A mitochondrial-associated link between an effector caspase and autophagic flux

It has become evident that caspases function in nonapoptotic cellular processes in addition to the canonical role for caspases in apoptotic cell death. We recently demonstrated that the Drosophila effector caspase Dcp-1 localizes to the mitochondria and positively regulates starvation-induced autophagic flux during mid-oogenesis. Loss of Dcp-1 leads to elongation of the mitochondrial network, increased levels of the adenine nucleotide translocase sesB, increased ATP levels, and a reduction in autophagy. We found that sesB is a negative regulator of autophagic flux, and Dcp-1 interacts with sesB in a nonproteolytic manner to regulate its stability, uncovering a novel mechanism of mitochondrial associated, caspase-mediated regulation of autophagy in vivo.     

Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy

How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type–specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn^S641,731D or the caspase-resistant Acn^D527A, exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels.     

Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.     

Mitochondria regulate autophagy by conserved signalling pathways

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

Synergistic induction of apoptosis and caspase-independent autophagic cell death by a combination of nitroxide Tempo and heat shock in human leukemia U937 cells

We have shown that heat stress or a superoxide dismutase mimic nitroxide, Tempo, induces apoptosis, while their combination causes nonapoptotic cell death; however, the underlying mechanism for this switch remains unclear. Here we identified for the first time that 10 mM Tempo present during heating at 44°C for 30 min rapidly induced autophagy in U937 leukemic cells in spite of Bax activation and mitochondrial outer membrane (MOM) permeabilization. This co-treatment inhibited the processing of heat-activated procaspases-2, -8, -9 and -3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy. The inactivation of caspases, a key event, could result from oxidation of active-site-CysSH of all caspases by a prooxidant oxo-ammonium cation, an intermediate derived Tempo during dismutation of heat-induced superoxide anion. In addition, the co-treatment caused mitochondrial calcium overloads, the mitochondrial inner membrane permeabilization, profound mitochondrial dysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction of autophagy. These autophagic cells underwent propidium iodide-positive necrosis in a delayed fashion, leading to the complete proliferative inhibition. Remarkably, ruthenium red and BAPTA, which interfere with mitochondrial calcium uptake, facilitated autophagic necrotic death. Cyclosporin A, which binds to cyclophilin D, had a similar necrotic effect. 3-Methyladenine facilitated the necrosis of autophagic cells. In contrast, 5 mM Tempo-44°C/10 min or 44°C/30 min induced Bax-mediated MOM permeabilization and caspase-dependent apoptosis more potently than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.     

Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Autophagic degradation of HAS2 in endothelial cells: A novel mechanism to regulate angiogenesis

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.     

UBE2D3 contributes to myocardial ischemia-reperfusion injury by regulating autophagy in dependence of p62/SQSTM1

The impairment of autophagic flux has been widely recognized in myocardial ischemia-reperfusion (I/R) injury, but its underlying mechanism contributing to impaired autophagic flux is poorly understood. As celluar major degradation systems, autophagy and ubiquitin proteasome system (UPS) participate in the multitudinous progression of disease by interactive relationship. Especially UBE2D3, one of the ubiquitin-binding enzyme E2 family, is closely related to the regulation impairment of autophagic flux under I/R in our study. Therefore, this study aims to further explore the regulatory mechanism of UBE2D3 in I/R induced autophagy. We determined interference with UBE2D3 alleviated injury of myocardial cells both in vivo and in vitro. Conversely, when inhibiting proteasome function by injecting MG-132, myocardial infarct size of rats became increasingly enhanced, along with the high expression levels of LDH and CK-MB in serum, compared with myocardial I/R injury without treatment of MG-132. This had been caused by UBE2D3 promoting p62/SQSTM1(p62) ubiquitination(Ub), which lead to worsen the impairment of autophagic flux induced by myocardial I/R injury. In addition, UBE2D3 could also participate in the regulation of autophagy by negatively regulating mTOR. But more surprisingly, this mechanism was independent of the known mTOR-beclin1 pathway. These results suggested that in myocardial I/R injury, UBE2D3 promoted p62 ubiquitination to aggravate the impairment of autophagic flux. Moreover, mTOR was also involved in its regulation of autophagic flux in a way escaped from beclin1.     

Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine

We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.

Addendum to: Berry DL, Baehrecke EH. Growth arrest and autophagy are required for programmed salivary gland cell degradation in Drosophila. Cell 2007; 131:1137-48.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.     

A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.     

Impaired autophagy contributes to hepatocellular damage during ischemia/reperfusion: Heme oxygenase-1 as a possible regulator

Liver ischemia and reperfusion (I/R) injury is characterized by oxidative stress that is accompanied by alterations of the endogenous defensive system. Emerging evidence suggests a protective role for autophagy induced by multiple stressors including reactive oxygen species. Meanwhile, heme oxygenase-1 (HO-1) has long been implicated in cytoprotection against oxidative stress in vitro and in vivo. Therefore, we investigated the impact of autophagy in the pathogenesis of liver I/R and its molecular mechanisms, particularly its linkage to HO-1. By using transmission electron microscopic analysis and biochemical autophagic flux assays with microtubule-associated protein 1 light chain 3-II, and beclin-1, representative autophagy markers, and p62, a selective substrate for autophagy, we found that reperfusion reduced autophagy both in the rat liver and in primary cultured hepatocytes. When autophagy was further inhibited with chloroquine or wortmannin, I/R-induced hepatocellular injury was aggravated. While livers that underwent I/R showed increased levels of mammalian target of rapamaycin and calpain 1 and 2, inhibition of calpain 1 and 2 induced an autophagic response in hepatocytes subjected to hypoxia/reoxygenation. HO-1 increased autophagy, and HO-1 reduced I/R-induced calcium overload in hepatocytes and prevented calpain 2 activation both in vivo and in vitro. Taken together, these findings suggest that the impaired autophagy during liver I/R, which is mediated by calcium overload and calpain activation, contributes to hepatocellular damage and the HO-1 system protects the liver from I/R injury through enhancing autophagy.