The in vitro cleavage of the hAtg proteins by cell death proteases

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.     

Pivotal role of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in apoptosis and autophagy

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C(2)-ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21(+/+) mouse embryonic fibroblasts (MEFs). On the other hand, C(2)-ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21(-/-) MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21(+/+) MEFs treated with C(2)-ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C(2)-ceramide-treated p21(-/-) MEFs. gamma-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.     

Epistatic interaction of Arg72Pro TP53 and −710 C/T VEGFR1 polymorphisms in breast cancer: predisposition and survival

Arginine deprivation is a promising strategy for treating ASS-negative malignant tumors including melanoma. However, autophagy can potentially counteract the effectiveness of this treatment by acting as a pro-survival pathway. By combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with arginine deprivation using ADI-PEG20 (pegylated arginine deiminase), we achieved enhanced apoptosis and accelerated cell death in melanoma cell lines. This implies a switch from autophagy to apoptosis. In our current investigation, we found that TRAIL could induce the cleavage of two key autophagic proteins, Beclin-1 and Atg5, in the combination treatment. Using specific inhibitors for individual caspases, we found that caspase-8 inhibitor could completely abolish the cleavage. Furthermore, caspase-8 inhibitor was able to fully reverse the enhanced cytotoxicity induced by TRAIL. Inhibitors for caspase-3, 6, 9, and 10 were able to block the cleavage of these two autophagic proteins to some extent and correspondingly rescue cells from the cytotoxicity of the combination of TRAIL and arginine deprivation. In contrast, calpain inhibitor could not prevent the cleavage of either Beclin-1 or Atg5, and was unable to prevent cell death. Overall, our data indicate that the cleavage of Beclin-1 and Atg5 by TRAIL-initiated caspase activation is one of the mechanisms that lead to the enhancement of the cytotoxicity in the combination treatment.     

Autophagy delays apoptotic death in breast cancer cells following DNA damage

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.     

A Complex between Atg7 and Caspase-9: A NOVEL MECHANISM OF CROSS-REGULATION BETWEEN AUTOPHAGY AND APOPTOSIS*

Several cross-talk mechanisms between autophagy and apoptosis have been identified, in which certain co-regulators are shared, allowing the same protein to participate in these opposing processes. Our studies suggest that caspase-9 is a novel co-regulator of apoptosis and autophagy and that its caspase catalytic activity is dispensable for its autophagic role. We provide evidence that caspase-9 facilitates the early events leading to autophagosome formation; that it forms a complex with Atg7; that Atg7 is not a direct substrate for caspase-9 proteolytic activity; and that, depending on the cellular context, Atg7 represses the apoptotic capability of caspase-9, whereas the latter enhances the Atg7-mediated formation of light chain 3-II. The repression of caspase-9 apoptotic activity is mediated by its direct interaction with Atg7, and it is not related to the autophagic function of Atg7. We propose that the Atg7·caspase-9 complex performs a dual function of linking caspase-9 to the autophagic process while keeping in check its apoptotic activity.     

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress,Autophagy, and Apoptosis

Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.     

Atg14L and Rubicon: Yin and yang of Beclin 1-mediated autophagy control

Emerging evidence suggests that Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, functions to coordinate two important cellular pathways: autophagy and apoptosis. Beclin 1 is a component of the Vps34/class III phosphatidylinositol 3-kinase (PtdIns3K) protein complex. However, the Beclin 1-Vps34/PtdIns3K protein complex formation and its function in autophagy regulation remain to be elucidated. Through an integrated approach that combines mouse genetics and biochemistry, we identified two novel Beclin 1 interacting proteins, Atg14L and Rubicon. We found that Atg14L and Rubicon play opposing roles in autophagy regulation by forming distinct complexes with Beclin 1, modulating the Vps34/PtdIns3K activity and targeting distinct steps of the autophagic process.     

Human hAtg2A protein expressed in yeast is recruited to preautophagosomal structure but does not complement autophagy defects of atg2Δ strain

Yeast Atg2, an autophagy-related protein, is highly conserved in other fungi and has two homologues in humans, one of which is hAtg2A encoded by the hATG2A/KIAA0404 gene. Region of homology between Atg2 and hAtg2A proteins comprises the C-terminal domain. We used yeast atg2D strain to express the GFP-KIAA0404 gene, its fragment or fusions with yeast ATG2, and study their effects on autophagy. The GFP-hAtg2A protein localized to punctate structures, some of which colocalized with Ape1-RFP-marked preautophagosomal structure (PAS), but it did not restore autophagy in atg2Δ cells. N-terminal fragment of Atg2 and N-terminal fragment of hAtg2A were sufficient for PAS recruitment but were not sufficient to function in autophagy. Neither a fusion of the N-terminal fragment of hAtg2A with C-terminal domain of Atg2 nor a reciprocal fusion were functional in autophagy. hAtg2A, in contrast to yeast Atg2, did not show interaction with the yeast autophagy protein Atg9 but both Atg2 proteins showed interaction with Atg18, a phospholipid-binding protein, in two-hybrid system. Moreover, deletion of ATG18 abrogated PAS recruitment of hAtg2A. Our results show that human hAtg2A can not function in autophagy in yeast, however, it is recruited to the PAS, possibly due to the interaction with Atg18.     

Beclin 1-Independent Pathway of Damage-Induced Mitophagy and Autophagic Stress: Implications for Neurodegeneration and Cell Death

Growing evidence supports an active role for dysregulated macroautophagy (autophagic stress) in neuronal cell death and neurodegeneration. Alterations in mitochondrial function and dynamics are also strongly implicated in neurodegenerative diseases. Interestingly, whereas the core autophagy machinery is evolutionarily conserved and shared among constitutive and induced or selective autophagy, recent studies implicate distinct mechanisms regulating mitochondrial autophagy (mitophagy) in response to general autophagic stimuli. Little is known about pathways regulating selective, damage-induced mitophagy. We found that the parkinsonian neurotoxin MPP+ induces autophagy and mitochondrial degradation that is inhibited by siRNA knockdown of autophagy proteins Atg5, Atg7 and Atg8, but occurs independently of Beclin 1, a component of the class III (PIK3C3/Vps34) phosphoinositide 3-kinase (PI3K) complex. Instead, MPP+-induced mitophagy is dependent upon MAPK signaling. Interestingly, all treatments that inhibited autophagy also conferred protection from MPP+-induced cell death. A prior human tissue study further supports a role for ERK/MAPK-regulated autophagy in Parkinson's and Lewy body diseases. As competition for limiting amounts of Beclin 1 may serve to prevent harmful overactivation of autophagy, understanding mechanisms that bypass or complement a requirement for PI3K-Beclin 1 activity could lead to strategies to modulate harmful autophagic stress in injured or degenerating neurons.     

Beclin 1-independent pathway of damage-induced mitophagy and autophagic stress: implications for neurodegeneration and cell death

Growing evidence supports an active role for dysregulated macroautophagy (autophagic stress) in neuronal cell death and neurodegeneration. Alterations in mitochondrial function and dynamics are also strongly implicated in neurodegenerative diseases. Interestingly, whereas the core autophagy machinery is evolutionarily conserved and shared among constitutive and induced or selective autophagy, recent studies implicate distinct mechanisms regulating mitochondrial autophagy (mitophagy) in response to general autophagic stimuli. Little is known about pathways regulating selective, damage-induced mitophagy. We found that the parkinsonian neurotoxin MPP(+) induces autophagy and mitochondrial degradation that is inhibited by siRNA knockdown of autophagy proteins Atg5, Atg7 and Atg8, but occurs independently of Beclin 1, a component of the class III (PIK3C3/Vps34) phosphoinositide 3-kinase (PI3K) complex. Instead, MPP(+)-induced mitophagy is dependent upon MAPK signaling. Interestingly, all treatments that inhibited autophagy also conferred protection from MPP(+)-induced cell death. A prior human tissue study further supports a role for ERK/MAPK-regulated autophagy in Parkinson's and Lewy body diseases. As competition for limiting amounts of Beclin 1 may serve to prevent harmful overactivation of autophagy, understanding mechanisms that bypass or complement a requirement for PI3K-Beclin 1 activity could lead to strategies to modulate autophagic stress in injured or degenerating neurons.     

Cleaving Beclin 1 to suppress autophagy in chemotherapy-induced apoptosis

Autophagy is often found in apoptosis-defective cancer cells and contributes to chemotherapy resistance. However, it is far from clear how the coordination of apoptosis and autophagy determines sensitivity of cancer cells to chemotherapy. Our recent study showed that Beclin 1, a key regulator of autophagy, is cleaved by caspase 8 at the execution stage of chemotherapy-induced and mitochondria-mediated apoptosis. Perturbation of Beclin 1 cleavage, by knock-in of a mutation, phenocopies the autophagy observed in apoptosis-defective cancer cells, and renders chemotherapy resistance in vitro and in vivo. These results demonstrate an important role of caspases in suppressing autophagy by cleaving autophagic machinery.     

Cleaving Beclin 1 to suppress autophagy in chemotherapy-induced apoptosis

Autophagy is often found in apoptosis-defective cancer cells and contributes to chemotherapy resistance. However, it is far from clear how the coordination of apoptosis and autophagy determines sensitivity of cancer cells to chemotherapy. Our recent study showed that Beclin 1, a key regulator of autophagy, is cleaved by caspase 8 at the execution stage of chemotherapy-induced and mitochondria-mediated apoptosis. Perturbation of Beclin 1 cleavage, by knock-in of a mutation, phenocopies the autophagy observed in apoptosis-defective cancer cells, and renders chemotherapy resistance in vitro and in vivo. These results demonstrate an important role of caspases in suppressing autophagy by cleaving autophagic machinery.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death

The term "autophagic cell death" was coined to describe a form of cell death associated with the massive formation of autophagic vacuoles without signs of apoptosis. However, questions about the actual role of autophagy and its molecular basis in cell death remain to be elucidated. We recently reported that adult hippocampal neural stem (HCN) cells undergo autophagic cell death following insulin withdrawal. Insulin-deprived HCN cells exhibit morphological and biochemical markers of autophagy, including accumulation of Beclin 1 and the type II form of microtubule-associated protein 1 light chain 3 (LC3) without evidence of apoptosis. Suppression of autophagy by knockdown of Atg7 reduces cell death, whereas promotion of autophagy with rapamycin augments cell death in insulin-deficient HCN cells. These data reveal a causative role of autophagy in insulin withdrawal-induced HCN cell death. HCN cells have intact apoptotic capability despite the lack of apoptosis following insulin withdrawal. Our study demonstrates that autophagy is the default cell death mechanism in insulin-deficient HCN cells, and provides a genuine model of autophagic cell death in apoptosis-intact cells. Novel insight into molecular mechanisms of this underappreciated form of programmed cell death should facilitate the development of therapeutic methods to cope with human diseases caused by dysregulated cell death.     

Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.