicle loss, which is very similar to human POI patients. Further investigation revealed that germ cell-specific Atg7 knockout resulted in germ cell over-loss at the neonatal transition period. In addition, our in vitro studies also demonstrated that autophagy could protect oocytes from over-loss by apoptosis in neonatal ovaries under the starvation condition. Taken together, our results uncover a new role for autophagy in the regulation of ovarian primordial follicle reservation and hint that autophagy-related genes might be potential pathogenic genes to POI of women.Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is an ovarian defect characterized by the premature depletion of ovarian follicles before the age of 40 years. POI is a common cause of infertility in women, affecting 1–2% of individuals aged <40 years and 0.1% of individuals aged <30 years.¹ Potential etiologies for POI are highly heterogeneous, which include iatrogenic, infectious, autoimmune, metabolic, chromosomal and genetic factors.² At present, about 25% of all forms of POF can be classified as iatrogenic and are related to cancer treatment, but >50% of the cases remain idiopathic. Though the pathogenic mechanism remains unexplained in the majority of the cases, several observations support a prevalent role of genetic mechanisms in the pathogenesis of idiopathic POI. It has been reported that mutations in FMR1, BMP-15, GDF-9, FOCL2, FSHR, LHR, INHA, GALT and AIRE are associated with POI.^{3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13} The genetic information of POI is very useful for family counseling, because it can predict the female relatives who may be at higher risk for POI and fertility loss in young age. The female carriers will be able to plan their conception before ovarian failure occurs. This requirement is becoming more and more important, because women nowadays tend to conceive ever more frequently in their thirties and forties,¹⁰ when the risk of POI in the general population is about 1–2%. However, still few genes could be identified that can explain a substantial proportion of the cases of POI.An important phenotype of POI is infertility, thus POI patients do not have large family histories, and therefore are difficult to study using traditional genetic methods, such as linkage analysis. Animal models of POI have been successfully used to identify candidate genes in this disease. The disruption of meiosis-specific genes, Bcl-2 family apoptotic-related genes, Pten-PI3K-Akt-Foxo3 pathway and Tsc1/2-mTOR signaling pathway result in POI-like phenotype in mice.^{14, 15, 16, 17} However, as a complex disorder, the genetic etiologies of POI still need to be further investigated to better understand the underlying molecular mechanisms.Macroautophagy (hereafter referred to as autophagy) is the primary intracellular catabolic mechanism for degrading and recycling long-lived proteins and organelles, which is evolutionarily conserved from yeast to mammals.¹⁸ During autophagy, isolation membrane enwraps parts of the cytoplasm and intracellular organelles, and fuse with each other forming a double membrane structure, known as the autophagosome. Then the outer membrane of the autophagosome fuses with the lysosome to form autolysosome, in which the cytoplasm-derived materials are degraded by resident hydrolases.¹⁹ The primary function of autophagy is to allow cells or organisms to survive nutrient starvation conditions by recycling either proteins or other cellular components. This process is important for cells to adapt their metabolism to starvation caused by decreased extracellular nutrients or by decreased intracellular metabolite concentrations. In addition to nutrient supply and adaptation to stress conditions, a number of observations have revealed that autophagy also functions in many physiological processes in mammalian systems, such as cell death, antiaging mechanisms, innate immunity, development and tumor suppression.^{20, 21, 22, 23, 24, 25}From the discovery of the molecular mechanism underlying autophagy, it was found that autophagy is required for the reproductive process in budding yeast.²⁶ In mammals, fertilization induces massive autophagy to degrade maternal proteins and messenger RNAs, and autophagy functions as a major nutrient-providing system for embryos before their implantation.²⁷ Our recent work indicates that autophagy is required for acrosome biogenesis during spermatogenesis in mice, thus essential to male fertility.²⁴ However, whether autophagy is involved in female gametogenesis or not is still unknown. Here, we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to POI in female mice, and the numbers of the oocytes and follicles were significantly declined in the adult mutant mice. Further investigation revealed that autophagy protected oocytes over-loss during the neonatal transition period. Our results suggest that autophagy-related genes might be pathogenic genes to POI.     

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.Vascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the biomechanical stimuli induced by blood flow, of which shear stress acting on the surface of EC has been recognized to be one of the most important vasoactive factors in EC.^{1, 2} A relatively high level of laminar shear stress is cytoprotective, whereas abnormal (low-magnitude or oscillatory) shear stress is a detrimental cellular stress to ECs.¹ Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions, such as gene expression, proliferation, migration, morphogenesis, permeability, thrombogenicity, and inflammation.²Autophagy (also known as macroautophagy) is an evolutionarily conserved cellular stress response.^{3, 4} Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12, for example.^{3, 5} Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment.^{4, 5} In ECs, an autophagic response can be initiated by different stress stimuli.^{6, 7, 8} It is noted that the cellular outcome following autophagy induction in ECs varies depending on the nature of stimuli and specific experimental settings.^{6, 7, 9, 10} Moreover, there is evidence showing that autophagy may also be involved in modulating other EC functions such as angiogenesis and cellular senescence.^{11, 12} Therefore, understanding the regulatory mechanisms of autophagy in ECs will be important for discovery of strategies to protect normal endothelial functions. Recently, Guo et al. provided some evidence indicating that the autophagic process in EC might be affected by shear stress.¹³ This argument, however, was only based on observations of changed expression levels of LC3 and beclin-1; further experimental evidence is needed to confirm such an effect of shear stress on autophagy. More importantly, the mechanisms underlying this phenomenon are not understood. Different signaling pathways may be involved in modulating autophagy in ECs.^{14, 15, 16} For example, inhibition of the mTOR (mammalian target of rapamycin) pathway by rapamycin-induced endothelial autophagy and prevented energy stress-triggered cell damage.¹⁶ There is also evidence indicating a potential role of Sirt1.¹⁴ Moreover, accumulating evidence has suggested that reactive oxygen species (ROS) are closely implicated in modulating autophagic responses via complex interactions with other autophagy-related factors.¹⁵ Despite of these results, the signaling mechanisms of shear stress-regulated autophagy in EC remain to be defined. Hence, here we aim to delineate the impacts and underlying mechanisms of shear stress on autophagy in human vascular ECs.     

Phosphatidylethanolamine positively regulates autophagy and longevity

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.Phosphatidylethanolamine (PE) is a phospholipid found in all living organisms. Together with phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI), PE represents the backbone of most biological membranes. PE is the second-most abundant phospholipid in mammalian membranes ranging from 20 to 50%.¹ In yeast, PE is essential for growth and is generated through four different enzymatic pathways:² PE can be produced by decarboxylation of PS, as a first option at the mitochondrial membrane via phosphatidylserine decarboxylase 1 (Psd1)^{3, 4} or, as a second, option at the Golgi and vacuolar membranes through phosphatidylserine decarboxylase 2 (Psd2).⁵ As a third possibility, PE can be produced from actively retrieved extracellular ethanolamine,^{6, 7} which is cytidine 5''-diphosphate-activated⁸ and then coupled to diacylglycerol to generate PE.⁹ The fourth, scarcely employed PE-generating pathway is based on the lysophospholipid acylation of lyso-PE. Importantly, PE does not spontaneously assemble in bilayers and rather incorporates into curved structures, such as the inverted hexagonal phase.¹⁰ The physiological function of non-bilayer lipids in membranes is considered to reside in their interaction with membrane proteins via the membrane lateral pressure¹⁰ and membrane tethering and fusion processes, which are relevant for autophagy.¹¹The term ‘autophagy'' describes a degradation process affecting intracellular components (for a review see, 12 13) which as an important cytoprotective mechanism, is closely linked to ageing. Autophagy mainly differs from the proteasomal pathway, the other major cellular degradation mechanism, in two aspects. First, autophagy can degrade large particles or whole organelles and second, the final degradation occurs in the lysosome/vacuole and not at the proteasome. Prior to the actual degradation, the cargo is gathered in autophagic particles, which are surrounded by a characteristic double-membrane. However, the origin of these autophagosomal membranes is still controversial and might actually depend on the mode of autophagy induction.^{14, 15} Among the discussed membrane sources are the Golgi apparatus, the endosplamic reticulum (ER) or the mitochondrion-associated membrane, which is formed at the interface between the ER.¹⁶ In higher eukaryotes autophagic membranes are enriched in PE with a high degree of unsaturation,¹⁷ similarly to the PE species found in mitochondria.^{14, 18} Moreover, the pre-autophagosomal structure or phagophore assembly site (PAS), which appears at the very beginning of autophagosome formation, already harbours Atg9, an autophagy-related transmembrane protein that shuttles between mitochondria and the PAS structure in yeast.¹⁹Importantly, PE also functions as an anchor to autophagosomal membranes for the autophagy-related protein Atg8 in yeast²⁰ and its mammalian orthologue LC3.^{21, 22} This PE anchor is provided to LC3/Atg8 post-translationally in a process called lipidation. First, LC3/Atg8 is carboxy-terminally cleaved by proteases from the Atg4 family.^{23, 24} Subsequently, the remaining C-terminal glycine is coupled to PE in a series of ubiquitination-like reactions involving diverse Atg-proteins.^{20, 25, 26, 27} In vitro, Atg8-PE causes hemifusion of vesicles, which argues for its potential role in autophagosomal phagophore expansion.^{11, 28} Consistently, semisynthetic LC3-PE has recently been described to stimulate membrane tethering and fusion.²⁹ We thus reasoned that the overall abundance of PE might be critical for PE-lipidation of LC3/Atg8 and could thus regulate autophagosomal membrane formation. Therefore, we tested whether increasing cellular PE levels might have an impact on autophagy and lifespan regulation.Here, we report that knock-out of PSD1 or PSD2 shortens the chronological lifespan of S. cerevisiae, whereas PSD1-overexpression enhances the autophagic capacity and increases longevity. Furthermore, external administration of ethanolamine increases endogenous PE levels, enhances autophagic flux and extends the lifespan of yeast, mammalian cells in culture and flies (Drosophila melanogaster).     

Pdcd4 deficiency enhances macrophage lipoautophagy and attenuates foam cell formation and atherosclerosis in mice

Macrophage foam cells, a major component of the atherosclerotic lesion, have vital roles in the development of atherosclerosis. Lipoautophagy, a type of autophagy characterized by selective delivery of lipid droplet for lysosomal degradation, may impact atherosclerosis by regulating macrophage foam cell formation. Previously, we reported that programmed cell death 4 (PDCD4), a tumor suppressor, negatively regulated autophagy in tumor cells. However, its roles in macrophage lipoautophagy, foam cell formation and atherosclerosis remain to be established. Here we found that Pdcd4 deficiency clearly improved oxidized low-density lipoproteins-impaired autophagy efflux, promoted autophagy-mediated lipid breakdown in murine macrophages and thus prevented macrophage conversion into foam cells. Importantly, Pdcd4 deficiency in mice significantly upregulated macrophage autophagy in local plaques along with attenuated lipid accumulation and atherosclerotic lesions in high-fat-fed Apolipoprotein E knockout mice. Bone marrow transplantation experiment demonstrated that PDCD4-mediated autophagy in hematopoietic cells contributed to the development of atherosclerosis. These results indicate that endogenous PDCD4 promotes for macrophage foam cell formation and atherosclerosis development via inhibiting autophagy and provides new insights into atherogenesis, suggesting that promoting macrophage autophagy through downregulating PDCD4 expression may be beneficial for treating atherosclerosis.Atherosclerosis is a lipid dysfunction-derived chronic inflammatory process in large and medium arterial wall.¹ Macrophage foam cell, as a major component in the lesion of atherosclerosis, has vital role in the development of atherosclerosis. In the initial step of atherosclerotic development, circulating monocytes migrate into arterial wall via dysfunctional endothelial cells and differentiate into macrophages.^{2, 3, 4} The infiltrated macrophages ingest and digest oxidized low-density lipoprotein (ox-LDL), and then transport lipid out of vascular wall.⁵ However, macrophage with overloaded lipids stored in the form of lipid droplets (LDs) will transform into foam cells. Macrophage foam cell formation could promote the development of atherosclerosis.⁶ Thus, decreasing the formation of macrophage foam cell would be an attractive strategy to reverse plaque lipid buildup.⁷The macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved and well-controlled cellular catabolic process. During the process, cytoplasmic components are sequestered in double-membrane vesicles (which is called autophagosome) and degraded by fusion with lysosomal compartments (autophagolysosome) for recycling application.⁸ The process of autophagy is regulated by several autophagy-related genes (ATGs) encoded proteins, such as ATG5, ATG6 (also known as BECN1), ATG8 (also known as microtubule-associated protein 1 light chain 3, LC3) and ATG12. ATG5 is involved in the early stage of autophagosome formation. ATG5 is conjugated with ATG12 and ATG16L to form ATG12–ATG5–ATG16L complex, which contributes to the elongation and closure of the autophagosomes in the generation of lipidated forms of LC3 family proteins.⁹ Lipoautophagy, a type of autophagy that selectively delivers LDs for lysosomal degradation,¹⁰ regulates lipid metabolism and is involved in the process of atherosclerosis.^{11, 12, 13, 14} In advanced atherosclerosis, macrophage autophagy becomes dysfunctional. However, the basic autophagy deficiency in macrophage by specific Atg5 knockout accelerates atherosclerotic plaques in high-fat-fed ldlr^−/− mice via promoting oxidative stress, plaque necrosis¹² or inflammasome hyperactivation.¹³ More interestingly, autophagy can enhance brokendown of lipid in LD, cholesterol efflux from macrophage foam cells and further inhibit atherogenisis.¹⁴ Stent-based delivery of everolimus (mTOR inhibitor) in atherosclerotic plaques of cholesterol-fed rabbits leads to a marked reduction of macrophages via autophagic cell death.¹⁵ Therefore, regulating the level of macrophage autophagy and macrophage conversion into foam cells would be a potential target for preventing the atherosclerotic plaques formation.¹⁶Programmed cell death 4 (PDCD4), an inhibitor of protein translation, inhibits translation initiation via binding to the translation initiation factor eIF4A or translation elongation by direct or indirectly binding to the coding region of specific RNAs.^{17, 18} Accumulated evidence has demonstrated PDCD4 as a tumor suppressor.¹⁹ PDCD4 can inhibit promotion and progression of tumors, such as lung cancer,²⁰ hepatocellular carcinoma cells,²¹ colon cancer,²² ovarian cancer²³ and glioma.²⁴ In addition, it has been reported that PDCD4 is also involved in the development of inflammatory diseases.^{25, 26, 27, 28, 29, 30} For example, Pdcd4-deficient mice are resistant to experimental allergic encephalitis,²⁵ LPS-induced endotoxin shock²⁶ and type-1 diabetes.²⁷ In addition, Pdcd4-deficient mice are sensitive to LPS/D-galactosamine-induced acute liver injury.²⁸ Recently, we reported that Pdcd4 deficiency attenuated adipocyte foam cells, diet-induced obesity, obesity-associated inflammation and insulin resistance,²⁹ and increased IL-10 expression by macrophages that partly involved in atherosclerosis in hyperlipidemic mice,³⁰ suggesting that PDCD4 may be involved in the metabolism-related diseases. Furthermore, we found that PDCD4 negatively regulated autophagy by inhibiting ATG5 expression in tumor cells.³¹ However, its role in macrophage lipoautophagy and foam formation, and association with atherosclerosis remain to be investigated.In the present study, we found that Pdcd4 deficiency improved ox-LDL-impaired autophagy efflux in murine macrophage and subsequently attenuated macrophage conversion into foam cells in an autophagy-dependent manner and further attenuated the formation of atherosclerotic lesions in hyperlipidemia mice. These results indicate that PDCD4 is critical for macrophage foam cell formation in atherosclerosis development and provides new insights into atherogenesis, and potential therapeutic avenues to treat atherosclerosis-associated diseases.     

Spermidine induces autophagy by inhibiting the acetyltransferase EP300

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.Macroautophagy (herein referred to as ‘autophagy'') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.¹ This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 ‘puncta''), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1).²There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity.^{3, 4, 5, 6} This applies to caloric restriction or intermediate fasting,⁷ continuous or intermittent medication of rapamycin,^{8, 9, 10} administration of the sirtuin 1-activator resveratrol,^{11, 12} external supply of the polyamine spermidine,¹³ or genetic ablation of p53.¹⁴ In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan.^{13, 14, 15, 16, 17} Moreover, direct induction of autophagy by transgenic expression of autophagy-relevant genes such as ATG5 in mice is sufficient to increase lifespan.¹⁸Recently, acetyltransferases have emerged as a potential target for the pharmaceutical induction of autophagy. Thus, depletion of the sole donor of acetyl groups, acetyl-coenzyme A (acetyl-CoA), is sufficient to reduce the acetylation of cytoplasmic and nuclear proteins coupled to the induction of autophagy.^{19, 20, 21, 22} Culture of mammalian cells in nutrient-free (NF) conditions or starvation of mice for 24 h reduced the intracellular nucleocytosolic concentrations of acetyl-CoA at the same time as autophagy was induced, and replenishment of acetyl-CoA by external sources (for instance, by providing a membrane-permeant precursor of α-ketoglutarate for anaplerotic reactions or by microinjection of acetyl-CoA) was sufficient to inhibit starvation-induced autophagy.^{19, 20, 21, 22} Beyond the inhibition of acetyltransferases by acetyl-CoA depletion, direct pharmacological inhibition of acetyltransferases might also contribute to the induction of autophagy. A close correlation between autophagy induction and deacetylation of cytoplasmic proteins was observed in a screen conceived to identify autophagy-stimulating polyphenols²³ as well as in in vivo experiments designed to explore the health-improving effects of coffee.²⁴ Spermidine turned out to be an efficient inhibitor of histone acetyltransferases in vitro¹³ and reduced the global protein acetylation levels in cultured cells.^{25, 26}Driven by these premises, we investigated the hypothesis that several health-related compounds including anacardic acid, curcumin, garcinol and spermidine might induce autophagy by inhibition of acetyltranferases. Here we report results supporting this hypothesis. Moreover, we demonstrate that one particular acetyltransferase, EP300 (E1A-binding protein p300), negatively controls autophagy and that anacardic acid, curcumin, garcinol and spermidine may induce autophagy by directly inhibiting EP300.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson''s disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.Nowadays, Parkinson''s disease (PD) constitutes the main motor disorder and the second neurodegenerative disease after Alzheimer''s disease. Etiology of PD remains unknown, but both environmental and genetic factors have been implicated. Among the genes associated with PD is the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) encoding gene encoded by PARK8. Indeed, LRRK2 mutations have been described in a substantial number of idiopathic late-onset PD patients without a known family history of the disease.^{1, 2, 3}The physiologic function remains unknown. It localizes in the cytosol as well as in specific membrane subdomains, including mitochondria, autophagosomes and autolysosomes,⁴ and interacts with a whole array of proteins, including both α- and β-tubulin,^{5, 6} tau,⁷ α-synuclein⁸ and F-actin.⁹ LRRK2 gene mutations, including the most common G2019S,³ are associated with increases in toxic putative kinase activity.^{1, 10} LRRK2 overexpression is toxic to cultured cells,^{11, 12} and LRRK2 loss did not cause neurodegenerative changes (for a review see Tong and Shen¹³). However, LRRK2 transgenic mice lack obvious PD-like behavioral phenotypes.¹⁴ LRRK2-associated PD patients show degeneration of dopaminergic neurons in the substantia nigra.¹⁵ Data from our own group and others have associated mitochondrial apoptotical pathways with PD,^{16, 17, 18} and, in this context, LRRK2 mutant-mediated toxicity could be due to mitochondria-dependent apoptosis.¹⁹ There is considerable evidence for impaired mitochondrial function and morphology in both early-onset, autosomal recessive inherited PD and late-onset sporadic PD.Mitochondrial dynamics include several mechanisms, such as fission, fusion and mitophagy.^{20, 21} Altered fission/fusion dynamics might be a common pathogenic pathway of neurodegenerative diseases. It is well documented that mitochondrial dynamics constitute a relevant issue in some experimental neurodegenerative models.^{20, 22, 23, 24, 25} Mitochondrial dynamics is tightly regulated by cellular pathways including those participated by the dynamin-related protein-1 (Drp1). Drp1 mostly locates in the cytoplasm, but is stimulated after fission stimuli to migrate to the mitochondria. Once there, Drp1 forms ring-like structures, which wrap around the scission site to constrict the mitochondrial membrane resulting in mitochondrial fission.^{26, 27} Interestingly, a functional interaction between PD-associated LRRK2 and members of the dynamin GTPase superfamily has been described.²⁸Macroautophagy (hereafter referred to as autophagy) is an active cellular response, which functions in the intracellular degradation system of cellular debris such as damaged organelles. Whether autophagy promotes cell death or enhances survival is still controversial.^{29, 30} It requires the formation of autophagosomes where cellular content is to be degraded by the action of lysosomal enzymatic content. Autophagosome formation is regulated by an orderly action of >30 autophagy-related (Atg) proteins. Among them is the microtubule-associated protein 1A/1B-light chain 3 (LC3), a homolog of Apg8p, which is essential for autophagy in yeast and is associated with autophagosome membranes.³¹ Interestingly, these vesicles are mostly highly mobile in the cytoplasm.³² Wild-type and mutant LRRK2 expression has been related to autophagy.^{4, 33, 34, 35, 36} Reactive oxygen species (ROS) function as relevant second messengers after several stimuli, including mitochondrial disruption. Exacerbated ROS increases might result in overactivation of antioxidant systems and yield harmful oxidative stress. Among oxidative stress hallmarks is the accumulation of α,β-unsaturated hydroxyalkenal 4-hydroxy-2-nonenal (4-HNE), whose accumulation has been reported in PD post-mortem patient brains,^{37, 38} thus giving a significant relevance to ROS in the pathogenesis of PD.All these results indicate LRRK2 as a promising pharmacologic target in PD treatment.³⁹ Several LRRK2 inhibitor drugs have been synthetized, such as the potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide (GSK2578215A). GSK2578215A exhibits biochemical IC₅₀s of 10.9 nM against wild-type LRRK2, and possesses a high ratio of brain to plasma distribution.⁴⁰ This study provides key insights into the mechanisms downstream of LRRK2 inhibition, and spreads light onto an underexplored, yet potentially tractable therapeutic target for treating LRRK2-associated PD. We demonstrate how inhibition of this kinase results in the activation of cellular death pathways such as the mitochondrial fission machinery, and how cells reply by activating a protective autophagic response. Our results show the presence of oxidative stress hallmarks, thus pointing to a key function for ROS, placed downstream of mitochondrial fission.     

Autophosphorylation Within the Atg1 Activation Loop Is Required for Both Kinase Activity and the Induction of Autophagy in Saccharomyces cerevisiae

Autophagy is an evolutionarily conserved degradative pathway that has been implicated in a number of physiological events important for human health. This process was originally identified as a response to nutrient deprivation and is thought to serve in a recycling capacity during periods of nutritional stress. Autophagy activity appears to be highly regulated and multiple signaling pathways are known to target a complex of proteins that contains the Atg1 protein kinase. The data here extend these observations and identify a particular phosphorylation event on Atg1 as a potential control point within the autophagy pathway in Saccharomyces cerevisiae. This phosphorylation occurs at a threonine residue, T226, within the Atg1 activation loop that is conserved in all Atg1 orthologs. Replacing this threonine with a nonphosphorylatable residue resulted in a loss of Atg1 protein kinase activity and a failure to induce autophagy. This phosphorylation required the presence of a functional Atg1 kinase domain and two known regulators of Atg1 activity, Atg13 and Atg17. Interestingly, the levels of this modification were found to increase dramatically upon exposure to conditions that induce autophagy. In addition, T226 phosphorylation was associated with an autophosphorylated form of Atg1 that was found specifically in cells undergoing the autophagy process. In all, these data suggest that autophosphorylation within the Atg1 activation loop may represent a point of regulatory control for this degradative process.MACROAUTOPHAGY (hereafter referred to as autophagy) is a highly conserved process of self-degradation that is essential for cell survival during periods of nutrient limitation (Tsukada and Ohsumi 1993). During autophagy, a double membrane grows out from a specific nucleation site, known as the pre-autophagosomal structure, or PAS, in Saccharomyces cerevisiae and the phagophore assembly site in mammals (Suzuki and Ohsumi 2007). This membrane encapsulates bulk protein and other constituents of the cytoplasm and ultimately targets this material to the vacuole/lysosome for degradation (Xie and Klionsky 2007). Recent studies have linked this pathway to a number of processes important for human health, including tumor suppression, innate immunity, and neurological disorders, like Huntington''s disease (Rubinsztein et al. 2007; Levine and Kroemer 2008). Determining how this pathway is regulated is therefore important for our understanding of these processes and our attempts to manipulate autophagy in clinically beneficial ways.Most of the molecular components of the autophagy pathway were initially characterized in the budding yeast, S. cerevisiae, but orthologs of many of these Atg proteins have since been found in other eukaryotes (Tsukada and Ohsumi 1993; Meijer et al. 2007). A complex of proteins that contains the Atg1 protein kinase is of special interest and appears to be a key point of regulatory control within this pathway (Kamada et al. 2000; Budovskaya et al. 2005; He and Klionsky 2009; Stephan et al. 2009). In S. cerevisiae, genetic and biochemical data indicate that this complex is targeted by at least three different signaling pathways. Two of these pathways, involving the Tor and cAMP-dependent protein kinases, inhibit this process, whereas the AMP-activated protein kinase is needed for the full induction of autophagy (Noda and Ohsumi 1998; Wang et al. 2001; Budovskaya et al. 2004; Stephan and Herman 2006; Kamada et al. 2010). The manner in which these signaling pathways regulate Atg1 activity and the precise role of this kinase in the autophagy process are presently matters of intense scrutiny.Although Atg1 kinase activity is required for the induction of autophagy, relatively little is known about how this enzyme is regulated in vivo. Two proteins associated with Atg1, Atg13 and Atg17, have been shown to be required for full Atg1 kinase activity both in vitro and in vivo (Kamada et al. 2000; Stephan et al. 2009). The roles of these proteins appear to be conserved through evolution as functional homologs of both have been identified in fruit flies and/or mammals (Hara et al. 2008; Chan et al. 2009; Chang and Neufeld 2009; Ganley et al. 2009; Hosokawa et al. 2009; Jung et al. 2009; Mercer et al. 2009). However, it is not yet clear precisely how these proteins stimulate Atg1 activity. In this study, we show that Atg1 is autophosphorylated within the activation loop and that this phosphorylation is required for both Atg1 kinase activity and the induction of autophagy. The activation loop is a structurally conserved element within the kinase domain and phosphorylation within this loop is often a necessary prerequisite for efficient substrate binding and/or phosphotransfer in the catalytic site (Johnson et al. 1996; Nolen et al. 2004). This loop generally corresponds to the sequence between two signature elements within the core kinase domain, the DFG and APE motifs (Hanks and Hunter 1995). Phosphorylation within this loop tends to result in a more ordered structure for this region and the proper positioning of key elements within the catalytic core of the kinase domain (Knighton et al. 1991; Johnson and O''reilly 1996; Huse and Kuriyan 2002). We found that Atg1 activation loop phosphorylation was correlated with the onset of autophagy and that replacing the site of phosphorylation with a phosphomimetic residue led to constitutive Atg1 autophosphorylation in vivo. In all, the data here suggest that Atg1 phosphorylation within its activation loop may be an important point of regulation within the autophagy pathway and models that discuss these data are presented.     

IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.Autophagy is a degradative process in eukaryotic cells that removes or turns over bulk cytoplasmic constituents through the endosomal and lysosomal fusion system (i.e., autophagosomes).^{1, 2} Autophagy is induced by stressful conditions such as starvation and pathogenic invasion.²Hypertrophic scar (HS) is a major skin fibrotic disorder caused by hypercellularity and extracellular matrix (ECM) component deposition.^{3, 4, 5} HS formation is usually recognized as the consequence of disturbed tissue repair processes and/or disrupted homeostasis in the skin after traumatic injury: HS negatively impacts on patient appearance, skeletal muscle function, and quality of life in general.^{6, 7, 8, 9} About 40–70% of surgeries and over 91% of burn injuries result in HS.¹⁰ A key feature of HS is a metabolic disorder of collagen-based ECM proteins.^{11, 12, 13} Autophagy has an important role in homeostasis of tissue structure and function.^{2, 14, 15} Skin autophagic capability is associated with HS and with the pathogenesis of many human diseases.^{16, 17, 18, 19, 20, 21, 22, 23}Existing studies suggest that cytokines are important regulators of the autophagic process in both immune and non-immune cells.^{24, 25, 26} Interleukin-10 (IL10), expressed by a variety of mammalian cell types, was first described as a cytokine-synthesis-inhibitory factor with immunosuppressive and anti-inflammatory functions.^{27, 28} IL10 has a pivotal role in wound healing^{29, 30} and is a promising therapeutic agent for scar improvement in both animal models and human cutaneous wounds.^{9, 31, 32}Fibroblasts are one of the most important effector cells responsible for HS formation.^{12, 33, 34} Thus, we were prompted to elucidate the mechanisms underlying the interactions among IL10, autophagy, and HS formation, with the aim of providing a molecular foundation for the therapeutic efficacy IL10. We used HS tissue, HS-derived fibroblasts (HSFs), and starvation-induced autophagy in HSFs as our research platform.Here, we report that IL10 inhibited autophagy by interfering with IL10R-mediated activation of IL10R-STAT3, as well as by activating the AKT-mTOR pathway. In addition, cross talk among STAT3, AKT, and mTOR and between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways collaboratively regulated starvation-induced autophagy in HSFs.     

Lysosomal membrane permeabilization and autophagy blockade contribute to photoreceptor cell death in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a group of hereditary retinal dystrophies that normally result in photoreceptor cell death and vision loss both in animal models and in affected patients. The rd10 mouse, which carries a missense mutation in the Pde6b gene, has been used to characterize the underlying pathophysiology and develop therapies for this devastating and incurable disease. Here we show that increased photoreceptor cell death in the rd10 mouse retina is associated with calcium overload and calpain activation, both of which are observed before the appearance of signs of cell degeneration. These changes are accompanied by an increase in the activity of the lysosomal protease cathepsin B in the cytoplasm of photoreceptor cells, and a reduced colocalization of cathepsin B with lysosomal markers, suggesting that lysosomal membrane permeabilization occurs before the peak of cell death. Moreover, expression of the autophagosomal marker LC3-II (lipidated form of LC3) is reduced and autophagy flux is blocked in rd10 retinas before the onset of photoreceptor cell death. Interestingly, we found that cell death is increased by the induction of autophagy with rapamycin and inhibited by calpain and cathepsin inhibitors, both ex vivo and in vivo. Taken together, these data suggest that calpain-mediated lysosomal membrane permeabilization underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.Autophagy is a cellular self-degradative pathway that mediates the recycling of damaged or disposable cell components and is activated in situations of nutritional, oxidative and other forms of stress.¹ This process begins with the formation of the autophagosome, an intracellular double-membrane organelle that surrounds parts of the cytoplasm containing organelles and protein aggregates. Autophagosomes subsequently fuse with lysosomes to initiate the degradation of the engulfed cellular components. Autophagy dysfunction has been implicated in many pathological conditions including infections, cancer and muscular and degenerative diseases.² In the nervous system, autophagy has a key role in preventing intracellular accumulation of misfolded and/or aggregated proteins, and its pharmacological upregulation through the administration of rapamycin and other drugs exerts protective effects against a wide range of proteinopathies.³ Moreover, defects in different stages of the autophagy pathway, including autophagosome formation, cargo recognition and lysosomal fusion and degradation, have been often implicated in neurodegeneration.⁴In addition to their degradative role, lysosomes are emerging as key regulators of cellular homeostasis, acting as nutritional sensors or actively participating in cell death.^{5, 6, 7} Lysosomal alterations including increases in lysosomal pH and lysosomal membrane permeabilization (LMP) have been demonstrated in Alzheimer''s and Parkinson''s diseases,^{8, 9} and mutations in lysosomal enzymes cause defects in autophagy, inducing a marked neurodegenerative phenotype in patients with lysosomal storage disorders.¹⁰ LMP induces the selective translocation of cathepsins to the cytoplasm, triggering caspase-dependent and -independent cell death.^{11, 12, 13} LMP has been implicated in mammary gland involution in physiological conditions,¹⁴ indicating that lysosomal-mediated cell death is not merely a consequence of accidental lysosomal damage. As in vivo administration of cathepsin inhibitors attenuates cell death in this model, a similar approach could hold therapeutic potential for the treatment of diseases associated with LMP, including Parkinson''s disease, Niemann–Pick disease type A and stroke.^{7, 10, 15} Oxidative stress and calpain activation are some of the many stimuli that can induce LMP, and have been observed both in vitro and in vivo.⁷ Several pathological processes in the nervous system associated with cell death, including excitotoxicity and ischaemia–reperfusion, have been linked to increased calpain activation.¹⁶ Calpains have also been shown to cleave many intracellular substrates including autophagy and lysosomal proteins,^{17, 18} suggesting links between calcium levels, calpain activation, lysosomal damage and autophagy blockade.Recent findings have begun to shed light on the role of autophagy in the retina. We previously reported decreased autophagy flux in the retinas of aged mice,¹⁹ and demonstrated photoreceptor cell death and decreased dim-light vision in the neuronal-specific Atg5-deficient mouse, a phenotype that closely resembles that observed during physiological aging.¹⁹ We have also demonstrated the essential cytoprotective role of autophagy in vivo in response to retinal ganglion cell damage in experimental models of glaucoma.²⁰ A recent study described lysosomal basification and decreased autophagic flux in travecular meshwork cells in response to chronic oxidative stress, with important implications for the pathogenesis of glaucoma.²¹ Furthermore, specific Atg5 deletion in pigment epithelium leads to reduced levels of visual pigments and vision alterations,²² indicating that autophagy has an important role in sustaining retinal pigment epithelium function.Retinitis pigmentosa is a large group of genetic disorders that normally involves photoreceptor cell death and leads to vision loss in both animal models and affected patients. To date, no treatment for this devastating disease has been developed to clinic. The study of animal models is thus essential to unravel the mechanisms of photoreceptor degeneration involved in these disorders and to identify therapeutic targets. The rd10 mouse, which harbours a mutation in the rod-specific phosphodiesterase gene Pde6b, is a suitable model of human retinitis pigmentosa.^{23, 24} This mutation results in reduced enzymatic function leading to increased cGMP and rod cell death, peaking around postnatal day 25 (P25), with only residual vision remaining after P30.^{24, 25} Here we show that rd10 mice exhibit massive intracellular calcium accumulation and m-calpain (calpain-2) activation at early ages, before the peak of photoreceptor cell death, that correlate with the blockade of autophagic flux. Moreover, we demonstrate an increase in cathepsin B activity in the cytoplasm of rd10 photoreceptors that correlates with the activation of DNAse II-dependent cell death. Induced calcium overload in wild-type (Wt) retinal explants phenocopies the degenerative features seen in rd10 retinas: lysosomal damage, cathepsin translocation and cell death. Finally, we show that calpain and cathepsin inhibitors attenuate cell death both in vitro, ex vivo and in vivo. Taken together, these data suggest that calpain-mediated LMP underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.     

The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: functional linkage with autophagy

Autophagy is a major nutrient recycling mechanism in plants. However, its functional connection with programmed cell death (PCD) is a topic of active debate and remains not well understood. Our previous studies established the plant metacaspase AtMC1 as a positive regulator of pathogen-triggered PCD. Here, we explored the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered PCD and aging. We observed that autophagy acts as a positive regulator of pathogen-triggered PCD in a parallel pathway to AtMC1. In addition, we unveiled an additional, pro-survival homeostatic function of AtMC1 in aging plants that acts in parallel to a similar pro-survival function of autophagy. This novel pro-survival role of AtMC1 may be functionally related to its prodomain-mediated aggregate localization and potential clearance, in agreement with recent findings using the single budding yeast metacaspase YCA1. We propose a unifying model whereby autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants, when these functions are not masked by the cumulative stresses of aging, and negatively regulating senescence in older plants.An emerging theme in cell death research is that cellular processes thought to be regulated by linear signaling pathways are, in fact, complex. Autophagy, initially considered merely a nutrient recycling mechanism necessary for cellular homeostasis, was recently shown to regulate cell death, mechanistically interacting with components that control apoptosis. Deficient autophagy can result in apoptosis^{1, 2, 3} and autophagy hyper-activation can also lead to programmed cell death (PCD).⁴ In addition, the pro-survival function of autophagy is mediated by apoptosis inhibition and apoptosis mediates autophagy, although this cross-regulation is not fully understood.⁵In plants, autophagy can also have both pro-survival and pro-death functions. Autophagy-deficient plants exhibit accelerated senescence,^{6, 7, 8} starvation-induced chlorosis,^{6, 7, 9} hypersensitivity to oxidative stress¹⁰ and endoplasmic reticulum stress.¹¹ Further, autophagy-deficient plants cannot limit the spread of cell death after infection with tissue-destructive microbial infections.^{12, 13} The plant phytohormone salicylic acid (SA) mediates most of these phenotypes.⁸ Autophagy has an essential, pro-survival role in situations where there is an increasing load of damaged proteins and organelles that need to be eliminated, that is, during aging or stress. Autophagy has an opposing, pro-death role during developmentally regulated cell death^{14, 15} or during the pathogen-triggered hypersensitive response PCD (hereafter, HR) that occurs locally at the site of attempted pathogen attack.^{16, 17} The dual pro-death/pro-survival functions of plant autophagy remain a topic of active debate.Also under scrutiny are possible novel functions of caspases and caspase-like proteins as central regulators of pro-survival processes. Caspases were originally defined as executioners of PCD in animals, but increasing evidence indicates that several caspases have non-apoptotic regulatory roles in cellular differentiation, motility and in the mammalian immune system.^{18, 19, 20}Yeast, protozoa and plants do not have canonical caspases, despite the occurrence of morphologically heterogeneous PCDs.²¹ More than a decade ago, distant caspase homologs termed metacaspases were identified in these organisms using structural homology searches.²² Metacaspases were classified into type I or type II metacaspases based on the presence or absence of an N-terminal prodomain, reminiscent of the classification in animals into initiator/inflammatory or executioner caspases, respectively. Despite the architectural analogy between caspases and metacaspases, differences in their structure, function, activation and mode of action exist.^{23, 24, 25}Metacaspases mediate PCD in yeast,^{26, 27, 28, 29, 30, 31} leishmania,^{32, 33} trypanosoma³⁴ and plants.²⁴ We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically regulate HR in Arabidopsis thaliana.³⁵ Our work showed that AtMC1 is a positive regulator of HR and that this function is mediated by its catalytic activity and negatively regulated by the AtMC1 N-terminal prodomain. AtMC2 antagonizes AtMC1-mediated HR.Besides AtMC2, new examples of metacaspases with a pro-life/non-PCD role are emerging. Protozoan metacaspases are involved in cell cycle dynamics^{34, 36, 37, 38} and cell proliferation.³⁹ The yeast metacaspase Yca1 alters cell cycle dynamics⁴⁰ and interestingly, is required for clearance of insoluble protein aggregates, thus contributing to yeast fitness.⁴¹Here, we explore the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered HR and aging. Our data support a model wherein autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants and negatively regulating senescence in older plants.     

Vps15 is required for stress induced and developmentally triggered autophagy and salivary gland protein secretion in Drosophila

Autophagy is a catabolic process used to deliver cellular material to the lysosome for degradation. The core Vps34/class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of Atg6, Vps15, and Vps34, is highly conserved throughout evolution, critical for recruiting autophagy-related proteins to the preautophagosomal structure and for other vesicular trafficking processes, including vacuolar protein sorting. Atg6 and Vps34 have been well characterized, but the Vps15 kinase remains poorly characterized with most studies focusing on nutrient deprivation-induced autophagy. Here, we investigate the function of Vps15 in different cellular contexts and find that it is necessary for both stress-induced and developmentally programmed autophagy in various tissues in Drosophila melanogaster. Vps15 is required for autophagy that is induced by multiple forms of stress, including nutrient deprivation, hypoxia, and oxidative stress. Furthermore, autophagy that is triggered by physiological stimuli during development in the fat body, intestine, and salivary gland also require the function of Vps15. In addition, we show that Vps15 is necessary for efficient salivary gland protein secretion. These data illustrate the broad importance of Vps15 in multiple forms of autophagy in different animal cells, and also highlight the pleiotropic function of this kinase in multiple vesicle-trafficking pathways.Autophagy is an evolutionarily conserved process in which cytoplasmic proteins or organelles are packaged into lysosomes for degradation. This process can be initiated by a variety of stimuli, such as high levels of starvation or stress, to provide nutrients to the cells or to clear the cell of damaged organelles or protein aggregates.¹ In some circumstances, autophagy can promote an alternative form of cell death, such as in the clearance of larval tissues in Drosophila melanogaster.² As defects in autophagy have been implicated in several physiological and pathological conditions, such as cancer, neurodegenerative diseases, and aging,^3,4 it is important to obtain a complete understanding of the molecular mechanisms controlling autophagy.The induction of autophagy is regulated by the Atg1/Ulk1 complex, and this complex is regulated by mechanistic target of rapamycin (mTOR).⁵ Vesicle nucleation is controlled by the class III phosphoinositide 3-kinase (PI3K) complex that generates phosphatidylinositol 3-phosphate (PI3P).⁶ This conserved complex consists of vacuolar protein sorting 34 (Vps34; also known as Pik3c3), Atg6/Becn1 (also known as Vps30 in yeast), and the serine-threonine kinase Vps15/ird1 (p150 in mammals; also known as Pik3r4).^7,8 Localized production of PI3P by Vps34 can act to recruit proteins containing PX or FYVE domains to membrane compartments, such as the autophagosome isolation membrane.⁹ Vps34 is also required more broadly for several vesicular trafficking processes such as the sorting of hydrolytic enzymes to the yeast vacuole and mammalian lysosome, and endocytic trafficking.^{10, 11, 12} There is mounting evidence demonstrating the pleiotropic function of the PI3K/Vps34 complex, but this has not been well studied in the context of autophagy under different physiological and cell contexts in animals.Of the three core PI3K complex proteins, Vps15 remains an understudied kinase, and its function has not been rigorously investigated in multicellular organisms in vivo. Most of the focus on the role of this complex in autophagy regulation has been on nutrient deprivation-initiated autophagy. Indeed, previous studies determined Vps15 to be necessary for starvation-induced autophagy in the Drosophila larval fat body.^13,14 However, its role in hormone-regulated autophagy, a process that occurs in the intestine,¹⁵ salivary glands,¹⁶ and fat body¹⁷ of developing Drosophila, as well as its role in other stress-induced conditions have not yet been examined. In order to address the role of Vps15 in these and other processes regulated by autophagy, we utilized Vps15 knockdown as well as a previously described null mutant¹⁴ to examine its role in a multicellular organism in vivo. We found that Vps15 is required not only for stress-induced autophagy in multiple tissues, but it is also a broad regulator of developmentally programmed autophagy in Drosophila. In addition, Vps15 is necessary for efficient protein secretion, as indicated by its role in the secretion of glue proteins from the Drosophila salivary gland. Together, these results highlight the importance of Vps15 in multiple processes in vivo.