Table of Contents

Volume 7, Issue 12

  December 2011

  Pages 1405 - 1574

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 Issue Catalogue (MARC XML)

EDITOR'S CORNER

Open Access Article

Activation of autophagy is required for muscle homeostasis during physical exercise

Usha Nair and Daniel J. Klionsky
Pages 1405 - 1406
http://dx.doi.org/10.4161/auto.7.12.18315
Abstract | Full Text | PDF

Skeletal muscle fibers of collagen VI null (Col6a1−/−) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.




AUTOPHAGIC FLUX

Open Access Article

Highlights from this issue

Andriy Nemchenko
Pages 1407 - 1409
http://dx.doi.org/10.4161/auto.7.12.18505
Full Text | PDF




ARTOPHAGOSOME

Open Access Article

A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

Andy Bass, Dean Sauer and Daniel J. Klionsky
Pages 1410 - 1414
http://dx.doi.org/10.4161/auto.7.12.16991
Abstract | Full Text | PDF

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.




BASIC BRIEF REPORTS

Open Access Article

Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles

Paolo Grumati, Luisa Coletto, Alvise Schiavinato, Silvia Castagnaro, Enrico Bertaggia, Marco Sandri and Paolo Bonaldo
Pages 1415 - 1423
http://dx.doi.org/10.4161/auto.7.12.17877
Abstract | Full Text | PDF

Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1–/–) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1–/– mice, where autophagy flux is compromised. Autophagy was not induced in Col6a1–/– muscles after either acute or prolonged exercise, and this led to a marked increase of muscle wasting and apoptosis. These findings indicate that proper activation of autophagy is important for muscle homeostasis during physical activity.

Open Access Article

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Sebastian Alers, Antje S. Löffler, Florian Paasch, Alexandra M. Dieterle, Hildegard Keppeler, Kirsten Lauber, David G. Campbell, Birgit Fehrenbacher, Martin Schaller, Sebastian Wesselborg and Björn Stork
Pages 1424 - 1433
http://dx.doi.org/10.4161/auto.7.12.18027
Abstract | Full Text | PDF

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.




BASIC RESEARCH PAPERS

Open Access Article

Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation

Joseph G. Moloughney, Claude E. Monken, Hanlin Tao, Haiyan Zhang, Janice D. Thomas, Edmund C. Lattime and Shengkan V. Jin
Pages 1434 - 1447
http://dx.doi.org/10.4161/auto.7.12.17793
Abstract | Full Text | PDF

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

Open Access Article

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

Mario Mauthe, Anke Jacob, Sandra Freiberger, Katharina Hentschel, York-Dieter Stierhof, Patrice Codogno and Tassula Proikas-Cezanne
Pages 1448 - 1461
http://dx.doi.org/10.4161/auto.7.12.17802
Abstract | Full Text | PDF

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.

Open Access Article

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: Functional analysis of p62 and autophagy in hypoxic neuronal cells

Fumito Tanabe, Kazunori Yone, Naoya Kawabata, Harutoshi Sakakima, Fumiyo Matsuda, Yasuhiro Ishidou, Shingo Maeda, Masahiko Abematsu, Setsuro Komiya and Takao Setoguchi
Pages 1462 - 1471
http://dx.doi.org/10.4161/auto.7.12.17892
Abstract | Full Text | PDF

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

Open Access Article

Ins(1,4,5)P₃ receptor-mediated Ca²⁺ signaling and autophagy induction are interrelated

Jean-Paul Decuypere, Kirsten Welkenhuyzen, Tomas Luyten, Raf Ponsaerts, Michael Dewaele, Jordi Molgó, Patrizia Agostinis, Ludwig Missiaen, Humbert De Smedt, Jan B. Parys and Geert Bultynck
Pages 1472 - 1489
http://dx.doi.org/10.4161/auto.7.12.17909
Abstract | Full Text | PDF

The role of intracellular Ca²⁺ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca²⁺ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca²⁺ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca²⁺-store content and enhanced agonist-induced Ca²⁺ release. The mechanism involved the upregulation of intralumenal ER Ca²⁺-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca²⁺-buffering capacity and reduced the ER Ca²⁺ leak. Second, starvation led to Ins(1,4,5)P₃R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P₃Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P₃-binding domain of the Ins(1,4,5)P₃R. The starvation-induced Ins(1,4,5)P₃R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P₃Rs in ⁴⁵Ca²⁺-flux assays, indicating a direct regulation of Ins(1,4,5)P₃R activity by Beclin 1. Finally, we found that Ins(1,4,5)P₃R-mediated Ca²⁺ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P₃R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca²⁺ signaling and autophagy stimulation as a proximal event in response to starvation.

Open Access Article

The induction of autophagy by mechanical stress

Jason S. King, Douwe M. Veltman and Robert H. Insall
Pages 1490 - 1499
http://dx.doi.org/10.4161/auto.7.12.17924
Abstract | Full Text | PDF

The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.

Open Access Article

Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex

Koutaro Ishibashi, Naonobu Fujita, Eiko Kanno, Hiroko Omori, Tamotsu Yoshimori, Takashi Itoh and Mitsunori Fukuda
Pages 1500 - 1513
http://dx.doi.org/10.4161/auto.7.12.18025
Abstract | Full Text | PDF

A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.

Open Access Article

Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

Zhanying Hu, Jingpu Zhang and Qingyou Zhang
Pages 1514 - 1527
http://dx.doi.org/10.4161/auto.7.12.18040
Abstract | Full Text | PDF

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.

Open Access Article

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Lin Zheng, Alexei Terman, Martin Hallbeck, Nodi Dehvari, Richard F. Cowburn, Eirikur Benedikz, Katarina Kågedal, Angel Cedazo-Minguez and Jan Marcusson
Pages 1528 - 1545
http://dx.doi.org/10.4161/auto.7.12.18051
Abstract | Full Text | PDF

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.




PROTOCOL

Open Access Article

GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis

Usha Nair, Michael Thumm, Daniel J. Klionsky and Roswitha Krick
Pages 1546 - 1550
http://dx.doi.org/10.4161/auto.7.12.18424
Abstract | Full Text | PDF

Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.




AUTOPHAGIC PUNCTA

Open Access Article

A subunit of coatomer protein complex offers a novel tumor-specific target through a surprising mechanism

Michael Shtutman and Igor B. Roninson
Pages 1551 - 1552
http://dx.doi.org/10.4161/auto.7.12.17659
Abstract | Full Text | PDF

COPI, a coatomer protein complex of secretory vesicles, is involved in Golgi and endoplasmic reticulum traffic and in early endosome maturation. The loss of COPI results in the fragmentation of Golgi, accumulation of immature autophagosomes, inhibition of autophagy, and cell death. Since COPI is required by all cells, it would appear an unlikely target for cancer treatment. However, our recent function-based genomic screen unexpectedly identified a specific COPI subunit, ζ1, as a cancer-specific target. The existing cancer drugs kill only proliferating but not growth-arrested tumor cells, but the depletion of ζ1 induces cell death in both dividing and nondividing tumor cells, while sparing normal cells. The mechanism of this remarkable tumor selectivity turned out to be surprising and heretofore unprecedented.

Open Access Article

Evolution of the “autophagamiR”

Justin S. Gundara, Bruce G. Robinson and Stan B. Sidhu
Pages 1553 - 1554
http://dx.doi.org/10.4161/auto.7.12.17762
Abstract | Full Text | PDF

MicroRNAs (miRs) are increasingly important diagnostic and prognostic markers in cancer but have not been defined in medullary thyroid carcinoma (MTC). MiR microarray profiling was performed on 19 primary MTC tumors, validated with qPCR in 45 cases and correlated with clinical outcomes. MiRs-183 and 375 were overexpressed and miR-9* underexpressed in sporadic vs. hereditary MTC (SMTC; HMTC). MiR-183 and 375 overexpression predicted lateral nodal metastases, residual disease, distant metastases and mortality. MiR-183 knockdown in an MTC cell line (TT cells) reduced cellular proliferation in association with elevated LC3B expression. This is suggestive of increased autophagic flux and potential cell death via autophagy induction. MiRs may subsequently be shown to serve as efficacious therapeutic strategies in MTC with a mechanism based upon autophagy.

Open Access Article

Ambra1: A Parkin-binding protein involved in mitophagy

Cindy Van Humbeeck, Tom Cornelissen and Wim Vandenberghe
Pages 1555 - 1556
http://dx.doi.org/10.4161/auto.7.12.17893
Abstract | Full Text | PDF

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.

Open Access Article

NAC1 and HMGB1 enter a partnership for manipulating autophagy

Yi Zhang, Jay W. Yang, Xingcong Ren and Jin-Ming Yang
Pages 1557 - 1558
http://dx.doi.org/10.4161/auto.7.12.17910
Abstract | Full Text | PDF

Our recent study revealed a new role of nucleus accumbens-1 (NAC1), a transcription factor belonging to the BTB/POZ gene family, in regulating autophagy. Moreover, we found that the high-mobility group box 1 (HMGB1), a chromatin-associated nuclear protein acting as an extracellular damage associated molecular pattern molecule (DAMP), is the downstream executor of NAC1 in modulating autophagy. In response to stress such as therapeutic insults, NAC1 increases the expression, cytosolic translocation and release of HMGB1; elevated level of the cytoplasmic HMGB1 leads to activation of autophagy. The NAC1-HMGB1 partnership may represent a previously unrecognized pathway that regulates autophagy in response to various stresses such as chemotherapy.

Open Access Article

Phospholipid synthetic defect and mitophagy in muscle disease

Satomi Mitsuhashi and Ichizo Nishino
Pages 1559 - 1561
http://dx.doi.org/10.4161/auto.7.12.17925
Abstract | Full Text | PDF

Mitophagy, selective autophagy of mitochondria, has been extensively demonstrated in cultured cell models but has never been described in skeletal muscle in the context of muscle disease. We recently reported the first example of human muscle disease where mitophagy plays a role in the peculiar muscle pathology. This disease is caused by loss-of-function mutations in the CHKB gene encoding choline kinase β. “Patients” and rostrocaudal muscular dystrophy (rmd) mice, spontaneous Chkb mutants, develop congenital muscular dystrophy with a peculiar mitochondrial abnormality—mitochondria are markedly enlarged at the periphery of muscle fibers and absent from the center. Choline kinase is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. Our discovery demonstrates that a phosphatydilcholine biosynthetic defect leads to mitochondrial dysfunction and increased mitophagy.

Open Access Article

Primary lysosomal dysfunction causes cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy

SooYeon Lee, Yutaka Sato and Ralph A. Nixon
Pages 1562 - 1563
http://dx.doi.org/10.4161/auto.7.12.17956
Abstract | Full Text | PDF

Abnormally swollen regions of axons and dendrites (neurites) filled mainly with autophagy-related organelles represent the highly characteristic and widespread form of “neuritic dystrophy” in Alzheimer disease (AD), which implies dysfunction of autophagy and axonal transport. In this punctum, we discuss our recent findings that autophagic/lysosomal degradation is critical to proper axonal transport of autophagic vacuoles (AVs) and lysosomes. We showed that lysosomal protease inhibition induces defective axonal transport of specific cargoes, causing these cargoes to accumulate in axonal swellings that biochemically and morphologically resemble the dystrophic neurites in AD. Our findings suggest that a cargo-specific failure of axonal transport promotes neuritic dystrophy in AD, which involves a mechanism distinct from the global axonal transport deficits seen in some other neurodegenerative diseases.

Open Access Article

MAPKs regulate mitophagy in Saccharomyces cerevisiae

Kai Mao and Daniel J. Klionsky
Pages 1564 - 1565
http://dx.doi.org/10.4161/auto.7.12.17971
Abstract | Full Text | PDF

The autophagy-dependent selective degradation of mitochondria (mitophagy) plays an important role in removing excessive, damaged and dysfunctional mitochondria to maintain a proper cellular homeostasis. Relative to its significance in cell physiology, very little is known about the molecular machinery and regulatory mechanism of mitophagy in mammalian cells or yeast. We found that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is involved in both mitophagy and pexophagy (the selective degradation of peroxisomes through autophagy), whereas Hog1 functions specifically in mitophagy.

Open Access Article

E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells

Elodie Hatchi, Geneviève Rodier, Claude Sardet and Laurent Le Cam
Pages 1566 - 1567
http://dx.doi.org/10.4161/auto.7.12.17991
Abstract | Full Text | PDF

The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.

Open Access Article

Together we are stronger: Fusion protects mitochondria from autophagosomal degradation

Angelika S. Rambold, Brenda Kostelecky and Jennifer Lippincott-Schwartz
Pages 1568 - 1569
http://dx.doi.org/10.4161/auto.7.12.17992
Abstract | Full Text | PDF

Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.

Open Access Article

Autophagosome biogenesis requires SNAREs

Usha Nair and Daniel J. Klionsky
Pages 1570 - 1572
http://dx.doi.org/10.4161/auto.7.12.18001
Abstract | Full Text | PDF

We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.




VIEWS AND COMMENTARIES

Open Access Article

Impaired mitophagy at the heart of injury

Roberta A. Gottlieb, Robert M. Mentzer, Jr. and Phyllis-Jean Linton
Pages 1573 - 1574
http://dx.doi.org/10.4161/auto.7.12.18175
Abstract | Full Text | PDF

Recent publications link mitophagy mediated by PINK1 and Parkin with cardioprotection and attenuation of inflammation and cell death. The field is in need of methods to monitor mitochondrial turnover in vivo to support the development of new therapies targeting mitochondrial turnover.