Atg7 in development and disease: panacea or Pandora’s Box?

Macroautophagy is an evolutionarily conserved intracellular degradation system used by life ranging from yeasts to mammals. The core autophagic machinery is composed of ATG (autophagy-related) protein constituents. One particular member of the ATG protein family, Atg7, has been the focus of recent research. Atg7 acts as an E1-like activating enzyme facilitating both microtubule-associated protein light chain 3 (LC3)-phosphatidylethanolamine and ATG12 conjugation. Thus, Atg7 stands at the hub of these two ubiquitin-like systems involving LC3 and Atg12 in autophagic vesicle expansion. In this review, I focus on the pleiotropic function of Atg7 in development, maintenance of health, and alternations of such control in disease.


INTRODUCTION
As a cellular scavenger, autophagy is a fundamental catabolic process consisting of three primary classes of autophagy: macroautophagy (the most prevalent form of autophagy and hereafter referred to as autophagy), microautophagy, and chaperone-mediated autophagy (Feng et al., 2015). Central to the sequential events of autophagy is de novo formation of cupshaped isolation membranes (also known as phagophores) to sequester cytoplasmic components, expansion of this membrane to create a seal for a double membrane-bound vesicles called an autophagosome, and fusion of the autophagosome with a lysosome membrane to generate an autolysosome allowing degradation and recycling of the cargoes (Nakatogawa et al., 2009). Eukaryotic cells have evolved a well-organized autophagic machinery to adapt to and survive adverse microenvironmental conditions, including dwindling nutrient supplies (Galluzzi et al., 2014). Genetic screening of autophagy-deficient mutants in yeast provides us with almost 40 ATG (autophagy-related) genes, among which approximately 18 genes possess orthologues in higher eukaryotes. These ATGencoded products act as the core autophagy machinery and contribute to the sequential steps of autophagosome formation including (I) induction of autophagosome formation by Atg1 complex, (II) phagophore expansion by Atg9-related cycling system, (III) vesicle nucleation by the phosphatidylinositol 3-kinase complex, and (IV) vesicle expansion by two ubiquitin-like conjugation systems. One such protein is Atg7, which is uniquely shared by, and plays crucial roles in, the two ubiquitinlike conjugation systems of microtubule-associated protein light chain 3 (LC3, a mammalian homologue of Atg8) and Atg12 respectively (Feng et al., 2015).
The ubiquitin-like conjugation system of LC3 involves Atg3, Atg4, Atg7, and LC3 for LC3-phosphatidylethanolamine production, while the ubiquitin-like conjugation system of Atg12 involves the Atg5, Atg7, Atg10, Atg12, and Atg16 for Atg12-Atg5-Atg16 production. The common ubiquitin E1-like activating enzyme, Atg7, is essential for the assembly and function of these two conjugates in the expansion of autophagosomal membranes (Nakatogawa et al., 2009;Feng et al., 2015). Substantial progress has been made during the past decade revealing the pivotal roles of Atg7 in autophagyrelated cell homeostasis. Recent studies have unveiled the diverse and complex autophagy-dependent function of the evolutionarily conserved Atg7 in varying species, especially plants and animals. This review focuses on how this dynamic function is achieved and discusses the implications of altered Atg7-mediated autophagic activities in molecular, cellular, and organismal levels.

FUNCTION OF ATG7 IN PLANTS
Phenotypic analyses of Atg7 mutants indicates that Atg7 disruption renders Arabidopsis (Arabidopsis thaliana) cells hypersensitive to a shortage of nutrients with features of premature leaf senescence, though the mutant is otherwise normal (Doelling et al., 2002). Increased expression of multiple LC3 isoforms are observed in Atg7 mutants due to impaired control of the two ubiquitin-like conjugation systems of LC3 and Atg12 (Thompson et al., 2005). Deletion of Atg7 in Nicotiana benthamiana and Arabidopsis leads to unrestricted hypersensitive responses during plant innate immunity (Liu et al., 2005;Hofius et al., 2009). Arabidopsis Atg7 mutant is also more susceptible to fungal infection (Lenz et al., 2011). Minina and colleagues showed that the autotroph Arabidopsis can benefit from caloric restriction-induced lifespan extension via Atg7-regulated autophagy (Minina et al., 2013).

FUNCTION OF ATG7 IN INVERTEBRATES Nematode
A similar role of Atg7-regulated autophagy in dietary restriction-induced lifespan extension exists in Caenorhabditis elegans (C. elegans) . In addition, genetic inactivation of C. elegans Atg7 exacerbates accumulation of toxic polyglutamine expansion protein aggregates and accelerates progress of neurodegenerative disorders .

Fruit fly
Steroid-and radiation-triggered programmed cell death accompanies increased Atg7 transcripts in Drosophila cells (Lee et al., 2003). Normal levels of Atg7-modulated autophagy, albeit dispensable for metamorphosis, seem to be critical for preventing neurodegeneration, resisting stresses, and promoting longevity in Drosophila (Juhasz et al., 2007;Juhasz and Neufeld, 2008). Using the Drosophila eye as a model system, Chen et al. described Atg7 as a downstream effector of heat shock protein 27, and as a participant in the regulation of normal eye development, neuronal homeostasis, and lifespan . Such critical roles of fruit fly Atg7 has been reported not only in development but also in infection. Mycobacterium marinum infection is sufficient to counteract the effectiveness of antimycobacterial treatment, and thereby drastically affects the survival rate in Atg7 mutant Drosophila (Kim et al., 2012). In addition, a recent study reveals a novel example of Atg7-independent autophagy during the developmental shortening of Drosophila intestine (Chang et al., 2013).

FUNCTION OF ATG7 IN ZEBRAFISH AND RATS
In zebrafish, approximately one third of Atg7-knockdown morphants had ectopic expression of essential transcription factors and severe developmental defects in cardiac morphology encompassing heart looping, pericardial edema, and malformation of chamber and valve (Lee et al., 2014). Gain-and loss-of-function of Atg7 studies in the αB-crystallin R120G mutation (CryAB R120G ) model of rat desmin-related cardiomyopathy reveal the significant ability of ATG7 in reversing autophagic deficiency and maintaining physiological levels of basal autophagy (Pattison et al., 2011). As a consequence of Cathepsin B treatment, stimulated ATG7mediated autophagy aggravates lipotoxicity via induction of nod-like receptor 3 proinflammatory response in rat insulinoma cells (Li et al., 2013).

FUNCTION OF ATG7 IN MICE
To investigate the in vivo function of ATG7 in mammals, Komatsu et al. generated Atg7-deficient mice (Atg7 −/− ). As anticipated, Atg7 −/− mice exhibit impaired constitutive and starvation-induced autophagy; however, they die soon after birth (Komatsu et al., 2005). Therefore, Ubc-CreERT2 mice were crossed with Atg7-floxed (Atg7 flox/flox ) mice for the generation of tamoxifen-inducible whole body Atg7 knockout mice. Karsli-Uzunbas et al. further reported that acute systemic deletion of Atg7 in adult mice leads to perturbed glucose metabolism, but blocks the progression of non-small cell lung cancer (NSCLC) in vivo (Karsli-Uzunbas et al., 2014). Thus, cells from embryo/fetus of Atg7 −/− mice and certain cell/tissue-specific Atg7-knockout postnatal mice were extensively employed in the quest for understanding the mechanisms underlying the pleiotropic effects of ATG7 in development, physiology, and pathology (Table 1).

Embryonic fibroblasts
Wild-type (WT) mouse embryonic fibroblasts (MEFs) were used to recapitulate robust autophagy-mediated capability of bacteria clearance, which is absent in Atg7 −/− MEFs (Sun et al., 2008). Using WT and Atg7 −/− MEFs and small interference RNA (siRNA)-mediated silencing of Atg7 in BAX/ BAK-knockout MEFs, it has been demonstrated that Atg7regulated autophagy is dispensable for obatoclax-induced toxicity (McCoy et al., 2010). Subsequently, Lee et al. found a novel function of Atg7, independent of its E1-like enzymatic activity. Briefly, ATG7 coordinates tumor suppressor p53mediated cell division cycle and cell apoptosis via physical interaction with p53 under limited nutrients, providing an explanation for the simultaneous or sequential metabolic stress-induced events, including exit from cell cycle, induction of autophagy, and activation of cell death signaling. In addition, the augmented genomic instability in Atg7 −/− mice may be a reason for its postnatal death . To characterize the regulatory network of autophagy, quantita- autophagy depends on an intact cytoskeletal protein filamentous actin network (Zhuo et al., 2013).

Pancreatic β cells and skeletal muscle cells
RIP-Cre mice were crossed with Atg7 flox/flox mice for the generation of pancreatic β cell-specific Atg7 knockout (β cell-Atg7 −/− ) mice (Ebato et al., 2008;Jung et al., 2008;Wu et al., 2009). These mice display impaired glucose tolerance and degenerated islets accompanied by reduced β cell mass and insulin secretion levels. A series of morphological malformations occur in Atg7 mutant β cells, including accumulation of ubiquitinated inclusions, enlargement of mitochondria, and distension of the endoplasmic reticulum (Ebato et al., 2008;Jung et al., 2008). MCK-Cre mice were crossed with Atg7 flox/flox mice for the generation of skeletal muscle cellspecific Atg7 knockout (SMC-Atg7 −/− ) mice. Furthermore, Wu et al. observed a decrease of mitochondrial oxidation consumption and an increase of compensatory basal glycolytic rates and reactive oxygen species levels in cells derived from β cell-Atg7 −/− and SMC-Atg7 −/− mice .

Fat cells and mammary gland cells
Fab4 (aP2)-Cre mice were crossed with Atg7 flox/flox mice for the generation of adipocyte-specific Atg7 knockout (FC-Atg7 −/− ) mice. Targeted deletion of Atg7 in adipose tissues leads to a lean body mass with an elevated rate of β-oxidation and a low rate of lipolysis. The white adipose tissue in FC-Atg7 −/− mice acquired more features of brown adipose tissue, and its mass diminished (Zhang et al., 2009a;Singh et al., 2009b). Strikingly, disruption of ATG7 confers sensitivity to insulin stimuli (Zhang et al., 2009a). Additional evidence that ATG7 plays a vital role in adipogenesis has been obtained in 3T3-L1 preadipocytes, wherein inhibition of ATG7 hampered adipocyte differentiation and lipid accumulation (Singh et al., 2009b).
Interestingly, seemingly opposing effects of ATG7 in dopamine neurons have been delineated by different groups (Cheng et al., 2011;Inoue et al., 2012a;. DAT-Cre mice were crossed with Atg7 flox/flox mice for the generation of dopamine neuron (enriched in the substantia nigra pars compacta)-specific Atg7 knockout (DAT-Atg7 −/− ) mice (Inoue et al., 2012a;. Inoue et al. observed that DAT-Atg7 −/− exhibits an even more severe phenotype of age-dependent neurodegeneration than CamKII-Atg7 −/− mice (Inoue et al., 2012a). Conversely, conditional deletion of Atg7 in substantia nigra dopaminergic neurons of adult mice by intranigral injection of adenoassociated virus-Cre achieves unexpected protection in retrograde degeneration of dopaminergic axons. Additionally, this process is tightly controlled by Akt/Rheb/the kinase mammalian target of rapamycin (mTOR) signaling pathways (Cheng et al., 2011). Likewise, using DAT-Atg7 −/− mouse model, Hernandez et al. revealed that mTOR inhibitor rapamycin decreases evoked dopamine secretion and decelerates recovery in Dopamine neurons, which is an autophagy-dependent regulation of presynaptic neurotransmission .

Skin cells, kidney cells, and cardiomyocytes
K14-Cre mice were crossed with Atg7 flox/flox mice for the generation of epidermal keratinocyte-specific Atg7 knockout mice (Zhao et al., 2013;Rossiter et al., 2013). Using this mouse model, Zhao et al. highlighted the importance of ATG7 for the removal of reactive oxidized phospholipids and damaged protein aggregates in the epidermis exposed to environmental insults (Zhao et al., 2013). However, ATG7mediated autophagy appears to be nonessential to execute skin barrier function . ATG7 has also been documented as a core regulator in caspase-8 inhibition-induced autophagic cell death in mouse L929 skin fibroblast cells (Yu et al., 2004).
To test whether autophagy can ameliorate or restore proteinopathy in CryAB R120G cardiac model of cardiomyopathy, Bhuiyan et al. crossed ATG7-expressing mice and CryAB R120G mice to generate Atg7-crossed CryAB R120G mice. Indeed, the entire cohort of Atg7-crossed CryAB R120G mice acquire relatively sustained autophagy, leading to improved cardiac function (Bhuiyan et al., 2013).

FUNCTION OF ATG7 IN HUMAN
Prompted by the clues from model organisms, the architecture of the functional ATG7-mediated regulatory network has been explored in the settings of human biology and disease, Function of Atg7 REVIEW such as cancer, infectious disease, and neurodegenerative diseases (Fig. 1).

Cancer
As implied earlier, autophagy has been considered as both a pro-survival pathway and type 2 cell death (Kroemer and Levine, 2008). Two main hallmarks of cancer cells are unrestricted proliferation and suppressed cell death (Hanahan and Weinberg, 2011), raising the possibility of ATG7 as both an oncogene and a tumor suppressor. On one hand, ATG7 suppresses resistance of human breast cancer cells to photodynamic therapy (Xue et al., 2010). It also facilitates the anti-tumor actions of cytosolic FoxO1 and obatoclax in human NSCLC cells (Zhao et al., 2010;McCoy et al., 2010), compound 2-Methoxyestradiol in human osteosarcoma (Yang et al., 2013), and tetrandrine in human hepatocellular carcinoma (Gong et al., 2012). Moreover, the caspase-8 inhibition-initiated autophagic cell death program requires ATG7 via activation of receptor-interacting protein/c-Jun N-terminal kinase signaling in human U937 monocyte lymphoma cells (Yu et al., 2004). Additionally, two microRNAs, miR-17 and miR-137, have been shown to target ATG7 for acquisition of resistance to anticancer drugs and low-dose ionizing radiation treatments in human glioma cells  Torisu et al., 2013 Endothelial abnormalities Maintaining endothelial cell viability Han et al., 2012 Neural system Parkinson's disease Pathogenic polymorphism Metzger et al., 2010Metzger et al., , 2013Chen et al., 2013 Neuroblastoma Maintaining mitochondria levels Dagda et al., 2009 Glioma Tumor suppressor Comincini et al., 2013;Zeng et al., 2015 Figure 1. Schematic illustration of physiopathological roles of ATG7 in human.

REVIEW
Jianhua Xiong (Comincini et al., 2013;Zeng et al., 2015). On the other hand, ATG7 can also serve as an oncogene. Heat shock factor 1-controlled transcriptional expression of ATG7 is inversely correlated with the chemotherapeutic prognosis of breast cancer patients (Desai et al., 2013). Inhibition of redundant Atg7-mediated lysosome-autophagy pathway augments the anti-cancer effects of a proteasome inhibitor in some human prostate cancer cells , epidermal growth factor receptor-tyrosine kinase inhibitors in human lung cancer cells (Han et al., 2011), and cisplatin in human esophageal squamous cell carcinoma cells . Through reciprocal mechanical interaction, ATG7, rather than ATG5 and Beclin-1, represses caspase-9mediated apoptosis in human colon and cervical cancer cells. Caspase-9 promotes Atg7-mediated autophagy (Han et al., 2014). Notably, ATG7 has a relatively higher expression in human THP1 acute monocytic leukemia cells than a panel of human immune and epithelial cells (Rioux et al., 2007). Importantly, two physical interactions between ATG7 and acetyltransferase p300 and between ATG7 and transcription factor p53 have been depicted in human HeLa cervical and HCT116 colon cancer cells, respectively, in the context of limited nutrient availability (Lee and Finkel, 2009;Lee et al., 2012).

Infectious disease
Dual effects of ATG7-mediated autophagy intersection with human immunodeficiency virus (HIV) biogenesis fuel the viral yields as they do in human U937 monocytoid cells and in primary human macrophages (Kyei et al., 2009). A battery of morphological and biochemical assays have been conducted showing that hepatitis C virus (HCV) causes an unfolded protein response-dependent incomplete ATG7mediated autophagy during pathogenesis in human hepatoma cells (Sir et al., 2008). Also, it has been documented that disruption of ATG7-mediated autophagy can evoke the interferon signaling pathway resulting in apoptosis of HCVinfected immortalized human hepatocytes (Shrivastava et al., 2011), and dramatically enhanced infectivity of human papillomavirus in primary human keratinocytes (Griffin et al., 2013). Knockdown of Atg7 also interferes with the elimination of intracellular pathogen Mycobacterium tuberculosis by human immunity-related GTPase family M protein in U937 cells (Singh et al., 2006). ATG7-mediated autophagy is also involved in the constricting activity of HIV infection via release of the HIV-1 transactivator Tat in human embryonic kidney 293 cells and MAGIC5B cells (i.e., HeLa cells modified to express CD4 and CXCR4 together with β-galactosidase under the control of the HIV LTR promoter) (Sagnier et al., 2015). Silencing of ATG7 may delay the progression of Chikungunya virus-induced caspase-dependent cell death in human fibroblast cells and HeLa cells (Joubert et al., 2012), and suppress the colony stimulating factor-1-induced differentiation of human peripheral blood monocytes into macrophages (Jacquel et al., 2012).

Neurodegenerative disease
It has been shown that downregulation of ATG7 can compensate the loss of mitochondria in PTEN-induced kinase 1 (PINK1) deficient dopaminergic human neuroblastoma cells, likely supporting the potential role of ATG7 in PINK1 mutation-related familial Parkinson's disease (Dagda et al., 2009). By analyzing a large number of European Huntington disease patients, Metzger et al. found that the V471A polymorphism in ATG7 was significantly associated with the age at onset. More specifically, the V471A polymorphism in ATG7 correlates with an earlier disease onset of 4 years in a mixed group of Huntington disease populations (Metzger et al., 2010(Metzger et al., , 2013. In five patients with Parkinson's disease, four novel genetic variants including 11313449G>A, 11313811T>C, 11313913G>A, and 11314041G>A, were identified on the ATG7 gene promoter, implying the altered transcriptional activity of the ATG7 may be a risk factor .

Miscellaneous
Together with FOXO3-ATG101 complex, coupling of acetylated FOXO1 with ATG7, upon stimulation with prosecretory mitogen lacritin, can rescue the metabolic homeostasis in human corneal epithelial cells . In a similar manner, acetylated FOXO1 and ATG7 can preserve human umbilical vein endothelial cells (HUVECs) viability under circumstances of oxidative stress (Han et al., 2012). ATG7 is also essential for normal secretion of VWF in HUVECs (Torisu et al., 2013), and EndMT in both HUVECs and human pulmonary aortic endothelial cells (Singh et al., 2015).

CONCLUDING REMARKS
The word "autophagy", literally auto-, meaning "self", and phagein, meaning "to eat", in Greek, was originally coined by Belgian cytologist Christian de Duve in 1963 (Klionsky, 2008). More than 50 years have passed since autophagy was defined as a core mechanism underlying both elimination and recycling of intracellular materials in normal development and diverse disease categories (Mizushima and Komatsu, 2011;Choi et al., 2013;Murrow and Debnath, 2013). These include, but are not limited to, immunity (Virgin and Levine, 2009), metabolism (Codogno and Meijer, 2010;Rabinowitz and White, 2010), aging (Madeo et al., 2010), and the cardiovascular Nussenzweig et al., 2015), and nervous system (Komatsu et al., 2006b). Although still in the early stages, it appears to be almost clear how core machinery plays in Atg7-dependent and -independent autophagosome biogenesis (Nishida et al., 2009;Lamb et al., 2013). No less important than this ATG7mediated autophagic assembly is the function and regulation of Atg7 in natural and stressed pathophysiological conditions. Thanks to the dedication and contribution of numerous laboratories over the years, recent exciting findings on ATG7 have caused a paradigm shift in the field of ATG7-mediated autophagic regulation. A brief historical overview of select prior landmark investigations has been summarized in the timeline of Fig. 2. Given that these studies reflect the nature of ATG7's intrinsic double-edged sword in development and disease, it is likely that excessive or deficient Atg7-mediated autophagy is harmful. Following advances in therapeutic manipulation of autophagy (Kroemer, 2015), it will be important to determine the specific and safe methods for pharmacologic fine-tuning of ATG7 activity. Another cardinal question concerns personal medicine for accurate and rapid diagnosis of ATG7-related disease. The complex role of ATG7 seems to be highly structured in a spatiotemporal fashion rather than ad libitum (Behrends et al., 2010). The diverse roles of Atg7 in different settings summarized by this review may be attributed to selective Atg7-mediated autophagy at distinct organelle, cell, tissue, organ, and organism levels. An accurate understanding of the specified and delicate roles of Atg7-autophagy requires the in-depth knowledge of both the contextual extracellular cues and the intracellular responses. Thus, it will be interesting to search for the exact niches responsible for how Atg7 activity is encoded. It is also plausible that certain intricate forms of crosstalk interactions between Atg7-mediated autophagy and other autophagy-dependent or -independent pathways are responsible for shaping the versatile functions of Atg7. Therefore, it might be essential to identify and characterize the key coordinators of Atg7-mediated autophagy and other regulatory networks. Despite these challenges to be faced, academic and industry's research progress in Atg7 offers new avenues towards refined autophagic mechanism and Atg7-based clinical treatment.