Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

An Atg1/Atg13 Complex with Multiple Roles in TOR-mediated Autophagy Regulation

The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.     

Contribution of Atg1-Dependent Autophagy to TOR-Mediated Cell Growth and Survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.

Addendum to:

Direct Induction of Autophagy by Atg1 Inhibits Cell Growth and Induces Apoptotic Cell Death

R.C. Scott, G. Juhász and T.P. Neufeld

Curr Biol 2007; 17:1-11     

Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death

BACKGROUND: To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell-growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, resulting in part from the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1. RESULTS: We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild-type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself. CONCLUSIONS: Our results reveal a central role for Atg1 in mounting a coordinated autophagic response and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.     

Contribution of Atg1-dependent autophagy to TOR-mediated cell growth and survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.     

Regulation of cell growth by autophagy

Cell growth–the primary determinant of cell size–has an intimate relationship with proliferation; cells divide only after they reach a critical size. Despite its developmental and medical significance, little is known about cellular pathways that mediate the growth of cells. Accumulating evidence demonstrates a role for autophagy–a mechanism of eukaryotic cells to digest their own constituents during development or starvation–in cell size control. Increasing autophagic activity by prolonged starvation, rapamycin treatment inhibiting TOR (target of rapamycin) signaling, or genetic intervention, causes cellular atrophy in worms, flies and mammalian cell cultures. In contrast, we have shown that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes, unc-51/Atg1 and bec-1/Atg6, confers reduced cell size. We argue that physiological levels of autophagy are required for normal cell size, whereas both insufficient and excessive levels of autophagy lead to retarded cell growth. Furthermore, we discuss data suggesting that the insulin/IGF-1 (insulin-like growth factor receptor-1) and TGFβ (transforming growth factor-beta) signaling systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a central regulatory mechanism of cell growth.

Addendum to: Aladzsity I, Tóth ML, Sigmond T, Szabó E., Bicsák B, Barna J, Reg?s A, Orosz L, Kovács AL, Vellai T. Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans. Genetics 2007; 177:655-60, DOI: 10.1534/genetics.107.075762     

Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis

Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.     

The TOR Pathway Modulates the Structure of Cell Walls in Arabidopsis

Plant cell growth is limited by the extension of cell walls, which requires both the synthesis and rearrangement of cell wall components in a controlled fashion. The target of rapamycin (TOR) pathway is a major regulator of cell growth in eukaryotes, and inhibition of this pathway by rapamycin reduces cell growth. Here, we show that in plants, the TOR pathway affects cell wall structures. LRR-extensin1 (LRX1) of Arabidopsis thaliana is an extracellular protein involved in cell wall formation in root hairs, and lrx1 mutants develop aberrant root hairs. rol5 (for repressor of lrx1) was identified as a suppressor of lrx1. The functionally similar ROL5 homolog in yeast, Ncs6p (needs Cla4 to survive 6), was previously found to affect TOR signaling. Inhibition of TOR signaling by rapamycin led to suppression of the lrx1 mutant phenotype and caused specific changes to galactan/rhamnogalacturonan-I and arabinogalactan protein components of cell walls that were similar to those observed in the rol5 mutant. The ROL5 protein accumulates in mitochondria, a target of the TOR pathway and major source of reactive oxygen species (ROS), and rol5 mutants show an altered response to ROS. This suggests that ROL5 might function as a mitochondrial component of the TOR pathway that influences the plant''s response to ROS.     

Regulation of cell growth by autophagy

Cell growth-the primary determinant of cell size-has an intimate relationship with proliferation; cells divide only after they reach a critical size. Despite its developmental and medical significance, little is known about cellular pathways that mediate the growth of cells. Accumulating evidence demonstrates a role for autophagy-a mechanism of eukaryotic cells to digest their own constituents during development or starvation-in cell size control. Increasing autophagic activity by prolonged starvation, rapamycin treatment inhibiting TOR (target of rapamycin) signaling, or genetic intervention, causes cellular atrophy in worms, flies and mammalian cell cultures. In contrast, we have shown that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes, unc-51/Atg1 and bec-1/Atg6, confers reduced cell size. We argue that physiological levels of autophagy are required for normal cell size, whereas both insufficient and excessive levels of autophagy lead to retarded cell growth. Furthermore, we discuss data suggesting that the insulin/IGF-1 (insulin-like growth factor receptor-1) and TGF-beta (transforming growth factor-beta) signaling systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a central regulatory mechanism of cell growth.     

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.     

Regulation of Autophagy by the p300 Acetyltransferase

Autophagy is a regulated process of intracellular catabolism required for normal cellular maintenance, as well as serving as an adaptive response under various stress conditions, including starvation. The molecular regulation of autophagy in mammalian cells remains incompletely understood. Here we demonstrate a role for protein acetylation in the execution and regulation of autophagy. In particular, we demonstrate that the p300 acetyltransferase can regulate the acetylation of various known components of the autophagy machinery. Knockdown of p300 reduces acetylation of Atg5, Atg7, Atg8, and Atg12, although overexpressed p300 increases the acetylation of these same proteins. Furthermore, p300 and Atg7 colocalize within cells, and the two proteins physically interact. The interaction between p300 and Atg7 is dependent on nutrient availability. Finally, we demonstrate that knockdown of p300 can stimulate autophagy, whereas overexpression of p300 inhibits starvation-induced autophagy. These results demonstrate a role for protein acetylation and particularly p300 in the regulation of autophagy under conditions of limited nutrient availability.Macro-autophagy, herein referred to as autophagy, is an evolutionary conserved process first characterized in lower organisms (1). In yeast, over 20 separate genes (designated ATG1, ATG2, etc.) have been demonstrated to be essential to carry out the autophagy program. This process is thought to provide a mechanism for the efficient removal of both long lived proteins and damaged cellular organelles. This regulated degradation provides several essential functions for the cell. First, it allows for the removal of damaged and potentially harmful cellular contents. In addition, in breaking down various intracellular components, the autophagy process provides essential building blocks for the cell to use in the re-synthesis of necessary macromolecules. To accomplish this recycling effort, the coordinated actions of various Atg gene products are required. In particular, the Atg gene products together orchestrate the formation of a double membrane structure known as the autophagosome that engulfs the intended cellular cargo targeted for degradation. The autophagosome eventually fuses with the vacuole in yeast or the lysosome in mammals.In both yeast and mammalian cells, autophagy can be stimulated by the withdrawal of nutrients. Under these conditions, autophagic degradation of nonessential components may be essential to meet ongoing energetic needs in the presence of limited extracellular nutrients. This point was underscored by the analysis of mice containing a targeted deletion of Atg5 (2). In the absence of Atg5, there is a lack of both basal and starvation-induced autophagy. Mice lacking Atg5 are born normally but succumb within the 1st day of life. This post-natal lethality is thought to be due in large part for the requirement of autophagy to supply the energetic needs of neonates. These needs are particularly critical during the small window of time where the animal no longer has a placental circulation and before the pup can begin to nurse and thus obtain external nutrients.Relatively little is known regarding how signals such as nutrient availability are able to be transduced to ultimately regulate the level of cellular autophagy. One important pathway that impinges on the process is signaling thorough the target of rapamycin (TOR)2 network (3). Evidence suggests that TOR signaling inhibits autophagy, and indeed agents such as rapamycin that can inhibit TOR are known to result in increased autophagy. We recently have observed that in addition to this mode of regulation, the NAD-dependent deacetylase Sirt1 is also a regulator of autophagy in mammalian cells and tissues (4). In particular, we demonstrated that in the absence of Sirt1 levels of acetylation for various components of the autophagy machinery are increased and that starvation-induced autophagy is impaired. Interestingly, like the Atg5 knock-out animals, Sirt1^-/- mice are also born normally but die within the few hours to days after birth. Consistent with a defect in autophagy, electron micrographs of hearts from Sirt1^-/- mice demonstrated an accumulation of abnormal appearing organelles, including mitochondria, a phenotype previously observed in Atg-deficient animals (5). Here we have further characterized the role of acetylation in the regulation of autophagy, and in particular, we demonstrate a role for the p300 acetyltransferase in this process.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Small Molecule Enhancers of Rapamycin-Induced TOR Inhibition Promote Autophagy,Reduce Toxicity in Huntington’s Disease Models and Enhance Killing of Mycobacteria by Macrophages

Upregulation of autophagy may have therapeutic benefit in a range of diseases that include neurodegenerative conditions caused by intracytosolic aggregate-prone proteins, such as Huntington’s disease, and certain infectious diseases, such as tuberculosis. The best-characterized drug that enhances autophagy is rapamycin, an inhibitor of the TOR (target of rapamycin) proteins, which are widely conserved from yeast to man. Unfortunately, the side effects of rapamycin, especially immunosuppression, preclude its use in treating certain diseases including tuberculosis, which accounts for approximately 2 million deaths worldwide each year, spurring interest in finding novel drugs that selectively enhance autophagy. We have recently reported a novel two-step screening process for the discovery of such compounds. We first identified compounds that enhance the growth-inhibitory effects of rapamycin in the budding yeast Saccharomyces cerevisiae, which we termed small molecule enhancers of rapamycin (SMERs). Next we showed that three SMERs induced autophagy independently, or downstream of mTOR, in mammalian cells, and furthermore enhanced the clearance of a mutant huntingtin fragment in cell disease models. These SMERs also protected against mutant huntingtin fragment toxicity in Drosophila. We have subsequently tested two of the SMERs in models of tuberculosis and both enhance the killing of mycobacteria by primary human macrophages.

Addendum to:

Small Molecules Enhance Autophagy and Reduce Toxicity in Huntington's Disease Models

S. Sarkar, E.O. Perlstein, S. Imarisio, S. Pineau, A. Cordenier, R.L. Maglathlin, J.A. Webster, T.A. Lewis, C.J. O'Kane, S.L. Schreiber and D.C. Rubinsztein

Nat Chem Biol 2007; 3:331-8     

Regulation of macroautophagy by mTOR and Beclin 1 complexes

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.     

Axonal damage, autophagy and neuronal survival

In recent years autophagy modulation has been shown to reduce or increase neuronal cell death in several models of neurodegeneration. How autophagy exerts these dual effects is currently unknown. Here we review recent evidence from our laboratory demonstrating that autophagy can protect the cell soma after axonal traumatic injury. Damage in the optic nerve induces retinal ganglion cell (RGC) death in glaucoma and other retinal diseases and is often modeled by axotomy of the optic nerve in laboratory animals. Using this well-known model of RGC degeneration we show that autophagy is strongly upregulated following the insult and before cell death. Enhancement of autophagy by pharmacological treatment with rapamycin decreases the number of degenerating neurons. Conversely, axotomy in Atg4B (-/-) mice increases the number of dying cells in the retinal ganglion cell layer. Similar findings were observed in Atg5 (flox/flox) mice following specific downregulation of the autophagy regulator ATG5 in RGCs, by intravitreal injection of a cre-expressing vector. Taken together, these findings point to a cytoprotective role of autophagy following axonal damage in vivo.     

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells.     

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage

Premenopausal women have better survival than men after intracerebral hemorrhage, which is associated with iron overproduction and autophagy induction. To examine the participation of neuronal autophagy and estrogen receptor α (ERα) in the E₂–mediated protection, PC12 neurons treated with Atg7 (autophagy-related protein 7) siRNA, rapamycin (an autophagy inducer), or Erα siRNA were applied. To study whether autophagy involves in β-estradiol 3-benzoate (E₂)-mediated neuroprotection against iron-induced striatal injury, castration and E₂ capsule implantation were performed at 2 weeks and 24 h, respectively, before ferrous citrate (FC) infusion into the caudate nucleus (CN) of Sprague Dawley male and female rats. Furthermore, the role of neuronal autophagy in the sex difference of FC-induced CN injury was confirmed by using conditional knockout Atg7 in dopamine receptor 2 (DRD2)-containing neurons in mice. The results showed that the suppression of FC-induced autophagy by E₂ was abolished by Erα siRNA preincubation. Atg7 silencing simulates and rapamycin diminishes E₂-mediated neuroprotection against FC-induced neurotoxicity. In vivo, FC induced a lower degree of autophagy, autophagic cell death, injury severity, histological lesion and behavioral deficit in female rats than in males. E₂ implantation decreased the levels of both FC-induced autophagy and injury in ovariectomized rats. Moreover, the sex difference of FC-induced CN injury was diminished in Atg7 knockout mice. Thus, suppression of autophagy by E₂ via ERα contributes to less severity of iron-induced brain injury in females than in male. This finding opens up the prospect for a therapeutic strategy targeting autophagic inhibition for patients suffering from intracerebral iron overload.