Non-canonical autophagy: an exception or an underestimated form of autophagy?

Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.     

Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5(-/-) mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5( flox/flox);Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

IFN-α/β and autophagy

Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral non-structural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated anti-viral signaling protein (MAVS) and Toll/interleukin-1 receptor domain containing adaptor inducing IFN-β (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFN-α/β signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFN-α/β in the liver remains unclear.     

Does autophagy contribute to cell death?

Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process where the cytoplasmic contents of a cell are sequestered within double membrane vacuoles, called autophagosomes, and subsequently delivered to the lysosome for degradation. Autophagy can function as a survival mechanism in starving cells. At the same time, extensive autophagy is commonly observed in dying cells, leading to its classification as an alternative form of programmed cell death. The functional contribution of autophagy to cell death has been a subject of great controversy. However, several recent loss-of-function studies of autophagy (atg) genes have begun to address the roles of autophagy in both cell death and survival. Here, we review the emerging evidence in favor of and against autophagic cell death, discuss the possible roles that autophagic degradation might play in dying cells, and identify salient issues for future investigation.     

A bird’s-eye view of autophagy

Autophagy is a process in which a eukaryotic (but not prokaryotic) cell destroys its own components through the lysosomal machinery. This tightly regulated process is essential for normal cell growth, development, and homeostasis, serving to maintain a balance between synthesis and degradation, resulting in the recycling of cellular products. Here we try to expand the concept of autophagy and define it as a general mechanism of regulation encompassing various levels of the biosphere. Interestingly, one of the consequences of such an approach is that we must presume an existence of the autophagic processes in the prokaryotic domain.     

Are nidoviruses hijacking the autophagy machinery?

Autophagy is an intracellular catabolic transport route conserved among all eukaryotic cells. It has multiple important physiological functions, one of which is to act as an immune mechanism against intracellular microbes.(1,2) Bacteria and viruses targeted for destruction are sequestered into large double-membrane vesicles called autophagosomes and subsequently delivered to the lysosomes where they are consumed by resident hydrolases. Unfortunately, conserved cellular pathways are often exploited by pathogens to facilitate their entry or replication, and autophagy is no exception. It has become clear that certain bacteria and viruses subvert this process to their advantage.(3) Nidoviruses, which comprise coronaviruses and arteriviruses, might be among the successful. Long recognized as the cause of veterinary infections, this group of enveloped, positive-stranded RNA viruses is currently of considerable interest due to the emergence of new human coronaviruses, especially the severe acute respiratory syndrome (SARS)-coronavirus. In this mini-review, we will summarize the so far limited number of studies that have demonstrated that double-membrane vesicles resembling autophagosomes are the sites of nidovirus replication. In addition, we will discuss how the formation of these large vesicles might be induced.     

The Parkinson disease protein α-synuclein inhibits autophagy

Parkinson disease (PD) is the most common movement disorder affecting people. It is characterized by the accumulation of the protein α-synuclein in Lewy body inclusions in vulnerable neurons. α-Synuclein overexpression caused by gene multiplications is sufficient to cause this disease, suggesting that α-synuclein accumulation is toxic. Here we review our recent study showing that α-synuclein inhibits autophagy. We discuss our mechanistic understanding of this phenomenon and also speculate how a deficiency in autophagy may contribute to a range of pleiotropic features of PD biology.     

The Parkinson disease protein α-synuclein inhibits autophagy

Parkinson disease (PD) is the most common movement disorder affecting people. It is characterized by the accumulation of the protein α-synuclein in Lewy body inclusions in vulnerable neurons. α-Synuclein overexpression caused by gene multiplications is sufficient to cause this disease, suggesting that α-synuclein accumulation is toxic. Here we review our recent study showing that α-synuclein inhibits autophagy. We discuss our mechanistic understanding of this phenomenon and also speculate how a deficiency in autophagy may contribute to a range of pleiotropic features of PD biology.     

α-synuclein fate: Proteasome or autophagy?

The accumulation of α-synuclein is critical for the development of Parkinson disease (PD), and unraveling the mechanisms that regulate α-synuclein levels is key to understanding the pathophysiology of the disease. We recently found that USP9X deubiquitinates α-synuclein, and that this process determines the partition of α-synuclein between the proteasomal and autophagy pathways. By manipulating USP9X levels, we observed that monoubiquitinated α-synuclein is degraded by the proteasome, whereas deubiquitination of α-synuclein favors its degradation by autophagy. As USP9X levels and activity are decreased in α-synucleinopathy brains, USP9X may now represent a novel target for PD.     

p53: The Janus of autophagy?

The autophagy pathway functions in adaptation to nutrient stress and tumour suppression. The p53 tumour suppressor, previously thought to positively regulate autophagy, may also inhibit it. This dual interplay between p53 and autophagy regulation is enigmatic, but may underlie key aspects of metabolism and cancer biology.     

Why do we need autophagy? A cartoon depiction

In my role as an instructor I am constantly looking for ways to more effectively convey information to my audience, which is typically the students in my class. However, the same concerns apply to most of the people who attend a seminar. The approach you take to making the material easier to understand is likely to be influenced by the course you teach. That is, the same instructor may use different examples when teaching an upper division vs. a lower division course. I teach introductory biology, and my students may have little familiarity with cell biology, let alone autophagy. Accordingly, I have tried to consider how to illustrate the importance of autophagy in a way that can be comprehended by people who may not even be familiar with the term.     

Antigens and autophagy: the path less travelled?

The Epstein-Barr virus (EBV)-coded nuclear antigen (EBNA) 1, a latent cycle protein endogenously expressed in EBV-transformed B lymphoblastoid cell lines (LCLs), is reported to be processed for CD4(+) T cell recognition by an intracellular route involving antigen delivery to the endosome/lyosome (MHC class II loading) compartment via macroautophagy. In contrast we find that, in the same cell type, two other virus-coded nuclear proteins of the latent cycle, EBNA2 and EBNA3C, are processed by a different route that is unaffected by autophagy inhibition. This involves the intercellular transfer of an antigenic moiety, detectable in cell-free culture supernatants, and its uptake and processing as exogenous antigen by neighboring cells. The process is cumulative and leads over several days of LCL culture to high levels of CD4+ T cell epitope display. The presentation of certain EBV lytic cycle proteins to CD4+ T cells has also recently been found to involve a similar intercellular antigen transfer. It becomes important to know why, even in the same cell type, some antigens but not others appear to access the MHC class II presentation pathway by autophagy.     

Heat shock response and autophagy—cooperation and control

Protein quality control (proteostasis) depends on constant protein degradation and resynthesis, and is essential for proper homeostasis in systems from single cells to whole organisms. Cells possess several mechanisms and processes to maintain proteostasis. At one end of the spectrum, the heat shock proteins modulate protein folding and repair. At the other end, the proteasome and autophagy as well as other lysosome-dependent systems, function in the degradation of dysfunctional proteins. In this review, we examine how these systems interact to maintain proteostasis. Both the direct cellular data on heat shock control over autophagy and the time course of exercise-associated changes in humans support the model that heat shock response and autophagy are tightly linked. Studying the links between exercise stress and molecular control of proteostasis provides evidence that the heat shock response and autophagy coordinate and undergo sequential activation and downregulation, and that this is essential for proper proteostasis in eukaryotic systems.     

Crosstalk between translation and the aggresome–autophagy pathway

Many neurodegenerative disorders feature the presence of misfolded polypeptide-containing intracellular inclusion bodies biochemically and morphologically analogous to cellular aggresomes. However, it is largely unknown how misfolded polypeptides form aggresomes and are eventually cleared by the aggresome-macroautophagy/autophagy pathway, so-called aggrephagy. Our recent study revealed that when the ubiquitin-proteasome system is impaired, the accumulated misfolded polypeptides are selectively recognized and transported to the aggresome by a CED complex. This complex is composed of CTIF, originally identified as a specific factor for nuclear cap-binding protein complex (a heterodimer of NCBP1/CBP80 and NCBP2/CBP20)-dependent translation (CT), and its associated factors EEF1A1 and DCTN1. Aggresomal targeting of a misfolded polypeptide via the CED complex is accompanied by CTIF release from the CT complex and thereby inhibits CT efficiency. Therefore, our study provides new mechanistic insights into the crosstalk between translational inhibition and aggresome formation under the influence of a misfolded polypeptide.     

Ubiquitination of α-synuclein and autophagy in Parkinson's disease

α-synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of α-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of α-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates α-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of α-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated α-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated α-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated α-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing α-synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.

Addendum to: Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E, Engelender S. Monoubiquitination of α-synuclein by SIAH promotes its aggregation in dopaminergic cells. J Biol Chem 2007; Epub ahead of print.     

Cardioprotection through autophagy: Ready for clinical trial?

Interventions that reduce infarct size in animal models have largely failed to improve outcome in patients suffering acute myocardial infarction (MI), or 'heart attack'. Our group recently reported a reduction of infarct size by chloramphenicol treatment in a porcine in vivo model of acute MI, through a mechanism involving the induction of autophagy. Since 2005 several studies have implicated autophagy as a target for cardioprotection.     

Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity

All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.Macroautophagy (hereafter referred to as autophagy) is a conserved mechanism characterized by the formation of double-membrane structures. These so-called autophagosomes deliver cytoplasmic material to the lysosome for subsequent degradation.¹ Basal autophagy requires tight regulation as alterations in autophagy have been associated with many pathological conditions, including cancer.² In addition, autophagy has been linked to fundamental processes such as development and cellular differentiation. In these processes, autophagy contributes to cell remodeling as observed during erythrocyte, lymphocyte or adipocyte differentiation.³ In the context of cancer and cancer therapy, autophagy is a double-edged sword. Owing to its homeostatic role in the removal of potentially harmful damaged organelles and protein aggregates, it is suggested to be tumor suppressive under normal conditions.⁴ In cancer cells, however, autophagy can be oncogenic, enabling survival under stressful conditions.⁵ Hence, the role of autophagy in tumorigenesis is clearly dependent on the cellular context and the tumor stage. In some cases, therapeutic agents induce an autophagic response that can promote resistance to treatment. In other cases, autophagy contributes to the action of antitumor agents.⁶ Therefore, knowledge about the action exerted by autophagy in response to anticancer treatments is a prerequisite for the identification of patients benefiting from therapeutic strategies based on autophagy modulators.All-trans retinoic acid (ATRA), the active metabolite of vitamin A, exerts diverse functions in almost every cell and organ system. ATRA controls cell proliferation, differentiation as well as immune, and neuronal functions primarily via regulation of gene expression.⁷ Endogenous retinoid levels are altered in different diseases of the lung, kidney and central nervous system, and contribute to their pathophysiology.⁸ ATRA is successfully used in the treatment of acute promyelocytic leukemia (APL), where it induces granulocytic differentiation of the blast and subsequent cell death of the differentiated leukemic cells. Importantly, ATRA-induced differentiation of the APL cell line, NB4, involves induction of macroautophagy.^{9, 10, 11, 12} In addition to its cytodifferentiating capacity in APL, ATRA has been proposed as an antitumorigenic agent for other types of cancer. The antiproliferative, cytodifferentiating and proapoptotic effects of retinoids are predominantly mediated by the nuclear hormone retinoid acid receptors RARα, RARβ and RARγ.^{13, 14} In breast cancer, preclinical studies have shown that retinoids are promising therapeutic agents. However, the clinical trials conducted so far were somewhat disappointing, possibly as a consequence of the study designs.¹⁵ Breast cancer is a highly heterogeneous disease represented as a collection of diseases with distinct histopathological and molecular features. The most important clinical classification of this tumor is based on the determination of ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor-2) receptors. ER-positive breast cancer patients are eligible for hormonal therapies, whereas HER2 oncogenic activity can be blocked using targeted therapies.¹⁶ Approximately 15–20% of breast carcinomas overexpress HER2, which is associated with poor prognosis.¹⁷ Owing to the development of resistance to current HER2-targeted treatments such as trastuzumab and lapatinib alternative therapeutic strategies are required.^{18, 19} ATRA was recently shown to exert strong antitumor activity in cell lines representing a subgroup of HER2-positive breast tumors characterized by coamplification of the ERBB2 and RARα genes.²⁰ This antitumor activity is remarkably stimulated by simultaneous HER2 inhibition with lapatinib. In addition, autophagy is induced upon ATRA treatment of the APL-derived cell line NB4^{9, 10, 11} and retinoids have clinical relevance in breast cancer. Thus, we investigated whether and how autophagy is induced in breast cancer cells. In addition, we evaluated whether autophagy modulation represents a potential therapeutic strategy for potentiating ATRA cytotoxicity in breast cancer cells.     

AMPKα promotes basal autophagy induction in Dictyostelium discoideum

Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5′-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα⁻ cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα⁻ cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα⁻ cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK–ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, β-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα⁻ cells exhibit enhanced poly-ubiquitinated protein levels and allied large ubiquitin-positive protein aggregates. Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Does autophagy have a license to kill mammalian cells?

Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime self-destruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death.